Ethnobotany, phytochemistry and DNA metabarcoding studies on

Indian traditional medicine

Seethapathy Gopalakrishnan Saroja

Dissertation presented for the degree of Philoshiae Doctor (PhD)

Section for Pharmaceutical Chemistry, Department of Pharmacy

&

Department of Research and Collections, Natural History Museum

The Faculty of Mathematics and Natural Sciences

University of Oslo

2019

© Seethapathy Gopalakrishnan Saroja, 2019 Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo No. 2140 ISSN 1501-7710 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. Cover: Hanne Baadsgaard Utigard. Print production: Reprosentralen, University of Oslo.

Table of contents

List of papers……………………………………………………………………………....….1

Summary………………………………………………………………………………………2

Abbreviations………………………………………………………………………………....4

1) Introduction………………………………………………………………………………..6

1.1) Ethnobotany.……………………………………………………………………………...6

1.1.1) Ethnopharmacology and traditional medicine………………………………….8

1.1.2) Dioecy in angiosperms and traditional medicine……………………………….11

1.2) Herbal products and commercialization………………………………………...………..12

1.2.1) Authenticity issues of herbal products……………………………………….. .14

1.2.2) Regulatory status of herbal products…………………………………..……….16

1.2.3) Quality control methods………………………………………………………..17

1.2.3.1) Macroscopic and microscopic authentication………………………..18

1.2.3.2) Chromatographic and spectroscopic methods………………………..19

1.2.3.3) DNA based identification…………………………………………….21

2) Aim of the thesis…………………………………………………………………………..27

3) Materials and methods……………………………………………………………………28

3.1) International legislative framework……………………………………………...28

3.2) Ethnobotanical field studies……………………………………………………...29

3.2) Material collection…………………………………………………………….…29

3.3) Extraction, isolation, and characterization of chemical compounds………….…30

3.4) DNA barcoding methods…………………………………………………….…..31

4) Summary of results………………………………………………………………….……32

5) Discussion………………………………..………………………………………….…….40

6) Concluding remarks and perspectives……………………………………………….…50

Acknowledgements……………………………………………………………………….…52

References……………………………………………………………………………….…..54

Papers I-IV

1

List of papers

This thesis is based on the following four Papers, which are referred to in the text by their Roman

numerals (I-IV).

I. Gopalakrishnan Saroja Seethapathy, Kaliamoorthy Ravikumar, Berit Smestad Paulsen,

Hugo J. de Boer and Helle Wangensteen. (2018). Ethnobotany of dioecious species:

Traditional knowledge on dioecious plants in India. Journal of Ethnopharmacology, 221:

56-64. doi: 10.1016/j.jep.2018.04.011

II. Gopalakrishnan Saroja Seethapathy, Christian Winther Wold, Kaliamoorthy Ravikumar,

Hugo J. de Boer and Helle Wangensteen. (2019). Ethnopharmacology, biological activities,

and chemical compounds of Canarium strictum: an important resin-yielding medicinal tree

in India. Manuscript.

III. Gopalakrishnan Saroja Seethapathy, Margey Tadesse, Santhosh Kumar J.

Urumarudappa, Srikanth V. Gunaga, Ramesh Vasudeva, Karl Egil Malterud, Ramanan

Uma Shaanker, Hugo J. de Boer, Gudasalamani Ravikanth and Helle Wangensteen. (2018).

Authentication of Garcinia fruits and food supplements using DNA barcoding and NMR

spectroscopy. Scientific Reports, 8(1):10561. doi: 10.1038/s41598-018-28635-z

IV. Gopalakrishnan Saroja Seethapathy, Ancuta-Cristina Raclariu, Jarl Andreas Anmarkrud,

Helle Wangensteen and Hugo J. de Boer. (2019). DNA metabarcoding authentication of

Ayurvedic herbal products on the European market raises concerns of quality and fidelity.

Frontiers in Plant Science, 10(68). doi: 10.3389/fpls.2019.00068

2

Summary

Medicinal plants form the basis of traditional medicine health systems and play an important role

in meeting primary healthcare needs around the world. At the same time, traditional knowledge on

medicinal plants is eroding and deteriorating due to ongoing cultural, ecological and socio-

economical changes. This poses a serious threat to biodiversity-based cultural knowledge and can

cause an attrition of leads for drug discovery. On the other hand, it is also important to validate

traditional knowledge on plant use using scientific methods to promote or discourage their wider

usage. Therefore, the first objective of the thesis is to document traditional knowledge on medicinal

plants in Southern India, and validate traditional knowledge of selected medicinal plant using

phytochemical methods and biological assays. Paper I aimed to document traditional knowledge

on dioecious plants of India. Specifically it addressed the research questions: do folk healers have

preference for plants of a specific gender? If so, what are those plants? In addition, do folk healers

differentially utilize male and female plant of a particular species for food, medicines or timber?

The study found that informants recognize the phenomenon of dioecy in plants, and reported

gender preferences for a number of species with respect to uses as timber, food and medicine.

Paper II aimed to document the medicinal uses of Canarium strictum Roxb. (Burseraceae) by folk

healers in India, and to investigate the chemical constituents and biological activities of the resin

and stem bark. Our results revealed the presence of α-amyrin and β-amyrin as the major compounds

in the resin. Whereas gallic acid, methyl gallate, scopoletin, 3,3'-di-O-methylellagic acid 4-O-α-

arabinofuranoside, elephantorrhizol (3,3',4',5,6,7,8-heptahydroxyflavan) and procyanidins were

isolated from the bark methanol extract. It is noteworthy that the finding of elephantorrhizol in C.

strictum is of chemotaxonomic interest as it is the first report from the family Burseraceae.

Furthermore, radical scavenging and 15-lipoxygenase inhibitory activities were tested in resin and

bark extracts, but no toxicity towards Artemia salina nauplii was found. Similarly, dose dependent

inhibition of NO production was observed in resin and dichloromethane bark extracts.

Traditional medicine based herbal products have gained increasing popularity in developing

countries as complementary therapies. However, herbal products are prone to contamination,

adulteration and substitution, and this raises quality and safety concerns for the public. Therefore,

the second part of thesis focused on molecular authentication of raw herbal drugs and marketed

herbal products using NMR spectroscopic and DNA methods. The aim of Paper III was to assess

3

the adulteration of morphologically similar samples of Garcinia using DNA barcoding, and to

quantify the content of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone in raw herbal

drugs and Garcinia food supplements using NMR spectroscopy. Our DNA barcoding study

revealed that there was no adulteration in raw herbal drugs of Garcinia. Whereas, analysis of ten

Garcinia food supplements revealed a large variation in the content of (−)-hydroxycitric acid

content per capsule or tablet. Paper IV aimed to test the composition and fidelity of Ayurvedic

products marketed in Europe using DNA metabarcoding. Our analysis revealed that the fidelity for

single ingredient products was 67%, and the overall ingredient fidelity for multi ingredient products

was 21%, and for all products 24%.

4

Abbreviations

ABS Access and Benefit Sharing

ASE Accelerated Solvent Extraction

AFLP Amplified Fragment Length Polymorphism

API Ayurvedic Pharmacopoeia of India

ARMS Amplification Refractory Mutation System

CAM Complementary and Alternative Medicine

CBD Convention on Biological Diversity

CE Capillary Electrophoresis

CTAB Cetyl trimethyl ammonium bromide

DCM Dichloromethane

DNA Deoxyribonucleic acid

EFSA European Food Safety Authority

EMA European Medicines Agency

EtOAc Ethyl acetate

EU European Union

FDA Food and Drug Administration

GC Gas Chromatography

H2O Water

HPLC High Performance Liquid Chromatography

HPTLC High Performance Thin Layer Chromatography

HTS High-Throughput Sequencing

ITS Internal Transcribed Spacer

MeOH Methanol

MOTU Molecular Operational Taxonomic Unit

MS Mass Spectrometry

5

NIR Near-Infrared Spectroscopy

NMR Nuclear Magnetic Resonance

PCR Polymerase Chain Reaction

RAPD Random Amplified Polymorphic DNA

rDNA Ribosomal DNA

rbcL Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit

RFLP Restriction Fragment Length Polymorphism

SCAR Sequence Characterized Amplified Region

SSR Simple Sequence Repeat

TBGRI Tropical Botanical Garden and Research Institute of India

TLC Thin Layer Chromatography

TM Traditional Medicine

WHO World Health Organization

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1) Introduction

1.1) Ethnobotany

The term Ethnobotany was suggested by John William Harshberger in 1896. Originally it was

applied to the study of the utilitarian relationship between humans and the plant environment in

primitive settings, and Harshberger (1896) noted that “ethnobotany aids in elucidating the cultural

position of the tribes who used the plants for food, shelter or clothing”. However, ethnobotany has

been given various definitions over time, and has now evolved into a much broader meaning that

covers not only a utilitarian relationship, but also relationships that embrace the symbolic,

ecological and cognitive, as well as the human–plant relationship in a modern setting (Soejarto et

al., 2005; Leonti, 2011). Ethnobotany was defined by Catherine M Cotton in 1996 as

“encompassing all studies which concern the mutual relationships between plants and traditional

people” (Cotton, 1996). Albuquerque et al. (2017) defines ethnobotany as “the study of the direct

interrelationship between people of extant cultures and the plants in their environment”. In simple

terms ethnobotany refers “to study of the interactions between people and plants” (Martin, 2004).

Under the utilitarian approach ethnobotanical studies allow the documentation of the use of plants

and its associated traditional knowledge by ethnic communities (Berlin, 1992). Such

documentation is an important part in understanding and analyzing elements of traditional

knowledge, it serves as base line data for future research, and aids to preserve the traditional

knowledge for both communities and future generations (Awas et al., 2010; Leonti, 2011; de Boer

and Cotingting, 2014).

One of the sub-disciplines of ethnobotany is ethnomedicine, and ethnomedicine studies the plants

used as medicine by people (Neumann and Lauro, 1982; Lee and Balick, 2001). Knowledge of

traditional medicines are preserved mostly by oral tradition, but with the advent of writing systems,

different cultures have documented their knowledge into pharmacopoeias or materia medica

(Leonti, 2013). The first record of poppy uses dates back to 6000 B.C., cuneiform tablets from the

Mesopotamian basin refers the medicinal properties of opium calling the "plant of joy" (Brook et

al., 2017). Similarly, another written evidence on plant usage for the preparation of drugs dates

back to 3000 B.C. and has been found on a Sumerian clay slab comprising twelve recipes for

preparation of drugs referring to over 250 various plants (Petrovska, 2012). The Ebers Papyrus, an

Egyptian medical papyrus, dates back to 1550 B.C. and contains about 700 formulas and remedies

7

(Petrovska, 2012; Rahman et al., 2018). The earliest documentation of Chinese materia medica

dates back to around 1100 B.C. with several numbers of drug descriptions (Leung, 2006). Ayurveda

is one such materia medica and it dates back to at least 200 B.C. in India (Chaudhury and Rafei,

2001; Jaiswal and Williams, 2017). The Sanskrit word "Ayurveda" consists of two words ‘Ayu’

which means life and ‘Veda’ which means knowledge or science. Thus "Ayurveda" in totality

means ‘Science of life’. The genesis of Ayurveda is believed to be based on four eminent

compilations of knowledge (Vedas) called Yajur Veda, Rig Veda, Sam Veda and Atharva Veda

(Jaiswal and Williams, 2017; Mukherjee et al., 2017). Maharishi Charaka is an Indian sage who

compiled all aspects of Ayurvedic medicine as “Charaka Samhita”, and this compilation is one of

the first and the most important ancient authoritative writings on Ayurveda (1000 BC). A number

of compilations like Nighantu Granthas, Madhava Nidana and Bhava Prakasha are from the

contributions of various scholars (Jaiswal and Williams, 2017; Mukherjee et al., 2017). The

materia medica of Ayurveda describes the uses of over 1,500 medicinal plants and 10,000

formulations (Joshi et al., 2017). However, there are many ethnic groups around the globe that

continue to orally transmit their indigenous knowledge systems that are yet to be documented

(Totelin, 2009).

The World Health Organization (WHO) defines Traditional Medicine (TM) as a medical system

that “includes diverse health practices, approaches, knowledge and beliefs incorporating plant,

animal, and/or mineral based medicines, spiritual therapies, manual techniques and exercises,

applied singly or in combination to maintain well-being, as well as to treat, diagnose or prevent

illness” (WHO, 2002). Based on this definition, traditional medicine and practices can be classified

broadly into:

(a) codified systems of traditional medicine which have a systematic body of knowledge in the

form of ancient scriptures such as Ayurveda, Siddha, Unani, Tibetan medicine and Traditional

Chinese medicine (Ved and Goraya, 2007; Upadhya et al., 2014), or

(b) non-codified systems of traditional medicine or folk medicine in which the medicinal

knowledge, skills and practices are passed on orally from generation to generation (Ved and Goraya,

2007; Upadhya et al., 2014).

8

While these two types of TMs are found in India, the non-codified systems of traditional medicine

(folk medicine) are diverse and vary with geography, regional flora and culture, and these have

often developed in accordance with the primary needs and locally available resources of a particular

region (de Boer and Lamxay, 2009; Weckerle et al., 2009; de Boer et al., 2012). Each ethnic culture

has its own relationship with the environment and a medical knowledge that uses specific medicinal

species (Macía et al., 2005). However, the practice of TM is diminishing with time due to several

factors including cultural, ecological and socio-economical changes, the abandonment and

devaluation of traditional customs, particularly the influence of urbanization and influence of

western lifestyles, and the increasing reliance on conventional medicine (Benz et al., 2000; Byg

and Balslev, 2004; Srithi et al., 2009; Vandebroek and Balick, 2012).

1.1.1) Ethnopharmacology and traditional medicine

The term Ethnopharmacology was formally introduced in 1967 in the title of a book on

hallucinogens: Ethnopharmacological search for psychoactive drugs (Efron, 1967). After the

initial use of the term ethnopharmacology in the context of hallucinogenic plants, it was only used

occasionally until 1979. Ethnopharmacology was established more definitively when the Journal

of Ethnopharmacology was founded by Laurent Rivier and Jan Bruhn in 1979 (Rivier and Bruhn,

1979), and the scope ethnopharmacology was broadened to ‘a multidisciplinary area of research

concerned with the observation, description, and experimental investigation of indigenous drugs

and their biological activity’ (Rivier and Bruhn, 1979). Though the term ethnopharmacology is

relatively new, researchers in the past have dealt with traditional uses of pharmacologically active

compounds in a comprehensive way so that their investigations would nowadays be classified as

ethnopharmacological research (de Smet and Rivier, 1989; Heinrich and Jäger, 2015). For example

William Withering (1741-1799) systematically studied the medicinal properties of foxglove

(Digitalis purpurea L., Scrophulariaceae) which was traditionally used to treat dropsy. He used

orally transmitted knowledge to develop a medicine that can be used by physicians. Prior to such

studies, traditional medicine practices were more interested in the patient’s welfare and less focused

on the systematic study and chemical properties of the employed medicinal plants (Heinrich and

Jäger, 2015). Ethnobotany and ethnopharmacology have often been seen as a tool for drug

discovery (Heinrich et al., 2006). Ethnobotanical studies not only focus on medicinal plants, but

also on other products derived from plants, such as foods, shelter, coloring agents, fiber plants,

9

poison, fertilizer, ornamentals and oil. Ethnopharmacological studies focus only on the study of

indigenous medicines derived from natural resources with a special focus on the evaluation of such

therapeutic uses by empirical science (e.g. pharmacological, phytochemical and toxicological).

Ethnopharmacology is defined by Michael Heinrich as, “a scientific approach to the study of the

biological activities of any preparation used by humans, which have, in a very broad sense, either

beneficial or toxic or other direct pharmacological effects” (Heinrich, 2014).

Ethnopharmacological studies and/or traditional medicines guided drug discovery have been the

basis for modern science and several studies have highlighted the importance of

ethnopharmacological studies for drug development (Patwardhan and Mashelkar, 2009; Graziose

et al., 2010; Zhao et al., 2015). A noteworthy recent recognition of this drug discovery process is

the 2015 Nobel Prize in Physiology or Medicine which was awarded to Youyou Tu for the

discovery of artemisinin from Artemisia annua L., (Compositae). Artemisinin was isolated in 1972

using information from the well-documented Chinese compendium of materia medica written

by Shizhen Li (1518 -1593) and Ge Hong's 'handbook of prescriptions for emergencies' from 340

AD to combat malaria (as reviewed in Kong and Tan, 2015; Su and Miller, 2015).

Ethnopharmacology has provided some very notable successes in discovering drug molecules,

including artemisinin isolated in 1972, morphine isolated in 1804 (as reviewed in Brook et al.,

2017), quinine isolated in 1820 (as reviewed in Willcox et al., 2004), atropine isolated in 1833 (as

reviewed in Shutt and Bowes, 1979), podophyllotoxin isolated in 1880 (as reviewed in Cragg et

al., 2011), ephedrine isolated in 1897 (as reviewed in Preuss and Bagchi, 2012), and vinblastine

isolated in 1958 as reviewed in Raviña, 2011). These compounds, or their analogs and derivatives,

are still in widespread use. Morphine is an alkaloid derived from Papaver somniferum L. (opium

poppy), and is one of the best known examples of a drug discovered based on traditional medicine.

The poppy plant has a long history of medicinal use as pain killer and as sedative, with morphine

being a responsible compound for the pharmacological activity. The first record of poppy uses

dates back to 6000 B.C., cuneiform tablets from the Mesopotamian basin refers the medicinal

properties of opium calling the "plant of joy" (Brook et al., 2017). The Ebers papyrus, an Egyptian

scroll from approximately 1500 B.C., lists opium, along with other medicinal plants such as garlic,

mandrake and aloe. The Greek physician Hippocrates (460-370 BC) recommended whole poppy

heads, not just opium, soaked in water to treat dropsy. However, the first ethnopharmacological

study on poppy began in the 15th century AD, when Theophrastus Bombastus Von Hohenheim

10

developed a tincture of opium which he called laudanum (Brook et al., 2017). Later, Friedrich

Wilhelm Adam Serturner isolated opium’s active ingredient in 1805 and named the crystalline

powder ‘morphium’. With the help of J.L. Gay-Lussac, the name ‘morphium’ was changed to

‘morphine’. Furthermore in 1819 C.F. Wilhelm Meissner classified morphine as the first compound

known as an alkaloid (Brook et al., 2017). In 1925, Sir Robert Robinson uses the chemical principle

of aromatization to rationalize the structure of morphine, and he was awarded the Nobel Prize in

Chemistry in 1947 for his achievements in morphine research (Robinson and Sugasawa, 1931,

1932, 1933; Bentley, 1987). Apart from morphine, three other important alkaloids found in opium

are codeine, noscapine and papaverine (Bernhoft et al., 2010; Brook et al., 2017).

Some of the successful ethnopharmacology lead drug compounds from Ayurvedic medicinal plants

(Balachandran and Govindarajan, 2007) are andrographolide isolated from Andrographis

paniculata (Burm. F.) Nees in 1911 (Chakravarti and Chakravarti, 1951; Smith et al., 1982),

vasicine isolated from Justicia adhatoda L. (Syn. Adhatoda vasica Nees) in 1924 (Sen and Ghose,

1924), allicin isolated from Allium sativum L. in 1944 (Cavallito and Bailey, 1944), and reserpine

isolated from Rauvolfia serpentina (L.) Benth. ex Kurz in 1952 (Müller et al., 1952; Curzon, 1990).

Case study: Kani berries in India

An interesting case study from India demonstrates how traditional medicine guided drug

development has been a global model for recognizing the traditional knowledge and drug discovery.

According to a World Intellectual Property Organization report (Gupta, 2004), during an

ethnobotanical expedition in 1987 by the All India Coordinated Research Project on Ethnobiology,

in which members of the Kani ethnic group were guiding researchers on an expedition organized

by the Tropical Botanical Garden and Research Institute of India (TBGRI), the researchers noticed

that the Kani were not getting tired, despite significant physical exertion and they were constantly

chewing on some black berries. On inquiring with the Kanis, the researchers were given a few of

the black berries to chew on, after which they felt revitalized. The researchers realized that the

berries had properties that relieve fatigue (Chaturvedi, 2009). The plant was later identified as

arogyapacha (Trichopus zeylanicus Gaertn.). The researchers from TBGRI collected the berries

for laboratory research and after a decade of research they managed to isolate several active

chemical compounds with beneficial properties (Chaturvedi, 2009). Incidentally, during their

research it was discovered that the leaves were more beneficial than the berries in terms of

11

bioactivity (Gupta, 2004; Chaturvedi, 2009; Reddy and Lakshmikumaran, 2015). Subsequently, in

1994, the researchers at the TBGRI filed for patents and licensed the same to an Indian

pharmaceutical company for USD 50,000 plus 2 % royalties on all sales. In 1997 the TBGRI

assisted the Kani in setting up a trust to document their traditional knowledge that placed them in

control of the first payment of USD 12,500 for development (Moran, 2000; Gupta, 2004; Reddy

and Lakshmikumaran, 2015).

1.1.2) Dioecy in angiosperms and traditional medicine

Angiosperms (flowering plants) are the most widespread and diverse group of plants, and the major

sexual systems of angiosperms are bisexual (~90 %), including those with hermaphroditic flowers

and those that are monoecious (~5 %), i.e., with separate male and female flowers on the same

individual (Charlesworth, 2002). A minority of plant species are ‘sexually polymorphic’, which

are dioecious (having separate sexes containing either only male or female flowers in a plant),

gynodioecious (having both hermaphrodite and female only flowers), androdioecious

(hermaphrodite and male), and polygamodioecious (populations with bisexual individuals, male

individuals, and female individuals) (Charlesworth, 2002). In general, the three most important

components for the survival of a plant is the maintenance of vegetative growth, the struggle for

existence with competitors (defense), and the reproduction. However, each of these components

and their activities requires the expenditure of energy, which is in limited supply, and current

investments in each one of these activities result in losses in the potential investments in the other

(Obeso, 2002). Therefore, there is a trade-off at the physiological level of resource allocation to

vegetative growth, defense or reproduction in plants. The evolution of dioecy has long intrigued

evolutionary biologists (Renner, 2014), and there are a number of studies that have used dioecious

plants to understand the cost of reproduction and resource trade-offs in plants (Obeso, 2002).

Trade-offs between allocation of resource to defense, growth and reproduction among genders of

dioecious plants varies and has shown to affect the production and concentration of secondary

metabolites (Obeso, 2002; Simpson, 2013; Milet-Pinheiro et al., 2015; Bajpai et al., 2016).

Understanding the ecology of plant biodiversity has been highlighted as an important strategy for

drug discovery (Coley et al., 2003), as well as ethnobotanical studies and/or traditional medicines

for drug development (Patwardhan and Mashelkar, 2009). In India, it is estimated that 8,000 plants

have medicinal usages. Considerable evidence for sex-biased herbivory and variation in secondary

12

metabolites in dioecious plants is available in scientific studies, but little is known about traditional

concepts and preferences for dioecious plants, either male or female.

Tinospora cordifolia (Willd.) Miers) (Menispermaceae) is a widely distributed and commonly used

dioecious medicinal plant used in Ayurvedic medicine in India for several therapeutic properties

(Panchabhai et al., 2008). A significant variation in the concentration of alkaloids were reported

between male and female plants of T. cordifolia (Bajpai et al., 2016), and an in vitro cytotoxicity

evaluation revealed that the stem extracts of male T. cordifolia was more effective in inhibiting the

growth of cancerous cell lines compared to the female plant stem extracts (Bajpai et al., 2017a;

Bajpai et al., 2017b). On the contrary, female plant stem extract caused a significant up regulation

in the pro-inflammatory and anti-inflammatory cytokines and activated the peritoneal exudate cells

leading to significant higher release in reactive oxygen species and enhanced the in vitro

lymphocyte proliferation more than male stem extract (Bajpai et al., 2017a; Bajpai et al., 2017b).

These findings highlight the importance of studying the variation in secondary metabolites and

bioactivity of male and female plants in dioecious species.

1.2) Herbal products and commercialization

Traditionally, assuring the quality and safety of traditional medicines was the responsibility of the

traditional medicinal practitioner who collected and prepared the medicine in small amounts for

curing diseases (Valiathan, 2006; Weston, 2009). WHO defines herbal medicine as (i) the use is

well-established and widely acknowledged, i.e., the use represents the accumulated experience of

many practitioners over an extended period of time, (ii) the use of the herbal medicine, including

dosage, indication, and administration route is well-established and documented; and (iii) the use

is generally and currently regarded as safe (WHO, 1998). However, in recent decades traditionally

used herbal medicines have continued to become mainstream commodities driven by the health

industry from craft-based tradition to globalized industry (Jagtenberg and Evans, 2003; Moahi,

2007). Opening up and interconnectedness of the world via globalization have opened up economic

and internationalized trade markets worldwide (Jagtenberg and Evans, 2003; Moahi, 2007). Such

globalization has paved the way for the herbal industry and its market demand across nations.

Countries like India and China with a long history of traditional medicine are utilizing such

opportunities of globalization and internationalized trade markets to promote their traditional

medicinal products to improve their economy (Sen et al., 2011). Traded medicinal herbal products

13

are defined as “finished, labelled pharmaceutical products in dosage forms that contain one or

more of the following: powdered plant materials, extracts, purified extracts, or partially purified

active substances isolated from plant materials” (WHO, 1998).

Based on the historical and economic development in a country, a nation’s traditional system of

medicine is often complemented with conventional medicine. Conversely, in other regions,

alongside conventional medicine different traditional systems of medicine and alternatives

(complementary medicine) are established (Leonti, 2013). Complementary and alternative

medicine (CAM) is referred to as a broad set of healthcare practices that are not part of that

country's own tradition and are not integrated into the dominant healthcare system (WHO, 2013).

Use of herbal medicine is one among the practices of CAM (Fischer et al., 2014) and the usage of

TM based herbal medicine is increasing worldwide (WHO, 2013). According to the CAMbrella

consortium in Europe (Fischer et al., 2014), over 100 million Europeans are TM & CAM users and

the usage of herbal medicine is the most commonly reported CAM therapy in Europe (WHO, 2013;

Fischer et al., 2014; Hegyi et al., 2015). Based on the National Health Interview Survey from 2002

to 2012, one-third of adults in USA used some form of CAM (Clarke et al., 2015). With the

substantial growth, and increasing evidence for the usage of CAM in developed countries (WHO,

2013), the WHO traditional medicine strategy for the period of 2014-2023 aims to facilitate the

integration of TM & CAM into the national health care systems of WHO member’s states, and also

aims to strengthen the quality assurance, safety, efficacy, and proper use of herbal medicines by

various measures (WHO, 2013). Some of the reasons for the popularity of herbal products are in

addition to the belief that herbal products are safe and have no side effects because they are natural,

the desire for the personal therapeutic regime, as natural herbal products that can be freely

combined with pharmaceutical drugs (Ernst, 2004; Byard et al., 2017). Also access to traditional

medicines does not require a prescription and products can be purchased as over the counter

products, prominently advertised in the popular media, marketed and distributed via channels,

including pharmacies, natural herbal shops, and online retail stores (Ernst, 2004; Byard et al., 2017).

Ayurveda is one among the traditional medicines recognized as a CAM by WHO (WHO, 2013),

and among the core primary healthcare options in India. A predominant proportion of the Indian

population uses traditional medicines for their healthcare needs (Katoch et al., 2017; Srinivasan

and Sugumar, 2017). Ayurvedic medicines are commonly used as crude materials or extracts for

14

therapeutic purposes (Joshi et al., 2017). In India, it is estimated that approximately 7,000 plants

have medicinal usages in codified and non-codified Indian systems of medicine, of which

approximately 1,178 plants are reported to be actively traded. The total commercial demand for

herbal raw drugs in India for the year 2014-2015 is estimated to be 512,000 metric tons with a

value of more than 1 billion USD (Goraya and Ved, 2017). India has over 8,000 licensed medicinal

drug manufacturing units, and in order to bring uniformity among the manufacturers and to follow

the same formula of ingredients, the Ayurvedic formulary and Ayurvedic Pharmacopoeia of India

(API) was been compiled to implement the standard norms of the Drugs and Cosmetics Act

(Mukherjee et al., 2012; Katoch et al., 2017).

1.2.1) Authenticity issues of herbal products

Herbal products as a commodity are not without safety or quality concerns, and the growing

commercial interest in herbal products increases the incentive for adulteration and substitution in

the medicinal plants market (Raclariu et al., 2018). An increasing awareness among consumers,

and several media reports on herbal product adulteration and mislabeling are calling attention to

the quality of traded herbal products that directly affect consumer safety (Ouarghidi et al., 2012;

Walker and Applequist, 2012; Newmaster et al., 2013). The most important issues affecting the

quality of the herbal products is adulteration (de Boer et al., 2015). Herbal product adulteration can

be deliberate in order to maximize the profits, and deliberate adulteration leads to the use of

alternate plant parts other than the parts used traditionally or totally other plant materials of inferior

quality. For example, based on different pharmacopoeias, the roots of Withania somnifera (L.)

Dunal (Indian ginseng) are considered to be a medicinally potent plant part due to the

pharmacologically active withanolides (Mundkinajeddu et al., 2014). The root samples are

supposed to be marketed based on the content of withanolides; however, the aerial parts of the plant

also contain the marker compounds along with flavonoid glycosides specific to the aerial parts of

the plant. As a result of the increase in the global herbal trade, the roots of these plants are often

mixed with the aerial parts of W. somnifera and are then fraudulently marketed (Mundkinajeddu et

al., 2014). Addition of synthetic substances to herbal products is also common under fraudulent

adulteration (Calahan et al., 2016; Rocha et al., 2016). For instance, herbal products for sexual

enhancement have been found to be adulterated and counterfeited with sildenafil, tadafil, vardenafil,

and several other synthetic derivatives (Gratz et al., 2006; Balayssac et al., 2009; Balayssac et al.,

15

2012); slimming herbal products with sibutramine and fenfluramine (Balayssac et al., 2009; Vaysse

et al., 2010; Monakhova et al., 2012) and body-building products with anabolic steroids

(Klinsunthorn et al., 2011).

Herbal product adulteration is also often due to misidentification or substitution with allied

congeneric species and geographically co-occurring species (Mitra and Kannan, 2007; de Boer et

al., 2015). For example, phenotypically very similar Phyllanthus species and Berberis species that

could easily be misidentified and mixed within herbal products, are collected from the wild by local

farmers or collectors who often rely only on their experience in identifying the species, and the

services of specialists like taxonomists are rarely used for authentication (Srirama et al., 2010;

Srivastava and Rawat, 2013). A DNA barcoding study reported that 24 % of raw drugs obtained

from herbal markets were adulterated with six other morphologically similar species (Srirama et

al., 2010). In another study using microscopy, physicochemical parameters and high-performance

thin-layer chromatography (HPTLC), it was reported that different species of Berberis were traded

and adulterated in different herbal markets of India (Srivastava and Rawat, 2013). Species

adulteration might also arise due to the same vernacular name being applied to different species in

various indigenous systems of medicine, or incorrect use of scientific generic names for the raw

drugs (Wu et al., 2007; Begum et al., 2014; Bennett and Balick, 2014; Rivera et al., 2014). In 1993

several cases of renal failure were reported in Belgium resulting from the consumption of a weight-

loss supplement. This nephropathy was due to the misidentification of Stephania tetrandra

S.Moore and Aristolochia fangchi Y.C.Wu ex L.D.Chow & S.M.Hwang which is rich in

aristolochic acid. Apparently in traditional Chinese medicine both the herbs shared an identical

vernacular name (Wu et al., 2007; Debelle et al., 2008).

The consequences of unreported ingredients and undeclared fillers used in herbal products may

range from simple misleading of consumers on labelling to potential adverse drug reaction or

poisoning due to toxic contaminants (Chan, 2003; Ernst, 2004; Gilbert, 2011). However, the

pharmacovigilance of herbal products remains difficult because these products are sold over the

counter, without any medical prescription, and under no legislative framework that traces and

monitors the adverse reactions that may occur (de Boer et al., 2015).

16

1.2.2) Regulatory status of herbal products

According to WHO there is considerable variation from country to country in the quality control

of herbal materials and products. This variation not only has an impact on public health, as

contaminants in herbal medicines may represent avoidable risks for patients and consumers, but

also has effects on international trade (WHO, 2007, 2013). Though regulation is complex, it is

constantly developing. For example, until 2008 Ginkgo biloba L. was considered as food in the

United Kingdom, while it was consistently regulated as a medical product in Germany, whereas in

the USA it is a food supplement. However, in the United Kingdom and in many other European

countries ginkgo is now generally regulated as a traditional herbal medical product (Heinrich,

2015). In Europe the herbal products fall under two main categories, primarily depending on their

intended use (i) ‘herbal medicines’ that are regulated as medicinal products by the European

Medicines Agency, and (ii) ‘herbal food supplements’ which are regulated under the provision of

food legislation (European Union., 2002, 2004; Vlietinck et al., 2009).

Based on the European Directive 2004/24/EC criteria’s herbal medicinal products can be classified

into two categories: (i) well established medicinal herbal products; and (ii) traditional used herbal

medicinal products (European Union., 2004). The European Directive 2004/24/EC provides a

mechanism that allows manufacturers of herbal medicines to register medicinal herbal products

based on a tradition of use (European Union., 2004). The traditional use is based on evidence that

a corresponding herbal product is derived from the same botanical drug and prepared in a similar

way, and has been used traditionally for at least 30 years including 15 in non-European countries

and 15 years in Europe or more than 30 years in Europe. Evidence on biological assays or clinical

trials are not required to prove the safety and efficacy of traditional herbal medicine, however proof

that ‘harmful in specified conditions of use and that the pharmacological effects and the efficacy

of the medicinal product are plausible on the basis of longstanding use and experience’ are

mandatory (European Union., 2004; Vlietinck et al., 2009; Jütte et al., 2017). The requirements for

the well-established medicinal herbal products are the published scientific literature on recognized

efficacy and safety (European Union., 2004; Calapai, 2008). The Committee on Herbal Medicinal

Products is part of European Medicines Agency (EMA) and responsible for establishing

monographs on therapeutic and safe use of medicinal products and to provide an inventory of safe

herbal substances with a long history of use (Vlietinck et al., 2009; Jütte et al., 2017). However,

17

the pharmacovigilance of the marketed herbal medicines is the responsibility of manufacturers and

suppliers (Raclariu, 2017).

In India, the Ayurvedic Pharmacopoeia of India (API) is a legalized document of the Government

of India describing the quality, purity and strength of selected drugs that are manufactured,

distributed, and sold by the licensed manufacturers in pan India (Joshi et al., 2017; Katoch et al.,

2017). Once a Pharmacopoeia, or an article in it, has been approved by the Ayurvedic

Pharmacopoeia Committee, it has the compliance with the quality prescribed therein becomes

mandatory under the Drugs and Cosmetics Act 1940 (Joshi et al., 2017). The API is comprised of

two parts; part-I is the Ayurvedic Formulary of India and comprises the monographs of selected

formulations of natural origin, and part-II includes monographs on single drugs sourced from the

schedule-1 books under the Drugs and Cosmetics Act, 1940 comprising of popular Ayurvedic

classics of different period of time (API, 2001). Each monograph in API includes the drug title in

Sanskrit along with drug definition regarding its integrity in scientific nomenclature and concise

knowledge with respect to its source, occurrence, distribution and collection precautions.

Synonymies in Sanskrit and also names in other Indian regional languages are listed (API, 2001;

Joshi et al., 2017). The first edition of API has been published in 1978, and currently comprises

976 compound formulations of Ayurveda and 540 monographs of plant, animal and mineral

(including metals) origin (Joshi et al., 2017).

1.2.3) Quality control methods

One of the core interests of modern pharmacognosy refers to the identification and authentication

of drug substances and to the quality of the resulting herbal medicine (Trease and Evans. D., 2009;

Heinrich and Anagnostou, 2017). Alongside the guidelines and regulations, the lack of appropriate

analytical procedures and methods that vary largely between countries adversely complicate the

monitoring and quality assessment of herbal products along the entire value chain (Bent, 2008;

Booker et al., 2012; Heinrich, 2015). The quality of herbal products is directly reflected in safety

and efficacy of that product, and one of the major challenges in herbal pharmacovigilance is to

develop novel strategies and appropriate standards to exhaustively assess and monitor the quality

of both raw materials and herbal products (Barnes, 2003; de Boer et al., 2015; Raclariu, 2017).

Quality monitoring ensures that the products are of the appropriate quality required for their

indented use in order to protect the integrity of public health (de Boer et al., 2015; Raclariu, 2017).

18

Currently the WHO’s guidelines for assessing quality of herbal medicines include a series of

procedures that are mainly to ensure the identity of the raw plant materials, and screening a

specified marker compound, and the microbiological purity of the herbal product (WHO, 2007,

2011). Using authenticated raw material is the basic starting point in developing a herbal product

(Trease and Evans. D., 2009). Most herbal monographs specify the use of macroscopic and

microscopic characterization, phytochemistry based analysis of specific markers compounds,

assays for toxic constituents such as heavy metals, and the use of different chromatographic

approaches to detect adulteration (Raclariu, 2017). The herbal product manufacturers are supposed

to use at least one appropriate test to determine the identity of the ingredients before plant

ingredients are being used in the preparation of a herbal product (Pawar et al., 2016). However, it

is important to note that the appropriate quality assurance methods can be applied based on the

various stages of value chain and preceded on the basis of case-by-case evaluation, starting from

the plant material harvest, storage, and to the finished herbal products (WHO, 2007).

1.2.3.1) Macroscopic and microscopic authentication

Both macroscopic and microscopic investigations are the classical botanical authentication and

characterization techniques for whole plants, plant parts, and in some cases, the plant material that

has been dried and powdered (Smillie and Khan, 2010; Khan and Smillie, 2012). In macroscopic

technique the plant morphological characteristics are examined to aid in the authentication process.

The examination includes the plant traits such as habit (e.g., woody/suffruticose/herbaceous), leaf

shape, size, and morphology (e.g., leaf margins: entire, undulate, dentate, serrate, lobed, or

pinnatifid); flower characters such as type of inflorescence (e.g., spike, raceme, panicle, cyme,

corymb, helicoid cyme, head); floral morphology (e.g., epigynous, perigynous, hypogynous;

stamen number and shape; number of carpels in ovary; number of seeds per carpel); root

characteristics including surface texture, type (corm, bulb, rhizome, etc.), and tissue layering

(banding patterns). As part of authentication, macroscopic techniques are applicable only when a

plant still retains one or more of these above “key” characters, so it is possible to determine the

identity of target plant species and adulterants that have similar morphology yet having a certain

level dissimilarity in some morphological key characters (Applequist, 2006; Smillie and Khan,

2010; Khan and Smillie, 2012).

19

Microscopic approaches involve techniques such as fluorescence microscopy, scanning electron

microscopy or standard light microscopy to analyze characteristics such as the presence or absence

of trichomes, oil glands, particular cell types, seed morphology, pollen morphology and vascular

traces (Joshi and Khan, 2005; Joshi et al., 2005). Microscopic techniques are specifically more

useful than macroscopic techniques in specific cases such as while attempting to establish

authenticity of the samples which are ground plant material. Under such condition most

macroscopic characters are lost in such ground plant material (Joshi and Khan, 2005; Smillie and

Khan, 2010; Khan and Smillie, 2012).

The capability of macroscopic and microscopic authentication techniques are hampered while

analyzing the complicated multicomponent powdered samples, or when there is no cellular

distinction between closely related genera, or where a material is processed beyond the ability to

provide distinct morphological characterization (Joshi and Khan, 2005; Khan and Smillie, 2012).

Under such circumstances, it is necessary to utilize alternative techniques in order to effectively

identify and authenticate botanical samples (Khan and Smillie, 2012; Pawar et al., 2016). Another

demerit of these techniques is that highly qualified individuals are required for both macroscopic

and microscopic identification. The number of trained taxonomists are in decline in recent years,

and a lack of interest in the area from the current generation of students has put this skill at a

premium (Smith and Figueiredo, 2009).

1.2.3.2) Chromatographic and spectroscopic methods

Analytical techniques based on phytochemicals are the most reliable and applicable authentication

methods that are routinely used for the quality control of raw plant materials and herbal products

(Liang et al., 2004; Khan and Smillie, 2012). The basic analytical technique is chemical

fingerprinting by Thin Layer Chromatography (TLC) (Wagner and Bladt, 1996). This method is

based on the separation of compounds on a solid phase, after a sample is applied on the thin layer

of adsorbent material such as silica gel, and a solvent (known as the mobile phase) is drawn up in

the plate via capillary action. Compounds ascend on the TLC plate at different rates based on their

chemical properties, and this creates a fingerprint for the compounds that are present in the sample

when visualized under different conditions (Wagner and Bladt, 1996). TLC is a cost effective

method and applicable for the preliminary screening of the compounds, and to establish initial

identification and quality control of raw plant materials (Liang et al., 2004). TLC can also be used

20

as a semi quantitative technique (Liang et al., 2004). High Performance Thin Layer

Chromatography (HPTLC) is an advancement of TLC method, used in quality control of the raw

plant materials and the herbal products (Khan and Smillie, 2012). HPTLC is both a quantitative

and qualitative method with automation in different steps, along with development chambers,

digital imagery, and densitometry capabilities to analyze the qualitative and quantitative data (Khan

and Smillie, 2012).

High Performance Liquid Chromatography (HPLC) is both a qualitative and a quantitative method

and uses well characterized marker compounds for the quality control of the raw plant materials

and the herbal products (Lazarowych and Pekos, 1998; Liang et al., 2004; Fan et al., 2006).

However in order to establish identity of the plant material, hyphenated HPLC methods, such as

HPLC-MS or HPLC-NMR, are often used (Liang et al., 2004). Yet, HPLC is suitable for the

development of the fingerprints for raw plant material and herbal products (Liang et al., 2004). Gas

chromatography (GC) is a highly sensitive method used for fingerprint analysis of volatile chemical

compounds (Liang et al., 2004). The advantage of GC clearly lies in its high sensitivity of detection

of volatile chemical compounds, the extraction of the volatile oil is relatively straightforward and

can be standardized, and the components can be identified using hyphenated GC–MS analysis

(Liang et al., 2004; Ong, 2004; Zeng et al., 2007).

In order to use, adapt and validate any of these analytical fingerprint methods, a sufficient quantity

of the selected marker compounds that are readily available is required (Smillie and Khan, 2010;

Khan and Smillie, 2012). The lack of commercially available standard markers for many

compounds limits the application of these analytical chemical methods for authentication of the

raw plant materials and the herbal products (Smillie and Khan, 2010; Khan and Smillie, 2012).

However, fingerprinting methods and phytochemical identity techniques have been developed and

combined as hyphenated techniques (combination of chromatographic and spectroscopic methods)

such as GC-MS, HPLC-MS and CE-MS to vastly improve the authentication process of herbal

products (Patel et al., 2010; Heyman and Meyer, 2012; Khan and Smillie, 2012).

The combination of phytochemical identity methods and chemometric analysis have also been

proposed in order to cope with the major confinement of relying on prior established species-

specific marker compounds (Khan and Smillie, 2012; Booker et al., 2016). For instance, Nuclear

Magnetic Resonance (NMR) based chemometric profiling has been emerged as another

21

methodology for the evaluation of botanical extracts (Heyman and Meyer, 2012). 1H NMR is a

robust, reliable and non-destructive method that may be used to simultaneously detect, identify and

quantify chemical compounds in a plant sample due to the relatively high sensitivity and

widespread occurrence of protons in plant metabolites (Kim et al., 2010; Heyman and Meyer, 2012).

The spectroscopic evaluation of 1H NMR of herbal extracts provides a complete secondary

metabolite profile, however, the metabolites need to be solved in the suitable solvent medium. Thus

NMR is a valuable companion for quality control of raw herbal material and products. However, a

major delimitation of 1H NMR is considerable overlap in the complex spectra of plant extracts (van

der Kooy et al., 2009; Heyman and Meyer, 2012).

1.2.3.3) DNA based identification

Genetic markers are Deoxyribonucleic acid (DNA) sequences with a physical location on a

chromosome. DNA sequences are unique and specific to individual species and molecular markers

utilize these genetic variations in DNA sequences to identify individuals or species. One of the

advantages of DNA based identification methods is that DNA markers are least affected by age,

environmental factors and physiological conditions of the plant samples. Even though, DNA

markers do not correspond to the chemical profile, they are not tissue specific and thus can be

detected at any stage of development, with a small amount of sample, in any physical form (Zhang

et al., 2007). Broadly DNA based identification methods can be classified into:

(i) Hybridization based methods include Restriction Fragment Length Polymorphism (RFLP)

(Botstein et al., 1980) and variable number tandem repeats (Nakamura et al., 1987). RFLP is

considered to be one of the first developments in the field of DNA markers and are based on the

principle of genetic variation caused by mutation, insertion or deletion in restriction enzyme

binding and cleavage sites (Ganie et al., 2015). The digested fragments vary in size, have to be

separated using southern blot analysis and accordingly visualized by hybridization to specific

probes which could be homologous or heterologous in nature (Ganie et al., 2015). Labelled probes

such as random genomic clones, cDNA clones, probes for microsatellite and minisatellite

sequences are hybridized to filters containing DNA, which have been digested with restriction

enzymes (Neumann and Kumar, 2008). Polymorphisms are then detected by presence or absence

of bands upon hybridization (Jeffreys et al., 1985; Litt and Luty, 1989). However, the major

22

disadvantages of RFLP are time consuming, the usage of radioactive substances, laborious and

difficult to automate (Ganie et al., 2015).

(ii) Polymerase Chain Reaction (PCR) methods involve in vitro amplification of particular loci of

DNA using a specific or arbitrary oligonucleotide primers. The arbitrary primer methods include

Random Amplified Polymorphic DNA (RAPD), arbitrarily primed PCR and DNA amplification

fingerprinting (Joshi et al., 2004). Specific primer based polymorphism detection system includes

inter simple sequence repeats polymorphism where a terminally anchored primer specific to a

particular Simple Sequence Repeat (SSR) is used to amplify the DNA between two opposed SSRs

of the same type. Polymorphism occurs whenever one of the SSRs has a deletion or insertion that

modifies the distance between the repeats (Joshi et al., 2004; Passinho-Soares et al., 2006). Another

approach known as Amplified Fragment Length Polymorphism (AFLP) is a technique that is based

on the detection of genomic restriction fragments by PCR amplification. Adaptors are ligated to

the ends of restriction fragments followed by amplification with adaptor homologous primers.

AFLP has the capacity to detect thousands of independent loci and can be used for DNAs of any

origin or complexity (Passinho-Soares et al., 2006). Sequence characterization of amplified regions

(SCAR), and Amplification Refractory Mutation System (ARMS) are some of the most important

marker for authentication of medicinal plants (Zhu et al., 2004; Kiran et al., 2010). SCAR markers

can be designed using a specific gene such as nrITS region or a random DNA fragment in the

genome of an organism such as the AFLP, RAPD and ISSR DNA fragment linked to a trait of

interest (Kiran et al., 2010; Ganie et al., 2015). The designed SCAR primers sequence-specific and

are used to identify the target species from a pool of related species by the presence of a single,

distinct and bright band in the desired sample (Kiran et al., 2010). Similarly, ARMS is based on

the use of sequence-specific PCR primers that will amplify the test DNA only when the target DNA

allele is contained within the sample and will not amplify the non-target DNA allele (Zhu et al.,

2004). However, the disadvantages of markers such as RAPD and AFLP are less reproducible, and

require high molecular weight DNA. Whereas, the demerit of SCAR and ARMS markers is the

need for prior sequence data to design the PCR primers (Zhu et al., 2004; Ganie et al., 2015).

(iii) Sequencing based methods uses the genetic variations caused by transversion, insertion or

deletion that can be directly assessed and information on a defined locus can be obtained. DNA

barcoding is one of the sequencing based methods that aids in the identification of biological

23

organisms. The term DNA barcoding was coined by Hebert et al. (2003) and can be defined as use

of short nuclear or organelle DNA sequences for the identification of organisms. Since it was first

proposed, the technique has been found to be very useful in fingerprinting and identification of

species to a remarkable 98 to 100 per cent accuracy in many organisms (mostly in Kingdom

Animalia) including birds (Hebert et al., 2004b), fish (Ward et al., 2005), butterflies (Burns et al.,

2007), insects (Ojha et al., 2014), reptiles (Khedkar et al., 2016), and amphibians (Biju et al., 2014).

Further, DNA barcoding has helped in clarifying the taxonomic position of ‘apparent species

complex’ by revealing several cryptic species within a ‘single’ species described through

conventional taxonomy (Hebert et al., 2004a). In the last one decade, the technique has been found

to be very useful in not only fingerprinting and identification of species but has extended beyond

and currently used in rapid biodiversity assessment studies (Valentini et al., 2009), biomonitoring

of pests and diseases (Hajibabaei et al., 2011), in identification of prey species consumed by the

predators (Peters et al., 2015), in forensic studies especially in investigating illegal trade of

endangered species and their products (Dubey et al., 2011), and in detecting poisonous plants

(Bruni et al., 2010). DNA barcoding has also been used in authentication of herbal products

(Newmaster et al., 2013), in identification of mislabelled food products (Carvalho et al., 2015), and

also as an educational tool to assess biodiversity in school campuses (Naaum et al., 2014). These

examples point to a number of applications of the DNA barcoding technique from fundamental

research on biodiversity to enforcement of food laws, from teaching to quarantine and phyto-

sanitary laws and protection of wildlife (Bonants et al., 2010; Cawthorn et al., 2012; Yan et al.,

2013; Liu et al., 2014).

Crucial to all DNA barcoding studies is the choice of an ideal DNA barcode region. An ideal DNA

barcode is the one that allows unambiguous species identification by having sufficient sequence

variation among species (inter-specific) and low intra-specific variation, and yet be conserved to

be present across the > 400 million years of evolutionary divergence of land plants (Chase et al.,

2007). Mitochondrial DNA in animals is highly conserved in terms of gene content and order with

a high rate of nucleotide substitution. Whereas, higher plants exhibit frequent rearrangements in

genomes, transfer of genes to the nuclear genome, and incorporation of foreign genes, and

nucleotide substitution rates are much slower in plants compared to animals (Cowan et al., 2006;

Vijayan and Tsou, 2010). Therefore, a number of chloroplastic candidate gene regions have been

suggested as a barcode for plants, [coding regions accD, matK, ndhJ, rpoB2, rpoC1, and ycf5, rbcL;

24

trnL intron, UPA (Universal Plastid Amplicon), rpoB] and non-coding spacers (Taberlet et al., 2006;

Kress and Erickson, 2007; Lahaye et al., 2008; Newmaster et al., 2008; Hollingsworth et al., 2009).

In the absence of suitable single locus DNA barcode region for land plants, the Consortium for the

Barcode of Life has proposed combination of two locus (rbcL + matK) as the standard plant barcode

for land plants after screening a number of different chloroplast regions such as atpF–atpH spacer,

matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer based on

major criteria’s such as universality, easy amplification, sequence quality, coverage and species

discrimination ability (Hollingsworth et al., 2011). Several studies have suggested a combination

of trnH–psbA, matK, rbcl and nrITS as the most potential DNA barcodes (de Vere et al., 2012;

Zimmermann et al., 2013; Yan et al., 2015). A single DNA barcode usually have the species

discriminatory power of about 75-85 %, and a combination of multi-locus DNA barcode yield the

discriminatory power >95 % in most taxa (Chen et al., 2010; Burgess et al., 2011).

Apart from several applications of the DNA barcoding, more specifically it has been proved to be

useful method in dealing with authenticity issues of medicinal plants and herbal products (de Boer

et al., 2015; Parveen et al., 2016; Heinrich and Anagnostou, 2017; Sgamma et al., 2017). Several

investigations using DNA barcoding method to identify and authenticate various herbal products

have reported varying degree of adulteration and substitution. For example, using ITS2 DNA

barcode region Shi et al. (2017) reported 98 % (28 out of 38 samples) of adulteration in the

traditional Chinese medicine herbal product ‘Baitouweng’ which supposed to contain the authentic

species Pulsatilla chinensis (Bge.) Regel., Ghorbani et al. (2017) reported 26 % of adulteration (18

out of 68 samples) in raw plant materials purchased from the herbal markets of Iran. Similarly, 6 %

of saw palmetto, 16 % of gingko, and 25 % of black cohosh herbal dietary supplements were

adulterated in the samples purchased in New York (Baker, 2012; Little and Jeanson, 2013; Little,

2014). Likewise a number of studies have utilized DNA barcoding to audit the quality of herbal

products (de Boer et al., 2015; Sgamma et al., 2017; Raclariu et al., 2018). It is not worthy that the

conventional DNA barcoding exclusively applicable for the authentication of single ingredient

herbal products, and one of the major limitations with DNA barcoding is the inability of traditional

Sanger sequencing technique in detecting DNA in herbal products containing more than one

species such as polyherbal products (Ivanova et al., 2016; Wilkinson et al., 2017). Several

comprehensive reviews on DNA-based authentication of botanicals have highlighted the merits

25

and demerits of Sanger sequencing based DNA barcoding and its application in quality control of

herbal products (de Boer et al., 2015; Parveen et al., 2016; Sgamma et al., 2017).

The rapid development of high-throughput sequencing (HTS) methods offers new opportunities

for the identification and quality control of herbal products using the DNA barcoding approach.

DNA metabarcoding is a combination of DNA barcoding and high-throughput DNA sequencing

methods. It offers several key advantages over conventional DNA barcoding such as mass-

amplification and sequencing of barcodes from complex mixtures of multiple species, analyzes of

samples with varying levels of DNA degradation, products containing fillers or contaminants, and

superior sensitivity of the method (Taberlet et al., 2012; Staats et al., 2016). The two most widely

used sequencing platforms are Illumina and Ion Torrent for the quality control of herbal products

(Speranskaya et al., 2018). Several studies have utilized this approach in authenticating herbal

products. For example, (Raclariu et al., 2017a; 2017b) have evaluated 78 herbal products

containing Hypericum perforatum and 16 herbal products containing Veronica officinalis using

High-throughput sequencing based DNA metabarcoding, and revealed that DNA metabarcoding

detected the species of interest in 68 % and 15 % of the products, respectively. Though, HTS has

several advantages in the identification and quality control of herbal products. One of the

limitations of DNA metabarcoding is that it depends on the data integrity of the reference sequences

database and some species are hard to clearly distinguish by means of HTS and DNA barcoding.

Therefore it is recommended to use the integrative approach involving both chemical profiling and

DNA-based barcoding methods for identification of herbal products (Raclariu et al., 2018;

Speranskaya et al., 2018; Xu et al., 2018).

All the above mentioned methods have different roles in quality control and authentication, and

the appropriateness of applying such methods are based on the various stages of value chains i.e.,

from harvest of a plant to herbal product to consumers. Figure 1 illustrates the methods that can

be used during various stages of value chain for quality control and authentication.

26

Figure 1. Various stages involved in the production of regulated or unregulated herbal products

and the roles of different quality control (chemical and biological) methods. * Quality control

methods are also commonly used for authentication. Permission to use the figure is obtained from

Raclariu et al. (2018).

27

2) Aim of the thesis

The overall aim of the thesis is to provide an insight into the traditional knowledge of dioecious

medicinal plants in India, validate the traditional knowledge on Canarium strictum by utilizing

phytochemical methods and biological assays and to authenticate herbal products using analytical

chemical and DNA methods.

The aim of Paper I was to document the traditional knowledge on dioecious plants of India.

Specifically this paper tried to answer these research questions: do folk healers have preference for

plant genders? If so, what are those plants? And document those plants. Other addressed aspects

were: do folk healers differentially utilize male and female plant of a particular species for food,

medicines or timber? Furthermore this study addresses folk healers’ perceptions of what are

considered to be male and female plants in their community and traditions. Overall, the concept of

plant gender in Indian systems of medicine are studied.

The aim of Paper II was to document the medicinal uses of Canarium strictum

(Burseraceae) by Indian folk healers. Furthermore to investigate the constituents of the resin and

stem bark, and the radical scavenging and antioxidant properties of the resin and stem bark extracts.

In addition, the effects of the resin and stem bark extracts on the NO production in

lipopolysaccharide stimulated murine dendritic D2SC/I cells were investigated.

The aim of Paper III was to find out whether the herbal raw drugs of Garcinia species and

herbal products said to be based on this plant are substituted and/or adulterated with

morphologically similar species of Garcinia. In this study DNA barcoding is used to authenticate

herbal raw drugs of Garcinia species, and NMR spectroscopy to be used to identify and quantify

the principle organic acids, (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone in Garcinia

raw herbal drugs and in food supplements that are labelled to contain Garcinia extracts.

The aim of Paper IV is to authenticate and detect species diversity in Ayurvedic herbal

products in Europe using DNA metabarcoding. More specifically, it tests the presence of labelled

species in herbal products, and aims to detect adulteration and substitution, and/or presence of other

unlabelled plant species.

28

3) Material and methods

The materials and different methods used in this thesis are presented in detail in the Papers from I

to IV. In this section therefore only an overview of different methods and materials will be

presented.

3.1) International legislative framework

Prior to conducting any ethnobotanical or ethnopharmacological studies, it is mandatory that the

researchers are aware of Convention on Biological Diversity (CBD), which is a multilateral treaty

comprising international laws and agreements relating to all ecosystems, species and genetic

resources (CBD, 1992, 2000). CBD has three main goals: (i) the conservation of biodiversity; (ii)

sustainable use of the components of biodiversity; and (iii) sharing the benefits arising from the

commercial and other utilization of genetic resources in a fair and equitable way (CBD, 2000).

Equitable sharing is a prerequisite for achieving the two first objectives. Without ‘Access’ there

will be fewer benefits to share, and without ‘benefit sharing’ there will be fewer resources

conserved for future use. ‘Prior informed consent’ and ‘mutually agreed terms’ are meant to guide

access to genetic resource (Koester, 1997; Rosendal, 2006). Genetic resources refer to genetic

material of actual or potential value. It may be any material of plant, animal, microbial or other

origin containing functional units of heredity (Rosendal, 2006). Genetic resources constitute as an

important input factors to biotechnology companies for bioprospecting; the screening of

biodiversity and related traditional knowledge in search for commercially valuable genetic and bio-

chemical resources (Rosendal, 2006). CBD also provides legal protection to genetic resources

associated with traditional knowledge and the benefits arising from its utilization (CBD, 2011).

Therefore, being an ethnobotanical or ethnopharmacological researcher, it is necessary to be aware

of the third goal of CBD which deals with access and benefit sharing of genetic resources, and

supplementary agreement to the CBD ‘The Nagoya Protocol’.

The Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of

Benefits Arising from their Utilization (ABS) to the Convention on Biological Diversity is a

supplementary agreement to the Convention on Biological Diversity (CBD, 2011). It provides a

transparent legal framework for the effective implementation of the objective. In terms of access

to genetic resources, benefit-sharing and compliance, the Nagoya Protocol consists of several core

29

obligations, and some important obligations are to ‘establish clear rules and procedures for prior

informed consent and mutually agreed terms’, and the ‘utilization includes research and

development on the genetic or biochemical composition of genetic resources, as well as subsequent

applications and commercialization’ (CBD, 2011).

3.2) Ethnobotanical field studies

In order to conduct the ethnobotanical studies on dioecious plants in India, an application form for

access to biological resources and associated traditional knowledge was submitted to the National

Biodiversity Authority (NBA); a statutory autonomous body under the Ministry of Environment

and Forests, Government of India (http://nbaindia.org/). Permission was granted to access the

traditional knowledge of Indian plants. The Access and Benefit-sharing Clearing-House Unique

Identifier is ABSCH-IRCC-IN-237734-1.

The ethnobotanical and ethnopharmacological studies were conducted during the period of March

to August 2016, and February 2018. In total 50 folk healers were interviewed using semi-structured

questionnaires and the study participants were selected using the snowball sampling method

(Berlin and Berlin, 2005). Prior to the study, the purpose of the study was explained to the folk

healers and the prior informed consent to conduct the study was requested and agreed. A detailed

description of the study protocol, and the semi-structured questionnaire used in the study can be

found in the method sections and appendix section of Paper I and II.

3.2) Material collection

The resin and stem bark of Canarium strictum Roxb. (Paper II) were collected from a tree in Kolli

Hills, India, in 2016. C. strictum is a polygamodioecious species. The tree was tapped for the

oozing of resin prior to three days of collection.

For Paper III ten herbal products labeled as containing either Garcinia gummi-gutta (L.) Roxb. or

Garcinia indica (Thouars) Choisy (Clusiaceae) with hydroxycitric acid were purchased from

pharmacies and via e-commerce, and 21 raw herbal drugs being traded as Garcinia species were

collected from different herbal markets of South India. The lists of samples including label

information can be found in the supplementary information of Paper III.

30

For Paper IV a total of 79 Ayurvedic herbal products either food supplements or herbal medicines

were randomly purchased via e-commerce and pharmacies from Europe. The lists of samples

including label information can be found in the supplementary information of Paper IV.

The herbal products analyzed in Paper III and IV were imported into Norway for scientific analyses

under Norwegian Medicines Agency license no. 16/04551–2. Voucher specimens of plant

materials used in this study are deposited in the herbarium of Ashoka Trust for Research in Ecology

and the Environment, and in FRLH-Herbarium and Raw Drug Repository of The Institute of Trans-

Disciplinary Health Sciences and Technology, India. All the plant nomenclature in this study

follows The Plant List (The Plant List, 2013 http://www.theplantlist.org),

3.3) Extraction, isolation and characterization of chemical compounds

The stem bark and resin of C. strictum (Paper II) were air dried and powdered, and then extracted

with dichloromethane (DCM) followed by methanol using an Accelerated Solvent Extraction (ASE)

instrument (ASE350 Solvent Extractor).

The resin DCM crude extract was applied to a normal phase silica column and chromatographed

with a gradient of ethyl acetate/dichloromethane (10–100% EtOAc) to yield fractions of

compounds. The stem bark methanol extract was purified by using Sephadex LH-20 column

material and eluted with a gradient of H2O/MeOH (35-100% MeOH) and H2O/acetone (70%

acetone). The obtained fractions were each separately applied to a VersaPak C18 column with a

gradient of H2O/acetonitrile (5-100% acetonitrile) followed by H2O/acetone (70% acetone). In

parallel to the fractionation work, analytical TLC (normal phase silica plate or reverse phase C18-

plates), analytical HPLC coupled to a C18-column and a diode array detector, and/or NMR was

used to guide in method development and select fractions for further purification. Selected fractions

were purified by preparative HPLC (gradient of 0.1% trifluoroacetic acid in water and acetonitrile)

to obtain pure compounds for identification and structure elucidation. 1D and 2D NMR

spectroscopy was conducted on a Bruker AVII400 or Bruker AVI600 instrument. The detailed

methodology for antioxidant activity, NO production in lipopolysaccharide stimulated murine

dendritic D2SC/I cells and toxicity assay are provided in Paper II.

For Paper III, Garcinia raw drugs were freeze-dried for 24 hours due to the high hygroscopic nature

of Garcinia fruits, and pulverized in a commercial blender. Garcinia fruit powders were extracted

31

with Milli-Q water using an ASE instrument. Similarly, the extraction of Garcinia food

supplements were carried out using the same methodology as for the Garcinia water extracts. Prior

to the extraction of Garcinia food supplements, the capsules were opened and their contents

removed, whereas tablets were ground to a powder. 1H NMR spectra of Garcinia water extracts

and food supplement extracts were acquired on a Bruker AVII400 instrument. For quantification

reference solvent solution was prepared containing 2.0 mg/ml maleic acid (internal standard) and

0.1% 3-(trimethylsilyl) propionic 2,2,3,3-d4 acid sodium salt in deuterium oxide.

3.4) DNA barcoding methods

For the DNA barcoding study (Paper III), a reference database was compiled for 11 authentic

Garcinia species with 2–5 individuals in each species using nrITS, psbA-trnH and rbcL sequences.

DNA was extracted from fresh as well as silica dried plant material using the Cetyl trimethyl

ammonium bromide (CTAB) method optimized for Garcinia species (Asish et al., 2010), and the

details of the wild collected Garcinia species voucher specimens, geographic origin, and National

Center for Biotechnology Information GenBank accessions can be found in the supplementary

information provided in Paper III. Similarly, DNA from raw herbal drugs of Garcinia traded as

Kodampuli and Kokum were isolated using the same using the CTAB method optimized for

Garcinia species (Asish et al., 2010) and nrITS sequence was used to establish the identity of

Garcinia raw herbal drugs.

DNA metabarcoding method was utilized to authenticate herbal products in Paper IV. A detailed

description of the metabarcoding protocol can be found in the method sections of Paper IV. Seventy

nine herbal products labelled to contain a total of 159 plant species belonging to 132 genera and

60 families. Conventional CTAB DNA extraction method was used to isolate DNA from the herbal

products (Doyle and Doyle, 1987), and the markers nrITS1 and nrITS2 were used as barcodes to

identify the presence of labelled species, and to simultaneously detect substitutes and adulterants

as well as to check the presence of any off labelled species in the product.

32

4) Summary of results

Paper I: Ethnobotany of dioecious species: Traditional knowledge on dioecious plants in

India

More than 15,000 angiosperm species are dioecious. However, little is known about the

ethnobotany of dioecious species and whether preferences exist for a specific gender, e.g., in food,

medicine or timber. Therefore the specific objective of this study was (a) to study and document

whether Indian folk healers have preference for plant genders, and their knowledge on dioecious

species and uses. (b) to understand the concept of plant gender in Ayurveda and whether Ayurvedic

literature includes any references to gender preferences. In order to conduct the study, lists of

dioecious plants used in Indian systems of medicine and folk medicine were compiled.

Ethnobotanical data were collected from 40 folk healers of Malayali communities in Kolli Hills,

Servarayan Hills, and Sittlingi Valley of Eastern Ghats, Tamil Nadu India. Folk healers’

perceptions and awareness of dioecious plants and any preferences of use for specific genders of

dioecious species were documented using semi-structured interviews. In addition, twenty

Ayurvedic doctors were interviewed to gain insight into the concept of plant gender in Ayurveda.

Our results revealed that Indian systems of medicine viz., Ayurveda, Siddha, Unani, and Sowa-

Rigpa, and folk medicine, contain 5–7 % dioecious species, and this estimate is congruent with the

number of dioecious species in flowering plants in general. Folk healers recognized the

phenomenon of dioecy in 31 out of 40 species, and reported gender preferences for 13 species with

respect to uses as timber, food and medicine. Among folk healers’ different plant traits such as

plant size, fruit size, and visibility of fruits determines the perception of a plant being a male or

female. For example, informants were unaware of dioecy for two shrubs (Dodonaea angustifolia

L.f., Dodonaea viscosa (L.) Jacq.), two climbers (Cocculus hirsutus (L.) W.Theob., Cyclea peltata

(Lam.) Hook.f. & Thomson), and two lianas (Asparagus racemosus Willd., Cissampelos pareira

L.). They are all sourced from the wild in the study area, but the useful parts of these plants are not

fruits or seeds. On the contrary, informants were aware of dioecy for Celastrus paniculatus Willd.

(liana), and Embelia tsjeriam-cottam (Roem. & Schult.) A.DC. (shrub), because the seeds are used

as medicines from these plants, and informants were aware of a plant that did not produce seeds.

33

It was also observed that folk healers recognize a particular plant species either as male or female

based on different phenotypes, texture of different plant parts and morphological appearance of

closely related species by providing gender specific vernacular names, and such plant species are

not dioecious in nature. For example Artocarpus hirsutus Lam. (ayanipala; kattupala; peyppala) is

considered to be male plant due to its property of more durable timber than Artocarpus

heterophyllus Lam. (palamaram; narpala), which is a female plant.

Ayurvedic classical literature provides no straightforward evidence on gender preferences in

preparation of medicines or treatment of illness; however it contains details about reproductive

morphology and sexual differentiation of plants. From this study it is evident that people have

traditional knowledge on plant gender and preferential usages towards one gender.

34

Paper II: Ethnopharmacology, biological activities and chemical compounds of Canarium

strictum: an important resin-yielding medicinal tree in India

Canarium strictum Roxb. (Burseraceae) is a resin yielding, polygamodioecious tree distributed

across parts of India, Myanmar and China. The resin is used for rheumatism, asthma, venereal

disease and chronic cutaneous diseases; the bark is used as mosquito repellent. Despite the

widespread use of this plant in traditional systems of Indian medicine, no systematic surveys of its

use among healers or phytochemical studies on the stem bark have hitherto been performed.

Therefore, the aim of this paper was to document the medicinal uses of C. strictum by Indian folk

healers. Furthermore, to investigate the constituents of the resin and stem bark, and the radical

scavenging and antioxidant properties, the effects of NO production in lipopolysaccharide

stimulated murine dendritic D2SC/I cells.

Ethnopharmacological data were obtained from ten folk healers in parts of Western Ghats, India.

The resin and stem bark were extracted with DCM followed by MeOH by using an Accelerated

Solvent Extraction instrument. The extracts were fractionated using different chromatographic

methods, and isolated compounds were identified by NMR spectroscopy.

Our results revealed that the healers employed resin, and the reasons for the use were related to

cold, airways diseases and rheumatoid arthritis. α-Amyrin and β-amyrin were identified as the

major constituents in the resin. Compounds isolated from the stem bark MeOH extract were

identified as procyanidins, gallic acid, methyl gallate, scopoletin, 3,3'-di-O-methylellagic acid 4-

O-α-arabinofuranoside and elephantorrhizol (3,3',4',5,6,7,8-heptahydroxyflavan). The finding of

elephantorrhizol in C. strictum is of chemotaxonomic interest, and the first report for the family

Burseraceae. Furthermore, phloroglucinol degradation of procyanidins indicated the presence of

catechin as the terminal unit and epicatechin as the extender units.

Radical scavenging and 15-lipoxygenase inhibitory activity, and dose dependent inhibition of NO

production were observed in resin and bark extracts. No toxicity towards Artemia salina nauplii

was found.

35

Paper III: Authentication of Garcinia fruits and food supplements using DNA barcoding and

NMR spectroscopy

Garcinia L. (Clusiaceae) fruits are widely used in traditional medicine, and the fruits of several

Garcinia species are locally and commercially harvested from the wild. The fruits are a rich source

of (−)-hydroxycitric acid, a compound that has gained considerable attention as an anti-obesity

agent. Commercial Garcinia based products are popular as food supplements worldwide, and the

trade in Garcinia dried fruit rinds and its value-added products are constantly increasing. Increasing

demand and limited supply of medicinal plants have resulted in widespread adulteration of a

number of medicinal plants. Therefore, the specific objective of this study was to assess the

adulteration of morphologically similar samples of Garcinia using DNA barcoding, and to quantify

the content of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone in raw herbal drugs and

Garcinia food supplements using NMR spectroscopy.

A DNA barcode library for 41 individuals comprising of eleven different species was established

using taxonomically authenticated samples referred to as biological reference material. DNA

barcoding was carried out for these 41 samples using three DNA barcode regions, nrITS, and

plastid markers psbA-trnH and rbcL. Twenty-one major herbal markets of South India were visited

and data for all known vernacular and trade names of Garcinia species were obtained from the

vendors, and raw drug samples were collected. For NMR quantification, one-gram aliquots of fruit

powder and food supplements were extracted using an Accelerated Solvent Extraction system with

Milli-Q water, and 1H NMR spectra of Garcinia water extracts and food supplement extracts were

acquired for quantitative analysis on a Bruker AVII400 instrument equipped with a 5 mm BBOF

probe and operating at a frequency of 400 MHz at 298 K, and with number of scans = 128.

DNA barcoding results revealed that nrITS is a suitable DNA barcode region for Garcinia species

by exhibiting the high mean interspecific genetic distance, and low intraspecific genetic distance

followed by the plastid makers psbA-trnH and rbcL. Similarly, nrITS and psbA-trnH illustrated the

clear grouping within Garcinia species with well supported bootstrap values, whereas rbcL on the

other hand showed poor resolution in delineating the Garcinia species. Analyses of nrITS

sequences of G. gummi-gutta (Kodampuli) and G. indica (Kokum) along with BRM barcode

library revealed that there was no adulteration in the species, and also that the morphological

36

characters of fruits aided in the authenticity verification of collected raw drug samples from the

market.

1H NMR signals of (-)-hydroxycitric acid, (-)-hydroxycitric acid lactone and the internal standard

maleic acid were identified in water extracts of Garcinia and used for quantification. The 1H NMR

signals for H-1 in (-)-hydroxycitric acid appear as two closely spaced doublets at δ 3.07 ppm (d, J

= 16.6 Hz) and 3.16 ppm (d, J = 16.6 Hz), while H-3 appears as a singlet at δ 4.45 ppm (Figure 2).

In the same spectrum the H-4 protons in (-)-hydroxycitric acid lactone give rise to two doublets at

δ 2.92 ppm (d, J = 18.0 Hz) and 3.29 ppm (d, J = 18.0 Hz), H-2 gives a singlet at δ 5.00 ppm (Figure

2). The two vicinal protons of the internal standard maleic acid gave rise to the singlet at δ 6.37

ppm (Figure 2). A slight variation in the characteristic signals of (-)-hydroxycitric acid and (-)-

hydroxycitric acid lactone within triplicates of each sample and between samples was observed

(relative standard deviation < 0.03). These variations in chemical shifts values were possibly due

to minor pH differences or interactions with other molecules in the extracts. Further, the total

content of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone in G. gummi-gutta and G.

indica was found, and it varied from 1.7% to 16.3%, and 3.5% to 20.7% respectively. Analysis of

ten Garcinia food supplements revealed a large variation in the content of (−)-hydroxycitric acid,

from 29 mg (4.6%) to 289 mg (50.6%) content per capsule or tablet. Only one product contained

quantifiable amounts of (−)-hydroxycitric acid lactone, and this product was also labelled to

contain crude drug. Furthermore the study demonstrates that DNA barcoding and NMR could be

effectively used as a regulatory tool to authenticate Garcinia fruit rinds and food supplements.

37

Figure 2.1H NMR spectrum of (a) Garcinia gummi-gutta water extract, and (b) water extract of

Garcinia gummi-gutta food supplement (product 5). Orange and green labelled signals were used

for quantification of (–)-hydroxycitric acid and (–)-hydroxycitric acid lactone, respectively, while

blue labelled signals are from maleic acid (internal standard).

38

Paper IV: DNA metabarcoding authentication of Ayurvedic herbal products on the

European market raises concerns of quality and fidelity

Ayurveda is popular and used worldwide in complementary and alternative healthcare and medical

practices, but the growing commercial interest in Ayurveda based products has increased the

incentive for adulteration and substitution within this herbal market. Consequently, the quality and

safety of herbal products are affected. Therefore this study aimed to test the composition and

fidelity of Ayurvedic products marketed in Europe using DNA metabarcoding (HTS using Ion

Torrent), and to evaluate the ability of DNA metabarcoding to identify the presence of authentic

species, any substitution and adulteration and/or presence of other off labeled plant species.

Seventy-nine Ayurvedic herbal products sold as tablets, capsules, powders and extracts were

randomly purchased via e-commerce and pharmacies across Europe. After high throughput

sequencing 35 products out of 79 (44%) yielded no Molecular Operational Taxonomic Units

(MOTUs) in any of the replicates either for nrITS1 or nrITS2 that fulfilled our quality criteria, and

they were excluded from the results and the further discussion. These products consisted of 13

tablets, 11 capsules, and 11 powders. The products that yielded MOTUs were represented by 17

tablets, 19 capsules, 5 powders and 3 extracts. A total of 188 different plant species belonging to

154 genera and 65 families were identified from the retained MOTUs using BLAST. Another

quality selection criteria where a species was considered and validated as being present within the

product only if it was detected in at least 2 out of the 3 replicates was applied. It resulted in another

five additional products that failed to yield the same MOTU in any of the replicates which were

discarded. The remaining 39 products resulted in a total of 97 plant species belonging to 40 families

(62 species for nrITS1, and 60 species for nrITS2). The species detected for all the replicates for

both ITS1 and ITS2, were merged for each sample for further analyses. Further analysis revealed

that only two out of 12 single ingredient products contained only one species as labelled, eight out

of 27 multiple ingredient products contained none of the species listed on the label, and the

remaining 19 products contained 1 to 5 of the species listed on the label along with many other

species not specified on the label. The fidelity for single ingredient products was 67%, the overall

ingredient fidelity for multi ingredient products was 21%, and for all products 24%.

The DNA metabarcoding analysis also confirmed the presence of four threatened species, i.e.,

Celastrus paniculatus Willd., Glycyrrhiza glabra L., Gymnema sylvestre (Retz.) R.Br. ex Sm., and

39

Saraca asoca (Roxb.) Willd., whereas the remaining threatened species were not detected despite

being included as labelled ingredients. The following species were found in over 20% of the

products: Withania somnifera (L.) Dunal (3%), Tribulus terrestris L. (27%), Convolvulus

prostratus Forssk. (23%), Coriandrum sativum L. (23%), Ipomoea parasitica (Kunth) G. Don

(23%), Ocimum basilicum L. (23%) and Senna alexandrina Mill. (23%).

40

5) Discussion

In order to conduct any ethnobotanical or ethnopharmacological studies, researchers should be

aware and learn more about ethical issues and research permission. These two are the single most

important aspects that govern and regulate the genetic materials and its associated traditional

knowledge. Until 1990, most ethnobiological research was not regulated on a global scale, and the

collection of genetic material was governed by few restrictions like the Convention on International

Trade in Endangered Species of Wild Flora and Fauna (Sands and Bedecarre, 1989; Weckerle et

al., 2018). The import of genetic materials required a Phytosanitary Certificate that would certify

that biological material was not carrying any disease agents (Weckerle et al., 2018). However, the

Convention on Biological Diversity (CBD) coming into effect in 1993 (CBD, 2000), and the

Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits

Arising from their Utilization (Nagoya Protocol) in 2014 (CBD, 2011, 2014), have changed this

practice, and are implementing a legal framework and regulation on genetic materials and its

associated traditional knowledge on a global scale (Weckerle et al., 2018).

The CBD assigned genetic resources as a property to national states, and the Nagoya Protocol

clearly assigns the property rights of traditional knowledge to the respective knowledge holders.

Any community work should be performed under the Nagoya Protocol on Access to Genetic

Resources and Equitable distribution of benefits from their use. The right of use and ownership of

any traditional knowledge of all informants remains with them. Except for scientific publication,

use of any other information requires the additional consent of the traditional owners (CBD, 2011,

2014). Both these treaties have several implications for research ethics and permits towards

ethnobiological research.

Prior to the ethnobotanical and ethnopharmacological data collection on dioecious plants (Paper I)

and C. strictum (Paper II), ethical approval for these studies was obtained from the National

Biodiversity Authority, Government of India (http://nbaindia.org/). While not all countries have

ratified the Nagoya Protocol, India has ratified the protocol. Especially researchers in European

Union are legally required to comply with the Nagoya Protocol, because the European Union has

ratified it. As of January 2019, 114 countries have ratified the protocol. Researchers from the

countries that have not ratified the protocol are still required to comply with Nagoya Protocol when

it comes to the import of biological material and its associated knowledge. After obtaining the

41

ethical approval for this study, the ethnobotanical and ethnopharmacological surveys were

conducted. The purpose of the study was explained to the informants and the consent to conduct

the study was requested and agreed in written format. Weckerle et al. (2018) have highlighted that

signing the papers is simply not common for the folk healers or participants, and a request for

written consent may create distrust among participants. Therefore, it was recommended to provide

a statement on what kind of consent they obtained and to state if they followed a specific code of

ethics. For ethnobiological research, the current standard is the International Society of

Ethnobiology Code of Ethics (ISE, 2006).

In our study (Paper I), a significant knowledge gap was identified. It was estimated that

angiosperms include 352,000 species worldwide, and an enormous effort is being made to

document and systematically study the traditional uses of flowering plants. However, it is

noteworthy that angiosperms have different sexual systems, and reproductive physiology of each

sexual systems demands varying level of resources (Charlesworth, 2002; Obeso, 2002). Dioecy is

one such sexual system of angiosperms and documenting traditional uses of dioecious plants based

on their gender was lacking in ethnobotanical literature. Therefore, this study (Paper I) aimed to

contribute to the scientific community interested on ethnobotanical research. This study involved

the listing of dioecious species that occur in India followed by ethnobotanical data collection in

parts of the Eastern Ghats in Tamil Nadu, India. A list of dioecious species occurring in India were

derived from 15,600 dioecious angiosperm species compiled by Renner (2014), and the occurrence

of 5–7 % dioecious medicinal plants in Indian systems of medicines in general is congruent with

the diversification rate of dioecious species in flowering plants (Käfer et al., 2014; Renner, 2014).

Though considerable evidence for sex-biased herbivory and variation in secondary metabolites in

dioecious plants is available in scientific studies, little is known about traditional concepts and

preferences for dioecious plants, either male or female. Several studies on folk classification of

plants, and ethno-ecology reported the traditional knowledge associated with dioecious plants, and

the importance of how the different genders of plant species are named and classified in local

languages (Berlin et al., 1973; Khasbagan, 2008; Bjora et al., 2015). In our study, snowball

sampling method was used to collect ethnobotanical data for 40 dioecious plants. Snowball

sampling method was employed in order to find informants who have a rich knowledge on

traditional usage of plants. In order to document the informants’ knowledge on dioecy, examples

42

such as papaya and palm trees were used to make them understand the phenomenon of dioecy,

coconut trees and pumpkins were explained for monoecy, and goose berries for bisexual plants

before initiating the interview process. The study area (Kolli Hills, Servarayan Hills and Sittilingi

Valley) was chosen due to the diverse natural vegetation, and the main ethnic group in the study

area is the Malayali community.

The research findings show that the informants have knowledge about the existence of the

dioecious nature of many plants, and informants were aware of 31 out of 40 dioecious plants.

However, the informant’s perception on what is male and female plants are highly dependent on a

species-specific plant characteristic traits. For example, informants were unaware of dioecious

plans such as Dodonaea angustifolia L.f., Dodonaea viscosa (L.) Jacq., Cocculus hirsutus (L.)

W.Theob., Cyclea peltata (Lam.) Hook.f. & Thomson, Asparagus racemosus Willd. and

Cissampelos pareira L.. On the other hand, informants were aware of dioecy for Celastrus

paniculatus Willd. and Embelia tsjeriam-cottam (Roem. & Schult.) A.DC. this is due to whether

the useful part of the plants is fruits/seeds or other parts. Several studies have also shown that

human utilization of a plant is highly influenced by its species traits (Díaz et al., 2011; Díaz et al.,

2013). This has also been shown by Cámara-Leret et al. (2017) who demonstrated that people

preferentially use large, widespread species rather than small, narrow ranged species, and that

different traits are linked to different uses. Few other factors that might have affected the informants

perception of a plant being dioecy or not is sex ratio and plasticity in sex expression of a particular

plant (Mcarthur, 1977; Borges et al., 1997; Pathak and Shukla, 2004; Geetha et al., 2007; Sharma

et al., 2010; Field et al., 2013; Renner, 2014).

Our results also highlights that the male tree timbers are preferred for construction purposes such

as houses, huts and furniture because it is believed that male plants have expected size and more

durable timber than female trees. Such preference on one gender in timber could be explained with

plant resource allocation theory that the male plants comparatively allocate more resource to

vegetative growth than the female plants. On the other hand, informants also consider that the male

plants of Myristica dactyloides Gaertn. are not beneficiary for them and selectively chosen for

firewood. Similar information were documented for Carica papaya L., the informants do not prefer

the male plants to be grown in their garden since it yields no fruits. Such selective logging has been

shown to endanger the plant species (Martínez‐Garza and Howe, 2003). Further, any anthropogenic

43

activity that modifies the male–female distance, sex ratio, plant size and pollinator abundance or

behavior could affect the long-term viability of dioecious plants, and endangers the species

(Somanathan and Borges, 2000).

The phytochemical study (Paper II) aimed to document the medicinal uses of C. strictum and

investigate the phytochemical contents of resin and stem bark, and the radical scavenging and

antioxidant properties, and the effects of NO production in lipopolysaccharide stimulated murine

dendritic D2SC/I cells. The present ethnopharmacological study revealed that the folk healers

mostly utilized the resins of C. strictum for various medicinal usages. Among the documented

usage common cold, runny nose and sneezing were the most commonly mentioned indications. In

addition, healers reported that resin is burnt in in house to produce smoke, which in turn act as

insect repellent. Similarly, Canarium schweinfurthii resin also reported to act as insect repellent

(Youmsi et al. (2017). The phytochemical analysis showed that α-amyrin and β-amyrin were the

major compounds present in the resin. Whereas procyanidins, gallic acid, methyl gallate, scopoletin,

3,3'-di-O-methylellagic acid 4-O-α-arabinofuranoside, and elephantorrhizol (3,3',4',5,6,7,8-

heptahydroxyflavan) were isolated from the bark MeOH extract. It is noteworthy that the finding

of elephantorrhizol in C. strictum is the first report for the family Burseraceae, and it has

chemotaxonomic interest. In addition 3,3'-Di-O-methylellagic acid 4-O-α-arabinofuranoside is

also a rare natural product and first report from Burseraceae, and it has been previously reported

four plant species such as Cornus capitata Wall., Cornus kousa F.Buerger ex Hance, Euphorbia

humifusa Willd., Euscaphis japonica (Thunb.) Kanitz, (Tanaka et al., 2001; Tanaka et al., 2003;

Lee et al., 2007; Deng et al., 2008). However, ellagic acid derivatives have been reported from

other Canarium species (as reviewed in Mogana and Wiart, 2011). Scopoletin and gallic acids are

also known from Canarium. Radical scavenging and 15-lipoxygenase inhibitory activity were

observed in resin and bark extracts, whereas no toxicity towards A. salina nauplii was found. The

bark MeOH crude extract showed higher activity in the antioxidant assays, than the methanol and

DCM extracts of resin. This finding could be explained by the presence of a high content of

polyphenols in the bark. On the other hand, 15-LO inhibition of MeOH and DCM bark extracts

showed relatively high activity compared with the positive control quercetin. The 15-LO inhibitory

activity of the MeOH bark extract is probably due to the procyanidins (Bräunlich et al., 2013).

Methanol and DCM extracts of resin shows a dose dependent anti-inflammatory effect by reducing

levels of NO, without being toxic to the cells. Whereas, DCM bark extract also showed significant

44

inhibition of NO. Similarly, several studies have reported the anti-inflammatory properties of

Canarium species (Mogana et al., 2013; Kuo et al., 2016).

Several studies have highlighted the importance of gender in phytochemical research and its impact

on pharmacological properties of a species (Simpson, 2013). With this evidence that the medicinal

dioecious species harness a potential to be studied comparatively for their chemical composition

between male and female plants and the pharmacological activities, and also provides a platform

to document ethno-ecological knowledge, and traditional knowledge of dioecious plants with

special reference to its gender.

One of the parameters of pharmacognosy is identification and authentication of medicinal plant

drugs and monitoring the quality of the resulting herbal medicines (Heinrich and Anagnostou,

2017). Complex herbal formulations pose unique challenges to the established quality control

methods that include identity, purity and analyzing specific constituents within the herbal products.

Also the synergistic action of polyherbal products is fundamental to the practice of Ayurveda and

the use of such specific combinations of medicinal plants is expected to result in an enhanced

outcome. To ensure the quality, safety and efficacy of such polyherbal products each of the plants

included must be identified.

The pharmacognosy studies (Paper III and IV) aimed to contribute to all the challenging efforts of

the scientific community towards finding and proposing improved alternative and complimenting

quality control methods for the traded raw herbal drugs and marketed herbal products. These

studies involved the use of NMR spectroscopy together with DNA barcoding, and DNA

metabarcoding to assess 89 complex herbal products sold in five different countries, including three

European countries. These studies involved top selling herbs such Garcinia gummi-gutta, Garcinia

indica (Paper III), and a wide variety of highly traded Indian medicinal plants such as Piper spp.,

Phyllanthus spp., Ocimum spp., Sida spp., and Withania somnifera (Indian ginseng) (Paper IV).

NMR spectroscopy was chosen as an analytical method for this study (Paper III) considering its

advantage of being robust, reliable and non-destructive. In Paper III, 1H NMR quantification was

used to identify and quantify the principle organic acids, (−)-hydroxycitric acid and (−)-

hydroxycitric acid lactone in Garcinia raw herbal drugs, and in food supplements that claim to

contain Garcinia extracts respectively.

45

Quantitative 1H NMR revealed varying amounts of (−)-hydroxycitric acid and (−)-hydroxycitric

acid lactone in dried Kodampuli and Kokum raw drugs. The total content of (−)-hydroxycitric acid,

and (−)-hydroxycitric acid lactone varied between 1.7% to 16.3%, and 3.5% to 20.7%, respectively,

in the two species. Several other studies using HPLC methods have also reported the content of

(−)-hydroxycitric acid in Garcinia species (Jayaprakasha and Sakariah, 1998, 2002; Jena et al.,

2002). However, a challenge with HPLC is to obtain a good resolution between the organic acid

and its lactone in Garcinia extracts. There are examples showing no discrimination between (−) -

hydroxycitric acid and the lactone or methods that suffer from poor resolution (Jayaprakasha and

Sakariah, 1998, 2000, 2002). Another challenge is the low specificity at 210 nm, therefore sample

preparation may be needed to remove co−eluting interfering substances (Jayaprakasha and

Sakariah, 1998, 2000, 2002). Gas chromatography, and capillary zone electrophoresis have

previously been used to quantify the hydroxycitric acid and hydroxycitric acid lactone content in

Garcinia fruits and food supplements (Lowenstein and Brunengraber, 1981; Jayaprakasha and

Sakariah, 2002; Muensritharam et al., 2008). Comparatively quantitative 1H NMR employed in our

study has the advantage of being rapid (less than 8 minutes needed for 128 scans) with no necessity

for additional cleanup of extracts or derivatization. NMR makes it possible to detect several organic

molecules in the same sample, as long as they are soluble in the NMR solvent, and is also highly

reproducible with little instrument-instrument variation (Kruk et al., 2017).

The overlaps between the total content of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone

in Garcinia species suggest that these organic acids cannot not be used as marker compounds to

distinguish between Garcinia species. The variation in the concentration of the organic acids in

Kodampuli and Kokum raw drugs might be due to seasonal and geographic variations, and

concentration of organic acids can also vary based on the harvest time, as it has been shown in

other fruits (Raffo et al., 2004; Liu et al., 2006). Similarly, seasonal and geographic variations in

chemical composition have been reported for a number of medicinal plants and also chemical

variability along the value chains, harvesting pressure, harvest time, collection of immature plant

parts etc. have been reported to influence the composition and concentration of plant constituents

(Pandey and Shackleton, 2012; Booker et al., 2014).

After validating the quantitative 1H NMR method developed to quantify the organic acids in

Garcinia raw drugs, the method was applied to authenticate the validity of Garcinia food

46

supplements claiming to contain Garcinia extracts. Analysis of ten Garcinia food supplements

revealed that five products contained both (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone,

including only one product contained quantifiable amounts of (−)-hydroxycitric acid lactone.

Whereas (−)-hydroxycitric acid lactone was missing or only present in trace amounts in the

remaining products. The absence of (−)-hydroxycitric acid lactone in the supplements with

Garcinia extracts might be due to the extraction method employed for the herbal products (Majeed

et al., 1998). Previous quality control studies on Garcinia products have reported both adulteration

and no adulteration (Jayaprakasha and Sakariah, 2000; Muensritharam et al., 2008; Jamila et al.,

2016).

1H NMR can be useful in qualitative and quantitative analysis of constituents in medicinal plants

and herbal products (Hachem et al., 2016). This study has shown that quantitative NMR can

accurately detect the presence or absence of targeted plant metabolites, and is also as a fast and

sensitive enough method for the quantification of organic acids and lactone in raw herbal drugs

and food supplements. However, one of the disadvantages of the quantitative NMR method is its

inability in detecting infrageneric species and its substitution, and in providing information about

other plant ingredients present in the products. This limits the applicability of quantitative NMR in

quality control of complex herbal products.

The efficacy of DNA barcoding in dealing with authenticity issues of medicinal plants and herbal

products has been discussed in various studies (Smillie and Khan, 2010; de Boer et al., 2015;

Parveen et al., 2016; Sgamma et al., 2017). In Paper III, DNA barcoding was used as an analytical

approach in herbal raw drug authentication of Garcinia raw herbal drugs in combination with 1H

NMR. Such combination of DNA barcoding with spectroscopic methods for authentication of

herbal medicines increases the resolution in species identification and analysis of mixtures

(Santhosh Kumar et al., 2016). nrITS marker was utilized to authenticate the raw drugs traded as

Kodampuli and Kokum, and the analyses of nrITS sequences of Kodampuli and Kokum revealed

that there was no adulteration in the species. In addition, the morphological characters of fruits

aided to identify the collected raw drug samples from the market. This was also apparently due to

the absence of taxonomic complexities in fruit morphological characters of Kodampuli and Kokum

(Parthasarathy and Nandakishore, 2014).

47

DNA barcoding was utilized to test the authenticity of Garcinia fruits traded in the Indian herbal

markets. On the other hand, it was not applicable to use the same method to authenticate Garcinia

herbal products due to its major limitation of Sanger sequencing based DNA barcoding. Sanger

sequencing has the inability to distinguish constituent species in multi-ingredient herbal products.

One of the prerequisites for the DNA-based molecular analysis of herbal products is the isolation

of good quality DNA. In our study, the yield of isolated DNA from the Garcinia food supplements

ranged from 0.05 to 1 ng/ml, and therefore DNA metabarcoding was not feasible. Plant DNA can

also be removed or degraded during the manufacturing process of herbal products: extensive heat

treatment, irradiation, ultraviolet light exposure, filtration and extraction will affect DNA yield and

integrity (de Boer et al., 2015).

The efficacy of DNA metabarcoding to investigate species diversity of several sorts of samples has

been discussed in various studies (Taberlet et al., 2012; Staats et al., 2016). In our study (Paper IV)

DNA metabarcoding was used as an analytical approach to identify the presence of all the labelled

species in 79 Ayurvedic herbal products. Out of 79 products analyzed, 35 products (44 %) have

not yielded MOTUs in any of the replicates either for nrITS1 or nrITS2 that fulfilled our quality

criteria, and they were excluded from the results and the further discussion. The success rate in

generating raw sequences reads from the herbal products, and the number of products from which

MOTUs could be identified after applying strict trimming and filtering quality criteria are reported

to vary in several studies. For example, 53% success rate was obtained for dietary products derived

from Echinacea, Ginkgo, Hypericum, Trigonella and Veronica (Ivanova et al., 2016), and 100 %

success rate for individual unprocessed crude traditional Chinese medicine products (Cheng et al.,

2014). These differences are likely due to the different quality and type of raw materials, but also

due to the effect of different variables in the experimental and data processing that can be optimized

to improve the quality of the results.

DNA metabarcoding detected considerable incongruences between the detected species and those

listed on the product label. In ten out of twelve single ingredient products that were labeled as

containing only one species, we detected multiple species, from which six contained the species

labeled on the product together with other species, whereas four products did not contain the

species listed on the product label but contained several other non-listed species. Out of 27

successfully analyzed multiple ingredient products, 8 (29.6%) products contained none of the

48

species listed on the label, and the remaining 19 products contained between one to five species

listed on the label along with many other species not specified on the product label. The fidelity

rate for single ingredient products was 67% (8 out of 12), and the overall ingredient fidelity

(detected species from product label/total number of species on label) for multi ingredient products

was 21%, and for all products 24%.

The discrepancies between the species detected using DNA metabarcoding and those listed on the

products require a careful consideration of possible contamination, and the fact that DNA

metabarcoding is a highly sensitive detection method (Raclariu, 2017; Raclariu et al., 2018). Even

small traces of pollen grain or plant dust left in production equipment may be revealed from the

analysis. It is therefore important that the herbal product authentication using DNA metabarcoding

focuses on the presence and/or absence of target species. Alarms need not to be raised over trace

contamination from other species, plausible present in the cultivation, transport or production chain

(Raclariu, 2017; Raclariu et al., 2018). However this requires a case based analysis including the

experimental steps (e.g., sample preparation, library preparation, and HTS) and post-bioinformatics

analysis that may provide false positive or false negative results (Robasky et al., 2014; Ficetola et

al., 2015). The large number of non-listed plant species may thus be expected as contamination

bias, amplification bias, sequencing errors or false-positive taxonomic identifications due to error-

prone barcode sequences reference databases (Robasky et al., 2014). On the contrary, the

overlooked species can be explained by false negative detections, usually determined by the

availability and integrity of plant DNA that can be removed or highly degraded during harvesting,

drying, storage, transportation and processing (e.g., mode of extraction, exposure to ultraviolet

light, heat or pressure) (Novak et al., 2007). In addition, primer fit issues or primer-template

mismatches (Piñol et al., 2015), stochasticity due to low DNA concentration or incomplete barcode

sequences reference databases (Giguet-Covex et al., 2014). The use of replicates may considerably

reduce these risk of missing the present taxa at the expense of substantially increasing sequencing

costs and time (Ficetola et al., 2015). Nevertheless, the bioinformatics post processing of the

sequencing data may help to reduce the uncertainty of the results (Robasky et al., 2014). In paper

IV, efforts were made to reduce the amount of sequencing errors, which were known to affect the

Ion torrent sequencing platform (Loman et al., 2012; Salipante et al., 2014).

49

In the context of the quality control of herbal products, DNA barcoding and DNA metabarcoding

do not provide any quantitative nor qualitative information about the active plant metabolites in

the raw plant materials or in herbal products, thus it narrows its applicability only to identification

and authentication procedures. Whereas, quantitative NMR method is accurate in identifying and

quantifying the presence of targeted chemical compounds, but limited ability in identifying the

targeted species. The results of this study Paper III and IV reveal that there is a need for a better

quality control of herbal products. A novel analytical approach should eventually use a combination

of innovative high throughput methods that complement the recommended standard methods.

50

6) Concluding remarks and perspectives

At the start of this PhD research, the aim was to focus on documenting and comparatively studying

the medicinal plants used by various ethnic groups in different ecological and cultural traditions

in India, and validate the traditional knowledge using scientific methods such as phytochemical

characterization of plants and associated biological activities. Simultaneously, the aim was to

employ DNA barcoding and high-throughput sequencing methods for identification of medicinal

plants in trade, and medicinal herbal products. However during literature searches, the existence of

a significant knowledge gap in ethnobotanical and ethnopharmacological literature was identified,

i.e., substantial ethnobotanical knowledge that has been documented lacks the documentation of

plant usages based on their sexual systems. Therefore, the present study started with the

documentation of traditional knowledge and use of dioecious species by folk healers in Eastern

Ghats of India. The majority of the healers had knowledge on genders of plants species, and the

results from this study revealed that the folk healers have traditional knowledge on gender of plants

and preferential usages towards one gender for some species. With this evidence, we propose that

researchers conducting ethnobotanical and ethnopharmacological studies should consider

documenting traditional knowledge on sexual systems of plants, and test the existence of gender

specific usages in their conceptual framework and hypothesis testing. The incorporation of such

concepts could provide new dimensions of scientific knowledge with potential implications to

conservation biology, chemical ecology, ethnoecology and drug discovery. The presence of the

bioactive compounds might give give a rationale for the wide spread usage of the resin in India.

Furthermore, the identified compounds can be utilized to comparatively study the phytochemical

profile of male and female plants of Canarium strictum.

Developments in pharmacognosy analytical methods are leading towards finding new systematic

approaches for monitoring the safety of traded medicinal plants and marketed herbal products.

Hence, the second phase of the PhD research focused on comprehensive evaluation of quantitative 1H NMR, DNA barcoding and DNA metabarcoding as a viable approach for resolving current

limitations in authentication of herbal products in order to improve the quality control of

traded/marketed herbal products. The results revealed that different analytical methods have its

own merits and demerits, its specific strength lies, while applying appropriately in the value chain

of herbal products. These analytical methods, quantitative 1H NMR, DNA barcoding and DNA

51

metabarcoding have the potential to be applied in the context of quality control of both well and

poorly regulated supply systems. However, standardization of protocols is necessary before these

methods can be implemented as routine analytical approaches and approved by the competent

authorities for use in regulatory procedures.

In the context of pharmacognosy and pharmacovigilance, a combination of analytical methods is

compulsory for comprehensive authentication and quality control of raw material and resulting

products.

52

Acknowledgements

Financial support during the study time was obtained from the Norwegian State Loan Fund

(Lånekassen); Section for Pharmaceutical Chemistry, Department of Pharmacy and Plant

Evolution and DNA Metabarcoding Research Group, the Natural History Museum at the

University of Oslo (UiO). Norwegian PhD School of Pharmacy, and the Faculty of Mathematics

and Natural Sciences at UiO. I am debited to all these institutes for their financial support.

The most sincere and profound respect go to my supervisors Helle Wangensteen, Hugo de Boer

and Berit Smestad Paulsen. I am grateful to Helle Wangensteen for her willingness to accept me

as a PhD student and introducing me to this exciting research field ethnopharmacology. Her

everlasting knowledge, encouragement, optimism has been a great source of inspiration during

these years. She is always available for scientific discussion. You inspired me in many ways with

your care, thoughts and discussion. Thanks a lot.

Hugo de Boer: You have been a hero from the very beginning of my PhD journey. Thank you for

creating the opportunity and the enabling environment that makes this collaboration and project

successful. I am grateful for your rigorousness, astute criticism, experience and prowess that made

me progress in this journey productively.

Berit Smestad Paulsen: I am thankful to you for a well-structured supervision. I acknowledge your

help in organizing my fieldworks, and in acquiring necessary permission to conduct this study.

Your immense scientific knowledge, enthusiasm, prompt feedback on all the sub-projects helped

me to accomplish this study.

I wish to express my gratitude to Karl Egil Malterud for his enthusiasm, inspiration, valuable ideas

and knowledge.

I owe deep gratitude to Darshan Shankar, Ravikumar Kaliamoorthy and Prakash B N at the

university of Trans-Disciplinary Health Sciences and Technology, India for their immense support

during this project.

Special thanks to the International Education Office UiO and staff memebers, Michele Jeanette

Nysæte for their unflinching support and good coordination in matters of internationals students’

welfare.

53

Thankful for the all-valuable suggestions from my colleagues that have considerably improved the

quality of this work at the section for Pharmaceutical Chemistry and Plant Evolution and DNA

Metabarcoding Research Group (UiO). My deepest gratitude to Ancuta-Cristina Raclariu-

Manolica, Margey Tadesse, Ingvild Austarheim, Vincent Manzanilla, Christian Winther Wold

Giang Thanh Thi Ho, Desalegn Chala Gelete and Abel Gizaw Seid.

Thanking my friends Raja K, Santhosh Kumar JU, Ashoka B, Kumaravel M and Thasreefa K for

their moral support.

Special thanks goes to my family for their great support during these years. My parents,

Gopalakrishnan K and Saroja G. Your love, understanding and blessing were always following me

wherever I am. My sisters Gomathi Ashok and Gothainayaki Ananad are deeply thanked. I am

thankful to my wife Sarumathi Palanivel for her love, support and understanding.

54

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I

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journal homepage: www.elsevier.com/locate/jethpharm

Ethnobotany of dioecious species: Traditional knowledge on dioeciousplants in India

Gopalakrishnan Saroja Seethapathya,b,c,⁎, Kaliamoorthy Ravikumarc, Berit Smestad Paulsena,Hugo J. de Boerb,1, Helle Wangensteena,1

a Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, P.O. Box 1068, 0316 Oslo, NorwaybNatural History Museum, University of Oslo, P.O. Box 1172, 0318 Oslo, Norwayc The Institute of Trans-Disciplinary Health Sciences and Technology, Foundation for Revitalisation of Local Health Traditions (FRLHT), 74/2 Jarakabande Kaval, PostAttur via Yelahanka, Bangalore 560064, India

A R T I C L E I N F O

Keywords:AyurvedaDioecyEthnopharmacologyFolk classificationPlant genderVernacular taxonomy

A B S T R A C T

Ethnopharmacological relevance: More than 15,000 angiosperm species are dioecious, i.e., having distinct maleand female individual plants. The allocation of resources between male and female plants is different, and alsovariation in secondary metabolites and sex-biased herbivory is reported among dioecious plants. However, littleis known about the ethnobotany of dioecious species and whether preferences exist for a specific gender, e.g., infood, medicine or timber.Aim of the study: The aim of this study was: 1) to study whether Indian folk healers have preference for plantgenders, and to document their knowledge and use of dioecious species; 2) to understand the concept of plantgender in Indian systems of medicine and folk medicine, and whether Ayurvedic literature includes any refer-ences to gender preference.Materials and methods: Lists of dioecious plants used in Indian systems of medicine and folk medicine werecompiled. Ethnobotanical data was collected on perceptions and awareness of dioecious plants, and preferencesof use for specific genders of dioecious species using semi-structured interviews with folk healers in Tamil Nadu,India. In addition, twenty Ayurvedic doctors were interviewed to gain insight into the concept of plant gender inAyurveda.Results: Indian systems of medicine contain 5–7% dioecious species, and this estimate is congruent with thenumber of dioecious species in flowering plants in general. Informants recognized the phenomenon of dioecy in31 out of 40 species, and reported gender preferences for 13 species with respect to uses as timber, food andmedicine. Among informants different plant traits such as plant size, fruit size, and visibility of fruits determinesthe perception of a plant being a male or female. Ayurvedic classical literature provides no straightforwardevidence on gender preferences in preparation of medicines or treatment of illness, however it contains detailsabout reproductive morphology and sexual differentiation of plants.Conclusions: A knowledge gap exists in ethnobotanical and ethnopharmacological literature on traditionalknowledge of dioecious plants. From this explorative study it is evident that people have traditional knowledgeon plant gender and preferential usages towards one gender. Based on this, we propose that researchers con-ducting ethnobotanical and ethnopharmacological studies should consider documenting traditional knowledgeon sexual systems of plants, and test the existence of gender specific usages in their conceptual framework andhypothesis testing. Incorporating such concepts could provide new dimensions of scientific knowledge withpotential implications to conservation biology, chemical ecology, ethnoecology and drug discovery.

1. Introduction

Enormous efforts are being made to document and systematicallystudy the traditional uses of plants. Dioecy, where species have separate

female and male plants, is widespread among flowering plants, and anestimated 6% of species are dioecious (Renner, 2014). Resource allo-cation, including trade-offs between allocation to defense, growth andreproduction, is different between genders of dioecious plants (Obeso,

https://doi.org/10.1016/j.jep.2018.04.011Received 19 January 2018; Received in revised form 19 March 2018; Accepted 6 April 2018

⁎ Corresponding author at: Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, P.O. Box 1068, 0316 Oslo, Norway.

1 These authors contributed equally to this work.E-mail address: g.s.seethapathy@farmasi.uio.no (G.S. Seethapathy).

2002). Several studies have shown that differences in reproductivedemands between the genders of dioecious plants cause gender phy-siology divergence that in turn affects the production and concentrationof secondary metabolites (Bajpai et al., 2016; Milet-Pinheiro et al.,2015; Simpson, 2013). Herbivory has been suggested as a selectivepressure that has resulted in the evolution of dioecy (Bawa, 1980), andstudies have utilized the plant resource allocation theory (Levins, 1968)to understand the patterns of plant-herbivore interaction (Obeso,2002), herbivore plant gender preferences (Hjalten, 1992), plantbrowsers (Danell et al., 1991), folivores (Maldonado-López et al.,2014), pollinators (Milet-Pinheiro et al., 2015), and gall formers(Wolfe, 1997). Cornelissen and Stiling (2005) reviewed the evidence ofsex-biased herbivory in dioecious plants, and found that male plantsexhibited significantly higher number of herbivores and herbivory interms of plant damage compared to female plants, and showed thatmale plants exhibited significantly lower concentrations of secondarycompounds and other defenses than female plants. However, there arealso examples of the opposite, e.g., the male plants leaves of Rhamnusalpinus L. and Juniperus macrocarpa Sm. exhibited a higher concentra-tion of anthraquinones, phenolics and terpenoids respectively thanthose of females, which contrasts with the resource allocation theory(Banuelos et al., 2004; Massei et al., 2006). Hence it is evident thatresource allocation might have a profound effect on the compositionand concentration of secondary compounds between individuals ofdioecious species (Simpson, 2013).

Simpson, 2013 has highlighted the importance of gender in phyto-chemical research and its impact on pharmacological properties of aspecies. For example in the dioecious species Cannabis sativa L., thefemale plants are used for marijuana, whereas the male plants arepreferred for fiber (Fetterman et al., 1971). In Dodonaea polyandraMerr. & L.M.Perry (Sapindaceae), labdane diterpenoids have been re-ported as major phytoconstituents, whereas female individuals containclerodane diterpenoids (Simpson, 2013; Simpson et al., 2011, 2012).Similarly, a significant variation in the concentration of alkaloids wasshown for the dioecious medicinal plant Tinospora cordifolia (Willd.)Miers (Menispermaceae). The mean abundances of magnoflorine, ja-trorrhizine and oblongine were significantly higher in male plants whilemean abundances of tetrahydropalmatine, norcoclaurine and reticulinewere significantly higher in female plants (Bajpai et al., 2016). It hasbeen suggested that female plants of T. cordifolia might be preferred fortherapeutic use due to the higher accumulation of secondary metabo-lites and higher antioxidant activity (Choudhry et al., 2014).

The 15,600 dioecious angiosperms occur in 987 genera (6%) and175 families (38%), with a number of families being entirely dioecious,e.g., Menispermaceae, Moraceae, Myristicaceae, and Putranjivaceae(Renner, 2014). Many of these dioecious species are well documentedfor their medicinal values (de Boer and Cotingting, 2014). In India, it isestimated that 8000 plants have medicinal usages. Some of these arecodified in traditional pharmacopoeias, i.e., Ayurveda, Siddha, Unani,and Sowa-Rigpa, whereas others are part of oral traditions in differentbiocultural groups. Considerable evidence for sex-biased herbivory andvariation in secondary metabolites in dioecious plants is available inscientific studies, but little is known about traditional concepts andpreferences for dioecious plants, either male or female. Few studies onfolk classification of plants and ethno-ecology report traditionalknowledge associated with dioecy, and the importance of how thedifferent genders of plant species are named and classified in locallanguages and how this reflects perceptions of the environment (Berlinet al., 1973), cultural values of biodiversity (Bjora et al., 2015), andecological characteristics (Khasbagan, 2008). Bernstein et al. (1997)used plot survey inventories in Brunei to show that their informantswere able to accurately predict the gender of dioecious plants. InNorthern Morocco, it was reported that the vernacular taxonomy iscongruent with the biological classification of the dioecious speciesFicus carica L. among three communities inhabited in three socio-geo-graphic regions who speaks Arabic, Berber, and both Arabic and Berber,

respectively (Hmimsa et al., 2012).Several studies have highlighted the importance of understanding

the ecology of plant biodiversity as a strategy for drug discovery (Coleyet al., 2003), as well as ethnobotanical studies and/or traditionalmedicines for drug development (Patwardhan and Mashelkar, 2009). Atthe same time, erosion and deterioration of traditional knowledgethreatens biocultural diversity and limits resilience in healthcarechoices for local communities, which also can cause a loss in leads fordrug discovery (de Boer and Cotingting, 2014; Srithi et al., 2009). In thecontext of ongoing cultural, ecological, and socio-economical changes,particularly the influence of urbanization and influence of westernlifestyles, the increasing reliance on biomedical healthcare, the deva-luation of traditional practices, and diminishing cultural cohesion areweakening the frequency and scope of traditional plant use and thisposes a serious threat to biodiversity-based cultural knowledge (Srithiet al., 2009; Vandebroek and Balick, 2012). Documenting the use ofplants by ethnic communities is an important part in understanding andanalyzing elements of traditional medicines, and also a way to perpe-tuate knowledge at risk of being lost (de Boer and Cotingting, 2014).

The aim of this study was: 1a) to document traditional knowledgeon dioecious plants among folk healers and 1b) to understand whetherfolk healers have preference for plant genders in food, medicines ortimber; 2) to understand folk healers’ perceptions of what are con-sidered to be male and female plants in their community and traditions;and 3) to understand the concept of plant gender in Indian systems ofmedicine and folk medicine, and whether Ayurvedic literature containsany references to plant gender and preferences.

2. Methodology

2.1. Selection of Indian dioecious plants

Dioecious species in India were derived from the list of 15,600dioecious angiosperms compiled by Renner (2014) by limiting to spe-cies occurring in India. Dioecious species in codified and non-codifiedIndian traditional medicine were mined from the Indian MedicinalPlant Database, National Medicinal Plants Board, Government of India,and full lists are provided in Supplementary Data S1. Nomenclaturefollows The Plant List (The Plant List, 2013 http://www.theplantlist.org) and Angiosperm Phylogeny Group IV (Byng et al., 2016). Theethnobotanical study focused on 40 dioecious plants in 30 genera and20 families (Table 1), which occurred in the study area (see below) andwere reported to be used in traditional medicine in previous studies.

2.2. Study area for ethnobotanical prospection

The present survey was conducted in the Kolli Hills (11.105°N,78.150°E), Servarayan Hills (11.455°N, 78.175°E), and Sittlingi Valley(11.543°N, 78.365°E), all of which are part of the Eastern Ghats inTamil Nadu, India. The natural vegetation of the study area is cate-gorized into shola (tropical montane forest), evergreen, semi-evergreen,deciduous, scrub, and plantation (Jayakumar et al., 2002). The mainethnic group in the study is the Malayali communities (lit. malai = hill,ali = dwells and/or malai = hill, alu = person), one of 36 scheduledtribal communities in Tamil Nadu. The Malayali is spread along thecontiguous hills of the Eastern Ghats from Pachamalai, Kollimalai, Si-theri, Palamalai, Javvadhu to the Servarayan Hills (Xavier et al., 2015).The major livelihood and local economy of these Malayali communitiesare cattle farming, agriculture, fuel-wood and collection of non-timberforest products such as herbal medicines, honey and some edible fruitsand tubers (Xavier et al., 2015).

2.3. Ethnobotanical data collection

Forty folk healers aged 40 to 80 (33 males and 7 females) wereinterviewed in 2016, and their knowledge documented using a semi-

G.S. Seethapathy et al.

Table1

Details

ofdioe

ciou

splan

tsstud

iedin

KolliHills(N

amak

kaldistrict),Sittlin

giValley(D

harm

apuridistrict)an

dSe

rvaray

anHills(Salem

district)of

TamilNad

u,Indiawithpreferen

cestowards

gend

eran

duses.

Spec

ies;

Family;

Vou

cher

Tamil

name

Hab

itPlan

tsreco

gnized

asdioe

ciou

s(%

)Gen

der

preferen

ceSp

ecifi

cus

ea

YN

Unc

ertain

Ana

mirta

cocculus

(L.)Wight

&Arn.;

Men

ispe

rmaceae;

Cl77

6Kak

amari,Nan

cukk

ottai

Lian

a35

588

Yes

O:F

ruitsan

dseed

sarepo

ison

ouswhich

areused

topo

ison

fishes

1,9,17,38(Tag

etal.,20

05)

R:♂

and♀

leav

esareused

todo

blackmag

ic1,4,7,13,14,31

Aph

anam

ixispolystachy

a(W

all.)

R.Parke

r;Meliaceae;C

l77

7Cem

maram

,Civap

purm

aram

Tree

3358

10Yes

M:L

eave

sareused

tocu

reskin

diseases,an

dstom

achpa

in2,3,18,24,32(Sen

etal.,20

11)

R:F

lowersareco

llected

forfrag

ranc

e,in

which

♀flow

erstend

toco

ntainmorefrag

ranc

e,an

doc

casion

ally

offered

inrituals2

,8,13,29

Asparagus

racemosus

Willd.;A

sparag

aceae;

Cl

778

Tann

irvitta

nkizha

ngu

Lian

a0

8813

No

M:T

ubersareused

tocu

rewhite

discha

rge,

stom

achpa

in,red

uces

body

heat,rejuv

inative,

anden

hanc

eslactation1

–40(Bop

anaan

dSa

xena

,20

07)

Bischo

fiajavanica

Blum

e;Ph

yllantha

ceae;Cl

779

Rom

aviruk

shapa

ttai,

Mila

chad

ayan

Tree

3558

8No

M:S

tem

bark

isused

tocu

rebo

dyache

,stom

achulcers,mou

thulcers

andinflam

matory

cond

itions

4,9,13,17,26,30,31,36

R:S

tem

andleav

esareused

toblackmag

icin

term

sof

remov

ingfear

1,4,7,9,13,14,31,36

Borassus

flab

ellifer

L.;A

recaceae;C

l78

0Pa

naim

aram

Tree

100

00

Yes

F:♂

plan

ttodd

yis

morevibran

tthan

♀1,3,13,17,22,25,27,38,40.F

ruitsan

dtube

rous

seed

lings

areed

ible

1–4

0(D

avis

andJo

hnson,

1987

)M:F

ruitsan

drootsareused

asdiuretic,a

ndan

tidiab

etic

1,11,23,31(D

avisan

dJo

hnson,

1987

)Can

arium

strictum

Rox

b.;B

urseraceae;C

l78

1ka

ruku

nkiliya

mTree

6815

18Yes

M:R

esin

isused

asan

ti-infl

ammatoryan

dto

cure

skin

diseases,a

gainst

poison

ousbites1–1

0,

31,35,37.(N

amsa

etal.,20

09).

O:♀

tree

yields

moreresinthan

♂plan

t1–1

0,23,33,36,37

Caricapa

paya

L.;C

aricaceae;

Cl78

2Pa

ppalip

azha

mTree

100

00

No

F:Fruits

areed

ible

1–4

0(K

rishna

etal.,20

08)

M:L

atex

areused

toco

ntroltoo

thache

,fruitsused

asrejuve

native

andpreg

nanc

yab

ortive

agen

t1,4,15,21,24,27,29,34(K

rishna

etal.,20

08)

Cassine

glau

ca(R

ottb.)Kun

tze;

Celastraceae;

Cl

783

Karuvali

Tree

830

18No

M:L

eave

san

dstem

areused

againstdy

sentery,

forwou

ndhe

aling,

againstpo

ison

ousbites,

head

ache

,fev

er5,11,12,13,21,23,26,29(M

oinet

al.,20

14)

Celastrus

paniculatusWilld.;C

elastraceae;

Cl

784

Valuluvai

Lian

a70

238

No

M:S

eeds

areused

inmen

talprob

lems,

jointpa

in,a

rthritis,s

kindiseases,w

ound

healing1

,5,10,14,18,20,31,36(R

ajku

mar

etal.,20

07)

Cissampelosp

areira

L.;M

enispe

rmaceae;

Cl7

85Vattattiruppi

Lian

a0

7328

No

M:R

oota

ndwho

leplan

tare

used

asap

petizer,an

tidiarrhoe

al,a

ntihelmintics,a

ntiulcer,a

ndto

cure

dige

stiveco

mplaints.1,3,7,13,16,19,21,27,34,36(A

mresh

etal.,20

07)

Cocciniagran

dis(L.)Voigt;C

ucurbitaceae;Cl

786

Kovai,K

ovaikk

ayVine

060

40No

F:Fruits

areed

ible

andused

asve

getable

1–4

0(A

ddis

etal.,20

09)

Cocculushirsutus

(L.)W.The

ob.;

Men

ispe

rmaceae;

Cl78

7Kattukk

oti

Clim

ber

083

18No

M:L

eave

san

drootsareused

tocu

reskin

diseases,s

kinirritation

,and

stom

achache

1,4,7,9,14,15,29,30,34(Patilet

al.,20

14)

Cycleapelta

ta(Lam

.)Hoo

k.f.&

Thom

son;

Men

ispe

rmaceae;

Cl78

8Malaithan

gi,Vattattiruppi

Clim

ber

078

23No

M:L

eave

san

drootsareused

tocu

repo

ison

ousbites,

indige

stion,

stom

achpa

in,b

oils

and

blisters

2,5,7,8,14,25,28,34(X

avieret

al.,20

15)

Dioscorea

alataL.;D

ioscoreaceae;Cl78

9Vettilai-valli

Vine

680

33No

F:Coo

kedtube

ris

used

asfood

,and

rejuve

native

1–4

0(K

umar

etal.,20

17)

D.bu

lbife

raL.;D

ioscoreaceae;Cl79

0Verrilaiv

alli

Vine

600

40No

D.esculenta(Lou

r.)Bu

rkill;D

ioscoreaceae;Cl

791

Mucila

mvalli

Vine

650

35No

D.hispidaDen

nst.;

Dioscoreaceae;Cl79

2Kavalak

odi

Vine

630

38No

D.oppositifolia

L.;D

ioscoreaceae;Cl79

3Maruvalli

Vine

680

33No

D.pentap

hylla

L.;D

ioscoreaceae;Cl79

4Kattuvalli

kalangu

Vine

780

23No

Diospyros

ebenum

J.Koenigex

Retz.;E

bena

ceae;

Cl79

5Karun

kali

Tree

100

00

No

F:Fruits

areed

ible

12,13,19,22,29,30,32,35(M

allava

dhan

iet

al.,19

98;R

aufet

al.,20

17)

D.melan

oxylon

Rox

b.;E

bena

ceae;C

l79

6Kattupa

laTree

100

00

No

F:Fruits

areed

ible

12,13,19,22,29,30,32,35(M

allava

dhan

iet

al.,19

98;R

aufet

al.,20

17)

M:L

eave

sareused

tocu

restom

achpa

in7,11,25,28(M

allava

dhan

iet

al.,19

98;Rau

fet

al.,

2017

).Le

aves

areused

toas

region

alciga

retteforpsycho

active

effects

21,23,27,33(R

atho

re,

1972

)D.mon

tana

Rox

b.;Eb

enaceae;

Cl79

7;Vak

kana

i,Vak

kana

thi

Tree

100

00

No

F:Fruits

areed

ible

12,13,19,22,29,30,32,35(M

allava

dhan

iet

al.,19

98;R

aufet

al.,20

17)

M:B

arkan

dstem

areused

tocu

refracturedbo

nes,acta

san

tico

agulan

t,an

dto

relie

vebo

dypa

in11,18,19,21,23,25(M

allava

dhan

iet

al.,19

98;R

aufet

al.,20

17)

Dod

onaeaan

gustifo

liaL.f.;

Sapind

aceae;

Cl7

98Virali

Shrub

093

8No

M:L

eave

sareused

aswou

ndhe

aling,

relie

vesbo

dypa

in,a

nti-inflam

matory.

Preg

nanc

yab

ortive

agen

t,cleansethewom

b1,2,5,8,11,14,17,18,21,24,26,37,38(G

etie

etal.,20

03;v

anHeerden

etal.,20

00)

D.viscosa(L.)Jacq

.;Sa

pind

aceae;

Cl79

9Velari

Shrub

093

8No

(con

tinuedon

next

page)

G.S. Seethapathy et al.

Table1(con

tinued)

Spec

ies;

Family;

Vou

cher

Tamil

name

Hab

itPlan

tsreco

gnized

asdioe

ciou

s(%

)Gen

der

preferen

ceSp

ecifi

cus

ea

YN

Unc

ertain

Drypetessepiaria

(Wight

&Arn.)Pa

x&

K.H

offm.;Pu

tran

jivaceae;

Cl80

0Kalvirai

Tree

100

00

No

F:Fruits

areed

ible

11–3

0(A

rina

than

etal.,20

07)

Embelia

tsjeriam

-cottam

(Roe

m.&

Schu

lt.)

A.DC.;Prim

ulaceae;

Cl80

1Vaivilangam

Shrub

780

23No

M:S

eeds

arean

thelmintic1

,4,5,9,16,19,21,27,34(V

enka

tasubram

anianet

al.,20

13)

Euph

orbiatirucalliL.;E

upho

rbiaceae;Cl80

2Tiruku

kalli

Tree

010

00

No

M:L

atex

isused

tocu

rene

ural

dysfun

ction,

jointpa

ins,

skin

disease,

andactas

neural

stim

uli2,3,4,5,11,13,17(Ban

iet

al.,20

07)

FicushispidaL.f.;

Moraceae;

Cl80

3Peiatth

iTree

1865

18No

M:F

ruitsareeatento

cure

maleim

potent

andalso

toincrease

fertility

1,8,12,18,24,30,35

(Lan

skyet

al.,20

08)

Hyd

nocarpus

pentan

drus

(Buc

h.-H

am.)

Oke

n;Ach

ariaceae;C

l80

4Neeradimuthu

Tree

7023

8No

F:Fruits

areed

ible

3,8,32,35(Sah

ooet

al.,20

14)

M:L

eave

san

dseed

sareused

tocu

reskin

diseases,c

hestpa

ins,jointp

ains

1,33,34,37,38(Sah

ooet

al.,20

14)

Lann

eacoroman

delica(H

outt.)Merr.;

Ana

cardiaceae;Cl80

5Odiya

maram

Tree

5835

8No

M:L

eave

san

dstem

bark

areused

tocu

refeve

r,dy

sentery,

andan

ti-infl

ammatory7

,9,15,28,33

Mallotusph

ilippensis(Lam

.)Müll.A

rg.;

Euph

orbiaceae;

Cl80

6Kam

ala,

Man

jana

thi

Tree

5313

35No

M:L

eave

san

dstem

bark

areused

tocu

restom

achache

.Fruitsareused

asan

tidiab

etic

3,6,12,15,20,32,35,38,39

Mom

ordica

dioica

Rox

b.ex

Willd.;Cuc

urbitaceae;C

l80

7Pa

karkoti,Pa

karkai

Clim

ber

2373

5No

F:Fruits

areused

asve

getable

1–4

0.(Ta

lukd

aran

dHossain,20

14)

M:F

ruitsareused

asan

tidiab

etic

1–4

0.(Ta

lukd

aran

dHossain,2

014)

Myristic

ada

ctyloidesGaertn.;M

yristicaceae;Cl

808

Jathikai

Tree

6820

13No

F:Macean

dke

rnel

isused

infood

1–4

0(Swetha

etal.,20

17)

PhoenixloureiroiK

unth;A

recaceae;Cl80

9Malai

eecham

Tree

100

00

Yes

F:Fruits

areed

ible

1–4

0,♂

plan

ttodd

yis

morevibran

tthan

♀plan

ttodd

y1,3,5,13,22,25,27,35,38,40(H

ayne

san

dMcLau

ghlin

,20

00;R

houm

aet

al.,20

10)

P.pu

silla

Gaertn.;A

recaceae;C

l81

0Icha

mTree

100

00

Yes

P.sylvestris(L.)Rox

b.;A

recaceae;C

l81

Icha

mTree

100

00

Yes

Piperbetle

L.;P

iperaceae;

Cl81

2Vettrila

iClim

ber

100

00

Yes

F:♀

leav

esarepreferred1

0,11,12,14,20,26,33,36

M:♂

plan

tleave

sarepreferredto

increase

malepo

tenc

y,an

d♀

plan

tleave

sareprescribed

tomalean

d♂

leav

esto

femaleto

actas

sexu

alstim

uli.♂

and♀

leav

esaregive

nin

combina

tion

forho

rmon

alba

lanc

eam

ongtran

sgen

ders

1,2,8,13,25,27,29,34,35,37,39,40.(Sa

rkar

etal.,20

00)

R:♀

leav

esarepreferredin

rituals2

,8,29,39

Semecarpu

san

acardium

L.f.;

Ana

cardiaceae;C

l81

3Senk

ottai

Tree

780

23No

M:S

eeds

arepo

ison

ous,

curesmusclespasm,skin

diseases,a

ndactas

wou

ndhe

aling9

,32,33,38(V

ijaya

lakshm

iet

al.,20

00)

Streblus

asperLo

ur.;Moraceae;

Cl81

4Kuttip

ilaa

Tree

048

53No

M:L

eave

san

dstem

bark

areused

tocu

reurinaryinfections

8,13(R

astogi

etal.,20

06)

Tino

sporacordifo

lia(W

illd.)Miers;

Men

ispe

rmaceae;

Cl81

5Cintilikko

tiLian

a38

063

Yes

M:S

tem

acts

asan

tidiab

etic,immun

omod

ulatory1

,9,12,15,40,♂

and♀

rootsareused

inco

mbina

tion

totreatmen

strual

disorders2

,8,16,19,21,39.(Grove

ret

al.,20

00)

aSu

perscriptnu

mbe

rsaretheiden

tifiersof

inform

ants

asshow

nin

Supp

lemen

tary

DataS3

;F:F

ood,

M:M

edicine,

O:O

thers,

R:R

itua

l.

G.S. Seethapathy et al.

structured questionnaire aided by props consisting of live specimensand photo galleries of the selected 40 dioecious species (Table 1). Studyparticipants were selected using the snowball sampling method (Berlinand Berlin, 2005), and we particularly focused on local people who areolder than 40 years, regularly use plants for medicinal purposes, fuel-wood and non-timber forest product collectors, and plant harvesters.Sampling was initiated through the indication of community leaders.The semi-structured questionnaire assessed the informants’ perceptionof dioecious plants, awareness about dioecious plants and, if aware, istheir preference for choosing a specific gender of dioecious plants(Supplementary Data S2). Additional information such as the folkhealers’ perspective on gender in plants and its roles in their traditionswere also recorded. The interviews were conducted in the informants’native language Tamil. Following the interviews, the plants mentionedduring the interviews were collected and confirmed for identification.Prior to the ethnobotanical survey, the purpose of the study was ex-plained to the informants and the consent to conduct the study wasrequested and agreed. The documented medicinal plants were collectedand pressed for herbarium vouchers, and identified with the help ofvalid references. All collected specimens were vouchered and depositedin the FRLH-Herbarium and Raw Drug Repository of The Institute ofTrans-Disciplinary Health Sciences and Technology, India (Table 1).Prior to the ethnobotanical data collection, ethical approval for thisstudy was obtained from the National Biodiversity Authority, Govern-ment of India.

2.4. Plant gender and Ayurveda

Twenty Ayurvedic doctors, who were formally educated and qua-lified to practice Ayurveda, were interviewed using a semi structuredquestionnaire in order to gain insight into the concept of plant gender inAyurveda and its literature. Before initiating the interview process, itwas explained to the doctors that the biological classification of plantsclassifies plant gender on the basis of their floral sexual characters i.e.the presence or absence of the androecium and gynoecium.

3. Results and discussion

3.1. Indian dioecious plants

Among dioecious species sex ratios deviate from the mean, andspecies with a male bias are associated with long-lived growth forms(e.g., trees), biotic seed dispersal and fleshy fruits, whereas female biasis associated with herbaceous species, and abiotic pollen dispersal(Field et al., 2013). Plasticity in sex expression has also been reportedfor a number of species (Borges et al., 1997; Geetha et al., 2007;Mcarthur, 1977; Renner, 2014). In this study, out of 40 dioecious plantsused in the ethnobotanical data collection, 31 plants belong to generaor families that are either strictly or completely dioecious (cf. Dioscoreaand Menispermaceae). The sex ratio of these species is not well studiedin the study area, but for example, Mallotus philippensis has been shownto be male-biased under low light environments and female-biasedunder more light environments in India (Pathak and Shukla, 2004).Biased sex ratios and plasticity in sex expression of a given dioeciousplants might have a significant effect on informants observation andclassification of a plant as male and female.

Supplementary Data S1 shows the list of dioecious plants that aredocumented for its medicinal values in folk medicine, Ayurveda,Siddha, Unani, and Sowa-Rigpa and it was found that 5–7% of medic-inal plants in Indian systems of medicines are dioecious plants, and thisestimate is congruent with the diversification rate of dioecious speciesin flowering plants (Käfer et al., 2014; Renner, 2014). Based on this, wepropose that these lists of species harness a potential to be studiedcomparatively for their chemical composition between male and femaleplants and the pharmacological activities, and also provides a platformto document ethno-ecological knowledge, and traditional knowledge of

dioecious plants with special reference to its gender.

3.2. Traditional knowledge and plant gender preference

To elicit knowledge on dioecy, informants were explained thephenomenon of dioecy in flowering plants, as correct knowledge wasdecisive for the outcome of the survey. They were informed that maleand female plants exist separately as individual plants, that male plantsonly bear flowers that will not yield fruits and seeds, whereas femaleplants bear flowers, fruits, and viable seeds. The existence of mono-ecious and bisexual plants was explained as well, and they were ex-plained that if the same plant bears male and female flowers it ismonoecious, and if the same flower contains both reproductive organsit is bisexual. Plants such as papaya, palm trees, coconut trees,pumpkin, and goose berries were given as examples to explain the re-productive systems of flowering plants before initiating the interviewprocess. Table 1 shows the details of 40 dioecious plants used in theethnobotanical study, and it was found that the informants were awareof existence of the dioecious nature of many plants. Out of 40 plantsused in the study, informants recognized the phenomenon of dioecy in31 species (Table 1), and no significant variation was found betweenthe 33 male and 7 female informants about their knowledge on theexistence of dioecious species and the number of dioecious species re-ported for usages. Therefore male and female informants were con-sidered as one category of informants for further analysis(Supplementary Data S3). However, Table 2 shows a significant varia-tion among the age groups of informants. The informants below the age50 had less knowledge on dioecious species, and used less number ofdioecious species. On the other hand, a linear growth was observedbetween the age groups for preferring any one gender of dioeciousplants, while using the plants which suggests that the age older in-formants had better perception on gender of plants and their uniqueuses (Table 2).

A number of studies has documented the lack of traditionalknowledge among younger people, and this has been attributed to theexpansion of modern education, cultural change, and the influences ofmodernization (Srithi et al., 2009; Voeks and Leony, 2004). As a resultof changing realities, traditional knowledge of medicinal plants thatwas once embedded in numerous indigenous cultures, is rapidly dis-appearing. It has been suggested that to avoid the loss of this in-tellectual heritage, it is necessary to either keep it alive, or at least todocument and describe the traditional use of plants (Bussmann andSharon, 2006).

Table 3 shows the overview of informants awareness, gender pre-ference and the habit of the dioecious plants. Since, fruits being in-formed and considered as the main identity to distinguish male andfemale plants among the informants, it was observed that the visibilityof fruit size, plant size and plant traits based uses of a particular plantdetermines the perception of a plant being male or female. For example,informants were unaware of dioecy for two shrubs (Dodonaea angusti-folia, Dodonaea viscosa), two climbers (Cocculus hirsutus, Cyclea peltata),and two lianas (Asparagus racemosus, Cissampelos pareira) they are allsourced from the wild in the study area, but the useful part of these

Table 2Total average of dioecious plants recognized, used, and preferred for its genderby different age group of informants.

Informantage cohorts

Totalnumber ofinformants

Speciesrecognized asdioecious byinformants

Average no.of speciesreported forusages

Average no.of speciespreferred forgender

41–50 17 19.35 21.47 2.7151–60 10 24.2 23.1 3.561–70 6 23.5 21.17 4.1771–80 7 24.14 22.14 5.71

G.S. Seethapathy et al.

plants are not fruits or seeds. On the contrary, informants were aware ofdioecy for Celastrus paniculatus (liana), and Emebelia tsjeriam-cottam(shrub), because the seeds are used as medicines from this plants, andinformants were aware of a plant that did not produced seeds. Plant-based ecosystem services are crucial for satisfying human needs, andhuman utilization of a plant is highly influenced by its species traits. Forexample, humans have selected plant species with traits that maximizecrop yield, such as large fruits or height, or large grain size (Díaz et al.,2013, 2011). Cámara-Leret et al. (2017) tested the relationship betweenplant traits and its perceived value by people through an inter-disciplinary perspective on the linkages between ecosystem services,human needs and species’ traits. It was demonstrated that people pre-ferentially use large, widespread species rather than small, narrow-ranged species, and that different traits are linked to different uses. Forexample, one would expect a species to possess traits that satisfy humanbasic needs such as food and health. Such traits are plant size, con-stantly high yielding subsistence, widespread and cost effective togather, and in contrast a species trait have strong link to easy avail-ability and weaker link to plant size for medicinal usages (Cámara-Leretet al., 2017).

3.3. Gender preference in food and medicinal usages

Table 1 shows the usages of plants under the categories of plantsbeing utilized as food, medicine, rituals, and a category of others.Among the 40 dioecious species, informants have gender preference for13 species (10 trees, 2 lianas, and 1 shrub), and it was found that theinformants have better knowledge about the toddy (palm wine) pre-pared out of male and female palm trees (Borassus flabellifer and Phoenixspecies). Toddy is a traditional alcoholic drink prepared by the fer-mentation of sap or exudate collected by slicing off the tip of unopenedflowers of palm trees (Davis and Johnson, 1987). Informants reportedthat male palms yield comparatively less toddy than female palms, andthe former are in higher demand among consumers because it is be-lieved to be more potent. In this study, we observed that this knowledgeis particular to elder informants, the reason for this was that in mid-20th century in India due to the increasing demand of toddy's, it wasreported that the toddy often was adulterated with chemical sub-stances, such as chloral hydrate and diazepam, and the adulteration haddetrimental health consequences for toddy consumers (Rao et al.,2004). Therefore, the consumption and sale of toddy were prohibitedfrom time to time in India, and the production of Indian-made foreignliquor such as whisky and brandy was promoted through in-dustrialization (Mahal, 2000).

It has been reported that harvesters in Nilgiri Biosphere Reserve,India were aware of male and female trees of Canarium strictum, andthat resin yielding trees were female trees (Varghese and Ticktin,2008). A similar case was observed in Canarium strictum in Kolli Hillsand Servarayan Hills where the informants reported that male treesproduce less resin than the female trees, and when inquired furtherabout the quality variation between the two gender the informants didnot comment on any quality variation in male and female plant resins,but informed about a general variation that based on the dryness of the

resin that the fragrance it produces varies. For example, resin compo-sition of male and female trees of Austrocedrus chilensis (D. Don) Florin& Boutelje (Cupressaceae) is reported to vary between genders andduring different seasons of the year (Olate et al., 2014).

Interestingly, for medicinal usages informants reported a genderpreference for Piper betle and Tinospora cordifolia, and the usage wasrather complex and dependent on spiritual beliefs and medication. Forexample, informants believe that the Piper betle leaves of any onegender can be used to balance the hormonal imbalance of people withtransgender sign. i.e., if a man is showing a sign of woman, prescribinga male leaf extract along with goat or sheep milk may cure the illnessand vice versa. Similarly male plant leaves are prescribed to woman,and female plant leaves are prescribed to men with the purpose to actboth as a sexual stimuli and to foster a good relationship between menand women. The informants reported male leaves as harder to chewthan the female leaves, therefore female leaves are prepared to makepaan (paan is a combination of betle leaves with areca nut or tobacco,chewed for its stimulant and psychoactive effects). However, whenenquired about the taxonomic identity of male and female Piper betleleaves, it was found that informants segregate male and female leavesbased on the venation pattern and number of veins in a leaf, i.e., theharder the venation pattern, and a minimum of 5 veins in a leaf isbelieved to be male leaf, and the softer venation pattern and less than 5veins in a leaf is a female leaf. In India, despite its availability in thewild, Piper betle is vegetatively propagated for cultivation and noflowering is observed in the subtropics due to the lack of inductivephotoperiods. The female plants rarely produce any flower or fruit inthe Indian climate (Bajpai et al., 2012; Guha, 2006). Despite the ab-sence of flower and fruiting to identify male and female Piper betle,sexual dimorphism for leaf character was reported in terms of lengthand breadth ratio of leaves. Male leaves are reported to be narrowlyovate with 1.84 ± 0.21 length: breadth ratio and female leaves arecordate or ovate to round leaves with 1.26 ± 0.13 length: breadthratio. Leaves of the female plants are mostly pungent and male plantsare less pungent (Krishnamurthy et al., 2008). However the congruencybetween folk healers identification, and biological identification ofmale and female Piper betle is yet to be documented. Jing and Coley,1990 have reported that male and female trees of Acer negundo (Acer-aceae) could be distinguished from one another solely based on leafcharacters, and the largest difference between the sexes was thetoughness of leaves. Leaves from female trees were on average tougherthan those from male trees, and suggests that male trees commonlysuffered greater herbivory than females due to toughness of leaves.Sexual dimorphism in vegetative growth for several dioecious plantswere also reported (Jing and Coley, 1990).

3.4. Timber plants and gender preference

Table 4 shows the plants used for its timber and preferential genderusages. Out of 21 tree species used in the study, informants have re-ported 9 species for various construction purposes, and among these 6species are preferred based on the gender (Table 4). Timber of malepalm trees (Borassus flabellifer and Phoenix species) and Drypetes sepiariais preferred for construction purposes such as houses, huts and furni-tures because it is believed that male plants have expected size andmore durable timber than female trees. On the contrary, female planttimber of Diospyros ebenum is preferred over male plants and it is be-lieved that carving in male plant timber is tough. Informants preferenceon one gender in timber could be explained with plant resource allo-cation theory that the male plants comparatively allocates more re-source to vegetative growth than the female plants (Obeso, 2002).Obeso (1997) have reported that mean annual tree-ring width of Ilexaquifolium L. was greater in males than in females for a 30-year periodand that the male plants grew more than females. Similarly, male treesof Bursera morelensis Ramírez, and Dacryodes excelsa Vahl were sig-nificantly taller and larger than female trees (Forero-Montaña et al.,

Table 3Overview of dioecious plants grouped on their habit, and the informantsawareness and gender preference.

Habit Dioeciousspecies

Species reported asdioecious byinformants

Species preferred forspecific gender byinformants

Tree 21 19 10Vine 7 6 0Liana 5 3 2Climber 4 2 1Shrub 3 1 0

G.S. Seethapathy et al.

2010; Pavón and de de Luna Ramírez, 2008). On the contrary male andfemale trees belonging to 16 species of Myristicaceae and Cecropiaschreberiana Miq. showed no differences in annual growth rates im-plying that females can compensate the higher cost of reproduction(Forero-Montaña et al., 2010; Queenborough et al., 2007).

On the other hand informants reported that male plants of Myristicadactyloides are selectively chosen for fire wood considering that it hasno other benefits for them. Similar information was documented forCarica papaya that the informants do not prefer the male plants to begrown in their garden since it yields no fruits to them. Selective loggingis reported to be the far most common management strategies to exploitcommercial timber trees in tropical regions (Putz et al., 2012), andwoody plants are especially vulnerable due to selective logging, giventheir economic value as timber and their long regeneration time(Martínez-Garza and Howe, 2003). Thus, selective logging and eco-nomic value increases the threat to dioecious taxa because of an un-derlying correlation between woodiness and dioecy (Martínez-Garzaand Howe, 2003). Among the threatened plants included in the IUCNRed List of Threatened Species, woody growth habit of dioecious spe-cies is contributing to the higher risk of extinction (Vamosi and Vamosi,2005). Any anthropogenic activity that modifies the male–female dis-tance, sex ratio, plant size and pollinator abundance or behavior couldaffect the long-term viability of dioecious plants, and endangers thespecies (Somanathan and Borges, 2000).

Apart from dioecious plants, informants had knowledge about theoccurrence of monoecious plants (i.e., with separate male and femaleflowers on the same individual plant), especially about Cocos nucifera(coconut), and Cucurbita species (pumpkins). Informants aware of maleflowers in coconut tree and pumpkins which will not bear fruits, andfew informants have informed that male coconut flowers can be used asmedicine to increase fertility for both men and women. On the otherhand, the female informants specified their tradition of using maleflowers of pumpkins as an ornamental.

3.5. Vernacular names and plant gender

In this study, it was observed that based on different phenotypes,texture of different plant parts and morphological appearance of closelyrelated species, people have the tendency to represent a particular plantspecies either as male or female by providing gender specific vernacularnames, and such plant species are not dioecious. For example, thephenotypic variations in the flowers such as blue and white in Clitoriaternatea L., Leguminosae (Shankapusphi) is attributed to gender in KolliHills. They consider white flower phenotype as female (resembles theIndian female god Lakshmi) and blue flower phenotype as male (re-sembles the Indian male god Krishna). They prefer either one pheno-type during the rituals and the choice of phenotype is based on theritual process and whether the spiritual god is male or female.Similarly, Mimosa pudica L., Leguminosae (thottasinungi; thottasuringi)was also categorized into male and female based on the characteristicobservations in movements in the pulvini of leaves, pinnae and pinnules

of the plants in response to touch. If the leaf movement of shrinkagestarts from top to bottom basal end upon the touch, it is called malevariety (munsuringi), and if the shrinkage starts from bottom to top, it iscalled female variety (pinsuringi). On the other hand, two closely relatedmonoecious species in Moraceae, Artocarpus hirsutus Lam. (ayanipala;kattupala; peyppala) and Artocarpus heterophyllus Lam. (palamaram;narpala), are considered to be male and female plants respectivelybased on the fruit texture and timber quality. Informants reported thatthe fruit of Artocarpus hirsutus is watery and mushy in nature, whereasthe fruit of Artocarpus heterophyllus is fibrous. In addition, it was re-ported that the timber of Artocarpus hirsutus was more durable than thatof Artocarpus heterophyllus.

3.6. Plant gender and Ayurveda

The interaction with Ayurvedic doctors indicated that the Ayurvedicclassical literature has no straight forward evidence on gender pre-ference to prepare medicine or to treat illness. However the concept ofplant gender is mentioned in Ayurvedic literatures such as CharakaSamhita, Vrikshayurveda, and Rajanighantu. For example, CharakaSamhita describes the morphological appearance, properties, and usesof a particular medicinal plant called Kutaja, and the plant is describedas male plant (Pum-Kutaja) and female plant (Stri-Kutaja), and these twoplants are decoded as Holarrhena pubescens Wall. ex G.Don (Syn.Holarrhena antidysenterica (Roth) Wall. ex A.DC.) and Wrightia tinctoriaR.Br. both belonging to the Apocynaceae family (Samhita, 2001). Thesetwo species are not biologically dioecious. Therefore, it appears that theconcept of gender differentiation in Charaka Samhita for Kutaja is notbased on the floral sexual characters of the plants, rather based on themorphological appearance and properties of the plants. On the con-trary, Vrikshayurveda and Rajanighantu describe the concept of re-productive morphology and sexual differentiation of plants (Prasad andNarayana, 2007; Sengupta, 2010). For example, Rajanighantu mentionsthe existence of male and female individuals of a dioecious species plantcalled Ketaki (Pandanus odoratissimus L.f. (Syn. of Pandanus odorifer(Forssk.) Kuntze) (Adkar and Bhaskar, 2014), but includes no indicationon gender preferential usage.

4. Conclusions

During the last century, substantial ethnobotanical knowledge hasbeen documented, and ethnobotanical studies have evolved to de-monstrate the importance of traditional ecological knowledge to live-lihoods around the globe, but also highlighted the rapid rate at whichknowledge is being forgotten and lost. From this study, we identifiedthe existence of a significant knowledge gap in ethnobotanical andethnopharmacological literature on traditional knowledge of dioeciousplants. Hence, an explorative study was conducted, and from this studyit is evident that people have traditional knowledge on gender of plantsand preferential usages towards one gender for some species. Based onthis, we propose that researchers conducting an ethnobotanical and

Table 4Timber species with preferential gender usage.

Species Preferential gender usagesa

Borassus flabellifer L. ♂ timber is preferred for construction 4,7,11,16,19,21,27,30,34

Diospyros ebenum J.Koenig ex Retz. ♀ timber is preferred for construction 4,7,11,18,25,28,30,32,35,38

Drypetes sepiaria (Wight & Arn.) Pax & K.Hoffm. ♂ timber is preferred for construction 13,15,16,18,22,23,26

Lannea coromandelica (Houtt.) Merr. ♂ is preferred to make wooden vessels (mara-kuduvai) to farm animals12,13,15. ♂ preferred to be grown in farm land in orderto avoid seedings 2,6,7,9,14,17,22,28,33,35

Myristica dactyloides Gaertn. ♂ preferably cut for firewood 1,2,3,7,10

Phoenix loureiroi Kunth ♂ timber is preferred for construction 5,6,12,13,17,24,28,33,40

P. pusilla Gaertn.P. sylvestris (L.) Roxb.

a Superscript numbers are the identifiers of informants as shown in Supplementary Data S3.

G.S. Seethapathy et al.

ethnopharmacological study should consider documenting traditionalknowledge on sexual systems of plants, and test the existence of genderspecific usages in their conceptual framework and hypothesis testing.The incorporation of such concepts could provide new dimensions ofscientific knowledge with potential implications to conservationbiology, chemical ecology, ethnoecology and drug discovery.

Acknowledgements

The authors would like to thank all the people who participated inthis study and shared their knowledge on medicinal plants andAyurveda. We are thankful to Padmashree Darshan Shankar, PadmaVenkatasubramanian, Hariramamurthi G, Prakash BN, Sarin NS,Noorunnisa Begum S, Prasan Shankar (The Institute of Trans-Disciplinary Health Sciences and Technology, India) and ThangarajFrancis Xavier and Moorthy Kannan (St. Joseph's college, Trichy, India)for facilitating the study. There has been no financial support for thiswork that could have influenced its outcome.

Author contributions

GSS, KR, BSP, HdB and HW synthesized the study concept and de-signed the study methodology. GSS with the guidance of KR conductedthe field study and collected the data. GSS, KR, BSP, HdB, and HWanalyzed and interpreted the data. GSS wrote the manuscript, and allauthors have contributed to the preparation and finalization of thearticle. All authors have read and approved the final version of themanuscript.

Conflicts of interest

The authors have no conflicts of interest.

Appendix A. Supporting information

Supplementary data associated with this article can be found in theonline version at http://dx.doi.org/10.1016/j.jep.2018.04.011.

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G.S. Seethapathy et al.

Graphical abstract

Ethnobotany of dioecious species: Traditional knowledge on dioecious plants in India.

Gopalakrishnan Saroja Seethapathy, Kaliamoorthy Ravikumar, Berit Smestad Paulsen, Hugo

J. de Boer and Helle Wangensteen.

Supplementary Data

Supplementary Data S1. List of possible dioecious plants used in Indian systems of codified

and non-codified medicine.

Supplementary Data S2. Study questionnaire on ethnobotany of dioecious plants.

Supplementary Data S3. Background details of informants, their knowledge on dioecious

plants, and the category of preference for plant gender.

Supp

lem

enta

ry D

ata

S1. L

ist o

f pos

sibl

e di

oeci

ous

plan

ts u

sed

in In

dian

sys

tem

s of

cod

ified

and

non

-cod

ified

med

icin

e. T

he li

st w

as p

repa

red

usin

g th

e N

atio

nal M

edic

inal

Pla

nts

Boa

rd, G

over

nmen

t of

Indi

a m

edic

inal

pla

nts

data

base

(ht

tp://

ww

w.m

edic

inal

plan

ts.in

/), a

nd th

e lit

erat

ure

com

pile

d by

R

enne

r 201

4, (R

enne

r, S.

S., 2

014.

The

rela

tive

and

abso

lute

freq

uenc

ies

of a

ngio

sper

m s

exua

l sys

tem

s: d

ioec

y, m

onoe

cy, g

ynod

ioec

y, a

nd a

n up

date

d on

line

data

base

. Am

. J. B

ot. 1

01(1

0), 1

588-

1596

.).

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us p

lant

s Fa

mily

In

dian

folk

m

edic

ine

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rved

a Si

ddha

U

nani

So

wa-

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pa

Acal

ypha

aln

ifolia

Kle

in e

x W

illd.

Eu

phor

biac

eae

+

Ac

alyp

ha h

ispi

da B

urm

.f.

Euph

orbi

acea

e +

+

Actin

odap

hne

angu

stifo

lia N

ees

Laur

acea

e +

+

Actin

odap

hne

dich

otom

a Fl

orsk

. La

urac

eae

+

Ac

tinod

aphn

e ho

oker

i Mei

sn.

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acea

e +

+

Actin

odap

hne

obov

ata

(Nee

s)

Blu

me

La

urac

eae

+

Aerv

a ja

vani

ca (B

urm

.f.) J

uss.

ex

Schu

lt A

mar

anth

acea

e +

+ +

Aerv

a la

nata

(L.)

Juss

. A

mar

anth

acea

e +

+ +

Aerv

a to

men

tosa

For

ssk.

A

mar

anth

acea

e +

+ +

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stis

tach

ys in

dica

Dal

zell.

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phor

biac

eae

+

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rost

ista

chys

mee

bold

ii Pa

x &

K

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fm.

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orbi

acea

e +

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nthu

s alti

ssim

a (M

ill.)

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gle

Sim

arou

bace

ae

+

Ai

lant

hus e

xcel

sa R

oxb.

Si

mar

ouba

ceae

+

+ +

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nthu

s gla

ndul

osa

Des

f. Si

mar

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+

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nthu

s tri

phys

a (D

enns

t.) A

lsto

n Si

mar

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ceae

+

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orne

a ru

gosa

(Lou

r.) M

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Arg

. Eu

phor

biac

eae

+

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orne

a til

iaea

folia

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ll-A

rg.

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acea

e +

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is g

ilead

ensi

s L.

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sera

ceae

+

+

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irta

coc

culu

s (L.

) Wig

ht &

A

rn.

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ispe

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+ +

+ +

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esm

a ac

idum

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z.

Phyl

lant

hace

ae

+

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tides

ma

alex

iteri

a L.

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ylla

ntha

ceae

+

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esm

a bu

nius

(L.)S

pren

g.

Phyl

lant

hace

ae

+

+

An

tides

ma

dian

drum

Rox

b.

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lant

hace

ae

+

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tides

ma

ghae

sem

billa

Gae

rtn.

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lant

hace

ae

+

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tides

ma

men

asu

(Tul

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l.Arg

. Ph

ylla

ntha

ceae

+

+

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esm

a ze

ylan

icum

Lam

. Ph

ylla

ntha

ceae

+

Apha

nam

ixis

pol

ysta

chya

(Wal

l.)

R. P

arke

r M

elia

ceae

+

Arun

cus d

ioic

us (W

alte

r) F

erna

ld

Ros

acea

e +

Bala

noph

ora

dioi

ca R

.BR

. B

alan

opho

race

ae

+

Ba

lano

phor

a fu

ngos

a Su

bsp.

indi

ca

(Arn

.) H

ans.

Bal

anop

hora

ceae

+

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noph

ora

invo

lucr

ata

Hoo

k.f.

Bal

anop

hora

ceae

+

Bala

noph

ora

poly

andr

a G

riff.

Bal

anop

hora

ceae

+

Balio

sper

mum

axi

llare

Blu

m

Bal

anop

hora

ceae

+

+ +

+ +

Balio

sper

mum

cal

ycin

um M

uell.

B

alan

opho

race

ae

+

Ba

uhin

ia m

alab

aric

a R

oxb.

Le

gum

inos

ae

+ +

+

Ba

uhin

ia ra

cem

osa

Lam

. Le

gum

inos

ae

+

+ +

Ba

uhin

ia v

arie

gata

L.

Legu

min

osae

+ +

Bisc

hofia

java

nica

Blu

me

Phyl

lant

hace

ae

+

Bl

yxa

octa

ndra

(Rox

b.) P

lanc

h.

ex.T

hwai

tes

Hyd

roch

arita

ceae

+

Bora

ssus

flab

ellif

er L

. A

reca

ceae

+

+ +

+ +

Bora

ssus

flab

ellif

orm

is L

. A

reca

ceae

+

+ +

+ +

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elia

cre

nula

ta R

oxb.

Ph

ylla

ntha

ceae

+

Can

ariu

m b

enga

lens

e R

oxb.

B

urse

race

ae

+

C

anar

ium

com

mun

e L.

B

urse

race

ae

+

+

C

anar

ium

eup

hyllu

m K

urz

Bur

sera

ceae

+

Can

ariu

m re

sini

feru

m B

ruce

ex

Kin

g B

urse

race

ae

+ +

+

Can

ariu

m st

rict

um R

oxb.

B

urse

race

ae

+ +

+

C

anar

ium

vul

gare

Lee

nh.

Bur

sera

ceae

+

+

Can

nabi

s ind

ica

L.

Can

naba

ceae

+

+ +

+ +

Can

nabi

s sat

iva

L.

Can

naba

ceae

+

+ +

+ +

Can

nabi

s sat

iva

L.

Can

naba

ceae

+

Car

ica

cand

amar

cens

is H

ook.

f. C

aric

acea

e +

Car

ica

papa

ya L

. C

aric

acea

e +

+ +

Car

ica

papa

ya L

. C

aric

acea

e

+

Cas

sine

gla

uca

(Rot

tb.)

Kun

tze

Cel

astra

ceae

+

+ +

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aton

ia si

liqua

L.

Legu

min

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+

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mae

rops

ritc

hiea

na G

riff.

Are

cace

ae

+

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issa

mpe

los p

arei

ra L

. M

enis

perm

acea

e +

+ +

+ +

Cla

oxyl

on in

dicu

m H

assk

. Eu

phor

biac

eae

+

C

laox

ylon

pol

ot (B

urm

.f.) M

err.

Euph

orbi

acea

e +

Coc

cini

a co

rdifo

lia (L

.)Cog

n.

Cuc

urbi

tace

ae

+

+ +

+ C

occi

nia

glau

ca S

avi

Cuc

urbi

tace

ae

+ +

C

occi

nia

gran

dis (

L.) V

oigh

t C

ucur

bita

ceae

+ +

+ +

Coc

cini

a in

dica

Wig

ht &

Arn

. C

ucur

bita

ceae

+ +

+ +

Coc

colo

ba u

vife

ra L

. C

ucur

bita

ceae

+

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culu

s cor

difo

lius (

Will

d.) D

C.

Men

ispe

rmac

eae

+ +

+ +

+ C

occu

lus h

irsu

tus (

L.) D

IELS

M

enis

perm

acea

e +

+ +

+

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culu

s lau

rifo

lius D

C.

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+

C

occu

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eaeb

a D

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Men

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+

C

occu

lus m

acro

carp

us W

. & A

. PR

OD

R.

Men

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rmac

eae

+

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culu

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dulu

s (JR

. & G

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RST

) DIE

LS.

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+

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culu

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eros

us D

C.

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+ +

+ +

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occu

lus v

illos

us D

C.

Men

ispe

rmac

eae

+ +

+ +

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odia

eum

var

iega

tum

(L.)

Rum

ph.

ex A

.Juss

. Eu

phor

biac

eae

+

Cor

dia

dich

otom

a Fo

rst.

f. B

orag

inac

eae

+ +

+

+ C

osci

nium

fene

stra

tum

(Gae

rtn.)

Col

eb.

Men

ispe

rmac

eae

+ +

+

+

Cte

nole

pis c

eras

iform

is (S

tock

s)

C.B

.Cla

rke

Cuc

urbi

tace

ae

+ +

+

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lea

arno

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iers

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enis

perm

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+ +

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lea

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ista

ta G

riff.

Men

ispe

rmac

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+

C

ycle

a bu

rman

ni A

rn. e

x W

ight

M

enis

perm

acea

e +

+ +

Cyc

lea

fissi

caly

x D

unn

Men

ispe

rmac

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+

C

ycle

a pe

ltata

(Lam

.) H

ook.

f. &

Th

omso

n M

enis

perm

acea

e +

+ +

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mon

orop

s dra

co B

lum

e A

reca

ceae

+ +

+

Dap

hnip

hyllu

m h

imal

ayen

se

(Ben

th.)

Mül

l.Arg

. D

aphn

iphy

llace

ae

+

Dap

hnip

hyllu

m n

eilg

herr

ense

(W

ight

) K.R

osen

thal

D

aphn

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llace

ae

+

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isca

can

nabi

na L

. D

atis

cace

ae

+

D

ebre

geas

ia lo

ngifo

lia (B

urm

.f.)

Wed

d.

Urti

cace

ae

+

+

Den

droc

nide

sinu

ata

(Blu

me)

C

hew

U

rtica

ceae

+

+

Dim

orph

ocal

yx g

labe

llus T

hwai

tes

Euph

orbi

acea

e +

Dio

scor

ea a

cule

ata

L.

Dio

scor

eace

ae

+ +

+

D

iosc

orea

ala

ta L

. D

iosc

orea

ceae

+

+ +

Dio

scor

ea b

elop

hylla

(Pra

in) V

oigt

ex

Hai

nes

Dio

scor

eace

ae

+ +

Dio

scor

ea b

ulbi

fera

L.

Dio

scor

eace

ae

+ +

+ +

+ D

iosc

orea

cri

spat

a R

oxb.

D

iosc

orea

ceae

+

+ +

+ +

Dio

scor

ea d

aem

ona

Rox

b.

Dio

scor

eace

ae

+ +

D

iosc

orea

del

toid

ea W

all.

ex

Kun

th

Dio

scor

eace

ae

+

Dio

scor

ea e

scul

anta

(Lou

r.)

Bur

kill

Dio

scor

eace

ae

+ +

+

Dio

scor

ea g

labr

a R

oxb.

D

iosc

orea

ceae

+

+

Dio

scor

ea g

lobo

sa R

oxb.

D

iosc

orea

ceae

+

+ +

Dio

scor

ea h

amilt

onii

Hoo

k.f.

Dio

scor

eace

ae

+

D

iosc

orea

hir

suta

Den

nst.

Dio

scor

eace

ae

+ +

D

iosc

orea

his

pida

Den

nst.

Dio

scor

eace

ae

+ +

D

iosc

orea

opp

ositi

folia

L.

Dio

scor

eace

ae

+ +

+

D

iosc

orea

pen

taph

ylla

L.

Dio

scor

eace

ae

+ +

+

D

iosc

orea

pra

zeri

Pra

in &

Bur

kill

Dio

scor

eace

ae

+

D

iosc

orea

pub

er B

lum

e D

iosc

orea

ceae

+

Dio

scor

ea p

urpu

rea

Rox

b.

Dio

scor

eace

ae

+ +

+

D

iosc

orea

rube

lla L

. D

iosc

orea

ceae

+

+ +

Dio

scor

ea sa

tiva

L.

Dio

scor

eace

ae

+ +

+ +

+ D

iosc

orea

tom

ento

sa J.

Koe

nig

ex

Spre

ng.

Dio

scor

eace

ae

+

Dio

scor

ea tr

iphy

lla L

. D

iosc

orea

ceae

+

+

Dio

scor

ea w

allic

hii H

okk.

f. D

iosc

orea

ceae

+

Dio

spyr

os b

ourd

illon

i Bra

ndis

Eb

enac

eae

+

D

iosp

yros

bux

ifolia

(Blu

me)

Hie

rn

Eben

acea

e +

Dio

spyr

os c

ando

llean

a W

ight

Eb

enac

eae

+ +

+

D

iosp

yros

chl

orox

ylon

Rox

b.

Eben

acea

e +

+

Dio

spyr

os c

ordi

folia

Rox

b.

Eben

acea

e +

Dio

spyr

os e

benu

m K

oeni

g Eb

enac

eae

+

+ +

D

iosp

yros

em

bryo

pter

is P

ers.

Eben

acea

e +

+ +

+

Dio

spyr

os e

xscu

lpta

Buc

h.-H

am.

Eben

acea

e

+ +

+

Dio

spyr

os fe

rrea

(Wild

d.) B

atch

. F.

Eb

enac

eae

+

+

Dio

spyr

os g

lutin

osa

Koe

nig

Eben

acea

e

+

Dio

spyr

os in

sign

is T

hwai

tes

Eben

acea

e +

Dio

spyr

os k

aki L

.f.

Eben

acea

e +

Dio

spyr

os la

ncae

folia

Rox

b.

Eben

acea

e +

Dio

spyr

os lo

tus L

. Eb

enac

eae

+

D

iosp

yros

mal

abar

ica

(Des

r.)

Kos

tel.

Eben

acea

e +

+ +

+

Dio

spyr

os m

elan

oxyl

on R

oxb.

Eb

enac

eae

+ +

+ +

D

iosp

yros

mon

tana

Rox

b.

Eben

acea

e +

+ +

Dio

spyr

os o

ocar

pa T

hwai

tes

Eben

acea

e

+

Dio

spyr

os p

anic

ulat

a D

alz.

Eb

enac

eae

+ +

+

D

iosp

yros

per

egri

na (G

aertn

.) G

urke

Eb

enac

eae

+ +

+ +

Dio

spyr

os q

uaes

ita T

hwai

tes

Eben

acea

e +

Dio

spyr

os ra

cem

osa

Rox

b.

Eben

acea

e +

Dio

spyr

os sy

lvat

ica

Rox

b.

Eben

acea

e +

Dio

spyr

os to

men

tosa

Rox

b.

Eben

acea

e

+ +

+

Dio

spyr

os to

posi

a B

uch.

-Ham

. Eb

enac

eae

+

D

iplo

clis

ia g

lauc

esce

ns (B

lum

e)

Die

ls

Men

ispe

rmac

eae

+

+

Dod

onae

a vi

scos

a (L

.) Ja

cq.

Sapi

ndac

eae

+ +

+

D

rype

tes c

onfe

rtifl

ora

(Hoo

k. F

.) Pa

x &

K.H

offm

. Pu

tranj

ivac

eae

+

Dry

pete

s mac

roph

ylla

(Blu

me)

Pax

&

K.H

offm

Pu

tranj

ivac

eae

+

Dry

pete

s rox

burg

hii (

Wal

l.) H

urus

Pu

tranj

ivac

eae

+ +

+

+ D

rype

tes z

eyla

nica

(Thw

aite

s)

Hur

us.

Putra

njiv

acea

e +

+

Embe

lia ri

bes B

urm

.f.

Prim

ulac

eae

+ +

+ +

+ Em

belia

subc

oria

cea

(C.B

.Cla

rke.

) M

ez.

Prim

ulac

eae

+

Embe

lia ts

jeri

am-c

otta

m (R

oem

. &

Schu

lt.) A

.DC

. Pr

imul

acea

e +

+

Eury

a ac

umin

ata

DC

. Pe

ntap

hyla

cace

ae

+

Eury

a ja

poni

ca T

hunb

. Pe

ntap

hyla

cace

ae

+

Eu

rya

nitid

a K

oth.

Pe

ntap

hyla

cace

ae

+

Ex

coec

aria

ace

rifo

lia D

idr

Euph

orbi

acea

e +

Exco

ecar

ia a

gallo

cha

L.

Euph

orbi

acea

e +

+

Exco

ecar

ia c

renu

lata

Wig

ht

Euph

orbi

acea

e +

Ficu

s exa

sper

ata

Vah

l M

orac

eae

+ +

+

Fi

cus f

ulva

Rei

nw. e

x B

lum

e M

orac

eae

+

+

Fi

cus h

eter

ophy

lla L

. f.

Mor

acea

e +

+ +

Ficu

s his

pida

L.f.

M

orac

eae

+ +

+ +

+ Fi

cus o

ppos

itifo

lia R

oxb.

M

orac

eae

+ +

+ +

+ Fi

cus t

inct

orea

For

st. f

. Sub

sp.

para

sitic

a (W

illd.

) Cor

ner

Mor

acea

e

+

Ficu

s tin

ctor

ia F

orst

.f.

Mor

acea

e +

+

Frax

inus

exc

elsi

or L

. O

leac

eae

+

G

aliu

m e

lega

ns W

all.

ex R

oxb.

R

ubia

ceae

+

Gar

cini

a at

rovi

ridi

s Grif

f. ex

T.

And

erso

n C

lusi

acea

e +

Gar

cini

a co

wa

Rox

b. e

x C

hois

y C

lusi

acea

e +

+

Gar

cini

a ec

hino

carp

a Th

wai

tes

Clu

siac

eae

+

G

arci

nia

gum

mi-g

utta

(L.)

Rox

b.

Clu

siac

eae

+ +

+

G

arci

nia

indi

ca (D

up.)

Cho

isy

Clu

siac

eae

+ +

+ G

arci

nia

kydi

a R

oxb.

C

lusi

acea

e +

+

Gar

cini

a m

orel

la (G

aertn

.) D

esr.

Clu

siac

eae

+ +

+ +

+ G

arci

nia

pedu

ncul

ata

Rox

b.

Clu

siac

eae

+ G

arci

nia

purp

urea

G.D

on.

Clu

siac

eae

+ +

+ G

arci

nia

rubr

o-ec

hina

ta K

oste

rm.

Clu

siac

eae

+

G

arci

nia

spic

ata

Hoo

k.f.

Clu

siac

eae

+

G

arci

nia

talb

otii

Rai

zada

ex

Sant

apau

C

lusi

acea

e +

Gar

cini

a tr

avan

cori

ca B

edd.

C

lusi

acea

e +

Gar

cini

a w

ight

ii T.

And

erso

n C

lusi

acea

e +

Gar

cini

a xa

ntho

chym

us H

ook.

f. ex

T.

And

erso

n

Clu

siac

eae

+ +

+

Gir

ardi

nia

hete

roph

ylla

(Vah

l) D

ecne

. U

rtica

ceae

+

+

Giv

otia

rottl

erifo

rmis

Grif

f. Eu

phor

biac

eae

+

+

G

ynoc

ardi

a od

orat

a R

.BR

. A

char

iace

ae

+ +

+

G

ynos

tem

ma

pent

aphy

llum

(T

hunb

.) M

akin

o C

ucur

bita

ceae

+

Hag

enia

aby

ssin

ica

(Bru

ce e

x St

eud.

) J.F

.Gm

el.

Ros

acea

e +

Hal

ophi

la o

valis

(R.B

r.) H

ook.

f. H

ydro

char

itace

ae

+

H

ippo

phae

rham

noid

es su

bsp.

sa

licifo

lia (D

. Don

) Ser

vetta

z El

aeag

nace

ae

+

Hip

poph

ae sa

licifo

lia D

.Don

El

aeag

nace

ae

+

H

ippo

phae

tibe

tana

Sch

ltdl.

Elae

agna

ceae

+

Hod

gson

ia m

acro

carp

a (B

lum

e)

Cog

n.

Cuc

urbi

tace

ae

+

Hom

onoi

a re

tusa

(Gra

ham

ex

Wig

ht) M

üll.A

rg.

Euph

orbi

acea

e +

Hom

onoi

a ri

pari

a Lo

ur.

Euph

orbi

acea

e

+ +

Hor

sfie

ldia

gla

bra

(Rei

nw. e

x B

lum

e) W

arb.

M

yris

ticac

eae

+

Hor

sfie

ldia

irya

(Gae

rtn.)

War

b.

Myr

istic

acea

e +

Hor

sfie

ldia

kin

gii (

Hoo

k.f.)

War

b.

Myr

istic

acea

e +

Hyd

noca

rpus

alp

ina

Wig

ht

Ach

aria

ceae

+

+

Hyd

noca

rpus

ant

helm

inth

ica

Pier

re

ex G

agne

p.

Ach

aria

ceae

+

Hyd

noca

rpus

cas

tane

a H

ook.

f. &

Th

omso

n A

char

iace

ae

+

Hyd

noca

rpus

ineb

rian

s Vah

l A

char

iace

ae

+ +

+

H

ydno

carp

us k

urzi

i (K

ing)

War

b.

Ach

aria

ceae

+

+ +

Hyd

noca

rpus

laur

ifolia

(Den

nst.)

St

eum

. A

char

iace

ae

+ +

+

Hyd

noca

rpus

mac

roca

rpa

War

b.

Ach

aria

ceae

+

Hyd

noca

rpus

oct

andr

a Th

wai

tes

Ach

aria

ceae

+

Hyd

noca

rpus

pen

tand

ra (B

uch.

-H

am.)

Oke

n A

char

iace

ae

+ +

+

Hyd

noca

rpus

ven

enat

a G

aertn

. A

char

iace

ae

+

+

H

ydno

carp

us w

ight

iana

Blu

me

Ach

aria

ceae

+

+ +

Hyd

rilla

ver

ticill

ata

(L.f.

) Roy

le

Hyd

roch

arita

ceae

+

+

Hyd

roch

aris

dub

ia (B

lum

e) B

acke

r H

ydro

char

itace

ae

+

Ile

x de

ntic

ulat

a W

all.

ex W

ight

A

quifo

liace

ae

+

Ile

x go

daja

m C

oleb

r. ex

Hoo

k.f.

Aqu

ifolia

ceae

+

Ilex

para

guar

iens

is A

.St.H

il.

Aqu

ifolia

ceae

+

Ilex

wig

htia

na W

all.

ex W

ight

A

quifo

liace

ae

+

Ja

teor

hiza

pal

mat

a (L

am.)

Mie

rs

Men

ispe

rmac

eae

+ +

+

K

nem

a an

gust

ifolia

(Rox

b.) W

arb.

M

yris

ticac

eae

+

K

nem

a at

tenu

ata

(Wal

l.) W

arn.

M

yris

ticac

eae

+

+

K

nem

a er

ratic

a (H

ook.

f. &

Th

omso

n) J.

Sin

clai

r M

yris

ticac

eae

+

Kne

ma

linifo

lia (R

oxb.

) War

b.

Myr

istic

acea

e +

Laur

us c

assi

a B

urm

.f.

Laur

acea

e

+

Laur

us c

inna

mom

um (

L.)

Laur

acea

e +

+ +

+ +

Laur

us n

obili

s L.

Laur

acea

e +

+

Lind

era

caud

ata

(Nee

s) H

ook.

f.

Laur

acea

e +

Lind

era

nees

iana

(Wal

l. ex

Nee

s)

Kur

z

Laur

acea

e +

Lind

era

pulc

herr

ima

(Nee

s) H

ook.

f.

Laur

acea

e +

Lits

ea c

hine

nsis

Lam

. La

urac

eae

+ +

+ +

Li

tsea

citr

ata

Blu

me

Laur

acea

e +

+

Lits

ea c

oria

cea

Hoo

k.f.

La

urac

eae

+

Lits

ea c

ubeb

a (L

our.)

Per

s.

Laur

acea

e +

+

Lits

ea g

lutin

osa

(Lou

r.) C

.B. R

ob.

Laur

acea

e +

+ +

+

Lits

ea la

ncifo

lia (R

oxb.

ex

Nee

s)

Fern

.-Vill

. La

urac

eae

+

Lits

ea m

onop

etal

a (R

oxb.

) Per

s. La

urac

eae

+ +

+

Li

tsea

pol

yant

ha Ju

ss.

Laur

acea

e +

+ +

Lits

ea q

uinq

ueflo

ra (D

enns

t.)

Sure

sh

Laur

acea

e +

Lits

ea se

bife

ra P

ers.

Laur

acea

e +

+ +

+

Lits

ea st

ocks

ii H

ook.

f. La

urac

eae

+

Li

tsea

zeyl

anic

a N

ees &

T. N

ees

Laur

acea

e +

Lodo

icea

cal

lypi

ge C

omm

. ex

J.St.H

il.

Are

cace

ae

+

Lodo

icea

mal

divi

ca (J

.F.G

mel

.) Pe

rs.

Are

cace

ae

+ +

+ +

Lodo

icea

seyc

hella

rum

Lab

ill

Are

cace

ae

+

+ +

Lu

ffa e

chin

ata

Rox

b.

Cuc

urbi

tace

ae

+ +

+

+ M

acar

anga

den

ticul

ata

(Blu

me)

M

üll.A

rg.

Euph

orbi

acea

e +

Mac

aran

ga in

dica

W.

Euph

orbi

acea

e +

+

Mac

aran

ga n

icob

aric

a N

.P.B

alak

r. &

Cha

krab

. Eu

phor

biac

eae

+

Mac

aran

ga p

elta

ta (R

oxb.

) Mue

ll.-

Arb

. Eu

phor

biac

eae

+

+

Mac

aran

ga ro

xbur

ghii

Wig

ht

Euph

orbi

acea

e +

+

Mac

aran

ga ta

nari

us (L

.) M

üll.A

rg.

Euph

orbi

acea

e +

Mac

aran

ga te

trac

occu

s (R

oxb.

) K

urz

Euph

orbi

acea

e +

Mac

lura

pom

ifera

(Raf

.) C

.K.S

chne

id.

Mor

acea

e +

Mal

lotu

s fer

rugi

neus

(Rox

b.)

Euph

orbi

acea

e +

Mül

l.Arg

. M

allo

tus p

elta

tus (

Gei

sele

r)

Mül

l.Arg

. Eu

phor

biac

eae

+

Mal

lotu

s phi

lippe

nsis

(Lam

.) M

uell.

-Arg

. Eu

phor

biac

eae

+ +

+ +

Mal

lotu

s rep

andu

s (W

illd.

) Mul

l.-A

rg.

Euph

orbi

acea

e +

+

Mal

lotu

s rha

mni

foliu

s (W

illd.

) M

üll.A

rg.

Euph

orbi

acea

e +

Mal

lotu

s tet

raco

ccus

(Rox

b.) K

urz

Euph

orbi

acea

e +

May

tenu

s sen

egal

ensi

s (La

m.)

Exel

l C

elas

trace

ae

+ +

+

Men

ispe

rmum

coc

culu

s L.

Men

ispe

rmac

eae

+ +

+ +

M

enis

perm

um c

olum

ba R

oxb.

M

enis

perm

acea

e +

+ +

Men

ispe

rmum

cor

difo

lium

Will

d.

Men

ispe

rmac

eae

+ +

+ +

+ M

enis

perm

um g

labr

um B

urm

.f.

Men

ispe

rmac

eae

+

M

omor

dica

coc

hinc

hine

nsis

(L

our.)

Spr

eng.

C

ucur

bita

ceae

+

+

Mom

ordi

ca d

ioic

a R

oxb.

ex

Will

d.

Cuc

urbi

tace

ae

+ +

+

M

omor

dica

um

bella

ta (K

lein

ex

Will

d.) R

oxb.

C

ucur

bita

ceae

+

Mor

us a

cido

sa G

riff.

Mor

acea

e +

Mor

us a

lba

L.

Mor

acea

e +

+ +

+

Mor

us in

dica

L.

Mor

acea

e

+

Myr

ica

escu

lent

a B

uch.

-Ham

. M

yric

acea

e +

+ +

+

Myr

istic

a ar

gent

ea W

arb

Myr

istic

acea

e

+

Myr

istic

a da

ctyl

oide

s Gae

rtn.

Myr

istic

acea

e

+ +

Myr

istic

a fr

agra

ns H

outt.

M

yris

ticac

eae

+ +

+ +

+ M

yris

tica

laur

ifolia

Spr

uce

ex

A.D

C.

Myr

istic

acea

e +

Myr

istic

a m

alab

aric

a La

m.

Myr

istic

acea

e +

+ +

Myr

istic

a m

osch

ata

Thun

b.

Myr

istic

acea

e +

+ +

+ +

Myr

istic

a of

ficin

alis

L.f.

M

yris

ticac

eae

+ +

+ +

+ N

atsi

atum

her

petic

um B

uch.

-Ham

. ex

Arn

. Ic

acin

acea

e +

Neo

litse

a ca

ssia

(L.)

Kos

term

. La

urac

eae

+

N

eolit

sea

chin

ensi

s (G

ambl

e) C

hun

Laur

acea

e +

Neo

litse

a sc

robi

cula

ta (M

eiss

ner)

G

ambl

e La

urac

eae

+

+

Neo

litse

a um

bros

a (N

ees)

Gam

ble

Laur

acea

e +

Neo

litse

a ze

ylan

ica

(Nee

s & T

. N

ees)

Mer

r. La

urac

eae

+

Nep

enth

es d

istil

lato

ria

Auc

t. ex

St

eud.

N

epen

thac

eae

+

Nep

enth

es k

hasi

ana

Hoo

k.f.

Nep

enth

acea

e +

Osy

ris q

uadr

ipar

tita

Salz

m. e

x D

ecne

. Sa

ntal

acea

e +

Otte

lia a

lism

oide

s (L.

) Per

s. H

ydro

char

itace

ae

+

Pa

chyg

one

ovat

a (P

oir.)

Die

ls

Men

ispe

rmac

eae

+

Pa

chyg

one

zeyl

anic

a Sa

ntap

au &

W

agh

M

enis

perm

acea

e +

Pand

anus

fasc

icul

aris

Lam

. Pa

ndan

acea

e +

+ +

+

Pand

anus

foet

idus

Rox

b.

Pand

anac

eae

+

Pa

ndan

us fu

rcat

us R

oxb.

Pa

ndan

acea

e +

+

Pand

anus

kai

da K

urz.

Pa

ndan

acea

e +

+

Pand

anus

odo

ratis

sim

us L

.f.

Pand

anac

eae

+ +

+ +

Pa

ndan

us te

ctor

ius P

arki

nson

ex

Du

Roi

Pa

ndan

acea

e +

+ +

+

Pand

anus

thw

aite

sii M

arte

lli

Pand

anac

eae

+

Pa

ndan

us u

nipa

pilla

tus D

enns

t. Pa

ndan

acea

e +

+

Pass

iflor

a as

sam

ica

Cha

krav

. Pa

ssifl

orac

eae

+

Pa

ssifl

ora

edul

is S

ims

Pass

iflor

acea

e +

Pass

iflor

a fo

etid

a L.

Pa

ssifl

orac

eae

+ +

+

Pa

ssifl

ora

perp

era

Mas

t. Pa

ssifl

orac

eae

+

Pa

ssifl

ora

quad

rang

ular

is L

. Pa

ssifl

orac

eae

+

Pe

rica

mpy

lus g

lauc

us (L

am.)

Mer

r. M

enis

perm

acea

e +

Peri

cam

pylu

s inc

anus

(Col

ebr.)

M

iers

ex

Hoo

k. f.

& T

hom

son

Men

ispe

rmac

eae

+

Phoe

nix

acau

lis R

oxb.

A

reca

ceae

+

+

Phoe

nix

aspe

rula

tus H

utch

. A

reca

ceae

+

Phoe

nix

dact

ylife

ra L

. A

reca

ceae

+

+ +

+

Phoe

nix

fari

nife

ra R

oxb.

A

reca

ceae

+

+

Phoe

nix

hum

ilis (

L.) C

av.

Are

cace

ae

+

Ph

oeni

x lo

urei

rii K

unth

A

reca

ceae

+

Phoe

nix

palu

dosa

Rox

b.

Are

cace

ae

+

Ph

oeni

x pu

silla

Gae

rtn.

Are

cace

ae

+ +

Ph

oeni

x sy

lves

tris

Rox

b.

Are

cace

ae

+ +

+

+ Pi

per b

arbe

ri G

ambl

e.

Pipe

race

ae

+

Pi

per b

etle

L.

Pipe

race

ae

+ +

+ +

+ Pi

per b

etle

oide

s C. D

C.

Pipe

race

ae

+

Pi

per l

ongu

m L

. Pi

pera

ceae

+

+ +

+

Pipe

r nig

rum

L.

Pipe

race

ae

+ +

+ +

+ Pi

per p

edic

ella

tum

C. D

C.

Pipe

race

ae

+

Pi

per s

arm

ento

sum

Rox

b.

Pipe

race

ae

+

Pi

per s

ylva

ticum

Rox

b.

Pipe

race

ae

+

+

Pi

per t

hom

soni

(C. D

C.)

Hoo

k.f.

Pipe

race

ae

+

Pi

per w

allic

hii (

Miq

.) H

and-

Maz

z Pi

pera

ceae

+

+ +

+

Pipt

urus

vel

utin

us (D

ecne

.) W

edd.

U

rtica

ceae

+

Poik

ilosp

erm

um su

aveo

lens

(B

lum

e) M

err.

Urti

cace

ae

+

Poly

scia

s fru

ticos

a (L

.) H

arm

s A

ralia

ceae

+

Popu

lus a

lba

L.

Salic

acea

e +

Popu

lus c

aspi

ca (B

ornm

.) Bo

rnm

. Sa

licac

eae

+

Po

pulu

s cili

ata

Wal

l. Ex

Roy

le

Salic

acea

e +

Popu

lus e

uphr

atic

a O

liv.

Salic

acea

e +

Popu

lus n

igra

L.

Salic

acea

e +

Prot

ium

serr

atum

(Wal

l. Ex

C

oleb

r.) E

ngl.

Bur

sera

ceae

+

+

Pseu

duva

ria

prai

nii (

Kin

g) M

err.

Ann

onac

eae

+

Rh

amnu

s dah

uric

us P

. Law

son

Rha

mna

ceae

+

Rhus

chi

nens

is M

ill

Ana

card

iace

ae

+

+

Rh

us h

ooke

ri K

.C. S

ahni

&

Bah

adur

A

naca

rdia

ceae

+

Rhus

pun

jabe

nsis

J.L.

Ste

war

t ex

Bra

ndis

A

naca

rdia

ceae

+

Rhus

succ

edan

ea L

. A

naca

rdia

ceae

+ +

+

Rhus

wal

lichi

i Hoo

k.f.

Ana

card

iace

ae

+

Ri

bes o

rien

tale

Des

f. G

ross

ular

iace

ae

+

Sa

lix a

cmop

hylla

Boi

ss.

Salic

acea

e +

Salix

acu

tifol

ia W

illd.

Sa

licac

eae

+

Sa

lix a

lba

L.

Salic

acea

e +

Salix

bab

ylon

ica

L.

Salic

acea

e +

Salix

cap

rea

L.

Salic

acea

e

+

+

Salix

dap

hnoi

des V

ill.

Salic

acea

e +

Salix

ele

gans

Wal

l. Sa

licac

eae

+

Sa

lix p

ychn

osta

chya

And

erss

on.

Salic

acea

e +

Salix

tetr

aspe

rma

Rox

b.

Salic

acea

e +

+ +

+

Sarc

ostig

ma

klei

nii W

ight

& A

rn.

Icac

inac

eae

+ +

Sa

ssaf

ras a

lbid

um (N

utt.)

Nee

s La

urac

eae

+

Sa

ssaf

ras o

ffici

nale

Nee

s. La

urac

eae

+

Sc

hinu

s mol

le L

. La

urac

eae

+

Sc

lero

cary

a bi

rrea

(A.R

ich.

) H

ochs

t. A

naca

rdia

ceae

+

Scle

ropy

rum

wal

lichi

anum

Arn

. A

naca

rdia

ceae

+

Sem

ecar

pus a

naca

rdiu

m L

. F.

Ana

card

iace

ae

+ +

+ +

Sk

imm

ia a

nque

tilia

N.P

.Tay

lor &

A

iry S

haw

R

utac

eae

+

Skim

mia

arb

ores

cens

T. A

nder

son

ex G

ambl

e R

utac

eae

+

Skim

mia

laur

eola

Fra

nch.

R

utac

eae

+

Sm

ilax

aspe

ra L

. Sm

ilaca

ceae

+

Smila

x ch

ina

L.

Smila

cace

ae

+

+ +

Sm

ilax

glab

ra R

oxb.

Sm

ilaca

ceae

+

Smila

x gl

auco

phyl

la K

lotz

sch

Smila

cace

ae

+

Sm

ilax

lanc

eifo

lia R

oxb.

Sm

ilaca

ceae

+

Smila

x m

acro

phyl

la P

oepp

. ex

A.D

C.

Smila

cace

ae

+ +

Smila

x oc

reat

a A

.DC

. Sm

ilaca

ceae

+

Smila

x ov

alifo

lia R

oxb.

Sm

ilaca

ceae

+

+

Smila

x pa

rvifo

lia W

all.

Smila

cace

ae

+

Sm

ilax

perf

olia

ta L

our.

Smila

cace

ae

+

Sm

ilax

prol

ifera

Rox

b.

Smila

cace

ae

+

Sm

ilax

pseu

do-c

hina

Will

d.

Smila

cace

ae

+

+

Sm

ilax

wig

htii

L.

Smila

cace

ae

+

Sm

ilax

zeyl

anic

a L.

Sm

ilaca

ceae

+

+ +

Spin

acia

ole

race

a L.

A

mar

anth

acea

e +

+ +

+

Spin

ifex

litto

reus

(Bur

m.f.

) Mer

r. Po

acea

e +

+

Step

hani

a gl

abra

(Thu

ng.)

Mie

rs.

Men

ispe

rmac

eae

+

St

epha

nia

glan

dulif

era

Mie

rs.

Men

ispe

rmac

eae

+

St

epha

nia

hern

andi

folia

(Will

d.)

Wal

p.

Men

ispe

rmac

eae

+ +

+

Step

hani

a ja

pani

ca (T

hunb

.) M

iers

V

ar. d

isco

lour

(Blu

me.

) For

man

M

enis

perm

acea

e +

+ +

Step

hani

a ja

poni

ca (T

hunb

.) M

iers

M

enis

perm

acea

e +

+ +

Step

hani

a ja

poni

ca (T

hunb

.) M

iers

V

ar. j

apon

ica

M

enis

perm

acea

e +

+ +

Step

hani

a ro

tund

a Lo

ur

Men

ispe

rmac

eae

+

St

epha

nia

wig

htii

Dun

n M

enis

perm

acea

e +

Stre

blus

asp

er L

our

Mor

acea

e +

+ +

Stre

blus

taxo

ides

(Rot

h) K

urz

Mor

acea

e +

Sure

gada

mul

tiflo

ra (A

.Juss

.) Ba

ill.

Euph

orbi

acea

e +

Tetr

amel

es n

udifl

ora

R. B

R.

Tetra

mel

acea

e +

+

Tetr

astig

ma

lanc

eola

rium

(Rox

b.)

Plan

ch.

Vita

ceae

+

Tetr

astig

ma

leuc

osta

phyl

um

(Den

nst.)

Als

ton

Vita

ceae

+

+

Tetr

astig

ma

plan

icau

le (H

ook.

f.)

Gag

nep.

V

itace

ae

+

Tetr

astig

ma

serr

ulat

a (R

oxb.

) Pl

anch

. V

itace

ae

+

Thon

ning

ia a

xilla

ris (

L.) K

untz

e B

alan

opho

race

ae

+

+

Ti

liaco

ra a

cum

inat

a M

iers

M

enis

perm

acea

e +

+

Tilia

cora

race

mos

a C

oleb

r M

enis

perm

acea

e +

+

Tino

spor

a an

dam

anic

a D

iels

M

enis

perm

acea

e +

Tino

spor

a co

rdifo

lia (W

illd.

) Mie

rs

Men

ispe

rmac

eae

+ +

+ +

+ Ti

nosp

ora

cris

pa (L

.) H

ook.

f. &

Th

omso

n

Men

ispe

rmac

eae

+

Tino

spor

a m

alab

aric

a (L

am.)

Hoo

k. F

. & T

hom

s M

enis

perm

acea

e +

+ +

Tino

spor

a si

nens

is (L

our.)

Mer

r. M

enis

perm

acea

e +

+ +

Tino

spor

a to

men

tosa

(Col

ebr.)

H

ook.

f. &

Tho

ms.

Men

ispe

rmac

eae

+ +

+

Trac

hyca

rpus

mar

tianu

s (W

all.

ex

Mar

t.) H

.Wen

dl.

Are

cace

ae

+

Trew

ia n

udifl

ora

L.

Euph

orbi

acea

e +

+ +

Trew

ia p

olyc

arpa

Ben

th.

Euph

orbi

acea

e +

Tric

hosa

nthe

s bra

ctea

ta (L

am.)

Voi

gt

Cuc

urbi

tace

ae

+ +

+ +

Tric

hosa

nthe

s dio

ica

Rox

b.

Cuc

urbi

tace

ae

+ +

+

Tr

ophi

s asp

era

Ret

z.

Mor

acea

e +

+ +

Urt

ica

arde

ns L

ink

Urti

cace

ae

+

U

rtic

a di

oica

L.

Urti

cace

ae

+

U

rtic

a hy

perb

orea

Jacq

. ex

Wed

d.

Urti

cace

ae

+

Va

leri

ana

jata

man

si Jo

nes

Cap

rifol

iace

ae

+ +

+ +

+ Va

leri

ana

wal

lichi

i DC

. C

aprif

olia

ceae

+

+ +

+ +

Valli

sner

ia sp

iral

is L

. H

ydro

char

itace

ae

+

+

Vi

scum

alb

um L

. Sa

ntal

acea

e +

+

+

Vitis

hey

nean

a R

oem

. & S

chul

t. V

itace

ae

+

+

Xy

losm

a lo

ngifo

lium

Clo

s Sa

licac

eae

+

Za

noni

a in

dica

L.

Cuc

urbi

tace

ae

+ +

Za

ntho

xylu

m a

cant

hopo

dium

DC

. R

utac

eae

+

+ +

Za

ntho

xylu

m a

rmat

um D

C.

Rut

acea

e

+ +

+

Zant

hoxy

lum

rhet

sa (R

oxb.

) DC

. R

utac

eae

+ +

+

+

indi

cate

s the

pre

senc

e of

that

par

ticul

ar sp

ecie

s in

diff

eren

t Ind

ian

tradi

tiona

l sys

tem

s of m

edic

ine.

Supp

lem

enta

ry D

ata

S2. S

tudy

que

stio

nnai

re o

n et

hnob

otan

y of

dio

ecio

us p

lant

s.

Ques

tionn

aire d

eals

with

Dioe

cious

plan

ts Id

entif

icatio

n, P

refe

renc

e and

use

s by E

thni

c Peo

ple

Form

1 In

form

ants

’ con

sent

for t

he p

artic

ipat

ion

in th

e stu

dy:

I.......

........

........

........

........

........

......

(nam

e of

infor

mant)

her

eby g

ive m

y full

cons

ent a

nd co

nscio

us to

par

ticipa

te in

this s

tudy a

nd d

eclar

e tha

t to

the b

est o

f my

kno

wled

ge th

e inf

orma

tion

that I

hav

e pr

ovide

d ar

e tru

e, ac

cura

te an

d co

mplet

e. I a

m als

o aw

are

that t

his c

onve

rsatio

n is

being

reco

rded

; I h

ave

also

been

fore

told t

hat th

e inf

orma

tion f

rom

this s

tudy w

ill be

shar

ed w

ith th

em

(

) Pre

fer no

t to be

reco

rded

. Da

te.....

........

........

........

........

... (S

ignatu

re/T

humb

impr

essio

n of In

forma

nt)

1. De

tails

of L

ocat

ion

1. Co

de of

the C

ommu

nity B

ased

orga

nizati

on:__

____

____

____

____

____

__

2. Na

me of

the V

illage

:____

____

____

____

____

____

____

____

____

____

___

3. Na

me of

the P

anch

ayat:

____

____

____

____

____

____

____

____

____

____

2. De

tails

of t

he in

terv

iewee

1. Na

me:__

____

____

____

____

____

____

____

____

____

____

____

____

____

_ 2.

Gend

er: (

M) (

F)

Age:_

____

____

_ 3.

Tribe

:____

____

____

____

____

____

____

____

____

4.

Occu

patio

n:___

____

____

____

____

____

__ &

Edu

catio

n___

____

____

____

____

____

____

____

___

3. Ex

istin

g Kn

owled

ge o

n Me

dicin

al Pl

ants

1. Do

you u

se lo

cally

avail

able

plants

to tr

eat/p

reve

nt an

y illn

esse

s?

Ye

s ( )

No

( )

2.) F

or ho

w ma

ny ye

ars h

ave y

ou be

en pr

actic

ing tr

aditio

nal m

edici

ne?

------

------

------

------

------

------

------

------

------

---

3.) A

re yo

u spe

cializ

ed in

trea

ting a

ny pa

rticula

r illne

sses

?

---

------

------

------

------

------

------

------

------

------

4. K

nowl

edge

on

Dioe

cy P

lants

1.) H

ave y

ou ob

serve

d a ge

nera

l phe

nome

non t

hat p

lants

can e

xist a

s male

plan

t and

fema

le pla

nt?

i.e

., tha

t a gr

oup o

f plan

ts be

ar se

eds/f

ruits

and o

ther f

rom

same

spec

ies/ki

nd do

not.

Yes

( )

No

( )

2.) If

yes,

Could

you l

ist th

e spe

cies n

ame?

For

exam

ple: P

apay

a, Pa

nai, J

athika

i,

2.1.)

after

writi

ng th

e plan

t nam

es, a

sk. D

o you

use t

hese

plan

ts for

any p

urpo

ses?

2.1

.1. If

yes,

fill in

Tab

le 1 g

ener

al us

es re

spec

tive t

o the

ir plan

t nam

es

2.1

.2) af

ter w

riting

the u

ses,

ask.

Wou

ld yo

u pre

fer to

use a

ny on

e gen

der in

the l

isted

plan

ts?

2.1.3)

Ask

for p

arts

used

, then

avail

abilit

y, pla

nt typ

e and

if the

re is

anyth

ing el

se of

impo

rtanc

e for

the s

pecie

s?

Table

1. S.no

Pl

ant n

ames

(Loc

al)

Gene

ral U

ses1

(M,F

,T,A

,H,O

) Ge

nder

2 (M,

F,B,

N)

Parts

used

Av

ailab

ility3

(CL,C

F,R,

CR)

Plan

t type

4 (H

,S,C

,V,T

) Re

marks

1

2

3

4

5

6

7

8

9

1 M=M

edici

ne; F

=Foo

d; T=

Timbe

r; A=

Agric

ultur

e; H=

Hand

craft;

O=Ot

her-s

pecif

y) 2 M

=Male

; F=F

emale

, B=B

oth (b

oth pl

ants

are e

quall

y pote

ntial)

, N=N

o (No

cons

idera

tion o

f dioe

cy)

3 CL=

Comm

on lo

cal; C

F=Co

mmon

For

est; R

= Ra

re; V

E=Ve

ry Ra

re

4 H=

Herb

; S=S

hrub

; C=

Clim

ber;

V=Vi

nes;

T=Tr

ee

5. Co

llect

ion,

use

s and

pre

para

tion

met

hods

for m

edici

nal p

lants

1) A

sk fo

r infor

matio

n abo

ut the

plan

ts us

ed fo

r med

icina

l pur

pose

s in

Table

2

Table

2

S.no

Pa

rts co

llecte

d and

us

ed

Colle

ction

stra

tegies

Mo

de of

prep

aring

the

medic

ines

Nam

e of

dise

ase(

s)

trea

ted/

Use

s M

ode

of a

dmin

istra

tion

and

Dosa

ge

Any

spec

ial p

roce

dure

s (i.

e..,

a pl

ant i

s po

isono

us o

r to

impr

ove

effic

acy)

6. Li

ve sp

ecim

ens a

nd p

hoto

gui

ded

inte

rview

1.) If

a per

son d

oes n

ot re

port

any d

iffere

ntiati

on be

twee

n male

and f

emale

plan

ts, th

en di

scus

s usin

g the

plan

t pho

tos or

herb

arium

s or c

ollec

ted liv

e spe

cimen

s!

Table

3

S.no

Pl

ant n

ames

Lo

cal N

ame

Gene

ral U

ses

Gend

er*

(M,F

,B,N

) Pa

rts u

sed

Avail

abilit

y**

(CL,

CF,R

,CR)

Pl

ant t

ype*

** (H

,S,C

,V,T

) Re

mar

ks

Supp

lem

enta

ry D

ata

S3. B

ackg

roun

d de

tails

of i

nfor

man

ts, t

heir

know

ledg

e on

dio

ecio

us p

lant

s, an

d th

e ca

tego

ry o

f pre

fere

nce

for

plan

t gen

der.

Info

rman

ts

iden

tity

num

ber

Gen

der a

nd a

ge o

f in

form

ants

A

rea

of th

e st

udy

Tota

l num

ber

of sp

ecie

s re

cogn

ized

as

dioe

ciou

s

Tota

l num

ber o

f di

oeci

ous

spec

ies r

epor

ted

for u

sage

s

Tota

l num

ber

of d

ioec

ious

sp

ecie

s pr

efer

red

for

its g

ende

r

Rep

orte

d di

oeci

ous s

peci

es

for p

refe

renc

e

1 M

ale,

75

Kol

li H

ills

25

28

8

Anam

irta

coc

culu

s Bo

rass

us fl

abel

lifer

C

anar

ium

stri

ctum

M

yris

tica

dact

yloi

des

Phoe

nix

lour

eiro

i Ph

oeni

x p

usill

a Ph

oeni

x sy

lves

tris

Pi

per b

etle

2 Fe

mal

e, 5

6 K

olli

Hill

s 25

24

6

Apha

nam

ixis

pol

ysta

chya

C

anar

ium

str

ictu

m

Lann

ea c

orom

ande

lica

M

yris

tica

dact

yloi

des

Pipe

r bet

le

Tino

spor

a co

rdifo

lia

3 M

ale,

48

Kol

li H

ills

15

22

6

Bora

ssus

flab

ellif

er

Can

ariu

m st

rict

um

Myr

istic

a da

ctyl

oide

s Ph

oeni

x lo

urei

roi

Phoe

nix

pusi

lla

Phoe

nix

sylv

estr

is

4 M

ale,

70

Kol

li H

ills

22

22

4

Anam

irta

coc

culu

s Bo

rass

us fl

abel

lifer

C

anar

ium

stri

ctum

D

iosp

yros

ebe

num

5

Mal

e, 5

7 K

olli

Hill

s 19

23

4

Can

ariu

m st

rict

um

Phoe

nix

lour

eiro

i Ph

oeni

x p

usill

a Ph

oeni

x sy

lves

tris

6 M

ale,

45

Kol

li H

ills

14

19

5

Can

ariu

m s

tric

tum

La

nnea

cor

oman

delic

a

Phoe

nix

lour

eiro

i Ph

oeni

x p

usill

a Ph

oeni

x sy

lves

tris

7 M

ale,

50

Kol

li H

ills

19

24

6

Anam

irta

coc

culu

s Bo

rass

us fl

abel

lifer

C

anar

ium

stri

ctum

D

iosp

yros

ebe

num

La

nnea

cor

oman

delic

a M

yris

tica

dact

yloi

des

8 Fe

mal

e, 4

8 K

olli

Hill

s 22

25

4

Apha

nam

ixis

pol

ysta

chya

C

anar

ium

str

ictu

m

Pipe

r bet

le

Tino

spor

a co

rdifo

lia

9 Fe

mal

e, 4

2 K

olli

Hill

s 22

23

2

Can

ariu

m s

tric

tum

La

nnea

cor

oman

delic

a

10

Mal

e, 4

7 K

olli

Hill

s 15

18

3

Can

ariu

m s

tric

tum

M

yris

tica

dact

yloi

des

Pipe

r bet

le

11

Mal

e, 5

5 Si

ttlin

gi V

alle

y 25

24

3

Bora

ssus

flab

ellif

er

Dio

spyr

os e

benu

m

Pipe

r bet

le

12

Mal

e, 4

2 Si

ttlin

gi V

alle

y 18

26

5

Lann

ea c

orom

ande

lica

Phoe

nix

lour

eiro

i Ph

oeni

x p

usill

a Ph

oeni

x sy

lves

tris

Pi

per b

etle

13

Mal

e, 8

0 Si

ttlin

gi V

alle

y 26

28

9

Anam

irta

coc

culu

s Ap

hana

mix

is p

olys

tach

ya

Bora

ssus

flab

ellif

er

Dry

pete

s sep

iari

a

Lann

ea c

orom

ande

lica

Phoe

nix

lour

eiro

i Ph

oeni

x p

usill

a Ph

oeni

x sy

lves

tris

Pi

per b

etle

14

Mal

e, 5

6 Si

ttlin

gi V

alle

y 26

25

3

Anam

irta

coc

culu

s La

nnea

cor

oman

delic

a

Pipe

r bet

le

15

Mal

e, 4

8 Si

ttlin

gi V

alle

y 23

21

2

Dry

pete

s sep

iari

a La

nnea

cor

oman

delic

a

16

Mal

e, 4

2 Si

ttlin

gi V

alle

y 19

19

3

Bora

ssus

flab

ellif

er

Dry

pete

s sep

iari

a

Tino

spor

a co

rdifo

lia

17

Mal

e, 6

0 Si

ttlin

gi V

alle

y 28

21

5

Bora

ssus

flab

ellif

er

Lann

ea c

orom

ande

lica

Ph

oeni

x lo

urei

roi

Phoe

nix

pus

illa

Phoe

nix

sylv

estr

is

18

Mal

e, 5

0 Si

ttlin

gi V

alle

y 26

22

2

Dio

spyr

os e

benu

m

Dry

pete

s sep

iari

a

19

Mal

e, 4

7 Si

ttlin

gi V

alle

y 19

21

2

Bora

ssus

flab

ellif

er

Tino

spor

a co

rdifo

lia

20

Mal

e, 4

8 Si

ttlin

gi V

alle

y 18

19

1

Pipe

r bet

le

21

Mal

e, 5

7 Si

ttlin

gi V

alle

y 25

24

2

Bora

ssus

flab

ellif

er

Tino

spor

a co

rdifo

lia

22

Mal

e, 7

0 Si

ttlin

gi V

alle

y 24

20

6

Bora

ssus

flab

ellif

er

Dry

pete

s sep

iari

a La

nnea

cor

oman

delic

a

Phoe

nix

lour

eiro

i Ph

oeni

x p

usill

a Ph

oeni

x sy

lves

tris

23

Mal

e, 6

1 Si

ttlin

gi V

alle

y 23

20

2

Can

ariu

m st

rict

um

Dry

pete

s sep

iari

a

24

Mal

e, 4

0 Si

ttlin

gi V

alle

y 18

20

3

Phoe

nix

lour

eiro

i Ph

oeni

x p

usill

a Ph

oeni

x sy

lves

tris

25

Mal

e, 8

0 Si

ttlin

gi V

alle

y 25

21

6

Bora

ssus

flab

ellif

er

Dio

spyr

os e

benu

m

Phoe

nix

lour

eiro

i Ph

oeni

x p

usill

a Ph

oeni

x sy

lves

tris

Pi

per b

etle

26

Mal

e, 4

5 Si

ttlin

gi V

alle

y 19

20

2

Dry

pete

s sep

iari

a

Pipe

r bet

le

27

Mal

e, 8

0 Si

ttlin

gi V

alle

y 26

20

5

Bora

ssus

flab

ellif

er

Phoe

nix

lour

eiro

i Ph

oeni

x p

usill

a Ph

oeni

x sy

lves

tris

Pi

per b

etle

28

Mal

e, 6

5 Si

ttlin

gi V

alle

y 25

20

5

Dio

spyr

os e

benu

m

Lann

ea c

orom

ande

lica

Ph

oeni

x lo

urei

roi

Phoe

nix

pus

illa

Phoe

nix

sylv

estr

is

29

Fem

ale,

60

Sittl

ingi

Val

ley

25

23

2 Ap

hana

mix

is p

olys

tach

y Pi

per b

etle

30

Mal

e, 5

8 Se

rvar

ayan

H

ills

25

21

2 Bo

rass

us fl

abel

lifer

D

iosp

yros

ebe

num

31

Mal

e, 4

0 Se

rvar

ayan

H

ills

18

19

1 An

amir

ta c

occu

lus

32

Mal

e, 5

0 Se

rvar

ayan

H

ills

23

23

1 D

iosp

yros

ebe

num

33

Mal

e, 6

0 Se

rvar

ayan

H

ills

21

21

6 C

anar

ium

stri

ctum

La

nnea

cor

oman

delic

a

Phoe

nix

lour

eiro

i

Phoe

nix

pus

illa

Phoe

nix

sylv

estr

is

Pipe

r bet

le

34

Fem

ale,

60

Serv

aray

an

Hill

s 24

21

2

Bora

ssus

flab

ellif

er

Pipe

r bet

le

35

Mal

e, 6

5 Se

rvar

ayan

H

ills

24

25

6

Dio

spyr

os e

benu

m

Lann

ea c

orom

ande

lica

Ph

oeni

x lo

urei

roi

Phoe

nix

pus

illa

Phoe

nix

sylv

estr

is

Pipe

r bet

le

36

Mal

e, 4

2 Se

rvar

ayan

H

ills

16

20

2 C

anar

ium

stri

ctum

Pi

per b

etle

37

Fem

ale,

65

Serv

aray

an

Hill

s 23

20

2

Can

ariu

m st

rict

um

Pipe

r bet

le

38

Mal

e, 7

7 Se

rvar

ayan

H

ills

22

22

5

Bora

ssus

flab

ellif

er

Dio

spyr

os e

benu

m

Phoe

nix

lour

eiro

i Ph

oeni

x p

usill

a,

Ph

oeni

x sy

lves

tris

39

Fem

ale,

75

Serv

aray

an

Hill

s 23

18

2

Pipe

r bet

le

Tino

spor

a co

rdifo

lia

40

Mal

e, 7

8 Se

rvar

ayan

H

ills

22

17

5

Bora

ssus

flab

ellif

er

Phoe

nix

lour

eiro

i Ph

oeni

x p

usill

a Ph

oeni

x sy

lves

tris

Pi

per b

etle

III

1SCIENTIFIC REPORTS | (2018) 8:10561

Authentication of Garcinia fruits and food supplements using DNA barcoding and NMR spectroscopyGopalakrishnan Saroja Seethapathy Tadesse Urumarudappa Srikanth Vasudeva Malterud Shaanker

Boer Ravikanth & Helle Wangensteen

Garcinia −

Garcinia− −

Garcinia G gummi-gutta G cambogia G indica

− −Garcinia food supplements

−−

used as a regulatory tool to authenticate Garcinia

Globalization in the trade of herbal products and an expanding commodity market have resulted in widespread consumption of medicinal plants as drugs, cosmetics and food supplements, both in developing and developed countries1,2. Quality, safety and efficacy of herbal medicines are key requirements for public health and a major concern for regulatory authorities3. In India, herbal raw materials for the herbal industry are predominately harvested from wild populations4 .Unsustainable harvest of medicinal plants from the wild can lead to severe ecological consequences, including reduced plant populations, habitat destruction, loss of genetic diversity, and local extinction of species5. India is the second largest exporter of medicinal plants and exports mainly in the form of dried plant products4. Several reports suggest that only 10% of medicinal plants traded in India are being cultivated2,4. Growing commercial demands for raw drug products increases the incentive for adulteration and substitution in the medicinal plants trade, and such adulteration can threaten consumer health, damage con-sumer confidence, and generally lower the trade value of such products1.

Garcinia L. (Clusiaceae) is a genus of evergreen polygamous trees and shrubs comprising 400 species with a pantropical distribution6. Many Garcinia species in tropical Asia, Southern Africa and Northern Australia are locally used, and the edible fruits are of interest worldwide, as well as having potential implication on the econ-omy of local communities7. Thirty-five species occur in India, of which 17 species are reported from the Western Ghats7. Garcinia fruits are widely collected and commercially exploited for their medicinal value, and the fruit rinds of Garcinia gummi-gutta (L.) Roxb. (syn. Garcinia cambogia (Gaertn.) Desr.) are traditionally used to treat constipation, piles, rheumatism, edema, irregular menstruation and intestinal parasites, and are also used as a food flavoring agent and preservative8. The fruit rinds of Garcinia indica (Thouars) Choisy are traditionally used to treat rheumatic pains, bowel complaints, hemorrhoids, ulcers, inflammations, sores, dermatitis, dysentery, to prevent hyperhidrosis, and are an important culinary agent, as well9. In India, G. gummi-gutta and G. indica are

Oslo, Norway.

Norway. Department of Forest Biology, College of Forestry,

gravikanth@atree.org helle.wangensteen@farmasi.uio.no

Received: 19 January 2018

Accepted: 26 June 2018

Published: xx xx xxxx

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2SCIENTIFIC REPORTS | (2018) 8:10561

the predominant Garcinia species occurring in the Western Ghats, and these are locally traded as Kodampuli and Kokum, respectively (Fig. 1). These species are traded throughout India and exported to other countries as raw drugs, juices and extracts7. Several other species, such as G. morella (Gaertn.) Desr., G. cowa Roxb. ex Choisy, G. mangostana L., G. kydia Roxb., G. lanceifolia Roxb. and G. pedunculata Roxb. ex Buch.-Ham, are also traditionally used and traded for various culinary and medicinal purposes7. The trade analysis of Garcinia species suggests that the demand for the dried fruit rinds (raw drugs) and its value-added products are increasing4,7.

Garcinia fruits are a rich source of (−)-hydroxycitric acid, anthocyanins and polyisoprenylated benzophe-none derivatives like garcinol, camboginol, guttiferones and xanthochymol10. (−)-Hydroxycitric acid, often abbreviated HCA in commercial products and advertisements, has gained considerable attention as a promising anti-obesity agent, and commercial Garcinia extracts claiming a high content of (−)-hydroxycitric acid are pop-ular food supplements worldwide11. In human and animal studies, (−)-hydroxycitric acid has been reported to effectively curb appetite, suppress food intake, increase the rates of hepatic glycogen synthesis, reduce fatty acid synthesis and lipogenesis and decrease body-weight gain12. However, contradictory results have been reported, suggesting that (−)-hydroxycitric acid can cause only short-term weight loss, the magnitude of the weight loss effect is small, and the clinical relevance is uncertain13. The safety related to the intake of extracts with a high content of (−)-hydroxycitric acid is also uncertain14.

A common problem in herbal products highlighted in recent media reports and in scientific literature, is adul-teration in high value raw drugs and finished products15–17. The underlying reasons for such adulterations may be due to confusion as morphologically similar material identified by vernacular names in different languages enters the chain of commercialization18, lack of authentic raw plant material19, and also adulteration for financial gain20. Adulteration may be considerable, given that there is no regulatory tool for quality control, no strict commercial testing for authentication of herbal products, and a lack of authentication along the value chains for highly traded medicinal plants21.

Value chain research focuses on the nature of the relationships among the various participants involved in the chain and on the implications of these relationships for development21. The demand for medicinal herbs or herbal products is growing at the rate of 15–25% annually21. Surprisingly, only a limited number of studies exist with the focus on value chains for herbal medicines22. Understanding and establishing the value chains and trade network for herbal medicines may reflect the reasons for the variation in quality of a particular herbal drug.

Figure 1. Places of collections of Garcinia raw drug samples in South India.

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3SCIENTIFIC REPORTS | (2018) 8:10561

To authenticate herbal medicines, different tools for identification are available depending on the plant species and processes involved. These range from straightforward morphological or microscopic identification of plant parts to more advanced genetic or chemical approaches23. Among molecular identification tools, DNA barcoding has been successfully used in detecting and quantifying adulteration in raw herbal trade of a variety of medicinal plants. However, the application of the technique is often constrained by the inability to extract good quality DNA from raw herbal materials ranging from simple dried leaves to powdered plant parts, for subsequent PCR-based amplification of DNA barcode markers19. Alternatively, spectroscopic methods such as NMR can be used for the analysis of chemical compounds in complex mixtures such as plant extracts, pharmaceuticals and herbal prepa-rations24. NMR spectroscopy is highly reproducible, robust and inherently quantitative without the need for prior chromatographic separation of multiple components. The technique has been used for detection, identification and quantitative determination of adulterants in weight loss food supplements24,25. Combining DNA barcoding with spectroscopic methods for authentication of herbal medicines increases the resolution in species identifica-tion and analysis of mixtures19.

In this study, the specific objectives were: (1) to understand the value chain of Garcinia species based herbal products; (2) to assess the extent of adulteration in Garcinia species in the herbal raw drug trade of Southern India using DNA barcoding; (3) to quantify the principle organic acids, (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone, in Garcinia raw herbal drugs using NMR spectroscopy; and (4) to identify and quantify (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone in food supplements that claim to contain Garcinia extract, using NMR spectroscopy.

Materials and MethodsInformal semi-quantitative interviews were conducted in villages of Sirsi (Fig. 1),

which is one among the five forest divisions of Uttara Kannada district in Western Ghats of India. Total forest area of Sirsi is 1737 km2, and the natural vegetation of Sirsi can be broadly divided into four different categories of forests, viz. tropical evergreen, semi-evergreen, moist deciduous and dry deciduous26. Twenty farmers were interviewed about the number of Garcinia species available in their region, how many species they collected for trade, and the amounts and which plant parts they collected. They were also asked if they used any post-harvest techniques to process the fruits and if they graded the fruits based on different qualities.

Forty-one taxonomically validated her-barium vouchers from eleven species of Garcinia L. were collected from the Western Ghats and Northeast India, (Supplementary Table S1). The collected samples were identified using morphological keys in The Flora of India27 as well as by Dr. Srikanth Gunaga, taxonomist at College of Forestry, University of Agricultural Sciences (Sirsi). Nomenclature follows The Plant List (The Plant List, 2013 http://www.theplantlist.org). For each of the species, herbarium specimens were prepared and deposited at the Herbarium of the Ashoka Trust for Research in Ecology and the Environment (ATREE), Bangalore (Supplementary Table S1). These taxonomically authenticated samples are referred to as Biological Reference Material (BRM). The BRM was used to amplify and sequence DNA bar-codes for three regions: the nuclear ribosomal nrITS and the chloroplast markers psbA-trnH and rbcL.

Raw drug samples were obtained from major raw drug markets in Southern India (Figs 1 and 2). During interviews with vendors we elicited data for all known vernacular and trade names of Garcinia species (Supplementary Table S2), but we found that only Kodampuli (G. gummi-gutta) and Kokum (G. indica) were traded. One hundred grams of dried fruit rinds were purchased from each shop, totally 21 shops, and the obtained samples were vouchered as Herbal Authentication Service (HAS) with all the necessary details of shops and place of collection and deposited in the herbarium of ATREE, Bangalore (Supplementary Table S3). A chain of custody protocol ensured that samples were not mixed from the time of collection to DNA extraction

Figure 2. (a) Raw drug samples collected as Kodampuli and Kokum from the raw drug markets of southern India. (b) Garcinia gummi-gutta fruit. (c) Garcinia indica fruit.

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4SCIENTIFIC REPORTS | (2018) 8:10561

and NMR spectroscopic analysis. Although most of the raw drugs collected were dried fruit rinds, they had retained significant morphological features of the fruits.

Garcinia Ten herbal products that included either G. gummi-gutta or G. indica were purchased in pharmacies in India (5), Romania (1), and via e-commerce from United States (2), Norway (1), and Sweden (1). According to the label information, five products contained G. gummi-gutta (syn. G. cambogia), two products contained G. indica, and three products contained G. gummi-gutta, among other ingredients. An overview of the samples including label information, but not the producer/importer name, lot number, expiration date or any other information that could lead to the identification of that specific product is presented in Supplementary Table S4.

DNA was extracted from the leaf tissues of BRM (n = 11 species with 2–5 individuals in each species) using a previously described protocol28. Extracted DNA was quantified, and polymerase chain reactions were performed to amplify nrITS, psbA-trnH and rbcL using specific primers following standard protocols19,29 (Supplementary Table S5). The amplified products of nrITS, psbA-trnH and rbcL were sequenced on an automated ABI 3100 Genetic Analyzer (Applied Biosystems, CA, United States) by Shrimpex Biotech, Chennai, India.

The obtained DNA sequence chromatograms were visualized and edited to remove ambiguous base calls and primer sequences using BioEdit v.5.0.630. The sequences of nrITS, psbA-trnH and rbcL were deposited in NCBI GenBank and the accession numbers for all the BRM samples are given in Supplementary Table S1. Each species sequence was used as a query sequence in a BLASTn search. Along with the best match sequences in BLASTn search, the available DNA sequences for nrITS and rbcL of Indian Garcinia species were downloaded in FASTA format from GenBank and included in the analysis. No psbA-trnH sequences for these Indian Garcinia species were found (Supplementary Table S1). Interspecific and intraspecific genetic distances were calculated in PAUP* v.4.0b.1031 using K2P model, as well as the number of parsimony characters per marker. A Maximum Likelihood tree was generated using RAxML32 available through the Cipres Web Portal (http://www.phylo.org)33. RAxML analyses were run with default parameters for the three markers separately, and Clusia species (Clusiaceae) were chosen as outgroup. Further, in order to test the possibilities of concatenating the three markers, incongruence between the nuclear and plastid markers were tested using incongruence length difference test (ILD) in PAUP* v.4.0b.1031, and also trees were visually evaluated whether there are any conflicting topologies in the gene trees, defined as ML bootstraps >85 on nodes.

In order to authenticate identities of the herbal raw drug samples procured as Kodampuli and Kokum, genomic DNA was isolated using a DNA extraction protocol optimized for Garcinia species28 and amplified for nrITS and sequenced as described above. A single-locus Maximum Likelihood tree was constructed using both the herbal raw drug samples nrITS sequences and the nrITS BRM barcode library using RAxML32 available through the Cipres Web Portal (http://www.phylo.org)33 (Fig. 3).

Garcinia Due to the highly hygroscopic nature of Garcinia fruits, the market samples of Kodampuli (n = 18) and Kokum (n = 6) were freeze-dried using an Alpha 1–4 LD plus laboratory freeze dryer (Martin Christ GmbH, Osterode am Harz, Germany) for 24 hours and then pulverized in a commercial blender (Waring Commercial, New Hartford, CT, United States). Aliquots of ca. 1 g, accurately weighed, of fruit powder were extracted with Milli-Q water at 120 °C using an Accelerated Solvent Extraction system (ASE350 Solvent Extractor, Dionex, Sunnyvale, CA, United States). Three grams of diatomaceous earth (Dionex) was mixed with the Garcinia fruit powder aliquots and loaded in 100 ml steel cartridges. The cartridges were fitted on to the system and exhaustive extraction was performed with three cycles. Preheating time was 5 min, static extraction per cycle was 5 min and the extraction was carried out under a pressure of ca. 1500 PSI (10 MPa). The extracts from each cycle was combined, and water was evaporated at 60 °C using a rotary evaporator (RV 10 Basic Rotavapor with vacuum controller, IKA-Werke GmbH & Co. KG, Staufen, Germany). Further the extract was dried by freeze-drying, yielding 462.2–772.3 mg (45.7–76.6%) of water extract.

For Garcinia food supplements analysis, the capsules were opened and their contents removed (7 capsules), whereas tablets were ground to powder (3 tablets). One to two grams of each food supplement was accurately weighed (Supplementary Table S4) and extracted (extraction yield was 0.8758–1.1854 g (53.8–98.3%)). Extraction and NMR analysis were carried out using the same methodology as for the Garcinia water extracts.

Apart from water extracts of fruit rinds and food supplements, methanol extracts of Garcinia food supple-ments and Garcinia fruit rind powder were prepared following a modified protocol34 in order to understand whether polyisoprenylated xanthones can be used as marker compounds for identification. Briefly, aliquots of capsule contents, powdered tablets or fruit rind powder were weighed into 2 ml Eppendorf tubes. CD3OD (1.5 ml) was added and the samples were vortexed for 1 min. The samples were then ultrasonicated for 20 minutes at room temperature before centrifugation at 1000 g for 30 minutes. To the supernatant was added 0.1% TFA and the acid-ified samples were transferred to NMR tubes for analysis.

− − 1H NMR spectra of Garcinia water extracts and food supplement extracts were acquired for quantitative analysis on a Bruker AVII400 (Bruker GmbH, Rheinstetten, Germany) instrument equipped with a 5 mm BBOF probe and operating at a frequency of 400 MHz at 298 K, and with number of scans = 128. A reference solvent solu-tion was prepared containing 2.0 mg/ml maleic acid (internal reference) (Sigma-Aldrich, St. Louis, Missouri, United States) and 0.1% 3-(trimethylsilyl) propionic 2,2,3,3-d4 acid sodium salt (TSP) (Sigma-Aldrich) in D2O (Sigma-Aldrich). For NMR analysis, extracts were dissolved in 500 μl of the NMR reference solvent solution

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under vortex agitation, giving a final concentration of 20 mg/ml of extract, and analyzed in triplicate. The pH of all Garcinia water extracts was measured to have a pH of ∼2 in the NMR solutions. For the Garcinia food supplement extracts, the NMR sample solutions were adjusted to a pH of ∼2 by adding trifluoroacetic acid-d (Sigma-Aldrich), which ensures that the carboxylic acid functional groups are in the protonated state and gives consistent shift values for (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone. Shift positions are relative to the TSP signal at δ 0.0 ppm.

The concentrations of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone were measured by comparing the NMR peak areas of selected protons of (−)-hydroxycitric acid (δ ca. 3.17 ppm, H-3a) and (−)-hydroxycitric acid lactone (δ ca. 3.29 ppm, H-4a) with those of the internal reference (δ 6.37 ppm). The area of each peak is directly proportional to the number of corresponding nuclei. The percentages of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone in the samples were calculated using the following equation, P (Sample) = (I(a) × N(ir) × M(a) × m(ir) × P(ir))/(I(ir) × N(a) × M(ir) × m(sample)), where I(a) and I(ir) are the areas of the ana-lytes ((−)-hydroxycitric acid/(−)-hydroxycitric acid lactone) and the internal reference, N(ir) and N(a) are the number of protons contributing to the signal of the internal reference (2 protons) and the analytes (1 proton each for (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone), M(a) and M(ir) are the molecular weights of (−)-hydroxycitric acid (208.12 g/mol) and (−)-hydroxycitric acid lactone (190.11 g/mol) and the internal refer-ence (116.07 g/mol), m(ir) and m(sample) is the mass of the internal reference (1 mg) and mass of the sample aliquot (10 mg), P(ir) is the purity of the internal reference (99.94%). The areas were measured by using the MNova v. 7.1.2 program (http://mestrelab.com/). In order to evaluate the NMR quantification method, test solutions containing Garcinia water extract in NMR reference solvent solution (0.18 to 40 mg/ml) were prepared and measured in triplicates. The S/N ratios were measured to determine Limit of Detection (LOD) and Limit of Quantification (LOQ)35, and linearity over the concentration range was also calculated.

Figure 3. Maximum Likelihood tree (RAxML) of biological reference material of Garcinia species and Garcinia raw drug market samples using nrITS region.

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GenBank (NCBI) [http://www.ncbi.nlm.nih.gov/genbank] Accession numbers of nucle-otide sequences are listed in Supplementary Table S1.

Results and DiscussionGarcinia The interviews with farmers from Western Ghats revealed that peo-

ple predominantly collected G. gummi-gutta and G. indica from the wild, with quantities of Garcinia fruits up to 15 to 20 kg per day. Fruits are the traditionally used plant part of Garcinia, but during the interview we noticed that resin from the trees was also locally collected36. Apart from these two species, G. morella was collected for culinary purposes and domestic consumption. The other Garcinia species such as G. mangostana and G. cowa, which grow sparsely in the area, were locally traded based on their seasonal availability. Farmers commented that not all trees produce fruits, and distinguished between “male” and “female” trees of Garcinia species. The preliminary understanding from the survey is that there is limited added value for Garcinia trade at the farmers end. However, people do process the fruit post-harvest and tend to sell seedless fruits, which fetch a higher price than the fruits with seeds. In the case of G. indica, the juice is extracted from the fruits by several small and large-scale industries.

DNA barcoding of Garcinia In recent years, a number of medicinal plants have been shown to be adulterated with other low cost plant materials15. Contamination in herbal products presents a potential health risk for consumers23. In the present study, DNA barcoding was used to analyze the extent of adulteration in herbal raw drug trade of Garcinia species. The first step in analyzing the presence of adulteration was the construction of a taxonomically authenticated BRM DNA barcode library for Garcinia species. This was assembled in order to provide a reliable DNA library of reference samples, to authenticate the herbal raw drug samples. A single-locus approach was used to delineate nine species collected from Western Ghats and two species from Northeast India based on genetic divergence of three regions (i.e., nrITS, psbA-trnH and rbcL) using the Kimura 2-Parameter model (Table 1). rbcL and psbA-trnH exhibited the lowest mean interspecific distance (0.023 and 0.129, respec-tively); in contrast, nrITS exhibited the highest mean interspecific distance (0.153). Similarly, the mean intraspe-cific distance for nrITS was 0.037, which was significantly lower than the mean interspecific distance. In the case of psbA-trnH and rbcL, the mean intraspecific distance was 0.011 and 0.037 respectively, showing that rbcL was not a suitable marker because that the interspecific distance, 0.023 is lower than the intraspecific distance, 0.037, and that psbA-trnH was considerably better with a significant mean inter- and intraspecific genetic dis-tance (0.129 and 0.037 respectively) (Table 1). nrITS had the most parsimony-informative characters with 278 characters. Similarly psbA-trnH and rbcL had 198 and 70 respectively (Table 1). The ILD test revealed significant incongruence between the nuclear and plastid markers (P > 0.01), and the visual inspection of phylogenetic trees suggests a topology conflict among the three markers, therefore the three matrices were not concatenated and single-locus trees were utilized for further analyses.

Authentication of herbal products has been a major challenge due to the fact that chemical studies have doc-umented high content variability of active ingredients of medicinal plants due to geographic conditions and also variability among products from diverse manufacturers of supplements16. Conventional techniques such as organoleptic analysis and microscopy are also limited for species authentication in herbal products37. There are a number of studies that have assessed adulteration in medicinal plants in raw drug trade and also have identified ingredients added in finished herbal products using DNA barcoding15,38–40.

However, a major limitation of Sanger DNA barcoding is the inability to distinguish constituent species in multi-ingredient herbal products20,41. Several comprehensive reviews on DNA-based authentication of botanicals have highlighted the merits and limitations of Sanger based DNA barcoding23,37,42. In this study, it is evident from the single-locus phylogenetic trees that nrITS and the psbA-trnH spacer are more suitable barcode regions for Indian Garcinia species than rbcL (Supplementary Figs S1, S2, and S3).

Tree-based methods are advantageous over sequence similarity based methods like BLAST that require a deci-sion on a threshold at which a sequence is considered to belong to a certain taxon, which could be subjective and may be applicable to certain taxa but not to others43. In tree-based methods, a clear advantage is that no cut-off value is necessary if the query sequence is considered to belong to a certain taxon, and if it is found in the same clade consisting of a reference sequences for that particular taxon38. In addition distance based methods (e.g., BLAST) have a tendency to produce false positive identifications if the reference sequences are not available in databases, whereas taxonomic identification based on tree-based methods are not as sensitive to incomplete data-bases and avoid false positive identification43. Supplementary Figs S1 and S2 of nrITS and psbA-trnH illustrate the clear grouping within Garcinia species with well supported bootstrap values, whereas rbcL on the other hand shows poor resolution in delineating the Garcinia species, and that can be explained by the low genetic divergence within this marker (Supplementary Fig. S3).

DNA regions

Reference PCR success (%)

Aligned sequence(bp)

Intraspecific distance(mean ± SD)

Interspecific distance(mean ± SD) p-value

Parsimony-informative sites

ITS 100 581 0.037 ± 0.010 0.153 ± 0.085 <0.01 278psbA-trnH 79 493 0.037 ± 0.010 0.129 ± 0.044 <0.01 198rbcL 93 710 0.011 ± 0.005 0.023 ± 0.007 <0.01 70

Table 1. Evaluation of the three DNA barcode regions used for Garcinia species.

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In order to authenticate the raw drugs traded as Kodampuli and Kokum, the marker nrITS was utilized due to its high amplification success rate from raw drugs (100%), whereas we were unable to amplify the chloroplast markers from the DNA isolated from the raw drug samples. Similarly, it was observed that the PCR amplification success rate in reference samples was 100% for nrITS compared to psbA-trnH (79%) and rbcL (93%) (Table 1). Analyses of nrITS sequences of Kodampuli and Kokum along with BRM barcode library revealed that there was no adulteration in the species (Fig. 3), and also that the morphological characters of fruits aided in the authentic-ity verification of collected raw drug samples from the market (Fig. 2). This was also apparently due to the absence of taxonomic complexities in fruit morphological characters of Kodampuli and Kokum44.

A prerequisite for DNA-based molecular analysis of herbal products is the isolation of good quality DNA23. In this study, the yield of isolated DNA from the Garcinia food supplements ranged from 0.05 to 1 ng/ml. Plant DNA can also be removed or degraded during the manufacturing process of herbal products: extensive heat treatment, irradiation, ultraviolet light exposure, filtration, extractive distillation or supercritical fluid extraction all affect DNA yield and integrity23.

Garcinia 1H NMR was used to quantify the (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone content in raw herbal drugs of Garcinia species. The chemical formu-las for (−)-hydroxycitric acid with the (2 S,3 S) configuration and its lactone form are shown in Fig. 4a. The (−)-hydroxycitric acid content was directly measured from areas of characteristic signals in the proton spectra of the water extracts (Fig. 4b). Limit of detection (LOD) and limit of quantification (LOQ) is the lowest concen-tration of an analyte in a sample which can be detected and quantified, respectively, for which the selected signals exhibit the minimal required S/N ratio. Table 2 shows LOD values of 0.06 and 0.04 mg/ml, and LOQ values of

Figure 4. (a) Chemical structures of (−)-hydroxycitric acid (1), (−)-hydroxycitric acid lactone (2) and maleic acid (3). (b) 1H NMR spectrum of Garcinia gummi-gutta (voucher no. HAS429) extract.

CompoundLOD (mg/ml)

LOQ (mg/ml)

Relative standard deviation Linear equation

(−)-Hydroxycitric acid in Garcinia fruit extracts 0.06 0.45 1.3% y = 1.0143 × +100.92 R² = 0.9374

(−)-Hydroxycitric acid lactone in Garcinia fruit extracts

0.04 0.71 2.1% y = 2.8848 × +163.45 R² = 0.9974

Table 2. Method validation data for quantification of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone.

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0.13 and 0.20 mg/ml for (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone respectively. Supplementary Fig. S4 shows NMR spectra of Garcinia fruit extracts in different concentrations with the resulting changes in signal intensities for (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone.

The 1H NMR spectrum of the water extract of G. gummi-gutta HAS429 depicted in Fig. 4b clearly shows the presence of characteristic signals from (−)-hydroxycitric acid (1). The 1H NMR signals for H-1 appear as two closely spaced doublets at δ 3.07 ppm (d, J = 16.6 Hz) and 3.16 ppm (d, J = 16.6 Hz), while H-3 appears as a singlet at δ 4.45 ppm. In the same spectrum the H-4 protons in (−)-hydroxycitric acid lactone (2) give rise to two dou-blets at δ 2.92 ppm (d, J = 18.0 Hz) and 3.29 ppm (d, J = 18.0 Hz), H-2 gives a singlet at δ 5.00 ppm. The two vicinal protons of the internal standard maleic acid (3) give rise to the singlet at δ 6.37 ppm. However, a slight variation in the characteristic signals of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone within triplicates of each sample and between samples was observed (relative standard deviation < 0.03). These variations in chemical shifts values are possibly due to minor pH differences or interactions with other molecules in the extracts.

Though no prior sample clean-up after extraction was necessary in order to quantify the (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone content in the Garcinia fruit extracts, it was observed that the extracts are rich in sugar compounds (Fig. 4b & Supplementary Fig. S5). Table 3 shows the percentage of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone (mass fraction) in the dried fruits of Kodampuli and Kokum raw drugs. The content of (−)-hydroxycitric acid in the dried Kodampuli fruits varied from 3.9% to 10.3% with an average of 7.9% and the content of (−)-hydroxycitric acid lactone varied from 7.4% to 19.1% with an average of 13.1%. On the other hand, the content of (−)-hydroxycitric acid in the dried Kokum fruits varied from 1.7% to 7.6% with an average of 4.3% and the content of (−)-hydroxycitric acid lactone varied from 3.5% to 11.7% with an average of 7.8%. Since the equilibrium state of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone in the Garcinia fruits is not known, the total content of the acid and the lactone was calculated. The total average content of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone in Kodampuli was 21.0% while the total content in Kokum was 12.1%. As far as we know, this is the first time that 1H NMR has been used for the quantification of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone. Gas chromatography45, high performance liquid chromatography46, and capillary zone electrophoresis47 have previously been used to quantify the hydroxycitric acid and hydroxycitric acid lactone content in Garcinia fruits and food supplements. HPLC with UV detection at 210–220 nm is commonly used today and has replaced the more laborious GC methods12,48. A challenge with HPLC is to obtain a good resolution between the organic acids and their lactones in Garcinia. There are examples showing no discrimination between (−)hydroxycitric acid and the lactone or methods that suffer from poor resolution46,49,50. Another challenge is the low selectivity at 210 nm, therefore sample preparation may be needed to remove co−eluting interfering substances46,49,50. Compared to these methods, quantitative NMR (qNMR)

Voucher of dried fruits

(−)-Hydroxycitric acid content, % (SD)

(−)-Hydroxycitric acid lactone content, % (SD)

Total (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone content, % (SD)

HAS370 8.5 (2.9) 14.8 (1.4) 23.3 (3.7)HAS404 8.2 (0.4) 14.4 (0.4) 22.6 (0.8)HAS391 10.3 (0.6) 19.1 (1.2) 29.4 (1.6)HAS468 7.6 (0.4) 11.7 (1.0) 19.4 (0.9)HAS378 6.3 (0.3) 7.8 (0.3) 14.1 (0.4)HAS288 7.5 (0.2) 11.9 (0.9) 19.5 (1.1)HAS470 8.8 (0.6) 14.9 (1.2) 23.6 (1.7)HAS395 7.9 (0.1) 12.5 (0.2) 20.3 (0.1)HAS388 8.1 (1.1) 14.1 (1.9) 22.2 (3.0)HAS379 6.7 (0.2) 10.0 (0.5) 16.8 (0.4)HAS443 8.1 (0.6) 11.3 (0.9) 19.4 (1.4)HAS422 8.9 (0.4) 17.0 (0.7) 25.9 (1.0)HAS414 7.8 (0.4) 12.5 (0.6) 20.3 (1.0)HAS389 8.2 (0.1) 14.4 (1.5) 22.6 (2.5)HAS409 7.6 (0.4) 13.1 (0.4) 20.7 (0.6)HAS399 8.9 (0.1) 14.1 (0.2) 23.0 (0.2)HAS429 8.9 (0.3) 14.5 (0.2) 23.4 (0.5)HAS203 3.9 (1.3) 7.4 (0.3) 11.3 (1.3)HAS469* 7.6 (0.3) 11.7 (0.4) 19.3 (0.6)HAS457* 3.7 (1.3) 11.3 (1.0) 15.0 (2.3)HAS473* 5.0 (0.8) 7.3 (0.7) 12.3 (1.3)HAS365* 2.7 (0.4) 5.4 (0.2) 8.2 (0.5)HAS369* 4.8 (0.1) 8.1 (0.1) 12.8 (0.1)HAS396* 1.7 (0.2) 3.5 (0.4) 5.2 (0.6)

Table 3. (−)-Hydroxycitric acid, and (−)-hydroxycitric acid lactone content (%) of Garcinia gummi-gutta (Kodampuli) and Garcinia indica (Kokum) fruit rinds using 1H NMR for quantification (*Garcinia indica samples).

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currently employed has the advantage of being rapid (less than 8 minutes needed for 128 scans) with no need for additional cleanup of extracts or derivatization. NMR makes it possible to detect of all kind of organic molecules in the same sample, as long as they are soluble in the NMR solvent, and is also highly reproducible with little instrument-instrument variation; in addition it is non-invasive and non-destructive51.

In previous studies, using an HPLC method, a (−)-hydroxycitric acid content of G. gummi-gutta of 16–18% was reported, and the leaves and rinds of G. indica were found to contain 4.1–4.6% and 10.3–12.7%, respectively, whereas only trace amounts of (−)-hydroxycitric acid lactone was reported46,50. For G. cowa, the (−)-hydroxycitric acid content was found to be 1.7, 2.3 and 12.7%, respectively in the fresh leaves, fruits and dried rinds48. These overlaps between the total content of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone in Garcinia species suggest that these organic acids should not be used as marker compound to distinguish between Garcinia species.

Seasonal and geographic variations in chemical composition have been reported for a number of medicinal plants52,53, and also chemical variability along the value chains, harvesting pressure, harvest time, collection of immature plant parts etc., have been reported to influence the composition and concentration of plant constit-uents54–56. The variation in the concentration of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone in Kodampuli and Kokum raw drugs can be explained since the raw drugs were obtained from different geographic locations of South India (Fig. 1), and concentration of organic acids can also vary based on the harvest time, as has been shown in other fruits57,58. The raw drugs stocked in the herbal markets are normally obtained from a single source or from collectors of a specific region. Enquiries with the traders indicated that supplies are made usually by wholesale agents who in turn obtain raw drugs from sub-agents and collectors, and it is likely that the collectors get the Kodampuli and Kokum from across the Western Ghats of India. This could explain the variation in the concentration of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone in the studied Garcinia fruits.

Validation of Garcinia G. cambogia (syn. G. gummi-gutta) or unspecified Garcinia extracts rich in (−)-hydroxycitric acid are examples of food supplements freely available in pharmacies, health food stores, and via the internet without any regulations and quality control. In order to authenticate the validity of Garcinia products, ten Garcinia food supplements were randomly purchased via e-commerce and pharmacies (Supplementary Table S4). One of the major challenges with the authentication of Garcinia food supplements is the lack of processing information. The majority of the supplements were not labeled with the solvent used for extraction, and thus the composition of the extracts was difficult to predict, and it is not obvious if they contain water-soluble consituents such as hydroxycitric acid or lipophilic compounds like garcinol and camboginol. The content of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone in the Garcinia supplements is shown in Table 4, and compared with the labeled contents. Product 1 and 5, labeled to contain 500 mg and 525 of extract with 60% HCA, were found to contain only 36 ± 2.9 mg (5.5%) and 29 ± 1.9 mg (4.6%) (−)-hydroxycitric acid, respectively. The other eight products contained more reasonable amounts of (−)-hydroxycitric acid com-pared to their labeled ingredients (59–289 mg (12.8–50.5%) per capsule or tablet). In comparison the analyzed G. gummi-gutta fruit rind water extracts were found to contain 32% of (−)-hydroxycitric acid and its lactone on average, and the average content found for G. indica water extracts was 19%. A previous study utilizing capillary zone electrophoresis concluded that one sample out of five did not contain G. atroviridis Griff extract as claimed on the label47. On the contrary, a study utilizing HPLC revealed no adulteration among G. gummi-gutta and G. indica commercial products59 and another study using HPLC revealed that G. gummi-gutta commercial products contained 51–55% of (−)-hydroxycitric acid with small quantities of tartaric, citric, and malic acids49. From quanti-tative NMR analysis of water extracts obtained from the supplements, it was found that five products contained both (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone. Among these, only one product contained quantifiable amounts of (−)-hydroxycitric acid lactone, whereas (−)-hydroxycitric acid lactone was missing or only present in trace amounts in the remaining products (Table 4 & Supplementary Fig. S6). The absence of (−)-hydroxycitric acid lactone in the supplements with Garcinia extracts might be due to the extraction method employed60.

Herbal products code no.

Labeled content per capsule/tablet (−)-Hydroxycitric acid content (mg/capsule) determined by qNMRGarcinia ingredients

mg HCA$

1 G. cambogia extract (60% HCA), 500 mg 300 36 ± 2.92 G. cambogia extract (50% HCA), 500 mg 250 149 ± 0.73 G. indica extract, 350 mg 122 ± 9.24 G. cambogia, 100 mg 59 ± 1.45 G. cambogia extract (60% HCA), 525 mg 315 29 ± 1.9

6 G. cambogia extract (65% HCA), 250 mg G. cambodia powder, fruit rind and leaf (1% HCA), 350 mg 166 144 ± 3.7#

7 G. cambogia extract, 300 mg 69 ± 1.8*8 G. indica powder, 400 mg 184 ± 7.39 G. cambogia extract, 350 mg 150 ± 4.310 G. cambogia fruit (60% HCA), 500 mg 300 289 ± 8.6

Table 4. (−)-Hydroxycitric acid, and (−)-hydroxycitric acid lactone content in Garcinia food supplements used in the study. $Calculated based on labeled content of HCA; #Contained 41 ± 0.6 mg (−)-hydroxycitric acid lactone, the other samples contained only detectable amounts or no lactone; *Samples were analyzed only twice.

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1H NMR spectra of G. indica and G. gummi-gutta (syn. G. cambogia) fruit rind methanol extracts showed characteristic signals from polyisoprenylated xanthones (garcinol or camboginol). Signals from aromatic pro-tons at δ 6.7, 7.0 and 7.2 ppm and unsaturated tertiary methyls groups at δ 1.5–1.7 ppm were observed61,62. These signals were also observed in product no. 6, labeled to contain both G. cambogia extract (250 mg) and powder of fruit rinds and leaves (350 mg), while methanol extracts of the other supplements did not give rise to signals from garcinol or camboginol. These signals were not present in spectra of the fruit rind or herbal supplement water extracts. Thus, garcinol or camboginol, which are likely not present in the Garcinia commercial extracts, should not be used as authentication compounds for Garcinia supplements.

In this study, we used DNA barcoding and 1H NMR spectroscopy to authenticate Garcinia fruits and food supple-ments. 1H NMR is useful for qualitative and quantitative analysis of constituents in medicinal plants and herbal products24. However, NMR has, so far, not received a wider application in the official food supplements testing and medicine control laboratories than other chromatographic methods, mostly because the NMR technique was judged to be complicated and instruments too expensive25. However, several improvements in instrumen-tation during the last decade have made NMR available for routine applications25. In the future, a metabolomic approach using NMR would be useful for large-scale analysis of Garcinia food supplements and this study has shown that qNMR is a fast and sensitive enough method for the quantification of (−)-hydroxycitric acid content. Several studies have advocated the use of DNA barcoding in herbal product authentication and pharmacovigi-lance19,23,29,63 due to its cost effectiveness and ability to identify plant species. In this study, DNA barcoding was used to demonstrate that there was no adulteration or substitution of Garcinia species in major herbal markets of south India. These results show the usefulness of DNA barcoding and 1H NMR spectroscopy for use in comple-menting traditional methods of quality control for consumer safety, and demonstrates that DNA barcoding can be used as a screening method for the identification of Garcinia species in herbal trade, and NMR for detection and quantification of hydroxycitric acid and (−)-hydroxycitric acid lactone in Garcinia fruits and food supplements.

Our 1H NMR results suggest that in order to increase consumer confidence by advocating and promoting a higher standard of quality in herbal products, there is an urgent need to study food supplements derived from traditional medicinal plants.

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This work was supported by Department of Biotechnology, Government of India (Grant number: No.BT/IN/ ISTP-EOI/2011). The first author of the paper acknowledges the Norwegian PhD School of Pharmacy’s research stay abroad grant. The NMR laboratory at the Chemistry Department, University of Oslo, Norway is acknowledged for the spectrometer facilities. GR acknowledges the Royal Norwegian Embassy grant for travel

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1 2SCIENTIFIC REPORTS | (2018) 8:10561

grant to Norway. We also acknowledge Anusha Bhat N, College of Forestry, University of Agricultural Sciences, Sirsi, India for the help in collection of samples and for her assistance in the laboratory. We thank Berit Smestad Paulsen and members of de Boer Lab for their constructive comments to improve the manuscript.

G.S.S., H.W., H.D.B., R.U.S., G.R. conceived the experiment. G.S.S. carried out molecular and phytochemical lab work. M.T. and H.W. developed phytochemical method, and M.T., G.S.S., K.E.M. were involved in phytochemical lab work. G.S.S., J.U.S., S.V.G., R.V. were involved in plant collection and interviewing the farmers. G.S.S. wrote the manuscript, and all authors have read and approved the final version of the manuscript.

Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-28635-z.Competing Interests: The authors declare no competing interests.Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or

format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per-mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018

Authentication of Garcinia fruits and food supplements using DNA barcoding and NMR

spectroscopy

Gopalakrishnan Saroja Seethapathy, Margey Tadesse, Santhosh Kumar J. Urumarudappa, Srikanth

Gunaga, Ramesha Vasudeva, Karl Egil Malterud, Ramanan Uma Shaanker, Hugo J. de Boer,

Gudasalamani Ravikanth and Helle Wangensteen

Supplementary Information

Supplementary Table S1. Details of biological reference material samples collected from different

parts of South India used in the analysis.

Supplementary Table S2. List of trade/vernacular names used during the collection of Garcinia raw

drugs samples from raw drug markets of South India.

Supplementary Table S3. Details of raw drug samples collected from different parts of South India

used in the analysis.

Supplementary Table S4. Details of Garcinia food supplements used in the study.

Supplementary Table S5. Details of the barcode primers used in the study.

Supplementary Table S6. 1H NMR shifts of (2S, 3S)-Hydroxycitric acid observed in the water extract

of G. gummi-gutta HAS429 depicted in Figure 4b.

Supplementary Table S7. 1H NMR shifts of (2S, 3S)-Hydroxycitric acid lactone observed in the

water extract of G. gummi-gutta HAS429 depicted in Figure 4b.

Supplementary Fig. S1. Maximum Likelihood tree (RAxML) of Garcinia species using nrITS.

Supplementary Fig. S2. Maximum Likelihood tree (RAxML) of Garcinia species using psbA-trnH.

Supplementary Fig. S3. Maximum Likelihood tree (RAxML) of Garcinia species using rbcL.

Supplementary Fig. S4. 1H NMR spectrum of (-)-hydroxycitric acid, and (-)-hydroxycitric acid

lactone in different concentration of Garcinia fruits.

Supplementary Fig. S5. 1H NMR spectrum of Garcinia fruits extract.

Supplementary Fig. S6. 1H NMR spectrum of analyzed Garcinia food supplements.

Supp

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0 V

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P318

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ka

KP3

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1 K

P318

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KP3

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2 A

TREE

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TREE

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KP3

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KP3

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3

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EE12

6 C

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dra

(GK

VK

, B

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lore

) K

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KP3

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4 K

P318

404

ATR

EE12

7 Si

rsi,

Kar

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ka

KP3

1833

7 -

KP3

1840

5 A

TREE

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4 G

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mor

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(Gae

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EE13

9

Sirs

i, K

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KP3

1834

9 K

P318

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KP3

1841

5 A

TREE

140

KP3

1835

0 K

P318

386

KP3

1841

6 A

TREE

141

KP3

1835

1 -

KP3

1841

7 A

TREE

142

KP3

1835

2 -

KP3

1841

8 A

TREE

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KP3

1835

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KP3

1841

9

5 G

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nia

man

gost

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L.

ATR

EE13

7 Si

rsi,

Kar

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ka

KP3

1834

7 K

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KP3

1841

3 A

TREE

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KP3

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8 K

P318

384

KP3

1841

4

6 G

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talb

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apau

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EE15

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K

P318

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KP3

1839

2 K

P318

434

ATR

EE15

1 Si

rsi,

Kar

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ka

KP3

1836

1 K

P318

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KP3

1842

5 A

TREE

152

KP3

1836

2 K

P318

394

KP3

1842

6

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EE15

3 K

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KP3

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427

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KP3

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0 A

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KP3

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5 K

P318

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KP3

1841

1 A

TREE

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KP3

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TREE

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KP3

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KP3

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1 A

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2 A

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eren

ces

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te T

J, B

runs

T, L

ee S

, Tay

lor J

(199

0) A

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and

dire

ct se

quen

cing

of f

unga

l rib

osom

al R

NA

gen

es fo

r phy

loge

netic

s.

In M

. A. I

nnis

, D. H

. Gel

fand

, J. J

. Sni

nsky

, & J

. Tho

mas

(Eds

.), P

CR

pro

toco

ls: A

gui

de to

met

hods

and

app

licat

ions

(pp.

315

).

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Die

go: A

cade

mic

Pre

ss.

2.

Shaw

J, L

icke

y EB

, Bec

k JT

, Far

mer

SB

, Liu

WS,

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er J

, Siri

pun

KC

, Win

der C

T, S

chill

ing

EE, S

mal

l RL

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1998

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on 4

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.

Supplementary Table S6. 1H NMR shifts of (2S, 3S)-Hydroxycitric acid observed in the water extract of G. gummi-gutta HAS429 depicted in Figure 4b.

Number δH (multiplicity, J in Hz)

1a 3.07 (d, J = 16.6 Hz)

1b 3.16 (d, J = 16.6 Hz)

3 4.45 (s)

Supplementary Table S7. 1H NMR shifts of (2S, 3S)-Hydroxycitric acid lactone observed in the water extract of G. gummi-gutta HAS429 depicted in Figure 4b.

Number δH (multiplicity, J in Hz)

2 5.00 (s)

4a 2.92 (d, J = 18.0 Hz)

4b 3.29 (d, J = 18.0 Hz)

Supplementary Fig. S1. Maximum Likelihood tree (RAxML) of Garcinia species using nrITS.

Supplementary Fig. S2. Maximum Likelihood tree (RAxML) of Garcinia species using psbA-trnH.

Supplementary Fig. S3. Maximum Likelihood tree (RAxML) of Garcinia species using rbcL.

Supp

lem

enta

ry F

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-hyd

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.

IV

ORIGINAL RESEARCH

published: 05 February 2019

doi: 10.3389/fpls.2019.00068

Edited by:Jiang Xu,

China Academy of Chinese Medical

Sciences, China

Reviewed by:Jianping Han,

Institute of Medicinal Plant

Development (CAMS), China

Zhigang Hu,

Hubei University of Chinese Medicine,

China

*Correspondence:Hugo J. de Boer

hugo.deboer@nhm.uio.no

Specialty section:This article was submitted to

Technical Advances in Plant Science,

a section of the journal

Frontiers in Plant Science

Received: 12 October 2018

Accepted: 17 January 2019

Published: 05 February 2019

Citation:Seethapathy GS,

Raclariu-Manolica A-C,

Anmarkrud JA, Wangensteen H and

de Boer HJ (2019) DNA

Metabarcoding Authentication

of Ayurvedic Herbal Products on

the European Market Raises

Concerns of Quality and Fidelity.

Front. Plant Sci. 10:68.

doi: 10.3389/fpls.2019.00068

DNA Metabarcoding Authenticationof Ayurvedic Herbal Products on theEuropean Market Raises Concernsof Quality and FidelityGopalakrishnan Saroja Seethapathy1,2, Ancuta-Cristina Raclariu-Manolica1,3,Jarl Andreas Anmarkrud1, Helle Wangensteen2 and Hugo J. de Boer1*

1 Natural History Museum, University of Oslo, Oslo, Norway, 2 Department of Pharmaceutical Chemistry, School

of Pharmacy, University of Oslo, Oslo, Norway, 3 Stejarul Research Centre for Biological Sciences, National Institute

of Research and Development for Biological Sciences, Piatra Neamt, Romania

Ayurveda is one of the oldest systems of medicine in the world, but the growing

commercial interest in Ayurveda based products has increased the incentive for

adulteration and substitution within this herbal market. Fraudulent practices such as

the use of undeclared fillers and use of other species of inferior quality is driven

both by the increased as well as insufficient supply capacity of especially wild plant

species. Developing novel strategies to exhaustively assess and monitor both the

quality of raw materials and final marketed herbal products is a challenge in herbal

pharmacovigilance. Seventy-nine Ayurvedic herbal products sold as tablets, capsules,

powders, and extracts were randomly purchased via e-commerce and pharmacies

across Europe, and DNA metabarcoding was used to assess the ability of this method

to authenticate these products. Our analysis reveals that only two out of 12 single

ingredient products contained only one species as labeled, eight out of 27 multiple

ingredient products contained none of the species listed on the label, and the remaining

19 products contained 1 to 5 of the species listed on the label along with many

other species not specified on the label. The fidelity for single ingredient products

was 67%, the overall ingredient fidelity for multi ingredient products was 21%, and

for all products 24%. The low level of fidelity raises concerns about the reliability of

the products, and detection of threatened species raises further concerns about illegal

plant trade. The study highlights the necessity for quality control of the marketed herbal

products and shows that DNA metabarcoding is an effective analytical approach to

authenticate complex multi ingredient herbal products. However, effort needs to be

done to standardize the protocols for DNA metabarcoding before this approach can

be implemented as routine analytical approaches for plant identification, and approved

for use in regulated procedures.

Keywords: Ayurvedic herbal products, botanical authentication, DNA barcoding, herbal medicines,

pharmacovigilance, quality control

Frontiers in Plant Science | www.frontiersin.org 1 February 2019 | Volume 10 | Article 68

Seethapathy et al. DNA Metabarcoding of Ayurvedic Products

INTRODUCTION

Ayurveda, or Ayurvedic medicine, is one of the oldest systemsof traditional medicine (TM), with origins in India more than3,000 years ago. Nowadays Ayurveda is popular and usedworldwide in complementary and alternative healthcare andmedical practices (CAM) (World Health Organization [WHO],2013). Ayurvedic formulations are obtained using an average of80% botanicals, 12% animals, and 8% minerals, and are usedas raw materials and preparations such as extracts (Joshi et al.,2017). About 7,000 plant species are used for medicinal purposesin India, from which, about 1,200 species have been reported tobe actively traded (Goraya and Ved, 2017). The total commercialdemand for herbal material in India, in 2014 and 2015, wasestimated to be in excess of 512,000 tons, with a market valueof 1 billion USD (Goraya and Ved, 2017). India has more than8,000 licensed manufacturing units for medicinal products andthe increasing level of consumption of herbal products exceed thesupply capacity for some plant species (Goraya and Ved, 2017).In order to ensure a level of uniformity of the therapeutic formulaand the ingredients used, the Ayurvedic formulary andAyurvedicPharmacopeia of India was published by the Government of Indiaas a legally binding document describing the quality, purity, andstrength of selected drugs that are manufactured, distributed andsold by the licensed manufacturers in India (Joshi et al., 2017).

As many other TMs, Ayurvedic herbal medicines, requirequality assurances for their wider usage and acceptability in CAMpracticing countries (World Health Organization [WHO], 2013).The growing demand for Ayurveda encourages an industryfor mass production of herbal products, leading to the use oflarge quantities of plant raw material, mainly harvested fromthe wild flora (Valiathan, 2006; Goraya and Ved, 2017; Joshiet al., 2017). Many of the Indian medicinal plant species arein short supply due to the lack of cultivation and several wildspecies are not available in sufficient quantities for commercialexploitation (Goraya and Ved, 2017). The intensive use ofherbal products increases the incentive for adulteration andsubstitution in the medicinal plant trade (Newmaster et al.,2013). This awareness of content irregularities calls attentionto the quality of the traded mass produced herbal productswith direct impact on their efficacy and safety (Leonti andCasu, 2013). One of the pharmacognostic parameters toassure quality, safety and efficacy of a herbal medicine is theutilization of correctly identified medicinal plants used as rawmaterial (Evans, 2009). Several new strategies and appropriatestandard methods have been proposed to exhaustively assessand monitor both the quality of raw materials and marketedherbal products (Barnes, 2003; De Boer et al., 2015). Standardmethods routinely used to assess herbal material, preparationsand products rely on morphological characters, microscopy,and chemical fingerprinting [i.e., thin–layer chromatography’,high–performance liquid chromatography (HPLC), and gaschromatography (GC)] (De Boer et al., 2015; Parveen et al.,2016). These methods are quick and cost-effective techniques forprimary qualitative analysis of raw material and derived herbalproducts. Alternatively, the use of more advanced methods foridentification and quantification of chemical marker compounds

is becoming popular [i.e., liquid chromatography (LC)–massspectrometry (MS), GC-MS, and LC-nuclear magnetic resonance(NMR)], but requires valuable instrumentation (Jiang et al., 2010;Wang et al., 2017; Zhang et al., 2017; Raclariu et al., 2018a).

Various important issues influence the quality of Ayurvedicherbal products and they need to be carefully taken intoconsideration when determining the analytical method of choicefor quality control. The herbal products are usually complexmixtures of plant material and/or extracts and excipients, andresults of manifold processing steps. To apply only standardanalytical methods may pose serious challenges to the accuracyof herbal product quality control. Furthermore, adulteration bythe deliberate use or admixture of substitutes and undeclaredplant fillers, fraudulent adulteration by using fillers of botanicalorigin or plant materials of inferior quality (Zhang et al., 2012),the addition of pharmaceuticals or other synthetic substances inorder to reach an expected effect or a certain level of markercompounds (Calahan et al., 2016; Rocha et al., 2016) raisesconcerns about the quality and safety of the herbal products.Multiple plant species as source for botanical drug as allowed indifferent pharmacopeias, as well as the accidental substitutions,all raise concerns ranging from simple misleading labeling topotential serious adverse drug reactions (Ernst, 1998; Heubl,2010; Gilbert, 2011) or poisoning due to toxic contaminants(Chan, 2003).

All the standard analytical approaches, including sensoryand chemical inspection may have a good resolution in qualitycontrol by detecting the quality and quantity of specific lead orphytochemical marker compounds. However, they are generallynot applicable in identifying target plant species within a complexherbal product, and show low ability to detect non-targetedplant ingredients in herbal products (De Boer et al., 2015).To overcome this limitation, DNA-based approaches have beenproposed as useful analytical tools for the quality control of herbsand herbal products (Parveen et al., 2016). DNA barcoding isa cost-effective, species-level identification based upon the useof short and standardized gene regions, known as ‘barcodes’(Hebert et al., 2003). Several reviews have corroborated thediverse applicability of DNA barcoding in the field of medicinalplant research (Techen et al., 2014; De Boer et al., 2015). Initiallyused as an identification tool, DNA barcoding is now applied inthe industrial quality assurance context to authenticate a widerange of herbal products (De Boer et al., 2015; Parveen et al., 2016;Sgamma et al., 2017).

The combination of High-Throughput Sequencing (HTS)and DNA barcoding, known as DNA metabarcoding, enablessimultaneous high-throughput multi-taxa identification by usingthe extracellular and/or total DNA extracted from complexsamples containing DNA of different origins (Taberlet et al.,2012). Several studies have utilized this approach in identifyingand authenticating medicinal plants and derived herbal products.For example, Echinacea species, Hypericum perforatum, andVeronica officinalis were detected in 89, 68 and 15%, respectively,of the investigated herbal products (Raclariu et al., 2017a,b,2018b). Similarly, Ivanova et al. (2016) found that 15 tested herbalsupplements contained non-listed, non-filler plant DNA, andCheng et al. (2014) showed that the quality of 27 tested herbal

Frontiers in Plant Science | www.frontiersin.org 2 February 2019 | Volume 10 | Article 68

Seethapathy et al. DNA Metabarcoding of Ayurvedic Products

preparations was highly affected by the presence of contaminants.Coghlan et al. (2012) revealed the species composition of 15highly processed traditional Chinese medicines using DNAmetabarcoding, and showed that the products contained speciesincluded on CITES appendices I and II. A number of studiesin India have surveyed herbal raw drug markets and tested theauthenticity of the herbal drugs using DNA barcoding. Thesestudies reported that 24% of raw drug samples of Phyllanthusamarus Schumach. & Thonn. were substituted with otherphenotypically similar Phyllanthus species (Srirama et al., 2010).Similar substitution were reported for other species, such asSida cordifolia L. (76%) (Vassou et al., 2015), Cinnamomumverum J.Presl (70%) (Swetha et al., 2014), Myristica fragransHoutt. (60%) (Swetha et al., 2017), Senna auriculata (L.) Roxb.(50%) (Seethapathy et al., 2015), Senna tora (L.) Roxb. (37%)(Seethapathy et al., 2015) and Senna alexandrina Mill. (8%)(Seethapathy et al., 2015). Furthermore, Vassou et al. (2016)reported that 21% of raw drugs in Indian herbal markets wereunauthentic. Shanmughanandhan et al. (2016) found that 60% of93 herbal products sold in the form of capsules and plant powdersin local stores in India were adulterated. Studies that combinedspectroscopic methods, such as NMR, with DNA barcodingor microscopy to authenticate herbal products, reported 80%adulteration in Saraca asoca (Urumarudappa et al., 2016), 80% inBerberis aristata (Srivastava and Rawat, 2013) and 22% in Pipernigrum (Parvathy et al., 2014). All these studies utilizing DNAbarcoding and metabarcoding have highlighted the concernsover the quality and good labeling practices of herbal products(Coghlan et al., 2012; Ivanova et al., 2016; Raclariu et al., 2017a,b;Veldman et al., 2017).

The aim of this study was threefold. First, we aimed to testthe composition and fidelity of Ayurvedic products marketedin Europe using DNA metabarcoding. Secondly, we aimed toanalyze the presence of any red listed species listed on the productlabel and used as ingredients using DNA metabarcoding. Ourfinal aim was to evaluate the ability of DNA metabarcodingto identify the presence of authentic species, any substitutionand adulteration and/or presence of other off labeled plantspecies.

MATERIALS AND METHODS

Sample CollectionSeventy-nine Ayurvedic herbal products sold as tablets (n = 30),capsules (n = 30), powders (n = 16), and extracts (n = 3) werepurchased via e-commerce (n = 53) and pharmacies (n = 26),from Norway (n = 21), Romania (n = 26), and Sweden (n = 32).Based on the label information, 26 were single plant ingredientproducts, 39 contained between two to ten plant ingredients, and14 products contained between eleven to 27 plant ingredients(Supplementary Table S1). The products contained a totalof 159 plant species belonging to 132 genera and 60 families(Supplementary Table S2). It was also confirmed that nrITSsequences of all the 159 plant species labeled in the analyzedherbal products were available within the NCBI/GenBankdatabase (Supplementary Table S2). The accepted binomial

names and authors of the plants species used as ingredients werevalidated using The Plant List (2013). The Ayurvedic herbalproducts were imported into Norway for scientific analysesunder Norwegian Medicines Agency license no. 16/04551–2.An overview of the products, including label information, butnot the producer/importer name, lot number, expiration dateor any other information that could lead to the identificationof that specific product, can be found in SupplementaryTable S1.

DNA Extraction, Amplicon Generation,

and High Throughput SequencingThe 79 Ayurvedic herbal products were processed depending ontheir pharmaceutical formulation, in addition to an extractionblank per DNA extraction round. A small amount of eachherbal product, about 200 mg, was homogenized using 3–5zirconium grinding beads in a Mini-Beadbeater-1 (BiospecProducts Inc., Bartlesville, Oklahoma, United States). The totalDNA from each product was extracted from homogenizedcontents using CTAB extraction (Doyle and Doyle, 1987).The final elution volume was 100 μl. Extracted DNA wasquantified using a Qubit 2.0 Fluorometer and Qubit dsDNAHS Assay Kit (Invitrogen, Carlsbad, California, United States).All amplicon libraries, defined as PCR amplified productsfrom a study sample, were prepared in three replicates.For each replicate two nuclear ribosomal target sequenceswere amplified, the internal transcribed spacers nrITS1 andnrITS2, respectively. The fusion primers included the annealingmotif from the Sun et al. (1994) plant-specific primer pairs17SE and 5.8I1, and 5.8I2 and 26SE. The forward primersincluded the Ion Torrent A adapter, a 10 bp multiplexidentifier tag following the IonXpress setup for Ion Torrent(Thermo Fisher Scientific, Carlsbad, California, United States).The reverse primer included the truncated P1 (trP1) tags inaddition to the annealing motif. Expected amplicon sizes were300–350 bp.

Polymerase chain reactions were carried out using DNAextracted from the herbal products in final reaction volumesof 25 μl including 0.5 μl of template DNA solution (rangingfrom 0.5 to 2 ng/μl), 1X Q5 reaction buffer (New EnglandBiolabs Inc., United Kingdom), 0.6 μM of each primer (BiolegioB.V., Netherlands), 200 nM dNTPs, 5 U Q5 High-FidelityDNA Polymerase (New England Biolabs Inc., United Kingdom)and 1X Q5 High GC enhancer. The PCR cycling protocolconsisted of initial denaturation at 98◦C for 30 s, followedby 35 cycles of denaturation at 98◦C for 10 s, annealing at56◦C for nrITS1 or 71◦C for nrITS2 for 30 s, and elongationat 72◦C for 30 s, followed by a final elongation step at 72◦Cfor 2 min. Three PCR negative controls of the extractionblanks were included per amplification to control for externaland cross sample contamination. After PCR, the ampliconswere purified using Illustra Exostar (GE Healthcare, Chicago,Illinois, United States) in accordance with the manufacturerprotocols. The molarity of each amplicon library was measuredusing a qPCR based assay (CFX96 Touch Real-Time PCRDetection System, Bio-Rad, Hercules, California, United States).The equimolar amounts of each amplicon library were merged

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and sequenced using an Ion Torrent Personal Genomic Machine(Thermo Fisher Scientific) as described by Raclariu et al.(2017a).

Bioinformatics AnalysisThe sequencing read data were analyzed and demultiplexedinto FASTQ files, per sample, using Torrent Suite version 5.0.4(LT), and each of the replicates was analyzed individually.FASTQ read files were processed using the HTS-barcode-checker pipeline available as a Galaxy pipeline at the NaturalisBiodiversity Center1 (Lammers et al., 2014). Using the HTSpipeline, nrITS1 and nrITS2 primer sequences were used todemultiplex the sequencing reads per sample and to filter outreads that did not match any of the primers. PRINSEQ wasused to determine filtering and trimming values based on readlengths and Phred read quality. All reads with a mean Phredquality score of less than 26 were filtered out, as well as readswith a length of less than 200 bp. The remaining reads weretrimmed to a maximum length of 380 bp. CD-HIT-EST wasused to cluster reads into molecular operational taxonomicunits (MOTUs) defined by a sequence similarity of >99% anda minimum number of ten reads. The consensus sequencesof non-singleton MOTUs were queried using BLAST againsta reference nucleotide sequence database, with a maximume-value of 0.05, a minimum hit length of 100 bp and sequenceidentity of >97%. The number of reads per MOTU, as wellas the BLAST results per MOTU, were compiled using customscripts from the HTS Barcode Checker pipeline (Lammers et al.,2014). The reference sequence database consisted of a localcopy of the NCBI/GenBank nucleotide database that is refreshedmonthly. These parameters were applied to each of the replicates.A species was considered and validated as being present withinthe product only if this was detected in at least 2 out of the 3replicates.

Presence and Abundance of Species

Across SamplesTo assess species diversity within each sample, and to obtaininsights into the dominant species within the Ayurvedic herbalproducts, the read abundances were normalized by dividingthe number of reads for a MOTU by the total number ofreads per sample. As a result, the read counts are transformedinto a proportion of reads found per species within eachsample (Supplementary Tables S3, S4). Furthermore, MOTUsdetected in at least two out of the three replicates, foreach sample, were categorized into expected-detected (MOTUscorresponding to species listed on the product label versusspecies detected in the analysis), expected-not detected (MOTUscorresponding to species listed on the product label but notdetected in the analysis), and not expected-detected (MOTUscorresponding to species non-listed on the product label butdetected in the analysis) (Supplementary Table S5). The totaloccurrences of MOTUs per category of expected and detectedwere evaluated (Supplementary Table S5), and a matrix of

1http://145.136.240.164:8080/

correlation was generated using ClustVis (Metsalu and Vilo,2015).

RESULTS

Fidelity of Ayurvedic ProductsThe genomic DNA extracts were highly variable in quantityand quality. Total DNA concentration for each of the 79 herbalproducts is provided in Supplementary Table S6. Table 1 showsthe average DNA yield for each of the investigated herbal producttypes. The result shows that three samples labeled as containingonly standardized extracts yielded an average of 0.5 ng/μl DNA,whereas tablets, capsules and powders yielded an average of 5.8,9.6, and 44.7 ng/μl DNA, respectively. Out of 79 products usedin the study, 10 tablets were also labeled to contain extractsin addition to crude plant material (#6, #12, #13, #14, #17,#18, #20, #21, #38, and #74). PCR amplification for nrITS1 andnrITS2 regions were performed for all 79 samples, and ampliconswere generated for all replicates for nrITS1 and nrITS2 (forsamples and concentrations see Supplementary Table S6). Theextraction blanks yielded no molecular operational taxonomicunits (MOTUs) with nrITS1 and nrITS2 primers.

The sequencing success rate was 44% for ITS1 and 41% forITS2 (Supplementary Table S6). Thirty-five products out of 79(44%) yielded no MOTUs in any of the replicates either fornrITS1 or nrITS2 that fulfilled our quality criteria, and they wereexcluded from the results and the further discussion (#11, #20–22,#28, #29, #33, #35, #37–39, #41–51, #53, #54, #56, #57, #59, #62,64, #65, #67, #71, #72, #76, and #78). These products consistedof 13 tablets, 11 capsules, and 11 powders (SupplementaryTable S6). The products that yieldedMOTUs were represented by17 tablets, 19 capsules, 5 powders, and 3 extracts (SupplementaryTable S7).

A total of 188 different plant species belonging to 154 generaand 65 families were identified from the retained MOTUs usingBLAST. The separate analyses resulted in 131 plant species (110genus and 53 families) for nrITS1, and 101 plant species (84 genusand 39 families) for nrITS2. The number of species detectedper sample ranged from one to 42. After applying our qualityselection criteria, where a species was considered and validatedas being present within the product only if it was detected in atleast 2 out of the 3 replicates, five additional products (#4, #15,#24, #25, and #26 includes 2 tablets and 3 extracts) that failed toyield the same MOTU in any of the replicates were discarded.The remaining 39 products resulted in a total of 97 plant speciesbelonging to 40 families (62 species for nrITS1, and 60 speciesfor nrITS2). The species detected for all the replicates for bothITS1 and ITS2, were merged for each sample for further analyses(Figure 1 and Supplementary Tables S3, S7).

Figure 2 illustrates the fidelity of herbal products betweenvarious product forms, country, and method of acquisition. Inten out of twelve single ingredient products that were labeledas containing only one species, we detected multiple species(exceptions #5 and #52), from which six contained the specieslabeled on the product together with other species, whereas fourproducts did not contained the species listed on the product

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FIGURE 1 | Discrepancies between listed species and detected species using

DNA metabarcoding in Ayurvedic herbal products. (A) Total number of

occurrences of expected species as labeled in the herbal products and

detected species using DNA metabarcoding. (B) Total number of detected

species occurred among expected species as labeled in herbal products

(expected-detected), the number of undetected species among the expected

species as labeled (expected-not detected), and the number of detected

unexpected species (not expected-detected) found in herbal products using

DNA metabarcoding. The overlapping numbers are the same species

detected in herbal products as expected, detected and unexpected detected.

label but contained several other non-listed species. Out of 27successfully analyzed multiple ingredient products, 8 (29.6%)products contained none of the species listed on the label, andthe remaining 19 products contained between one to five specieslisted on the label along with many other species not specified onthe product label. The fidelity rate for single ingredient productswas 67% (8 out of 12), and the overall ingredient fidelity (detectedspecies from product label/total number of species on label) formulti ingredient products was 21% and for all products 24%.Table 2 shows the top ten products with highest fidelity is alsorelatively high in the level of substitution, whereas Table 3 showsthe top ten products with highest adulteration and its fidelity.

Figure 3 depicts all 97 detected species based on the relativeabundance of read numbers in 39 herbal products per type underthe categories of expected-detected, expected-not detected, andnot expected-detected.

Plant Ingredients in Herbal ProductsA total of 159 plant species belonging to 132 genera and 60families were specified on the labels of the 79 Ayurvedic herbalproducts used in this study. Assessing the source and availabilityof these plants, we found that 83 plants species are solelyharvested from wild, and 31 of these are under various threatlevels, including critically endangered and protected species, such

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FIGURE 2 | Fidelity of herbal products (A) per product form; (B) per country;

(C) per acquisition method. n = total number of herbal products.

as Pterocarpus marsupium Roxb., Pterocarpus santalinus L.f.,Santalum album L., and Saraca asoca (Roxb.) Willd. (Figure 4and Supplementary Table S2; Ved and Goraya, 2007; EnvisFrlht, 2017; Goraya and Ved, 2017). The DNA metabarcodinganalysis confirms the presence of four of these threatened species,

i.e., Celastrus paniculatus,Glycyrrhiza glabra, Gymnema sylvestre,and Saraca asoca, whereas the remaining threatened specieswere not detected despite being included as labeled ingredients(Figure 3 and Supplementary Table S7). The following specieswere found in over 20% of the products:Withania somnifera (L.)Dunal (39%), Tribulus terrestris L. (27%), Convolvulus prostratusForssk. (23%), Coriandrum sativum L. (23%), Ipomoea parasitica(Kunth) G. Don (23%), Ocimum basilicum L. (23%) and SennaalexandrinaMill. (23%) (Figure 3 and Supplementary Table S3).Seventeen are present in more than 10% of samples are listed inthe Supplementary Table S3.

DISCUSSION

The British Pharmacopeia is one the first to publish a specificmethods section on DNA barcoding, and in the 2016 versionit included a new methods appendix on “Deoxyribonucleicacid (DNA) based identification techniques for herbal drugs”to create a framework for compliance of DNA barcoding withregulatory requirements (British Pharmacopeia Commission,2016; Sgamma et al., 2017). However, DNA barcoding andmetabarcoding are not yet widespread validated methods foruse in the regulatory context of quality control. Severalstudies advocate its usefulness for herbal product authenticationand pharmacovigilance either as a standard method or as acomplementary method (Ivanova et al., 2016; Raclariu et al.,2017a,b, 2018a; Sgamma et al., 2017). In this study, DNAmetabarcoding was used as an analytical approach in Ayurvedaherbal product authentication.

A number of studies have shown that the quality of theextraction substrate influences amplification and sequencingsuccess (Ivanova et al., 2016; Raclariu et al., 2017a, 2018b).In addition the presence of DNA in the extraction substratesis influenced by degradation during the harvesting, drying,storage, and industrial processing of plant material (Novak et al.,2007). The success rate in generating raw sequence reads fromthe herbal products, and the number of products from whichMOTUs could be identified per product after applying stricttrimming and filtering quality criteria, reduced the number of

TABLE 2 | Top ten products with the highest fidelity and their level of adulteration.

Herbal

product code

Product

type

No. species

on label

Detected by

DNA

metabarcoding

Fidelity

(Expected-

detected,

absolute)

Fidelity

(Expected-

detected,

relative)

Adulteration

(Detected-Not

expected,

absolute)

Adulteration

(Detected-Not

expected,

relative)

34 Tablets 8 15 5 63% 10 67%

31 Tablets 10 7 5 50% 2 29%

36 Tablets 13 7 4 31% 3 43%

73 Tablets 14 14 3 21% 11 79%

74 Tablets 9 5 3 33% 2 40%

66 Capsules 6 5 3 50% 2 40%

7 Capsules 6 5 2 33% 3 60%

75 Tablets 3 3 2 67% 1 33%

69 Capsules 1 13 1 100% 12 92%

3 Tablets 4 9 1 25% 8 89%

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TABLE 3 | Top ten products with the highest adulteration and their fidelity.

Herbal

product code

Product

type

No species on

label

Detected by

DNA

metabarcoding

Fidelity

(Expected-

detected,

absolute)

Fidelity

(Expected-

detected,

relative)

Adulteration

(Detected-Not

expected,

absolute)

Adulteration

(Detected-Not

expected,

relative)

69 Capsules 1 13 1 100% 12 92%

32 Tablets 6 12 0 0% 12 100%

73 Tablets 14 14 3 21% 11 79%

34 Tablets 8 15 5 63% 10 67%

3 Tablets 4 9 1 25% 8 89%

68 Capsules 1 8 0 0% 8 100%

40 Capsules 4 8 1 25% 7 88%

27 Capsules 1 8 1 100% 7 88%

6 Tablets 9 7 1 11% 6 86%

23 Tablets 4 7 1 25% 6 86%

samples yielding DNA metabarcoding results from 79 to 39samples. In this study, 44% of products did not yield MOTUsin any of the replicates either for nrITS1 or nrITS2. Also, inthe herbal products labeled to contain only extracts, no plantDNA was detected. The undetected MOTUs in these productscould be related to the methodological framework of DNAmetabarcoding such as DNA extraction protocol, suitabilityof primer pair sequences, amplification protocols in PCR forthe library preparation, sequencing platform, filtering, qualitythresholds, and chimera removal, and clustering thresholds (DeBoer et al., 2017; Sgamma et al., 2017; Raclariu et al., 2018b).In addition, extraction of crude herbal drugs either in pre-processing or manufacturing can reduce the availability of plantDNA from those species, especially if material is extractedin boiling water or alcohol, and evaporated or dried at hightemperatures.

Considerable incongruences were observed between thedetected species and those listed on the label of the products.Similarly, Raclariu et al. (2017b) demonstrated the abilityof DNA metabarcoding in detecting Hypericum species incomplex herbal formulations, and revealed the incongruencebetween constituent species and those listed on the label inall products. Also, De Boer et al. (2017) performed DNAmetabarcoding analyses on 55 commercial products based onorchids (salep) purchased in Iran, Turkey, Greece, and Germany,and concluded that there are significant differences in labeledand detected species. They also highlighted the applicabilityof DNA metabarcoding in targeted efforts for conservationof endangered orchid species. In our study, we detected atotal of 97 species in 39 products that passed our qualitycriteria, and most of the identified species are likely ingredientsof Ayurvedic herbal products. Detection of certain speciesis improbable given their distribution or unlikely use, andthese include Achillea millefolium L., Anchusa italica Retz.,Calluna vulgaris (L.) Hull, Damrongia cyanantha Triboun,Fraxinus albicans Buckley and Trigastrotheca molluginea F.Muell.The identification of these plant species may be explainedby (i) amplified PCR chimeras; (ii) false-positive BLASTidentifications due to incomplete or error-prone reference

databases; or (iii) presence of pollen from wind pollinatingspecies, and this confirms previously raised concerns aboutthe hypersensitivity of DNA metabarcoding (De Boer et al.,2017).

Out of 97 species detected in the DNA metabarcodinganalysis, 40 species are sourced from wild, 38 species arecultivated, and 15 species are sourced from both wild andcultivation. Similarly, among the 89 species which were notdetected in the analysis, 62 species are mainly sourced fromwild, including endangered species such as Embelia ribesBurm.f., Pterocarpus marsupium Roxb., Pterocarpus santalinusL.f., Pueraria tuberosa (Willd.) DC., and Santalum album L.Understanding, the discrepancies between the species detectedusing DNA metabarcoding and those listed on the label of theproducts require careful consideration. In DNA metabarcodinganalyses, the level of similarity clustering thresholds (>97,>99, and 100%) have an impact on the number and sizeof assigned MOTUs (Raclariu et al., 2017a). In this study,we used a 99% clustering threshold similar to previouslypublished studies (Raclariu et al., 2017a; Veldman et al., 2017).Furthermore, to limit the impact of sequencing errors, whichare known to affect the Ion Torrent sequencing platform(Salipante et al., 2014) and which could lead to the formationof false MOTUs, we used only the clusters that contained aminimum of 10 reads. In addition, by using three replicatesfor each sample and marker, we reduced further noise byaccepting MOTUs only if present in more than one replicate.Furthermore, the strict filtering and trimming thresholds forbase calling, length and quality, and strict clustering criteriafor MOTUs formation, increase confidence of the results. Asreported by previous studies (Ivanova et al., 2016; Raclariuet al., 2017b), the results related to the authentication ofherbal products using DNA metabarcoding need to focusprimarily on checking the presence of the labeled ingredientsand contaminants. The presence of non-listed species may beexplained by various factors, including but not limited to thedeliberate adulteration and unintentional substitution that mayoccur from the early stage of the supply chain of medicinal plants(i.e., cultivation, transport, and storage), to the manufacturing

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FIGURE 3 | Detection of species in Ayurvedic herbal products. Species (y-axis) are colored by relative abundance of normalized read numbers. Species are

categorized in expected-detected and not expected-detected, based on the total number of occurrences, whereas the category expected-not detected is based on

the number of times that the species is expected but not detected. Species are clustered by Euclidean distances. Ayurvedic samples (x-axis) are numbered with

product code and grouped by product type.

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FIGURE 4 | Source and conservation status of species in Ayurvedic products. (A) Source of plants labeled as ingredients in the herbal products studied.

(B) Conservation status of plants labeled as ingredients in the herbal products studied. N = total number of species. ∗Wild/Cultivation denotes that the plants

species are sourced both from wild and cultivation.

process and the commercialization of the final products. DNAmetabarcoding is a highly sensitive method and even traces ofDNA, e.g., contamination from grains of pollinating species orplant dust in the manufacturing process, can be detected andidentified.

The advantage of DNA metabarcoding is its ability tosimultaneously identify total species diversity within complexmulti-ingredient and processed mixtures. Importantly, DNAmetabarcoding data is used for qualitative evaluation only,to determine presence of taxa, and not for quantitativeassessment of relative species abundance based on readnumbers, as many variables considerably impact the obtainedsequence read results (Staats et al., 2016). In the context ofthe quality control of herbal products, DNA metabarcodingdoes not provide any quantitative nor qualitative informationof the active metabolites in the raw plant material orthe resulting preparation, and this narrows its applicabilityonly to identification and authentication procedures. Thus, ifproduct safety control relies on threshold levels of specificmarker compounds, absence of toxins, allergens and admixedpharmaceuticals, then other methods may be more relevantthan DNA-based composition analysis. On the other hand,if product fidelity, species substitution or adulteration issuspected then the latter method outperforms in terms ofresolution.

The results of this study reveal that there is a need for abetter quality control of herbal products. A novel analyticalapproach should eventually use a combination of innovativehigh throughput methods that complement the standard onesrecommended today.

CONCLUSION

Assessment of Ayurvedic herbal medicines using DNAmetabarcoding provides insight into species diversity inthese products and highlights a marked incongruence betweenspecies listed as ingredients on the product labels and thosedetected from DNA present in the samples. Detection ofnot-listed and not-expected species first and foremost suggestsirregularities in the manufacturing process. The presence offoreign plant material could be due accidental reasons, suchas contamination from insufficiently cleaned bags, containers,mills, conveyors, and other equipment, or co-occurrence ofweeds in cultivation, pollen from wind pollinated speciesor seeds from wind-dispersed species. However, foreignplant material could also result from fraud, i.e., substitution,adulteration and/or admixture of other species. Interpretationof incongruences should focus on the detected species in theproducts, and less on the failure to detect species as thereare many steps in manufacturing processes that could leadto degradation or loss of DNA beyond detectable limits, e.g.,alcoholic extraction, decoction and drying of material athigh temperatures. Our study showed that the investigatedherbal products contained species not listed on the productlabels, and this reveals a clear need for improved qualitycontrol. A novel analytical approach should eventually use acombination of advanced chemical methods and innovativehigh throughput sequencing to complement the standardones recommended today. The findings of our study showthat DNA metabarocoding is a promising tool for qualityevaluation of herbal products and pharmacovigilance, and

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Seethapathy et al. DNA Metabarcoding of Ayurvedic Products

a good candidate for an effective use as a regulatory tool toauthenticate complex herbal products. However, standardizationof protocols is necessary before DNA metabarcoding can beimplemented as a routine analytical approach and approved bycompetent authorities for use in a regulatory framework.

SUPPORTING INFORMATION

Ion-Torrent sequencing data is deposited in Zenodo doi: 10.5281/zenodo.2548681.

AUTHOR CONTRIBUTIONS

GS, ACRM, HW, and HdB conceived the experiment. GScollected the material and carried out the molecular lab work andanalysis together with ACRM. JA carried out high-throughputsequencing together with GS. GS wrote the manuscript togetherwith HdB. All authors contributed to and approved the finalversion of the manuscript.

FUNDING

GS was supported through the University of Oslo by the QuotaScheme of the Norwegian Centre for International Cooperationin Higher Education.

ACKNOWLEDGMENTS

The authors acknowledge the help received in the DNAlaboratory and genetic analyses from Audun Schrøder-Nielsenand Birgitte Lisbeth Graae Thorbek. Data storage was providedby UNINETT Sigma2 (project no. NS9080K) – the Norwegiannational infrastructure for high performance computing and datastorage.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found onlineat: https://www.frontiersin.org/articles/10.3389/fpls.2019.00068/full#supplementary-material

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Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.

Copyright © 2019 Seethapathy, Raclariu-Manolica, Anmarkrud, Wangensteen andde Boer. This is an open-access article distributed under the terms of the CreativeCommons Attribution License (CC BY). The use, distribution or reproduction inother forums is permitted, provided the original author(s) and the copyright owner(s)are credited and that the original publication in this journal is cited, in accordancewith accepted academic practice. No use, distribution or reproduction is permittedwhich does not comply with these terms.

Frontiers in Plant Science | www.frontiersin.org 11 February 2019 | Volume 10 | Article 68

DNA metabarcoding authentication of Ayurvedic herbal products

on the European market raises concerns of quality and fidelity

Gopalakrishnan Saroja Seethapathy, Ancuta-Cristina Raclariu, Jarl Andreas Anmarkrud,

Helle Wangensteen and Hugo J. de Boer

Supplementary data

Supplementary Table S1. Ayurvedic herbal products used in the study.

Supplementary Table S2. Details of the plants species used as ingredients in the herbal

products.

Supplementary Table S3. Details of total number sequencing reads per products.

Supplementary Table S4. Details of normalized sequencing reads per products.

Supplementary Table S5. Details of sum occurrences of a species in Ayurvedic herbal

products used to generate heatmap.

Supplementary Table S6. Details of concentration of genomic DNA, nrITS amplicon

concentration and success rate of sequence yield in herbal products after quality filtering of

sequences between nrITS1 and nrITS2, and among replicates, and number of MOTUs in

nrITS1 and nrITS2 after NCBI-BLAST identification.

Supplementary Table S7. Results of the DNA metabarcoding analysis of herbal products.

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eae

Boer

haav

ia d

iffus

a L.

* N

ycta

gina

ceae

Spha

eran

thus

indi

cus L

.*

Com

posit

ae

Tino

spor

a co

rdifo

lia (W

illd.

) Mie

rs *

M

enis

perm

acea

e

Swer

tia c

hira

ta B

uch.

-Ham

. ex

Wal

l.*

Gen

tiana

ceae

Trib

ulus

terr

estr

is L.

* Zy

goph

ylla

ceae

Phyl

lant

hus a

mar

us S

chum

ach.

& T

honn

.*

Phyl

lant

hace

ae

Gm

elin

a ar

bore

a R

oxb.

* La

mia

ceae

Gos

sypi

um h

erba

ceum

L.*

M

alva

ceae

Berb

eris

ari

stata

DC.

* B

erbe

ridac

eae

Aloe

bar

bade

nsis

Mill

. (=

Aloe

ver

a (L

.) B

urm

.f.)*

X

anth

orrh

oeac

eae

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Roxb

. C

ombr

etac

eae

Embl

ica

offic

inal

is G

aertn

. (=

Phyl

lant

hus e

mbl

ica

L.)

Phyl

lant

hace

ae

Term

inal

ia c

hebu

la R

etz.

C

ombr

etac

eae

Mom

ordi

ca c

hara

ntia

L.

Cuc

urbi

tace

ae

Oci

mum

tenu

iflor

um L

. La

mia

ceae

Abut

ilon

indi

cum

(L.)

Swee

t M

alva

ceae

Rum

ex m

ariti

mus

L.

Poly

gona

ceae

Cur

cum

a lo

nga

L.

Zing

iber

acea

e

Pipe

r nig

rum

L.

Pipe

race

ae

Pipe

r lon

gum

L.

Pipe

race

ae

Zing

iber

offi

cina

le R

osco

e Zi

ngib

erac

eae

15

27

Cap

sule

s

Sara

ca a

soca

(Rox

b.) W

illd.

Le

gum

inos

ae

Ash

oka

Aegl

e m

arm

elos

(L.)

Cor

rêa

Rut

acea

e D

asha

moo

la #

Prem

na in

tegr

ifolia

Will

d. (=

Pre

mna

serr

atifo

lia L

.) La

mia

ceae

D

asha

moo

la #

Oro

xylu

m in

dicu

m (L

.) K

urz

Big

noni

acea

e D

asha

moo

la #

Ster

eosp

erm

um su

aveo

lens

(Rox

b.) D

C. (

= S.

che

lono

ides

(L.f.

) DC

.) B

igno

niac

eae

Das

ham

oola

# G

mel

ina

arbo

rea

Rox

b.

Lam

iace

ae

Das

ham

oola

# So

lanu

m in

dicu

m L

. So

lana

ceae

D

asha

moo

la #

Sola

num

xan

thoc

arpu

m S

chra

d. &

H. W

endl

. (=

S. v

irgi

nian

um L

.) So

lana

ceae

D

asha

moo

la #

Des

mod

ium

gan

getic

um (L

.) D

C.

Legu

min

osae

D

asha

moo

la #

Ura

ria

pict

a (J

acq.

) DC

. Le

gum

inos

ae

Das

ham

oola

# Tr

ibul

us te

rres

tris

L.

Zygo

phyl

lace

ae

Das

ham

oola

# Sy

mpl

ocos

race

mos

a R

oxb.

Sy

mpl

ocac

eae

Lodh

ra

Tino

spor

a co

rdifo

lia (W

illd.

) Mie

rs

Men

ispe

rmac

eae

Gud

uchi

So

lanu

m n

igru

m L

.

Sola

nace

ae

Kak

amac

hi

Boer

haav

ia d

iffus

a L.

N

ycta

gina

ceae

Pu

narn

ava

Aspa

ragu

s rac

emos

us W

illd.

A

spar

agac

eae

Shat

avar

i Al

oe b

arba

dens

is M

ill. (

= Al

oe v

era

(L.)

Bur

m.f.

) X

anth

orrh

oeac

eae

Kum

ari

Sant

alum

alb

um L

. Sa

ntal

acea

e C

hand

ana

C

yper

us ro

tund

us L

. C

yper

acea

e M

usta

Ju

stic

ia a

dhat

oda

L.

Aca

ntha

ceae

V

asak

a Te

rmin

alia

bel

liric

a (G

aertn

.) Ro

xb.

Com

bret

acea

e Tr

ipha

la #

Embl

ica

offic

inal

is G

aertn

. (=

Phyl

lant

hus e

mbl

ica

L.)

Phyl

lant

hace

ae

Trip

hala

# Te

rmin

alia

che

bula

Ret

z.

Com

bret

acea

e Tr

ipha

la #

Pipe

r nig

rum

L.

Pipe

race

ae

Trik

atu

# Pi

per l

ongu

m L

. Pi

pera

ceae

Tr

ikat

u #

Zing

iber

offi

cina

le R

osco

e Zi

ngib

erac

eae

Trik

atu

# Bo

mba

x ce

iba

L.

Mal

vace

ae

Shal

mal

i #

16

8 Ta

blet

s

Zing

iber

offi

cina

le R

osco

e Zi

ngib

erac

eae

Gin

ger

Did

ymoc

arpu

s ped

icel

lata

(Ait.

) Ait.

F.

Asc

lepi

adac

eae

Shila

pusp

ha

Saxi

frag

a lig

ulat

a M

urra

y (=

Sax

ifrag

a sto

loni

fera

Cur

tis)

Saxi

fraga

ceae

Pa

sana

bhed

a Ru

bia

cord

ifolia

L.

Rub

iace

ae

Indi

an m

adde

r C

yper

us sc

ario

sus R

.Br.

Cyp

erac

eae

Um

brel

la's

edge

Ac

hyra

nthe

s asp

era

L.

Am

aran

thac

eae

Pric

kly

chaf

f flo

wer

O

nosm

a br

acte

atum

Wal

l B

orag

inac

eae

Sedg

e

Vern

onia

cin

erea

(L.)

Less

. (=

Cya

nthi

llium

cin

ereu

m (L

.) H

.Rob

.) C

ompo

sitae

Pu

rple

flea

bane

(S

ahad

evi)

#

17

6 Ta

blet

s

Sara

ca in

dica

L.

Legu

min

osae

A

shok

a As

para

gus r

acem

osus

Will

d.*

Asp

arag

acea

e Sh

atav

ari

Term

inal

ia c

hebu

la R

etz.

C

ombr

etac

eae

Har

itaki

Si

da c

ordi

folia

L.

Mal

vace

ae

Bal

a G

lycy

rrhi

za g

labr

a L.

Le

gum

inos

ae

Yas

htim

adhu

C

ente

lla a

siat

ica

(L.)

Urb

. A

piac

eae

Man

duka

parn

i

18

6 Ta

blet

s

Bosw

ellia

serr

ata

Rox

b. e

x Co

lebr

.*

Bur

sera

ceae

Sh

alla

ki

Com

mip

hora

wig

htii

(Arn

.) B

hand

ari*

B

urse

race

ae

Gug

gul

Alpi

nia

gala

nga

(L.)

Will

d.*

Zing

iber

acea

e Ja

va g

alan

gal

Gly

cyrr

hiza

gla

bra

L.*

Legu

min

osae

Li

coric

e

Trib

ulus

terr

estr

is L.

Zy

goph

ylla

ceae

Sm

all c

altro

ps

Tino

spor

a co

rdifo

lia (W

illd.

) Mie

rs

Men

ispe

rmac

eae

Tino

spor

a G

ulan

cha

19

4 Ta

blet

s Tr

ibul

us te

rres

tris

L.

Zygo

phyl

lace

ae

Smal

l cal

trop

Cae

salp

inia

bon

duce

lla (L

.) Fl

emin

g (=

C. b

ondu

c (L

.) R

oxb.

) Le

gum

inos

ae

Bon

duc

nut

Aspa

ragu

s rac

emos

us W

illd.

A

spar

agac

eae

Asp

arag

us

Cra

teva

nur

vala

Buc

h.-H

am

Cap

para

ceae

Th

ree

leav

ed c

aper

A

kik

pish

ti $

Pr

oces

sed

agat

e

20

6 Ta

blet

s

Com

mip

hora

wig

htii

(Arn

.) B

hand

ari

Bur

sera

ceae

In

dian

bed

elliu

m

Tino

spor

a co

rdifo

lia (W

illd.

) Mie

rs

Men

ispe

rmac

eae

Gul

anch

a tin

ospo

ra

Rubi

a co

rdifo

lia L

.*

Rub

iace

ae

Indi

an m

adde

r Em

blic

a of

ficin

alis

Gae

rtn. (

= Ph

ylla

nthu

s em

blic

a L.

)*

Phyl

lant

hace

ae

Indi

an g

oose

berry

M

orin

ga p

tery

gosp

erm

a G

aertn

.*

Mor

inga

ceae

H

orse

-radi

sh tr

ee

Gly

cyrr

hiza

gla

bra

L.*

Legu

min

osae

Li

coric

e

21

5 Ta

blet

s

Com

mip

hora

wig

htii

(Arn

.) B

hand

ari*

B

urse

race

ae

Gug

gulu

G

arci

nia

cam

bogi

a (G

aertn

.) D

esr.

(= G

. gum

mi-g

utta

(L.)

Rox

b.)*

C

lusi

acea

e

Gym

nem

a sy

lves

tre

(Ret

z.) R

.Br.

ex S

m.*

A

pocy

nace

ae

Mes

hash

ringi

Te

rmin

alia

che

bula

Ret

z.*

Com

bret

acea

e

Trig

onel

la fo

enum

-gra

ecum

L.*

Le

gum

inos

ae

Med

hika

22

7 Ta

blet

s

Tino

spor

a co

rdifo

lia (W

illd.

) Mie

rs

Men

ispe

rmac

eae

Zing

iber

offi

cina

le R

osco

e Zi

ngib

erac

eae

Men

tha

arve

nsis

L.

Lam

iace

ae

Mor

inga

pte

rygo

sper

ma

Gae

rtn.

Mor

inga

ceae

Car

ica

papa

ya L

. C

aric

acea

e

Citr

us li

mon

(L.)

Osb

eck

Rut

acea

e

Kae

mpf

eria

gal

anga

L.

Zing

iber

acea

e

23

4 Ta

blet

s

Aste

raca

ntha

long

ifolia

Nee

s (=

Hyg

roph

ila a

uric

ulat

a (S

chum

ach.

) H

eine

) A

cant

hace

ae

Prun

us a

myg

dalu

s Bat

sch

(= P

runu

s dul

cis (

Mill

.) D

.A.W

ebb)

R

osac

eae

Cro

cus s

ativ

us L

. Iri

dace

ae

Trib

ulus

terr

estr

is L.

Zy

goph

ylla

ceae

24

19

Extra

cts

Com

mip

hora

wig

htii

(Arn

.) B

hand

ari

Bur

sera

ceae

Vitis

vin

ifera

L.

Vita

ceae

Oci

mum

tenu

iflor

um L

. La

mia

ceae

Hys

sopu

s offi

cina

lis L

. La

mia

ceae

Tino

spor

a co

rdifo

lia (W

illd.

) Mie

rs

Men

ispe

rmac

eae

Just

icia

adh

atod

a L.

A

cant

hace

ae

Myr

istic

a fr

agra

ns H

outt.

M

yris

ticac

eae

Gly

cyrr

hiza

gla

bra

L.

Legu

min

osae

Ono

sma

brac

teat

um W

all

Bor

agin

acea

e

Viol

a od

orat

a L.

V

iola

ceae

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Roxb

. C

ombr

etac

eae

Trip

hala

# Em

blic

a of

ficin

alis

Gae

rtn. (

= Ph

ylla

nthu

s em

blic

a L.

) Ph

ylla

ntha

ceae

Tr

ipha

la #

Term

inal

ia c

hebu

la R

etz.

C

ombr

etac

eae

Trip

hala

# Pi

per n

igru

m L

. Pi

pera

ceae

Tr

ikat

u #

Pipe

r lon

gum

L.

Pipe

race

ae

Trik

atu

# Zi

ngib

er o

ffici

nale

Ros

coe

Zing

iber

acea

e Tr

ikat

u #

Embe

lia ri

bes B

urm

.f.

Prim

ulac

eae

So

lanu

m x

anth

ocar

pum

Sch

rad.

& H

. Wen

dl. (

= S.

vir

gini

anum

L.)

Sola

nace

ae

C

inna

mom

um c

assia

(L.)

J.Pre

sl La

urac

eae

25

19

Extra

cts

Com

mip

hora

wig

htii

(Arn

.) B

hand

ari

Bur

sera

ceae

Vitis

vin

ifera

L.

Vita

ceae

Oci

mum

tenu

iflor

um L

. La

mia

ceae

Hys

sopu

s offi

cina

lis L

. La

mia

ceae

Tino

spor

a co

rdifo

lia (W

illd.

) Mie

rs

Men

ispe

rmac

eae

Just

icia

adh

atod

a L.

A

cant

hace

ae

Myr

istic

a fr

agra

ns H

outt.

M

yris

ticac

eae

Gly

cyrr

hiza

gla

bra

L.

Legu

min

osae

Ono

sma

brac

teat

um W

all

Bor

agin

acea

e

Viol

a od

orat

a L.

V

iola

ceae

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Roxb

. C

ombr

etac

eae

Trip

hala

# Em

blic

a of

ficin

alis

Gae

rtn. (

= Ph

ylla

nthu

s em

blic

a L.

) Ph

ylla

ntha

ceae

Tr

ipha

la #

Term

inal

ia c

hebu

la R

etz.

C

ombr

etac

eae

Trip

hala

# Pi

per n

igru

m L

. Pi

pera

ceae

Tr

ikat

u #

Pipe

r lon

gum

L.

Pipe

race

ae

Trik

atu

# Zi

ngib

er o

ffici

nale

Ros

coe

Zing

iber

acea

e Tr

ikat

u #

Embe

lia ri

bes B

urm

.f.

Prim

ulac

eae

So

lanu

m x

anth

ocar

pum

Sch

rad.

& H

. Wen

dl. (

= S.

vir

gini

anum

L.)

Sola

nace

ae

C

inna

mom

um c

assia

(L.)

J.Pre

sl La

urac

eae

26

14

Extra

cts

Just

icia

adh

atod

a L.

A

cant

hace

ae

Alth

aea

offic

inal

is L

. M

alva

ceae

Gly

cyrr

hiza

gla

bra

L.

Legu

min

osae

Oci

mum

tenu

iflor

um L

. La

mia

ceae

Pipe

r lon

gum

L.

Pipe

race

ae

Zizi

phus

sativ

a G

aertn

. (=

Zizi

phus

juju

ba M

ill.)

Rha

mna

ceae

Alpi

nia

gala

nga

(L.)

Will

d.

Zing

iber

acea

e

Hys

sopu

s offi

cina

lis L

. La

mia

ceae

Viol

a od

orat

a L.

V

iola

ceae

Zing

iber

offi

cina

le R

osco

e Zi

ngib

erac

eae

Pipe

r nig

rum

L.

Pipe

race

ae

Cur

cum

a lo

nga

L.

Zing

iber

acea

e

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Roxb

. C

ombr

etac

eae

Men

tha

× p

iper

ita L

. La

mia

ceae

27

1 C

apsu

les

Gym

nem

a sy

lves

tre

(Ret

z.) R

.Br.

ex S

m.

Apo

cyna

ceae

28

1 C

apsu

les

Gar

cini

a ca

mbo

gia

(Gae

rtn.)

Des

r. (=

G. g

umm

i-gut

ta (L

.) R

oxb.

) C

lusi

acea

e

29

1 C

apsu

les

Andr

ogra

phis

pani

cula

ta (B

urm

.f.) N

ees

Aca

ntha

ceae

30

3 C

apsu

les

Embl

ica

offic

inal

is G

aertn

. (=

Phyl

lant

hus e

mbl

ica

L.)

Phyl

lant

hace

ae

Term

inal

ia c

hebu

la R

etz.

C

ombr

etac

eae

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Roxb

. C

ombr

etac

eae

31

10

Tabl

ets

Oci

mum

tenu

iflor

um L

. La

mia

ceae

Cur

cum

a lo

nga

L.

Zing

iber

acea

e

Embl

ica

offic

inal

is G

aertn

. (=

Phyl

lant

hus e

mbl

ica

L.)

Phyl

lant

hace

ae

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Roxb

. C

ombr

etac

eae

Term

inal

ia c

hebu

la R

etz.

C

ombr

etac

eae

Gly

cyrr

hiza

gla

bra

L.

Legu

min

osae

Zing

iber

offi

cina

le R

osco

e Zi

ngib

erac

eae

Pipe

r lon

gum

L.

Pipe

race

ae

Pipe

r nig

rum

L.

Pipe

race

ae

Cin

nam

omum

zeyl

anic

um B

lum

e (=

C. v

erum

J.Pr

esl)

Laur

acea

e

32

6 Ta

blet

s

Saxi

frag

a gr

anul

ata

L.

Saxi

fraga

ceae

Hem

ides

mus

indi

cus (

L.) R

. Br.

ex S

chul

t. A

pocy

nace

ae

Spha

eran

thus

indi

cus L

. C

ompo

sitae

Cur

cum

a lo

nga

L.

Zing

iber

acea

e

Sant

alum

alb

um L

. Sa

ntal

acea

e

Car

um c

optic

um (L

.) B

enth

. & H

ook.

f. ex

C.B

.Cla

rke

(=

Trac

hysp

erm

um a

mm

i (L.

) Spr

ague

) A

piac

eae

Cor

alliu

m ru

brum

$

33

6 Ta

blet

s

Embl

ica

offic

inal

is G

aertn

. (=

Phyl

lant

hus e

mbl

ica

L.)

Phyl

lant

hace

ae

Aspa

ragu

s rac

emos

us W

illd.

A

spar

agac

eae

Gly

cyrr

hiza

gla

bra

L.

Legu

min

osae

Muc

una

prur

iens

(L.)

DC

. Le

gum

inos

ae

Term

inal

ia c

hebu

la R

etz.

C

ombr

etac

eae

Zing

iber

offi

cina

le R

osco

e Zi

ngib

erac

eae

Myt

illus

mar

gare

tifer

ous $

34

8 Ta

blet

s

Gly

cyrr

hiza

gla

bra

L.

Legu

min

osae

Term

inal

ia a

rjun

a (R

oxb.

ex

DC.

) Wig

ht &

Arn

. C

ombr

etac

eae

Amom

um su

bula

tum

Rox

b.

Zing

iber

acea

e

Pipe

r lon

gum

L.

Pipe

race

ae

Pipe

r nig

rum

L.

Pipe

race

ae

Zing

iber

offi

cina

le R

osco

e Zi

ngib

erac

eae

Cin

nam

omum

zeyl

anic

um B

lum

e (=

C.v

erum

J.Pr

esl)

Laur

acea

e

Iris

× g

erm

anic

a L.

Iri

dace

ae

35

10

Tabl

ets

Bosw

ellia

serr

ata

Rox

b. e

x Co

lebr

. B

urse

race

ae

Com

mip

hora

muk

ul (H

ook.

ex

Stoc

ks) E

ngl.

Bur

sera

ceae

Cur

cum

a lo

nga

L.

Zing

iber

acea

e

Alliu

m sa

tivum

L.

Am

aryl

lidac

eae

Zing

iber

offi

cina

le R

osco

e Zi

ngib

erac

eae

Pipe

r nig

rum

L.

Pipe

race

ae

Pipe

r lon

gum

L.

Pipe

race

ae

Pipe

r cha

ba H

unte

r (=

Pipe

r ret

rofra

ctum

Vah

l) Pi

pera

ceae

Apiu

m g

rave

olen

s L.

Api

acea

e

Spha

eran

thus

indi

cus L

. C

ompo

sitae

36

13

Tabl

ets

Term

inal

ia c

hebu

la R

etz.

C

ombr

etac

eae

Andr

ogra

phis

pani

cula

ta (B

urm

.f.) N

ees

Aca

ntha

ceae

Cur

cum

a lo

nga

L.

Zing

iber

acea

e

Zing

iber

offi

cina

le R

osco

e Zi

ngib

erac

eae

Trig

onel

la fo

enum

-gra

ecum

L.

Legu

min

osae

Swer

tia c

hira

ta B

uch.

-Ham

. ex

Wal

l. G

entia

nace

ae

Car

um c

optic

um (L

.) B

enth

. & H

ook.

f. ex

C.B

.Cla

rke

(=

Trac

hysp

erm

um a

mm

i (L.

) Spr

ague

) A

piac

eae

Pipe

r lon

gum

L.

Pipe

race

ae

Myr

istic

a fr

agra

ns H

outt.

M

yris

ticac

eae

Lotu

s ara

bicu

s L.

Legu

min

osae

Bosw

ellia

serr

ata

Rox

b. e

x Co

lebr

. B

urse

race

ae

Cyp

raea

mon

eta $

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Roxb

. C

ombr

etac

eae

Embl

ica

offic

inal

is G

aertn

. (=

Phyl

lant

hus e

mbl

ica

L.)

Phyl

lant

hace

ae

37

8 Ta

blet

s C

omm

ipho

ra m

ukul

(Hoo

k. e

x St

ocks

) Eng

l. B

urse

race

ae

Bauh

inia

var

iega

ta L

. Le

gum

inos

ae

Hem

ides

mus

indi

cus (

L.) R

. Br.

ex S

chul

t. A

pocy

nace

ae

Cur

cum

a lo

nga

L.

Zing

iber

acea

e

Gly

cyrr

hiza

gla

bra

L.

Legu

min

osae

Saxi

frag

a gr

anul

ata

L.

Saxi

fraga

ceae

Hor

deum

vul

gare

L.

Poac

eae

Sant

alum

alb

um L

. Sa

ntal

acea

e

38

12

Tabl

ets

Com

mip

hora

muk

ul (H

ook.

ex

Stoc

ks) E

ngl.

Bur

sera

ceae

Hib

iscu

s ros

a-si

nens

is L.

M

alva

ceae

Cor

alliu

m ru

brum

$

Aspa

ragu

s rac

emos

us W

illd.

A

spar

agac

eae

Hem

ides

mus

indi

cus (

L.) R

. Br.

ex S

chul

t. A

pocy

nace

ae

Gly

cyrr

hiza

gla

bra

L.

Legu

min

osae

Cur

cum

a lo

nga

L.

Zing

iber

acea

e

Embl

ica

offic

inal

is G

aertn

. (=

Phyl

lant

hus e

mbl

ica

L.)

Phyl

lant

hace

ae

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Roxb

. C

ombr

etac

eae

Term

inal

ia c

hebu

la R

etz.

C

ombr

etac

eae

Zing

iber

offi

cina

le R

osco

e Zi

ngib

erac

eae

Pipe

r lon

gum

L.

Pipe

race

ae

Pipe

r nig

rum

L.

Pipe

race

ae

Alo

e m

ucila

gine

s $

39

6 Ta

blet

s

Baco

pa m

onni

eri (

L.) W

ettst

. Pl

anta

gina

ceae

Alpi

nia

gala

nga

(L.)

Will

d.

Zing

iber

acea

e

Gly

cyrr

hiza

gla

bra

L.

Legu

min

osae

Embl

ica

offic

inal

is G

aertn

. (=

Phyl

lant

hus e

mbl

ica

L.)

Phyl

lant

hace

ae

Sant

alum

alb

um L

. Sa

ntal

acea

e

Myr

istic

a fr

agra

ns H

outt.

M

yris

ticac

eae

Cor

alliu

m ru

brum

$

40

4 C

apsu

les

Term

inal

ia a

rjun

a (R

oxb.

ex

DC.

) Wig

ht &

Arn

. C

ombr

etac

eae

Arju

n

Cis

sus q

uadr

angu

laris

L.

Vita

ceae

H

arjo

r Ph

ylla

nthu

s em

blic

a L.

Ph

ylla

ntha

ceae

A

mal

aki

Oci

mum

gra

tissim

um L

. La

mia

ceae

V

ana

Tuls

i

41

4 C

apsu

les

Inul

a ra

cem

osa

Hoo

k.f.

Com

posit

ae

Push

karm

ool

Oci

mum

tenu

iflor

um L

. La

mia

ceae

K

rishn

a Tu

lsi

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Roxb

. C

ombr

etac

eae

Vib

hita

ki fr

uit

Pipe

r lon

gum

L.

Pipe

race

ae

Pipa

li fru

kt

42

1 C

apsu

les

Oci

mum

tenu

iflor

um L

. La

mia

ceae

K

rishn

a Tu

lsi,

Ram

a Tu

lsi,

Van

a Tu

lsi

43

3 C

apsu

les

Term

inal

ia a

rjun

a (R

oxb.

ex

DC.

) Wig

ht &

Arn

. C

ombr

etac

eae

Arju

na

Sapi

ndus

trifo

liatu

s L.

Sapi

ndac

eae

Ree

tha

Mor

inga

ole

ifera

Lam

. M

orin

gace

ae

Sahi

jan

blad

44

3 C

apsu

les

Coc

cini

a in

dica

Wig

ht &

Arn

. (=

Coc

cini

a gr

andi

s (L.

) Voi

gt)

Cuc

urbi

tace

ae

Bim

bi b

lad

Boug

ainv

illea

spec

tabi

lis W

illd.

N

ycta

gina

ceae

B

ougi

nvel

lea

blad

Vi

nca

rose

a L.

(= C

atha

rant

hus r

oseu

s (L.

) G.D

on)

Apo

cyna

ceae

Sa

daba

har b

lad

45

3 C

apsu

les

Aegl

e m

arm

elos

(L.)

Cor

rêa

Rut

acea

e B

el b

lad

Lepi

dium

sativ

um L

. B

rass

icac

eae

Cha

ndra

shoo

r frø

Pl

anta

go o

vata

For

ssk.

Pl

anta

gina

ceae

Ps

ylliu

m H

usk

46

3 C

apsu

les

Picr

orhi

za k

urro

a R

oyle

ex

Ben

th.

Plan

tagi

nace

ae

Kat

uki

Oci

mum

tenu

iflor

um L

. La

mia

ceae

K

rishn

a Tu

lsi

Oci

mum

gra

tissim

um L

. La

mia

ceae

V

ana

Tuls

i

47

3 C

apsu

les

Phyl

lant

hus n

irur

i L.

Phyl

lant

hace

ae

Bhu

mya

mal

aki h

el u

rt Pi

cror

hiza

kur

roa

Roy

le e

x B

enth

. Pl

anta

gina

ceae

K

atuk

i roo

t Bo

erha

avia

diff

usa

L.

Nyc

tagi

nace

ae

Pum

arna

va ro

ot

48

1 Po

wde

r Al

thae

a of

ficin

alis

L.

Mal

vace

ae

Mar

shm

allo

w ro

ot

49

1 Po

wde

r H

emid

esm

us in

dicu

s (L.

) R. B

r. ex

Sch

ult.

Apo

cyna

ceae

50

1 Po

wde

r C

ente

lla a

siat

ica

(L.)

Urb

. A

piac

eae

51

10

Pow

ders

Aegl

e m

arm

elos

(L.)

Cor

rêa

Rut

acea

e B

ilva

root

Pr

emna

inte

grifo

lia W

illd.

(= P

. ser

ratif

olia

L.)

Lam

iace

ae

Agn

iman

tha

root

O

roxy

lum

indi

cum

(L.)

Kur

z B

igno

niac

eae

Shyo

naka

root

Ster

eosp

erm

um su

aveo

lens

(Rox

b.) D

C. (

= S.

che

lono

ides

(L.f.

) DC

.) B

igno

niac

eae

Pata

la ro

ot

Gm

elin

a ar

bore

a R

oxb.

La

mia

ceae

K

ashm

ari r

oot

Sola

num

indi

cum

L.

Sola

nace

ae

Bru

hati

root

Sola

num

xan

thoc

arpu

m S

chra

d. &

H.W

endl

. (=

S. v

irgi

nian

um L

.) So

lana

ceae

K

anta

kari

root

Des

mod

ium

gan

getic

um (L

.) D

C.

Legu

min

osae

Sh

alap

arni

root

U

rari

a pi

cta

(Jac

q.) D

C.

Legu

min

osae

Pr

ushn

ipar

ni ro

ot

Trib

ulus

terr

estr

is L.

Zy

goph

ylla

ceae

G

oksh

ura

root

52

1

Pow

der

Eclip

ta a

lba

(L.)

Has

sk.

(= E

clip

ta p

rost

rata

(L.)

L.)

Com

posit

ae

Brin

gara

j 53

1

Pow

der

Baco

pa m

onni

eri (

L.) W

ettst

. Pl

anta

gina

ceae

B

rahm

i 54

1

Pow

der

Boer

haav

ia d

iffus

a L.

N

ycta

gina

ceae

Pu

narn

ava

55

1 Po

wde

r Si

da c

ordi

folia

L.

Mal

vace

ae

56

1 Po

wde

r Ru

bia

cord

ifolia

L.

Rub

iace

ae

Man

jisth

a 57

1

Pow

der

Gym

nem

a sy

lves

tre

(Ret

z.) R

.Br.

ex S

m.

Apo

cyna

ceae

M

adhu

nash

ini

58

1 Po

wde

r Ti

nosp

ora

cord

ifolia

(Will

d.) M

iers

M

enis

perm

acea

e G

uduc

hi

59

1 C

apsu

les

Gar

cini

a in

dica

(Tho

uars

) Cho

isy

Clu

siac

eae

60

1 C

apsu

les

Term

inal

ia a

rjun

a (R

oxb.

ex

DC.

) Wig

ht &

Arn

. C

ombr

etac

eae

61

1 C

apsu

les

Boer

haav

ia d

iffus

a L.

N

ycta

gina

ceae

Pu

narn

ava

62

1

Pow

der

Baco

pa m

onni

eri (

L.) W

ettst

. Pl

anta

gina

ceae

63

3 C

apsu

les

Phyl

lant

hus n

irur

i L.

Phyl

lant

hace

ae

Bhu

mia

mal

aki

Ti

nosp

ora

cord

ifolia

(Will

d.) M

iers

M

enis

perm

acea

e A

mal

aki

Phyl

lant

hus e

mbl

ica

L.

Phyl

lant

hace

ae

Gud

uchi

64

4 C

apsu

les

Baco

pa m

onni

eri (

L.) W

ettst

. Pl

anta

gina

ceae

B

rahm

i C

ente

lla a

siat

ica

(L.)

Urb

. A

piac

eae

Got

u K

ola

Con

volv

ulus

plu

rica

ulis

Cho

isy (=

C. p

rost

ratu

s For

ssk.

) C

onvo

lvul

acea

e Sh

ankp

ushp

i W

ithan

ia so

mni

fera

(L.)

Dun

al

Sola

nace

ae

Ash

wag

andh

a

65

4 C

apsu

les

Cyp

erus

rotu

ndus

L.

Cyp

erac

eae

Mot

ha rh

izom

e W

ithan

ia so

mni

fera

(L.)

Dun

al

Sola

nace

ae

Ash

wag

andh

a Ti

nosp

ora

cord

ifolia

(Will

d.) M

iers

M

enis

perm

acea

e G

uduc

hi

Oci

mum

tenu

iflor

um L

. La

mia

ceae

R

ama

Tuls

i

66

6 C

apsu

les

Rubi

a co

rdifo

lia L

. R

ubia

ceae

M

anjit

rot

Pt

eroc

arpu

s san

talin

us L

.f.

Legu

min

osae

Sa

ndel

trä

C

urcu

ma

long

a L.

Zi

ngib

erac

eae

Gur

kmej

a

Oci

mum

tenu

iflor

um L

. La

mia

ceae

R

ama

Tuls

i Az

adir

acht

a in

dica

A.Ju

ss.

Mel

iace

ae

Nee

m

Ti

nosp

ora

cord

ifolia

(Will

d.) M

iers

M

enis

perm

acea

e G

uduc

hi

67

4 C

apsu

les

Cyp

erus

rotu

ndus

L.

Cyp

erac

eae

Mot

ha

Azad

irac

hta

indi

ca A

.Juss

. M

elia

ceae

N

eem

C

urcu

ma

long

a L.

Zi

ngib

erac

eae

Gur

kmej

a O

cim

um te

nuifl

orum

L.

Lam

iace

ae

Ram

a Tu

lsi

68

1 C

apsu

les

Gly

cyrr

hiza

gla

bra

L.

Legu

min

osae

La

krits

rot /

Y

asht

imad

hu

69

1 C

apsu

les

Sara

ca in

dica

L.

Legu

min

osae

70

1 C

apsu

les

Aegl

e m

arm

elos

(L.)

Cor

rêa

Rut

acea

e B

ael

71

22

Tabl

ets

Asp

hala

tum

$

Shila

jit

With

ania

som

nife

ra (L

.) D

unal

So

lana

ceae

A

shw

agan

dha

Gym

nem

a sy

lves

tre

(Ret

z.) R

.Br.

ex S

m.

Apo

cyna

ceae

G

urm

aar

Azad

irac

hta

indi

ca A

.Juss

. M

elia

ceae

N

imba

Term

inal

ia c

hebu

la R

etz.

C

ombr

etac

eae

Har

ar c

hoti

Tino

spor

a co

rdifo

lia (W

illd.

) Mie

rs

Men

ispe

rmac

eae

Gilo

y H

olar

rhen

a an

tidys

ente

rica

(Rot

h) W

all.

ex A

.DC.

(= H

. pub

esce

ns

Wal

l. ex

G.D

on)

Apo

cyna

ceae

K

utaj

Trib

ulus

terr

estr

is L.

Zy

goph

ylla

ceae

G

okhr

udan

a Te

rmin

alia

bel

liric

a (G

aertn

.) Ro

xb.

Com

bret

acea

e B

aher

a Em

blic

a of

ficin

alis

Gae

rtn. (

= Ph

ylla

nthu

s em

blic

a L.

) Ph

ylla

ntha

ceae

A

mal

a

Aegl

e m

arm

elos

(L.)

Cor

rêa

Rut

acea

e B

elpa

tra

Cur

cum

a ze

doar

ia (C

hrist

m.)

Rosc

oe

Zing

iber

acea

e K

acho

or

Just

icia

adh

atod

a L.

A

cant

hace

ae

Vas

a Fi

cus b

engh

alen

sis L

. M

orac

eae

Bad

jata

Ac

acia

ara

bica

(Lam

.) W

illd.

(= A

. nilo

tica

(L.)

Del

ile)

Legu

min

osae

K

ikar

fali

Stry

chno

s nux

-vom

ica

L.

Loga

niac

eae

Kuc

hla

Shud

h C

entr

athe

rum

ant

helm

intic

um (L

.) G

ambl

e (=

Bac

char

oide

s an

thel

min

tica

(L.)

Moe

nch)

C

ompo

sitae

K

aali

jeer

i

Picr

orhi

za k

urro

a R

oyle

ex

Ben

th.

Plan

tagi

nace

ae

Kut

ki

Syzy

gium

cum

ini (

L.) S

keel

s M

yrta

ceae

Ja

mun

gut

hli

Swer

tia c

hira

ta B

uch.

-Ham

. ex

Wal

l. G

entia

nace

ae

Chi

raya

ta

Cur

cum

a lo

nga

L.

Zing

iber

acea

e H

aldi

Tr

igon

ella

foen

um-g

raec

um L

. Le

gum

inos

ae

Met

hi

Sala

cia

chin

ensis

L.

Cel

astra

ceae

Sa

ptra

ngi

72

9 Ta

blet

s

Baco

pa m

onni

eri (

L.) W

ettst

. Pl

anta

gina

ceae

Con

volv

ulus

plu

rica

ulis

Cho

isy (=

C. p

rost

ratu

s For

ssk.

) C

onvo

lvul

acea

e

Acor

us c

alam

us L

. A

cora

ceae

Ono

sma

brac

teat

um W

all

Bor

agin

acea

e

Cel

astr

us p

anic

ulat

us W

illd.

C

elas

trace

ae

With

ania

som

nife

ra (L

.) D

unal

So

lana

ceae

Tino

spor

a co

rdifo

lia (W

illd.

) Mie

rs

Men

ispe

rmac

eae

Prav

al p

isht

i $

Muk

ta p

ishti $

Nar

dost

achy

s jat

aman

si (D

.Don

) DC.

C

aprif

olia

ceae

Rauv

olfia

serp

entin

a (L

.) B

enth

. ex

Kur

z A

pocy

nace

ae

73

14

Tabl

ets

Ory

za sa

tiva

L.

Poac

eae

Sacc

haru

m m

unja

Rox

b. (=

Sac

char

um b

enga

lens

e R

etz.

) Po

acea

e

Sacc

haru

m o

ffici

naru

m L

. Po

acea

e

Echi

nops

ech

inat

us R

oxb.

C

ompo

sitae

Not

es. A

yurv

edic

her

bal p

rodu

cts 1

-26

wer

e pu

rcha

sed

from

pha

rmac

ies a

nd h

erba

l sho

ps in

Rom

ania

, 27-

47 fr

om N

orw

ay, a

nd 4

8-79

pur

chas

ed v

ia e

-com

mer

ce

from

Sw

eden

. $ Non

-pla

nt in

gred

ient

s. *

Scie

ntifi

c na

mes

that

indi

cate

the

use

of v

ario

us re

fined

/sta

ndar

dize

d he

rbal

subs

tanc

es w

ithin

the

prod

uct;

whe

reas

, all

othe

rs in

dica

te th

e us

e of

not

ext

ract

ed p

lant

mat

eria

l, in

clud

ing

simpl

y pr

oces

sed

and

com

min

uted

pla

nt m

ater

ial,

with

in th

e pr

oduc

t. # T

hese

ver

nacu

lar n

ames

in

dica

te th

at th

ese

spec

ies h

ave

mor

e sc

ient

ific

nam

es, a

nd A

yurv

edic

pha

rmac

opoe

ia o

f Ind

ia w

as u

sed

to c

hoos

e th

e co

rrect

pla

nt sp

ecie

s nam

e. A

ll ot

her p

lant

in

gred

ient

s list

ed o

n th

e pr

oduc

t lab

el, a

re p

rovi

ded

with

bot

h th

e sc

ient

ific

and

vern

acul

ar n

ame.

Tino

spor

a co

rdifo

lia (W

illd.

) Mie

rs

Men

ispe

rmac

eae

Prem

na m

ucro

nata

Rox

b. (=

Pre

mna

mol

lissim

a Ro

th)

Lam

iace

ae

Cas

sia

fistu

la L

. Le

gum

inos

ae

Sida

cor

difo

lia L

. M

alva

ceae

Aspa

ragu

s rac

emos

us W

illd.

A

spar

agac

eae

Puer

aria

tube

rosa

(Will

d.) D

C.

Legu

min

osae

Sola

num

sura

ttens

e B

urm

. f.

Sola

nace

ae

Sola

num

indi

cum

L.

Sola

nace

ae

Hor

deum

vul

gare

L.

Poac

eae

Picr

orhi

za k

urro

a R

oyle

ex

Ben

th.

Plan

tagi

nace

ae

74

9 Ta

blet

s

Baco

pa m

onni

eri (

L.) W

ettst

.*

Plan

tagi

nace

ae

Con

volv

ulus

plu

rica

ulis

Cho

isy (=

C. p

rost

ratu

s For

ssk.

)*

Con

volv

ulac

eae

Acor

us c

alam

us L

.*

Aco

race

ae

Lava

ndul

a st

oech

as L

. La

mia

ceae

Ono

sma

brac

teat

um W

all

Bor

agin

acea

e

Cel

astr

us p

anic

ulat

us W

illd.

* C

elas

trace

ae

Nar

dost

achy

s jat

aman

si (D

.Don

) DC.

C

aprif

olia

ceae

Foen

icul

um v

ulga

re M

ill.

Api

acea

e

With

ania

som

nife

ra (L

.) D

unal

So

lana

ceae

Cor

alliu

m ru

brum

$

Myt

illus

mar

gare

tifer

ous $

75

3 Ta

blet

s Ti

nosp

ora

cord

ifolia

(Will

d.) M

iers

M

enis

perm

acea

e G

iloy

Oci

mum

tenu

iflor

um L

. La

mia

ceae

Tu

lsi

Azad

irac

hta

indi

ca A

.Juss

. M

elia

ceae

N

eem

76

1

Pow

der

With

ania

som

nife

ra (L

.) D

unal

So

lana

ceae

A

shw

agan

dha

pulv

er

77

1 Po

wde

r As

para

gus r

acem

osus

Will

d.

Asp

arag

acea

e R

aw S

hata

vari

root

78

1

Pow

der

Trib

ulus

terr

estr

is L.

Zy

goph

ylla

ceae

R

aw G

oksh

ura

pulv

er

79

1 Po

wde

r Te

rmin

alia

arj

una

(Rox

b. e

x D

C.) W

ight

& A

rn.

Com

bret

acea

e R

aw A

rjuna

Pul

ver

Supp

lem

enta

ry T

able

S2.

Det

ails

of th

e pl

ants

spec

ies u

sed

as in

gred

ient

s in

the

herb

al p

rodu

cts.

Scie

ntifi

c na

mes

as i

ndic

ated

on

the

prod

uct l

abel

* Pl

ant f

amily

H

abit

Sour

ce#

Con

serv

atio

n st

atus

#

nrIT

S se

quen

ces

in G

enBa

nk

(NC

BI)$

Abut

ilon

indi

cum

(L.)

Swee

t M

alva

ceae

Sh

rub

Wild

Le

ast C

once

rn

Yes

Ac

acia

ara

bica

(Lam

.) W

illd.

(= A

caci

a ni

lotic

a (L

.) D

elile

) Le

gum

inos

ae

Tree

W

ild

Leas

t Con

cern

Y

es

Acac

ia c

atec

hu (L

.f.) W

illd.

Le

gum

inos

ae

Tree

W

ild

Leas

t Con

cern

Y

es

Achi

llea

mill

efol

ium

L.

Com

posi

tae

Her

b W

ild/C

ultiv

atio

n

Leas

t Con

cern

Y

es

Achy

rant

hes a

sper

a L.

A

mar

anth

acea

e H

erb

Wild

Le

ast C

once

rn

Yes

Ac

orus

cal

amus

L.

Aco

race

ae

Her

b W

ild/C

ultiv

atio

n

Leas

t Con

cern

Y

es

Aegl

e m

arm

elos

(L.)

Cor

rêa

Rut

acea

e Tr

ee

Wild

V

ulne

rabl

e Y

es

Albi

zia le

bbec

k (L

.) B

enth

. Le

gum

inos

ae

Tree

W

ild

Leas

t Con

cern

G

enus

is

repr

esen

ted

Alliu

m sa

tivum

L.

Am

aryl

lidac

eae

Her

b C

ultiv

atio

n Le

ast C

once

rn

Yes

Al

oe b

arba

dens

is M

ill. (

= Al

oe v

era

(L.)

Bur

m.f.

) X

anth

orrh

oeac

eae

Her

b C

ultiv

atio

n Le

ast C

once

rn

Yes

Alpi

nia

gala

nga

(L.)

Will

d.

Zing

iber

acea

e H

erb

Wild

/Cul

tivat

ion

Le

ast C

once

rn

Yes

Al

thae

a of

ficin

alis

L.

Mal

vace

ae

Her

b W

ild

Leas

t Con

cern

Y

es

Amom

um su

bula

tum

Rox

b.

Zing

iber

acea

e H

erb

Cul

tivat

ion

Leas

t Con

cern

Y

es

Andr

ogra

phis

pani

cula

ta (B

urm

.f.) N

ees

Aca

ntha

ceae

H

erb

Wild

/Cul

tivat

ion

Le

ast C

once

rn

Yes

Anet

hum

sowa

Rox

b. e

x Fl

emin

g

Api

acea

e H

erb

Cul

tivat

ion

Leas

t Con

cern

G

enus

is

repr

esen

ted

Apiu

m g

rave

olen

s L.

Api

acea

e H

erb

Cul

tivat

ion

Leas

t Con

cern

Y

es

Aspa

ragu

s rac

emos

us W

illd.

A

spar

agac

eae

Shru

b W

ild/C

ultiv

atio

n

Leas

t Con

cern

Y

es

Aste

raca

ntha

long

ifolia

Nee

s (=

Hyg

roph

ila

auric

ulat

a (S

chum

ach.

) Hei

ne)

Aca

ntha

ceae

H

erb

Wild

Le

ast C

once

rn

Yes

Azad

irach

ta in

dica

A.Ju

ss.

Mel

iace

ae

Tree

W

ild/C

ultiv

atio

n

Leas

t Con

cern

Y

es

Baco

pa m

onni

eri (

L.) W

etts

t. Pl

anta

gina

ceae

H

erb

Wild

/Cul

tivat

ion

Le

ast C

once

rn

Yes

Ba

uhin

ia v

arie

gata

L.

Legu

min

osae

Tr

ee

Wild

/Cul

tivat

ion

Le

ast C

once

rn

Yes

Be

rber

is ar

istat

a D

C.

Ber

berid

acea

e Tr

ee

Wild

V

ulne

rabl

e Y

es

Berg

enia

ligu

lata

Eng

l.

Saxi

frag

acea

e H

erb

Wild

Le

ast C

once

rn

Yes

Bo

erha

avia

diff

usa

L.

Nyc

tagi

nace

ae

Her

b W

ild

Leas

t Con

cern

Y

es

Bom

bax

ceib

a L.

M

alva

ceae

Tr

ee

Wild

Le

ast C

once

rn

Yes

Bosw

ellia

serr

ata

Rox

b. e

x C

oleb

r. B

urse

race

ae

Tree

W

ild

Vul

nera

ble

Gen

us is

re

pres

ente

d

Boug

ainv

illea

spec

tabi

lis W

illd.

N

ycta

gina

ceae

W

oody

V

ine

Wild

/Cul

tivat

ion

Le

ast C

once

rn

Yes

Caes

alpi

nia

bond

ucel

la (L

.) Fl

emin

g (=

Ca

esal

pini

a bo

nduc

(L.)

Rox

b.)

Legu

min

osae

H

erb

Wild

/Cul

tivat

ion

Le

ast C

once

rn

Yes

Cam

ellia

sine

nsis

(L.)

Kun

tze

Thea

ceae

Sh

rub

Cul

tivat

ion

Leas

t Con

cern

Y

es

Capp

aris

spin

osa

L.

Cap

para

ceae

Sh

rub

Wild

/Cul

tivat

ion

Le

ast C

once

rn

Yes

Ca

rica

papa

ya L

. C

aric

acea

e Tr

ee

Cul

tivat

ion

Leas

t Con

cern

Y

es

Caru

m c

optic

um (L

.) B

enth

. & H

ook.

f. ex

C

.B.C

lark

e (=

Tra

chys

perm

um a

mm

i (L.

) Sp

ragu

e)

Api

acea

e H

erb

Cul

tivat

ion

Leas

t Con

cern

Y

es

Case

aria

esc

ulen

ta R

oxb.

Sa

licac

eae

Shru

b W

ild

Leas

t Con

cern

G

enus

is

repr

esen

ted

Cass

ia fi

stula

L.

Legu

min

osae

Tr

ee

Wild

/Cul

tivat

ion

Leas

t Con

cern

Y

es

Cass

ia o

ccid

enta

lis L

. (=o

f Sen

na

occi

dent

alis

(L.)

Link

) Le

gum

inos

ae

Her

b W

ild

Leas

t Con

cern

Y

es

Cela

strus

pan

icul

atus

Will

d.

Cel

astra

ceae

W

oody

Li

ana

Wild

/Cul

tivat

ion

Vul

nera

ble

Yes

Cent

ella

asia

tica

(L.)

Urb

. A

piac

eae

Her

b W

ild/C

ultiv

atio

n Le

ast C

once

rn

Yes

Ce

ntra

ther

um a

nthe

lmin

ticum

(L.)

Gam

ble

(=

Bacc

haro

ides

ant

helm

intic

a (L

.) M

oenc

h)

Com

posi

tae

Her

b W

ild/C

ultiv

atio

n Le

ast C

once

rn

Yes

Chlo

roph

ytum

aru

ndin

aceu

m B

aker

A

spar

agac

eae

Her

b W

ild

End

ange

red

Yes

Ci

chor

ium

inty

bus L

. C

ompo

sita

e H

erb

Wild

/Cul

tivat

ion

Leas

t Con

cern

Y

es

Cinn

amom

um c

assia

(L.)

J.Pre

sl La

urac

eae

Tree

Im

port

Leas

t Con

cern

Y

es

Cinn

amom

um ze

ylan

icum

Blu

me

(=

Cinn

amom

um v

erum

J.Pr

esl)

Laur

acea

e Tr

ee

Wild

/Cul

tivat

ion

Leas

t Con

cern

Y

es

Ciss

us q

uadr

angu

laris

L.

Vita

ceae

V

ine

Wild

/Cul

tivat

ion

Leas

t Con

cern

Y

es

Citru

s lim

on (L

.) O

sbec

k R

utac

eae

Tree

C

ultiv

atio

n Le

ast C

once

rn

Yes

C

occi

nia

indi

ca W

ight

& A

rn. (

= Co

ccin

ia

gran

dis (

L.) V

oigt

) C

ucur

bita

ceae

V

ine

Cul

tivat

ion

Leas

t Con

cern

Y

es

Com

mip

hora

muk

ul (H

ook.

ex

Stoc

ks) E

ngl.

Bur

sera

ceae

Sh

rub

Wild

Le

ast C

once

rn

Yes

Co

mm

ipho

ra w

ight

ii (A

rn.)

Bha

ndar

i B

urse

race

ae

Shru

b W

ild

Enda

nger

ed

Yes

Co

nvol

vulu

s plu

ricau

lis C

hois

y (=

Co

nvol

vulu

s pro

stra

tus F

orss

k.)

Con

volv

ulac

eae

Her

b W

ild

Leas

t Con

cern

Y

es

Crat

eva

nurv

ala

Buc

h.-H

am

Cap

para

ceae

Tr

ee

Wild

/Cul

tivat

ion

Leas

t Con

cern

G

enus

is

repr

esen

ted

Croc

us sa

tivus

L.

Irid

acea

e H

erb

Cul

tivat

ion

Leas

t Con

cern

Y

es

Curc

uma

long

a L.

Zi

ngib

erac

eae

Her

b C

ultiv

atio

n Le

ast C

once

rn

Yes

Cu

rcum

a ze

doar

ia (C

hris

tm.)

Ros

coe

Zing

iber

acea

e H

erb

Wild

/Cul

tivat

ion

Leas

t Con

cern

Y

es

Cype

rus r

otun

dus L

. C

yper

acea

e H

erb

Wild

Le

ast C

once

rn

Yes

Cype

rus s

cario

sus R

.Br.

Cyp

erac

eae

Her

b W

ild

Leas

t Con

cern

G

enus

is

repr

esen

ted

Des

mod

ium

gan

getic

um (L

.) D

C.

Legu

min

osae

H

erb

Wild

Le

ast C

once

rn

Yes

Did

ymoc

arpu

s ped

icel

lata

(Ait.

) Ait.

F.

Asc

lepi

adac

eae

Her

b W

ild

End

ange

red

Gen

us is

re

pres

ente

d Ec

hino

ps e

chin

atus

Rox

b.

Com

posi

tae

Her

b W

ild

Leas

t Con

cern

Y

es

Eclip

ta a

lba

(L.)

Has

sk.

(= E

clip

ta p

rostr

ata

(L.)

L.)

Com

posi

tae

Her

b W

ild

Leas

t Con

cern

Y

es

Elet

taria

car

dam

omum

(L.)

Mat

on

Zing

iber

acea

e H

erb

Cul

tivat

ion

Leas

t Con

cern

Y

es

Embe

lia ri

bes B

urm

.f.

Prim

ulac

eae

Woo

dy

Clim

ber

Wild

V

ulne

rabl

e Y

es

Enic

oste

mm

a lit

tora

le B

lum

e G

entia

naca

e H

erb

Wild

Le

ast C

once

rn

No

(oth

er g

ener

a ar

e re

pres

ente

d)

Evol

vulu

s alsi

noid

es (L

.) L.

C

onvo

lvul

acea

e H

erb

Wild

Le

ast C

once

rn

Yes

Fi

cus b

engh

alen

sis L

. M

orac

eae

Tree

W

ild

Leas

t Con

cern

Y

es

Foen

icul

um v

ulga

re M

ill.

Api

acea

e H

erb

Cul

tivat

ion

Leas

t Con

cern

Y

es

Fum

aria

offi

cina

lis L

. Pa

pave

race

ae

Her

b W

ild/C

ultiv

atio

n Le

ast C

once

rn

Yes

G

arci

nia

cam

bogi

a (G

aertn

.) D

esr.

(=

Gar

cini

a gu

mm

i-gut

ta (L

.) R

oxb.

) C

lusia

ceae

Tr

ee

Wild

/Cul

tivat

ion

Leas

t Con

cern

Y

es

Gar

cini

a in

dica

(Tho

uars

) Cho

isy

Clu

siace

ae

Tree

W

ild/C

ultiv

atio

n Le

ast C

once

rn

Yes

G

lycy

rrhi

za g

labr

a L.

Le

gum

inos

ae

Her

b C

ultiv

atio

n Le

ast C

once

rn

Yes

G

mel

ina

arbo

rea

Rox

b.

Lam

iace

ae

Tree

W

ild

Leas

t Con

cern

Y

es

Gos

sypi

um h

erba

ceum

L.

Mal

vace

ae

Shru

b C

ultiv

atio

n Le

ast C

once

rn

Yes

G

ymne

ma

sylv

estre

(Ret

z.) R

.Br.

ex S

m.

Apo

cyna

ceae

H

erb

Wild

V

ulne

rabl

e Y

es

Hem

ides

mus

indi

cus (

L.) R

. Br.

ex S

chul

t. A

pocy

nace

ae

Her

b W

ild

Leas

t Con

cern

Y

es

Hib

iscus

rosa

-sin

ensis

L.

Mal

vace

ae

Shru

b C

ultiv

atio

n Le

ast C

once

rn

Yes

H

olar

rhen

a an

tidys

ente

rica

(Rot

h) W

all.

ex

A.D

C.(=

Hol

arrh

ena

pube

scen

s Wal

l. ex

G

.Don

) A

pocy

nace

ae

Tree

W

ild

Leas

t Con

cern

Y

es

Hor

deum

vul

gare

L.

Poac

eae

Her

b C

ultiv

atio

n Le

ast C

once

rn

Yes

H

umul

us lu

pulu

s L.

Can

naba

ceae

C

limbe

r C

ultiv

atio

n Le

ast C

once

rn

Yes

H

ysso

pus o

ffici

nalis

L.

Lam

iace

ae

Her

b W

ild

Vul

nera

ble

Yes

In

ula

race

mos

a H

ook.

f. C

ompo

sita

e H

erb

Wild

Le

ast C

once

rn

Yes

Ip

omoe

a di

gita

ta L

. (=

Ipom

oea

chei

roph

ylla

O

'Don

ell)

Con

volv

ulac

eae

Her

b W

ild

Leas

t Con

cern

G

enus

is

repr

esen

ted

Iris

germ

anic

a L.

Ir

idac

eae

Her

b Im

port/

Cul

tivat

ion

Leas

t Con

cern

Y

es

Justi

cia

adha

toda

L.

Aca

ntha

ceae

Sh

rub

Wild

/Cul

tivat

ion

Leas

t Con

cern

Y

es

Kae

mpf

eria

gal

anga

L.

Zing

iber

acea

e H

erb

Cul

tivat

ion

Leas

t Con

cern

Y

es

Lava

ndul

a sto

echa

s L.

Lam

iace

ae

Shru

b Im

port

Leas

t Con

cern

Y

es

Lepi

dium

sativ

um L

. B

rass

icac

eae

Her

b C

ultiv

atio

n Le

ast C

once

rn

Yes

Le

ptad

enia

retic

ulat

a (R

etz.

) Wig

ht &

Arn

. A

pocy

nace

ae

Shru

b W

ild

End

ange

red

Yes

Lo

tus a

rabi

cus L

. Le

gum

inos

ae

Her

b W

ild

Leas

t Con

cern

Y

es

Men

tha

pipe

rita

L.

Lam

iace

ae

Her

b C

ultiv

atio

n Le

ast C

once

rn

Yes

M

enth

a ar

vens

is L.

La

mia

ceae

H

erb

Wild

/Cul

tivat

ion

Leas

t Con

cern

Y

es

Mom

ordi

ca c

hara

ntia

L.

Cuc

urbi

tace

ae

Vin

e C

ultiv

atio

n Le

ast C

once

rn

Yes

M

orin

ga o

leife

ra L

am.

Mor

inga

ceae

Tr

ee

Cul

tivat

ion

Leas

t Con

cern

Y

es

Mor

inga

pte

rygo

sper

ma

Gae

rtn.

Mor

inga

ceae

Tr

ee

Cul

tivat

ion

Leas

t Con

cern

Y

es

Muc

una

prur

iens

(L.)

DC

. Le

gum

inos

ae

Clim

ber

Wild

En

dang

ered

Y

es

Myr

istic

a fra

gran

s Hou

tt.

Myr

istic

acea

e Tr

ee

Wild

/Cul

tivat

ion

Leas

t Con

cern

Y

es

Nar

dosta

chys

jata

man

si (D

.Don

) DC

. C

aprif

olia

ceae

H

erb

Wild

V

ulne

rabl

e Y

es

Oci

mum

gra

tissim

um L

. La

mia

ceae

H

erb

Wild

/Cul

tivat

ion

Leas

t Con

cern

Y

es

Oci

mum

tenu

iflor

um L

. La

mia

ceae

H

erb

Cul

tivat

ion

Leas

t Con

cern

Y

es

Ono

sma

brac

teat

um W

all

Bor

agin

acea

e H

erb

Wild

Le

ast C

once

rn

Gen

us is

re

pres

ente

d O

roxy

lum

indi

cum

(L.)

Kur

z B

igno

niac

eae

Tree

W

ild

Vul

nera

ble

Yes

O

ryza

sativ

a L.

Po

acea

e H

erb

Cul

tivat

ion

Leas

t Con

cern

Y

es

Phyl

lant

hus a

mar

us S

chum

ach.

& T

honn

. Ph

ylla

ntha

ceae

H

erb

Wild

Le

ast C

once

rn

Yes

Ph

ylla

nthu

s em

blic

a L.

Ph

ylla

ntha

ceae

Tr

ee

Wild

/Cul

tivat

ion

Vul

nera

ble

Yes

Ph

ylla

nthu

s niru

ri L.

Ph

ylla

ntha

ceae

H

erb

Wild

Le

ast C

once

rn

Yes

Ph

ylla

nthu

s urin

aria

L.

Phyl

lant

hace

ae

Her

b W

ild

Leas

t Con

cern

Y

es

Picr

orhi

za k

urro

a R

oyle

ex

Ben

th.

Plan

tagi

nace

ae

Her

b W

ild

Enda

nger

ed

Yes

Pi

per c

haba

Hun

ter (

= P.

retro

fract

um V

ahl)

Pipe

race

ae

Vin

e C

ultiv

atio

n Le

ast C

once

rn

Yes

Pi

per c

ubeb

a B

ojer

Pi

pera

ceae

V

ine

Cul

tivat

ion

Leas

t Con

cern

Y

es

Pipe

r lon

gum

L.

Pipe

race

ae

Vin

e C

ultiv

atio

n Le

ast C

once

rn

Yes

Pi

per n

igru

m L

. Pi

pera

ceae

V

ine

Cul

tivat

ion

Leas

t Con

cern

Y

es

Plan

tago

ova

ta F

orss

k.

Plan

tagi

nace

ae

Her

b C

ultiv

atio

n Le

ast C

once

rn

Yes

Pl

umba

go ze

ylan

ica

L.

Plum

bagi

nace

ae

Clim

ber

Wild

V

ulne

rabl

e Y

es

Prem

na in

tegr

ifolia

Will

d. (=

P. s

erra

tifol

ia

L.)

Lam

iace

ae

Shru

b W

ild

Leas

t Con

cern

Y

es

Prem

na m

ucro

nata

Rox

b. (=

P. m

ollis

sima

Rot

h)

Lam

iace

ae

Shru

b W

ild

Leas

t Con

cern

Y

es

Prun

us a

myg

dalu

s Bat

sch

(= P

. dul

cis (

Mill

.) D

.A.W

ebb)

R

osac

eae

Tree

W

ild

Leas

t Con

cern

Y

es

Pter

ocar

pus m

arsu

pium

Rox

b.

Legu

min

osae

Tr

ee

Wild

En

dang

ered

Y

es

Pter

ocar

pus s

anta

linus

L.f.

Le

gum

inos

ae

Tree

W

ild

Enda

nger

ed

Yes

Pu

erar

ia tu

bero

sa (W

illd.

) DC

. Le

gum

inos

ae

Clim

ber

Wild

V

ulne

rabl

e Y

es

Raph

anus

sativ

us L

. (=

Raph

anus

ra

phan

istru

m su

bsp.

sativ

us (L

.) D

omin

) B

rass

icac

eae

Her

b C

ultiv

atio

n Le

ast C

once

rn

Yes

Rauv

olfia

serp

entin

a (L

.) B

enth

. ex

Kur

z A

pocy

nace

ae

Shru

b W

ild

Enda

nger

ed

Yes

Rh

eum

em

odi L

. Po

lygo

nace

ae

Her

b W

ild

Enda

nger

ed

Yes

Ru

bia

cord

ifolia

L.

Rub

iace

ae

Clim

ber

Wild

V

ulne

rabl

e Y

es

Rum

ex m

ariti

mus

L.

Poly

gona

ceae

H

erb

Wild

Le

ast C

once

rn

Gen

us is

re

pres

ente

d Sa

ccha

rum

mun

ja R

oxb.

(= S

acch

arum

be

ngal

ense

Ret

z.)

Poac

eae

Shru

b W

ild/C

ultiv

atio

n Le

ast C

once

rn

Yes

Sacc

haru

m o

ffici

naru

m L

. Po

acea

e Sh

rub

Cul

tivat

ion

Leas

t Con

cern

Y

es

Sala

cia

chin

ensis

L.

Cel

astra

ceae

C

limbe

r W

ild

Leas

t Con

cern

Y

es

Sant

alum

alb

um L

. Sa

ntal

acea

e Tr

ee

Wild

En

dang

ered

Y

es

Sapi

ndus

trifo

liatu

s L.

Sapi

ndac

eae

Tree

W

ild

Leas

t Con

cern

Y

es

Sara

ca a

soca

(Rox

b.) W

illd.

Le

gum

inos

ae

Tree

W

ild

End

ange

red

Yes

Sa

raca

indi

ca L

. Le

gum

inos

ae

Tree

W

ild

Leas

t Con

cern

Y

es

Saxi

fraga

gra

nula

ta L

. Sa

xifr

agac

eae

Her

b W

ild

Leas

t Con

cern

Y

es

Saxi

fraga

ligu

lata

Mur

ray

(= S

axifr

aga

stolo

nife

ra C

urtis

) Sa

xifr

agac

eae

Her

b W

ild

Leas

t Con

cern

Y

es

Sida

cor

difo

lia L

. M

alva

ceae

H

erb

Wild

Le

ast C

once

rn

Yes

So

lanu

m in

dicu

m L

. So

lana

ceae

H

erb

Wild

Le

ast C

once

rn

Yes

So

lanu

m n

igru

m L

.

Sola

nace

ae

Her

b W

ild

Leas

t Con

cern

Y

es

Sola

num

sura

ttens

e B

urm

. f.

Sola

nace

ae

Her

b W

ild

Leas

t Con

cern

Y

es

Sola

num

xan

thoc

arpu

m S

chra

d. &

H. W

endl

. (=

Sol

anum

virg

inia

num

L.)

Sola

nace

ae

Her

b W

ild

Leas

t Con

cern

Y

es

Spha

eran

thus

indi

cus L

. C

ompo

sita

e H

erb

Wild

Le

ast C

once

rn

Yes

St

ereo

sper

mum

suav

eole

ns (R

oxb.

) DC

. (=

Ster

eosp

erm

um c

helo

noid

es (L

.f.) D

C.)

Big

noni

acea

e Tr

ee

Wild

V

ulne

rabl

e Y

es

Stry

chno

s nux

-vom

ica

L.

Loga

niac

eae

Tree

W

ild

Vul

nera

ble

Yes

Sw

ertia

chi

rata

Buc

h.-H

am. e

x W

all.

Gen

tiana

ceae

H

erb

Wild

En

dang

ered

Y

es

Sym

ploc

os ra

cem

osa

Rox

b.

Sym

ploc

acea

e Sh

rub

Wild

V

ulne

rabl

e Y

es

Syzy

gium

cum

ini (

L.) S

keel

s M

yrta

ceae

Tr

ee

Wild

/Cul

tivat

ion

Le

ast C

once

rn

Yes

Ta

mar

ix g

allic

a L

Tam

aric

acea

e Sh

rub

Wild

Le

ast C

once

rn

Yes

Te

phro

sia p

urpu

rea

(L.)

Pers

. Le

gum

inos

ae

Her

b W

ild/C

ultiv

atio

n

Leas

t Con

cern

Y

es

Term

inal

ia a

rjuna

(Rox

b. e

x D

C.)

Wig

ht &

A

rn.

Com

bret

acea

e Tr

ee

Wild

Le

ast C

once

rn

Yes

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Rox

b.

Com

bret

acea

e Tr

ee

Wild

Le

ast C

once

rn

Yes

Te

rmin

alia

che

bula

Ret

z.

Com

bret

acea

e Tr

ee

Wild

Le

ast C

once

rn

Yes

Ti

nosp

ora

cord

ifolia

(Will

d.) M

iers

M

enis

perm

acea

e C

limbe

r W

ild/C

ultiv

atio

n

Leas

t Con

cern

Y

es

Trib

ulus

terr

estri

s L.

Zygo

phyl

lace

ae

Her

b W

ild

Leas

t Con

cern

Y

es

Trig

onel

la fo

enum

-gra

ecum

L.

Legu

min

osae

H

erb

Cul

tivat

ion

Leas

t Con

cern

Y

es

Ura

ria p

icta

(Jac

q.) D

C.

Legu

min

osae

H

erb

Wild

En

dang

ered

Y

es

Vale

riana

offi

cina

lis L

. C

aprif

olia

ceae

H

erb

Wild

Le

ast C

once

rn

Yes

Va

leria

na w

allic

hii D

C. (

= Va

leria

na

jata

man

si Jo

nes)

C

aprif

olia

ceae

H

erb

Wild

V

ulne

rabl

e Y

es

Vern

onia

cin

erea

(L.)

Less

. (=

Cyan

thill

ium

ci

nere

um (L

.) H

.Rob

.) C

ompo

sita

e H

erb

Wild

Le

ast C

once

rn

Yes

Vinc

a ro

sea

L. (=

Cat

hara

nthu

s ros

eus (

L.)

G.D

on)

Apo

cyna

ceae

H

erb

Cul

tivat

ion

Leas

t Con

cern

Y

es

Viol

a od

orat

a L.

V

iola

ceae

H

erb

Cul

tivat

ion

Leas

t Con

cern

Y

es

Vitis

vin

ifera

L.

Vita

ceae

C

limbe

r C

ultiv

atio

n Le

ast C

once

rn

Yes

W

ithan

ia so

mni

fera

(L.)

Dun

al

Sola

nace

ae

Shru

b W

ild/C

ultiv

atio

n Le

ast C

once

rn

Yes

Zi

ngib

er o

ffici

nale

Ros

coe

Zing

iber

acea

e H

erb

Cul

tivat

ion

Leas

t Con

cern

Y

es

Zizip

hus s

ativ

a G

aertn

. (=

Zizip

hus j

ujub

a M

ill.)

Rha

mna

ceae

Tr

ee

Wild

/Cul

tivat

ion

Leas

t Con

cern

Y

es

Not

es. *

Scie

ntifi

c na

mes

as

indi

cate

d on

the

prod

uct l

abel

, whe

reas

sci

entif

ic n

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in th

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the

Plan

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tp://

ww

w.th

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lant

sou

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and

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ateg

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12

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56

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910

1213

1415

1617

1819

2324

2526

2730

3132

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4052

5558

6061

6366

6869

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7475

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Nam

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44 p

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Abel

mos

chus

esc

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tus

(L.)

Moe

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Mal

vace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

126

00

00

12

%Ab

utilo

n in

dicu

m (

L.) S

wee

tM

alva

ceae

00

00

00

00

00

00

00

00

011

70

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

Acan

thos

perm

um h

ispid

um D

C.Co

mpo

sitae

00

00

00

00

00

00

00

205

00

00

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

A grim

onia

eup

ator

ia L

.Ro

sace

ae0

00

00

00

00

00

00

500

00

00

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

Alce

a ro

sea

L.M

alva

ceae

00

00

00

00

00

00

00

00

00

00

00

00

027

40

00

00

00

00

00

00

00

00

01

2 %

Allo

casu

arin

a ze

phyr

ea L

.A.S

.John

son

Casu

arin

acea

e0

00

00

00

00

00

00

00

00

00

00

1685

00

00

00

00

00

00

00

00

00

00

00

12

%Al

loph

ylus

boj

eria

nus

(Cam

bess

.) Bl

ume

Sapi

ndac

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00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

044

00

00

00

12

%Al

thae

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ficin

alis

L.M

alva

ceae

00

00

00

00

00

00

00

026

3226

840

00

00

010

418

00

00

00

00

011

10

038

426

650

670

00

800

818

%Am

aran

thus

viri

dis

L.A

mar

anth

acea

e0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

120

00

00

00

00

00

00

01

2 %

Amm

i tri f

olia

tum

(H

.C.W

atso

n) T

rel.

Api

acea

e0

00

00

00

00

00

00

00

00

00

00

011

00

00

00

00

00

00

00

00

00

00

01

2 %

Anch

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italic

a Re

tz.

Bora

gina

ceae

00

00

00

00

023

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

A per

a sp

ica-

vent

i (L

.) P.

Beau

v.Po

acea

e0

00

00

00

00

00

00

00

00

00

00

930

00

00

00

00

00

00

00

00

00

00

01

2 %

Arac

his c

orre

ntin

a (B

urka

rt) K

rapo

v. &

W.C

.Gre

g.Le

gum

inos

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

120

00

01

2 %

Aspa

ragu

s rac

emos

us W

illd.

Asp

arag

acea

e0

00

00

023

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

024

570

25

%Az

adira

chta

indi

ca A

.Juss

.M

elia

ceae

00

510

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

012

00

00

00

155

00

37

%Ba

ccha

roid

es a

nthe

lmin

tica

(L.)

Moe

nch

Aste

race

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

2769

00

029

0310

9795

6978

30

00

04

9 %

Behn

ia re

ticul

ata

(Thu

nb.)

Did

r.A

spar

agac

eae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

4004

01

2 %

Berb

eris

asia

tica

Roxb

. ex

DC.

Berb

erid

acea

e0

00

00

00

00

00

00

00

00

00

00

00

00

850

00

00

00

00

00

00

00

00

01

2 %

Ber g

enia

ligu

lata

Eng

l.Sa

xifr

agac

eae

00

00

00

00

00

00

00

2657

00

00

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

Boer

havi

a di

ffusa

L.

Nyc

tagi

nace

ae0

00

00

00

00

00

00

00

00

00

00

017

00

022

50

169

00

00

1444

00

00

203

320

210

00

716

%Br

assic

a ni

gra

(L.)

K.K

och

Bras

sicac

eae

00

170

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

12

%Br

assic

a ol

erac

ea L

. Br

assic

acea

e0

00

00

00

00

00

021

278

00

00

00

00

043

4487

00

00

00

00

00

00

00

00

00

00

37

%Ca

lluna

vul

garis

(L.)

Hul

lEr

icac

eae

00

00

00

503

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

Cann

abis

sativ

a L.

Cann

abac

eae

00

00

00

00

4378

60

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

12

%Ca

ssia

sp.

Le

gum

inos

ae31

00

00

00

018

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

02

5 %

Cela

strus

pan

icul

atus

Will

d.Ce

lastr

acea

e0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

016

40

00

12

%Ce

nchr

us a

mer

ican

us (

L.) M

orro

nePo

acea

e0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

6229

00

00

12

%Ce

ntau

rea

cyan

us L

.Co

mpo

sitae

00

00

00

00

00

00

00

00

00

610

00

00

00

00

00

00

00

00

00

00

00

00

12

%Ce

ntel

la a

siatic

a (L

.) U

rb.

Api

acea

e0

00

00

00

00

00

00

00

320

00

00

038

00

692

00

00

00

00

00

00

00

362

00

04

9 %

Chae

roph

yllu

m re

flexu

m A

itch.

Api

acea

e0

00

00

00

00

00

00

00

00

00

00

049

00

00

00

00

00

00

00

00

00

00

01

2 %

Cham

aecr

ista

oles

i phy

lla (

Vog

el) H

.S.Ir

win

& B

arne

byLe

gum

inos

ae24

80

00

00

00

00

00

00

00

00

00

00

640

00

00

00

00

00

00

00

00

00

00

25

%Ci

chor

ium

int y

bus

L.Co

mpo

sitae

00

592

00

819

370

476

00

2334

00

00

00

00

00

00

00

00

00

00

00

00

8910

50

00

00

07

16 %

Cinn

amom

um c

assia

(L.

) J.P

resl

Laur

acea

e0

00

00

00

00

00

00

00

00

00

00

00

00

025

250

00

00

00

00

00

00

00

00

12

%Ci

nnam

omum

iner

s Re

inw

. ex

Blum

eLa

urac

eae

00

00

059

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

12

%Ci

ssus

qua

dran

gula

ris L

.V

itace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

054

00

00

00

00

00

00

00

01

2 %

Citru

llus l

anat

us (

Thun

b.) M

atsu

m. &

Nak

aiCu

curb

itace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

180

00

01

2 %

Citru

s cle

men

tina

hort.

Ruta

ceae

00

00

00

00

00

00

00

00

00

500

00

00

00

00

00

00

00

00

00

00

00

00

12

%Co

ccin

ia a

doen

sis (H

ochs

t. ex

A. R

ich.

) Cog

n.Cu

curb

itace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

430

00

00

00

00

00

01

2 %

Cocc

inia

gra

ndis

(L.)

Voi

gtCu

curb

itace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

588

00

660

00

00

00

00

25

%Co

ccin

ia se

ssili

folia

(Son

d.) C

ogn.

Cucu

rbita

ceae

00

00

00

110

00

00

00

00

00

00

00

690

00

00

00

00

00

00

00

00

00

00

25

%Co

mbr

etum

nig

rican

s Le

pr. e

x G

uill.

& P

err.

Com

bret

acea

e0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

148

00

00

12

%Co

nvol

vulu

s mic

roph

yllu

s Si

eber

ex

Spre

ng.

Conv

olvu

lace

ae0

00

00

00

00

1612

00

00

00

00

00

00

00

017

771

00

00

00

00

00

00

00

00

03

7 %

Conv

olvu

lus p

rostr

atus

For

ssk.

Conv

olvu

lace

ae64

00

00

00

00

2418

30

675

00

00

00

00

00

100

1839

719

00

00

00

00

00

00

526

4294

20

120

1023

%Co

nvol

vulu

s sub

hirs

utus

Re g

el &

Sch

mal

h.Co

nvol

vula

ceae

00

00

00

00

00

00

00

00

00

023

00

00

00

292

00

00

00

00

00

00

00

00

02

5 %

Corc

horu

s olit

oriu

s L.

Mal

vace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

390

00

00

00

00

00

00

00

00

01

2 %

Coria

ndru

m sa

tivum

L.

Api

acea

e75

00

00

870

00

00

050

40

00

00

014

00

131

00

6028

00

00

00

00

022

589

026

00

00

1023

%Cu

cum

is m

elo

L.

Cucu

rbita

ceae

00

00

00

00

00

00

00

455

00

074

00

00

00

00

00

00

00

00

00

00

043

00

03

7 %

Cucu

mis

sativ

us L

.Cu

curb

itace

ae0

00

00

00

013

00

00

230

075

40

2023

00

00

00

00

00

00

017

316

00

00

100

00

00

07

16 %

Culle

n co

r ylif

oliu

m (

L.) M

edik

.Le

gum

inos

ae0

034

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

120

00

02

5 %

Cum

inum

cym

inum

L.

Api

acea

e40

00

00

059

00

023

00

00

00

00

00

360

220

00

1049

430

00

00

088

00

00

00

00

00

818

%C y

amop

sis te

trago

nolo

ba (

L.) T

aub.

Legu

min

osae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

041

12

%C y

nanc

hum

stau

bii

Boss

erA

pocy

nace

ae0

00

00

00

00

00

00

00

00

00

00

056

00

00

00

00

00

00

00

00

00

00

01

2 %

Cyno

don

dact

ylon

(L.

) Per

s.Po

acea

e0

00

00

00

00

00

00

00

068

00

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

Dam

ron g

ia c

yana

ntha

Trib

oun

Ges

neria

ceae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

431

00

00

00

00

00

00

00

01

2 %

Don

ioph

yton

wed

delli

i K

atin

as &

Stu

essy

Com

posit

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

1865

00

00

00

00

00

12

%D

raba

niv

alis

Lil j

.Br

assic

acea

e0

00

00

00

00

00

00

00

5060

00

00

00

00

00

00

00

00

043

290

00

00

00

00

02

5 %

Dry

moc

allis

rupe

stris

(L.)

Soj k

Rosa

ceae

00

00

00

00

00

00

00

089

945

9113

50

00

00

00

00

00

00

00

00

00

00

00

00

03

7 %

Dr y

pete

s hoa

ensis

Gag

nep.

Putra

njiv

acea

e0

00

00

00

00

00

00

00

00

00

00

00

00

00

090

40

00

00

00

00

00

00

00

12

%D

ysch

orist

e sip

hona

ntha

(N

ees)

Kun

tze

Aca

ntha

ceae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

286

00

00

00

01

2 %

Echi

nace

a pu

rpur

ea (L

.) M

oenc

hCo

mpo

sitae

00

00

00

00

830

015

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

02

5 %

Echi

noch

loa

colo

na (

L.) L

ink

Poac

eae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

1313

00

00

00

00

01

2 %

Echi

noch

loa

pyra

mid

alis

(Lam

.) H

itchc

. & C

hase

Poac

eae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

720

00

00

00

00

12

%Ec

hium

vul

gare

L.

Bora

gina

ceae

00

00

025

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

12

%Ec

li pta

alb

a (L

.) H

assk

. (s

yn. E

clip

ta p

rostr

ata

L.)

Com

posit

ae0

00

00

00

00

00

110

00

00

00

00

00

00

00

00

086

853

00

7718

404

00

1615

00

00

00

06

14 %

Eleu

ther

ococ

cus s

essil

iflor

us (

Rupr

. & M

axim

.) S.

Y.H

uA

ralia

ceae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

205

062

00

02

5 %

Embe

lia ts

jeria

m-c

otta

m (R

oem

. & S

chul

t.) A

.DC.

Prim

ulac

eae

00

00

037

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

12

%Eu

odia

hor

tens

is J.R

. For

st. &

G. F

orst.

Ruta

ceae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

014

070

00

00

00

00

01

2 %

Evol

vulu

s alsi

noid

es (

L.) L

.Co

nvol

vula

ceae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

012

650

00

00

01

2 %

Fago

pyru

m e

scul

entu

m M

oenc

hPo

lygo

nace

ae0

00

00

00

00

00

00

00

00

00

00

025

00

00

00

00

00

00

00

00

00

00

01

2 %

Festu

ca g

igan

tea

(L.)

Vill

.Po

acea

e0

00

00

00

00

00

00

00

00

620

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

Foen

icul

um v

ulga

re M

ill.

Api

acea

e15

057

00

2645

00

022

10

00

00

00

00

00

041

00

00

00

00

00

00

00

012

860

352

00

00

716

%Fr

axin

us a

lbic

ans

Buck

ley

Ole

acea

e0

00

00

00

00

00

00

00

00

042

60

00

00

00

00

00

00

00

00

00

00

00

00

12

%Fr

axin

us p

enns

ylva

nica

Mar

shal

lO

leac

eae

00

00

00

00

00

00

00

00

412

00

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

Gly

cyrr

hiza

gla

bra

L.

Legu

min

osae

00

00

00

00

00

00

00

00

00

00

00

00

204

066

50

00

00

00

00

00

00

00

00

25

%G

oss y

pium

hirs

utum

L.

Mal

vace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

120

401

00

00

25

%G

reen

iops

is sib

uyan

ensis

Elm

erRu

biac

eae

00

00

00

00

021

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

12

%G

ymne

ma

sylv

estre

(Re

tz.)

R.Br

. ex

Sm.

Apo

cyna

ceae

00

00

00

00

00

00

00

00

00

00

00

760

00

00

00

00

00

00

120

00

00

00

25

%H

elwi

ngia

chi

nens

is Ba

talin

Hel

win

giac

eae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

038

00

00

01

2 %

Hib

iscus

sabd

ariff

a L

.M

alva

ceae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

048

80

312

00

00

25

%H

ibisc

us sy

riacu

s L.

Mal

vace

ae76

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

103

00

00

00

00

00

25

%H

iera

cium

fusc

um V

ill.

Com

posit

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

020

220

00

00

12

%H

iero

n ym

a al

chor

neoi

des

Alle

mão

Phyl

lant

hace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

121

00

00

00

00

00

12

%H

omal

ium

ce y

lani

cum

(Gar

dner

) Ben

th.

Salic

acea

e0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

120

00

01

2 %

Hor

deum

vul

gare

L.

Poac

eae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

059

00

00

12

%H

umul

us lu

pulu

s L.

Cann

abac

eae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

1344

00

00

00

00

00

00

12

%H

ygro

phila

aur

icul

ata

(Sch

umac

h.) H

eine

Aca

ntha

ceae

00

00

00

00

00

00

00

00

00

540

00

00

00

00

00

00

00

00

00

00

00

00

12

%H

ygro

phila

schu

lli M

.R.A

lmei

da &

S.M

.Alm

eida

Aca

ntha

ceae

00

820

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

12

%H

yosc

yam

us n

iger

L.

Sola

nace

ae0

00

00

00

00

00

00

00

00

028

20

00

00

00

00

00

00

00

00

00

00

00

00

12

%H

ypoc

haer

is m

acul

ata

L.Co

mpo

sitae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

107

107

00

00

25

%In

ula

race

mos

a H

ook.

f.Co

mpo

sitae

00

00

00

00

00

00

00

00

00

00

00

00

00

290

00

00

00

00

00

00

00

00

01

2 %

I pom

oea

nil

(L.)

Roth

Conv

olvu

lace

ae0

3945

10

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

25

%Ip

omoe

a pa

rasit

ica

(Kun

th) G

. Don

Conv

olvu

lace

ae60

8814

746

450

180

043

969

00

00

00

00

00

00

00

017

280

00

00

00

00

00

00

018

832

70

2442

00

00

1023

%Ja

troph

a cu

rcas

L.

Euph

orbi

acea

e0

00

00

330

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

300

370

00

03

7 %

Justi

cia

adha

toda

L.

Aca

ntha

ceae

00

00

00

00

00

00

00

00

00

00

00

00

00

185

00

023

00

00

042

9933

046

00

00

00

49

%La

ctuc

a sa

tiva

L.Co

mpo

sitae

00

00

00

00

011

062

00

00

00

00

00

00

00

00

00

00

00

00

00

3458

3458

00

00

49

%La

wson

ia in

erm

is L.

L yth

race

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

072

01

2 %

Loph

ocer

eus m

argi

natu

s (D

C.) S

.Aria

s & T

erra

zas

Cact

acea

e0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

571

2 %

Loxo

car p

us a

lata

R.B

r.G

esne

riace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

019

00

00

00

00

00

00

00

01

2 %

Luffa

ech

inat

a Ro

xb.

Cucu

rbita

ceae

00

00

00

00

00

00

00

013

20

00

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

Mel

issa

o ffic

inal

is L.

Lam

iace

ae0

00

00

00

00

00

00

00

062

10

7481

00

00

00

00

00

010

00

00

00

00

00

00

03

7 %

Men

tha

arve

nsis

L.La

mia

ceae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

021

00

00

00

12

%M

enth

a ca

nade

nsis

L.La

mia

ceae

00

00

00

00

033

00

00

00

00

00

00

00

00

00

00

00

00

00

00

059

00

00

25

%M

enth

a s p

icat

a L.

Lam

iace

ae0

00

00

00

00

00

00

00

00

00

680

00

00

010

40

00

00

00

00

00

00

00

230

37

%M

icro

berli

nia

braz

zavi

llens

is A

.Che

v.Le

gum

inos

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

140

00

01

2 %

Mom

ordi

ca c

hara

ntia

L.

Cucu

rbita

ceae

00

121

00

530

00

00

00

00

00

950

00

015

00

00

00

020

20

034

30

00

00

00

00

06

14 %

Mur

raya

koe

nigi

i (L

.) Sp

reng

.Ru

tace

ae0

110

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

Mus

a ac

umin

ata

Colla

Mus

acea

e0

00

00

00

00

00

00

00

00

00

00

457

00

00

00

00

00

00

00

00

00

00

00

12

%M

usa

late

rita

Chee

sman

Mus

acea

e0

00

00

500

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

Nard

osta

chys

jata

man

si (D

.Don

) DC.

Capr

ifolia

ceae

00

00

00

00

00

00

00

00

00

00

00

00

016

00

00

00

00

00

00

00

00

00

12

%Ne

lum

bo n

ucife

ra G

aertn

.N

elum

bona

ceae

00

00

00

00

00

00

00

051

70

00

00

00

00

5058

00

00

00

00

00

00

00

00

00

25

%Nu

xia

o ppo

sitifo

lia (H

ochs

t.) B

enth

.St

ilbac

eae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

052

00

00

01

2 %

Nyct

anth

es a

rbor

-trist

is L

.O

leac

eae

00

00

00

00

00

00

00

00

00

00

00

00

00

132

00

00

00

00

00

00

00

00

01

2 %

Oci

mum

bas

ilicu

m L

.La

mia

ceae

00

00

00

00

371

00

00

00

00

00

00

00

00

00

029

130

00

017

012

9247

761

040

010

6374

317

1023

%O

cim

um te

nui fl

orum

L.

Lam

iace

ae0

00

00

00

013

30

00

00

00

00

00

00

00

176

00

059

20

00

00

3429

20

328

020

40

793

00

818

%O

ddon

iode

ndro

n no

rman

dii

Aub

rev.

Legu

min

osae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

137

00

00

00

01

2 %

Oen

othe

ra d

rum

mon

dii

Hoo

k.O

nagr

acea

e0

00

00

00

00

00

00

018

00

00

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

Oen

othe

ra la

cini

ata

Hill

Ona

grac

eae

00

00

00

00

00

00

00

570

00

00

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

Ole

a eu

ropa

ea L

.O

leac

eae

00

00

00

00

00

00

00

00

00

00

00

00

00

045

50

00

00

00

00

00

00

00

01

2 %

Ory

za sa

tiva

L.Po

acea

e0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

179

00

00

12

%Pa

rthen

ium

hys

tero

phor

us L

.Co

mpo

sitae

00

00

049

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

12

%Pe

dicu

laris

flam

mea

L.

Oro

banc

hace

ae0

00

00

00

00

00

00

00

083

00

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

Peril

la se

toye

nsis

G.H

onda

Lam

iace

ae0

00

00

00

142

00

00

00

00

00

00

00

00

710

00

142

00

00

00

230

140

00

870

06

14 %

Phyl

lant

hus e

mbl

ica

L.Ph

ylla

ntha

ceae

00

00

00

00

00

00

00

00

00

00

00

180

00

00

00

00

00

00

00

00

00

00

12

%Ph

ylla

nthu

s mad

eras

pate

nsis

L.Ph

ylla

ntha

ceae

00

00

00

00

00

089

00

00

00

00

00

00

00

00

00

00

00

772

00

00

012

00

03

7 %

Tota

l num

ber

of n

rITS

1 an

d nr

ITS2

rea

ds (R

eplic

ate

1+2+

3)

Her

bal p

rodu

ct ID

Supp

lem

enta

ry T

able

S3.

Det

ails

of to

tal n

umbe

r seq

uenc

ing

read

s per

pro

duct

s.

Phyl

lant

hus t

enel

lus

Roxb

.Ph

ylla

ntha

ceae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

2742

00

00

00

00

01

2 %

Pipe

r lon

gum

L.

Pipe

race

ae0

00

00

00

00

00

00

00

126

00

00

00

00

3879

5156

4289

3117

00

00

00

00

104

00

00

00

06

14 %

Pipe

r nig

rum

L.

Pipe

race

ae0

00

00

00

00

00

032

123

00

00

00

00

00

013

275

1175

140

00

00

00

00

00

00

00

05

11 %

Pista

cia

atla

ntic

a D

esf.

Ana

card

iace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

012

00

00

00

00

00

00

00

00

01

2 %

Pisu

m sa

tivum

L.

Legu

min

osae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

014

12

%Pl

anta

go a

fra L

.Pl

anta

gina

ceae

140

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

12

%Pl

anta

go m

ajor

L.

Plan

tagi

nace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

010

70

00

00

00

00

00

00

00

00

12

%Pl

anta

go o

vata

For

ssk.

Plan

tagi

nace

ae0

012

270

00

010

10

00

00

00

050

00

00

00

00

00

00

00

00

00

00

228

00

00

05

11 %

Pleu

rope

talu

m d

arwi

nii

Hoo

k. f.

Am

aran

thac

eae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

012

00

00

12

%Pl

uken

etia

mad

a gas

carie

nsis

Lean

dri

Euph

orbi

acea

e0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

110

00

00

00

00

01

2 %

Pol y

pogo

n m

onsp

elie

nsis

(L.)

Des

f.Po

acea

e0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

013

20

00

00

00

00

00

00

12

%Ps

eudo

sela

go se

rrat

a (P

.J. B

ergi

us) H

illia

rdSc

roph

ular

iace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

138

00

150

25

%Pu

nica

gra

natu

m L

.Ly

thra

ceae

00

00

051

00

015

910

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

02

5 %

P yru

s cal

lery

ana

Dec

ne.

Rosa

ceae

)0

00

00

00

00

00

00

00

00

00

140

00

00

290

00

00

00

00

00

00

00

00

02

5 %

Raph

anus

sativ

us L

.Br

assic

acea

e0

052

00

00

00

00

00

00

00

00

00

00

00

00

340

00

00

00

00

00

00

00

02

5 %

Rauv

olfia

serp

entin

a (L

.) Be

nth.

ex

Kur

zA

pocy

nace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

840

00

01

2 %

Robi

nia

pseu

doac

acia

L.

Legu

min

osae

00

00

00

00

00

00

00

00

00

053

00

00

00

00

00

00

00

00

00

00

00

00

12

%Ro

sa ru

gosa

Thu

nb.

Rosa

ceae

00

018

00

00

00

00

00

00

00

00

00

00

051

100

00

00

00

00

00

00

00

00

37

%Sa

lvia

ple

beia

R.B

r.La

mia

ceae

00

00

00

00

00

00

00

00

00

240

00

00

00

00

00

00

00

00

543

850

00

00

03

7 %

Salv

ia ro

smar

inus

Sch

leid

.La

mia

ceae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

014

30

00

00

00

00

01

2 %

Sara

ca a

soca

(Rox

b.) W

illd.

Legu

min

osae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

1557

878

50

00

00

02

5 %

Seca

le c

erea

le L

.Po

acea

e0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

082

01

2 %

Sedu

m ja

poni

cum

Sie

bold

ex

Miq

.Cr

assu

lace

ae0

033

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

01

2 %

Senn

a al

exan

drin

a M

ill.

Legu

min

osae

4000

00

00

2968

00

048

870

00

00

157

00

850

00

4484

00

422

00

00

00

066

330

00

500

1570

00

00

1023

%Se

nna

auric

ulat

a (L

.) Ro

xb.

Legu

min

osae

00

00

00

00

00

00

00

00

00

00

00

00

055

90

00

00

00

00

00

00

00

00

01

2 %

Senn

a oc

cide

ntal

is (L

.) Li

nkLe

gum

inos

ae0

00

00

018

395

00

00

00

00

00

00

00

00

00

044

00

00

00

00

00

00

00

00

02

5 %

Senn

a to

ra (

L.) R

oxb.

Legu

min

osae

00

00

00

00

069

60

00

00

00

00

00

00

00

00

00

00

00

00

00

00

162

00

291

03

7 %

Sesa

mum

indi

cum

L.

Peda

liace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

2569

00

00

00

00

00

00

920

00

03

7 %

Sida

acu

ta B

urm

.f.M

alva

ceae

00

00

00

00

00

00

00

00

00

00

00

00

370

117

00

057

890

039

40

00

00

00

1622

20

614

%Si

da c

ordi

folia

L.

Mal

vace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

047

00

00

00

00

150

00

00

25

%Si

lene

ura

lens

is (R

upr.)

Boc

quet

Cary

ophy

llace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

130

00

01

2 %

Sola

num

l yco

pers

icum

L.

Sola

nace

ae0

00

00

00

00

300

018

917

00

154

00

017

10

00

00

00

00

00

120

00

032

00

023

10

00

818

%So

lanu

m n

i gru

m L

.So

lana

ceae

00

00

00

320

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

12

%So

lanu

m v

ir gin

ianu

m L

.So

lana

ceae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

124

00

00

00

00

00

00

01

2 %

Sor g

hum

bic

olor

(L.

) Moe

nch

Poac

eae

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

023

420

00

01

2 %

S pon

dias

tube

rosa

Arr

uda

Ana

card

iace

ae0

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

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m

List

of p

lant

spec

ies

Expe

cted

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tect

ed

Expe

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ot

dete

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Not

exp

ecte

d-de

tect

edTa

blet

1Ta

blet

2Ta

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3C

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Tabl

et 6

Cap

sule

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le 8

Cap

sule

9C

apsu

le 1

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12

Tabl

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3Ta

blet

14

Tabl

et 1

6Ta

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17

Tabl

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19

Tabl

et 2

3C

apsu

le 2

7C

apsu

le 3

0Ta

blet

31

Tabl

et 3

2Ta

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34

Tabl

et 3

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apsu

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0Po

wde

r 52

Pow

der

55Po

wde

r 58

Cap

sule

60

Cap

sule

61

Cap

sule

63

Cap

sule

66

Cap

sule

68

Cap

sule

69

Cap

sule

70

Tabl

et 7

3Ta

blet

74

Tabl

et 7

5Po

wde

r 77

Pow

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79

Abel

mos

chus

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Moe

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00

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00

00

00

00

00

00

00

00

00

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Abut

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t0

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00

00

00

02

00

00

00

00

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00

00

00

00

00

00

00

00

0Ac

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ara

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.) W

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03

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22

00

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00

00

00

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00

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00

00

00

00

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Achi

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ium

L.

01

00

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00

02

00

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00

00

00

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00

00

00

00

00

00

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Ach y

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00

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20

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00

00

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Aegl

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Corr

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20

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00

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00

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02

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00

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00

00

00

00

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00

00

00

00

Alth

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00

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01

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Anet

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para

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35

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02

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12

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02

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10

Azad

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21

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20

00

00

00

00

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00

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01

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01

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Bacc

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30

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22

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02

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00

00

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30

Berb

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01

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20

00

00

00

00

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00

00

00

00

00

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00

00

Ber g

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l.0

11

20

00

00

00

00

00

30

00

00

00

00

00

00

00

00

00

00

00

00

0Bo

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diff

usa

L.

15

02

00

02

00

02

02

20

00

00

00

00

00

00

00

01

00

00

00

00

00

Bosw

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20

00

00

00

00

00

00

00

20

00

00

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20

00

00

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20

00

00

00

00

00

30

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00

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00

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00

00

00

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00

Caes

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duc

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00

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00

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00

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00

02

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Capp

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Case

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20

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Cass

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01

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02

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Cela

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Chae

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Cham

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Chlo

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Cich

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20

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Cinn

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qua

dran

gula

ris L

.1

00

00

00

00

00

00

00

00

00

00

00

00

01

00

00

00

00

00

00

00

0Co

ccin

ia g

rand

is (L

.) V

oigt

00

20

00

00

00

00

00

00

00

00

00

00

00

00

03

00

30

00

00

00

00

Cocc

inia

sess

ilifo

lia (S

ond.

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n.0

01

00

00

00

00

00

00

00

00

03

00

00

00

00

00

00

00

00

00

00

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mm

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01

00

00

00

00

00

00

00

02

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00

00

00

00

00

00

00

00

00

00

00

Conv

olvu

lus

mic

roph

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s Si

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ex

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ng.

00

10

00

00

00

00

00

00

00

00

00

00

30

00

00

00

00

00

00

00

00

Conv

olvu

lus

pros

tratu

s Fo

rssk

.1

03

30

00

00

00

00

00

00

00

00

00

03

00

00

00

00

00

00

31

00

0Co

rcho

rus

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01

00

00

00

00

00

00

00

00

00

00

30

00

00

00

00

00

00

00

00

0Co

riand

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03

00

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00

00

00

00

00

00

00

30

00

00

00

00

00

30

00

00

0Cr

atev

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uch.

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02

02

00

00

00

00

00

00

00

20

00

00

00

00

00

00

00

00

00

00

00

Croc

us sa

tivus

L.

01

00

00

00

00

00

00

00

00

02

00

00

00

00

00

00

00

00

00

00

00

Culle

n c

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ifoliu

m (L

.) M

edik

.0

01

00

30

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0Cu

min

um c

ymin

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06

30

00

30

00

00

00

00

00

03

00

33

00

00

00

30

00

00

00

00

0Cu

rcum

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07

00

20

00

02

00

00

20

00

00

00

22

02

00

00

00

02

00

00

00

00

Cype

rus

scar

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s R.

Br.

01

00

00

00

00

00

00

02

00

00

00

00

00

00

00

00

00

00

00

00

00

Dam

ron g

ia c

yana

ntha

Trib

oun

00

10

00

00

00

00

00

00

00

00

00

00

00

30

00

00

00

00

00

00

00

Did

ymoc

arpu

s ped

icel

lata

(A

it.) A

it. F

.0

10

00

00

00

00

00

00

20

00

00

00

00

00

00

00

00

00

00

00

00

0D

onio

phyt

on w

edde

llii

Kat

inas

& S

tues

sy0

01

00

00

00

00

00

00

00

00

00

00

00

00

00

00

30

00

00

00

00

0D

raba

niv

alis

Lilj

.0

01

00

00

00

00

00

00

00

30

00

00

00

00

00

00

00

00

00

00

00

0D

rym

ocal

lis ru

pestr

is (L

.) So

j k0

01

00

00

00

00

00

00

03

00

00

00

00

00

00

00

00

00

00

00

00

0D

rype

tes

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nsis

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nep.

00

10

00

00

00

00

00

00

00

00

00

00

00

30

00

00

00

00

00

00

00

Dys

chor

iste

sipho

nant

ha (N

ees)

Kun

tze

00

10

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

30

00

00

00

Echi

noch

loa

col

ona

(L.)

Link

00

10

00

00

00

00

00

00

00

00

00

00

00

00

00

00

30

00

00

00

00

Echi

nops

ech

inat

us R

oxb.

01

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

02

00

00

Ecli p

ta p

rostr

ata

(L.)

L.1

21

00

00

20

00

20

00

00

00

00

00

00

00

10

00

30

00

00

00

00

0El

etta

ria c

arda

mom

um (L

.) M

aton

01

00

00

00

00

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Embe

lia ri

bes

Burm

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20

00

00

00

00

02

20

00

00

00

00

00

00

00

00

00

00

00

00

00

0En

icos

tem

ma

litto

rale

Blu

me

01

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Evol

vulu

s al

sinoi

des

(L.)

L.0

11

00

00

00

00

02

00

00

00

00

00

00

00

00

00

00

00

30

00

00

0Fo

enic

ulum

vul

gare

Mill

.0

14

00

30

30

00

00

00

00

00

03

00

00

00

00

00

00

00

30

02

00

0Fr

axin

us a

lbic

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Buck

ley

00

10

00

00

00

00

00

00

00

03

00

00

00

00

00

00

00

00

00

00

00

Fum

aria

offi

cina

lis L

.0

10

00

00

00

00

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

0G

lycy

rrhi

za g

labr

a L

.2

40

00

00

00

00

00

02

02

20

00

01

01

00

00

00

00

02

00

00

00

0G

mel

ina

arb

orea

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b.0

10

00

00

00

00

00

02

00

00

00

00

00

00

00

00

00

00

00

00

00

0G

ossy

pium

her

bace

um L

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10

00

00

00

00

00

02

00

00

00

00

00

00

00

00

00

00

00

00

00

0G

ossy

pium

hirs

utum

L.

00

10

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

03

00

00

Gym

nem

a sy

lves

tre (R

etz.

) R.B

r. ex

Sm

.1

20

02

00

00

00

00

02

00

00

01

00

00

00

00

00

00

00

00

00

00

0H

emid

esm

us in

dicu

s (L

.) R.

Br.

ex S

chul

t.0

10

00

00

00

00

00

00

00

00

00

00

20

00

00

00

00

00

00

00

00

0H

ibisc

us sa

bdar

i ffa

L.

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

03

00

30

00

Hib

iscus

syria

cus

L.0

01

30

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0H

orde

um v

ulga

re L

.1

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

10

00

0H

umul

us lu

pulu

s L.

01

00

00

00

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Hyg

roph

ila a

uric

ulat

a (S

chum

ach.

) Hei

ne1

00

00

00

00

00

00

00

00

00

10

00

00

00

00

00

00

00

00

00

00

0H

yosc

yam

us n

iger

L.

00

10

00

00

00

00

00

00

00

03

00

00

00

00

00

00

00

00

00

00

00

Inul

a ra

cem

osa

Hoo

k.f.

00

10

00

00

00

00

00

00

00

00

00

00

30

00

00

00

00

00

00

00

00

Ipom

oea

dig

itata

L.

01

00

00

00

00

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Ipom

oea

par

asiti

ca (K

unth

) G. D

on0

07

33

30

03

00

00

00

00

00

03

00

00

00

00

00

00

00

30

30

00

0Ir

is g

erm

anic

a L

.0

10

00

00

00

00

00

00

00

00

00

00

02

00

00

00

00

00

00

00

00

0Ju

stici

a a

dhat

oda

L.

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

33

00

00

00

Lava

ndul

a st

oech

as L

.0

10

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

02

00

0La

wson

ia in

erm

is L

.0

01

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

03

0Le

ptad

enia

retic

ulat

a (R

etz.

) Wig

ht &

Arn

.0

10

00

00

02

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0Lo

tus

arab

icus

L.

01

00

00

00

00

00

00

00

00

00

00

00

02

00

00

00

00

00

00

00

00

Mel

issa

offi

cina

lis L

.0

01

00

00

00

00

00

00

00

00

30

00

00

00

00

00

00

00

00

00

00

0M

omor

dica

cha

rant

ia L

.0

21

02

30

00

00

00

02

00

00

00

00

00

00

00

00

00

00

00

00

00

0M

ucun

a p

rurie

ns (L

.) D

C.0

10

00

00

00

00

02

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0M

yrist

ica

frag

rans

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tt.0

10

00

00

00

00

00

00

00

00

00

00

00

20

00

00

00

00

00

00

00

0Na

rdos

tach

ys ja

tam

ansi

(D.D

on) D

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20

00

00

00

00

02

00

00

00

00

00

00

00

00

00

00

00

00

02

00

0Ne

lum

bo n

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ra G

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01

00

00

00

00

00

00

00

00

00

00

30

00

00

00

00

00

00

00

00

0Ny

ctan

thes

arb

or-tr

istis

L.

00

10

00

00

00

00

00

00

00

00

00

00

30

00

00

00

00

00

00

00

00

Oci

mum

bas

ilicu

m L

.0

05

00

00

00

03

00

00

00

00

00

00

00

03

00

00

00

33

00

00

00

3O

cim

um g

ratis

simum

L.

01

00

00

00

00

00

00

00

00

00

00

00

00

20

00

00

00

00

00

00

00

Oci

mum

tenu

i flor

um L

.3

13

00

00

00

00

00

02

00

00

00

01

00

03

00

00

00

10

30

30

10

0O

lea

eur

opae

a L

.0

01

00

00

00

00

00

00

00

00

00

00

00

30

00

00

00

00

00

00

00

0O

nosm

a br

acte

atum

Wal

l0

20

00

00

00

00

00

00

20

00

00

00

00

00

00

00

00

00

00

02

00

0O

roxy

lum

indi

cum

(L.)

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z i

01

00

00

00

00

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Ory

za sa

tiva

L.

10

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

01

00

00

Peril

la se

toye

nsis

G.H

onda

00

40

00

00

03

00

00

00

00

00

00

30

00

30

00

00

03

00

00

00

00

Phyl

lant

hus

amar

us S

chum

ach.

& T

honn

.0

20

00

00

00

00

00

22

00

00

00

00

00

00

00

00

00

00

00

00

00

0Ph

ylla

nthu

s em

blic

a L

.0

80

00

00

00

00

02

22

00

00

00

22

00

22

00

00

02

00

00

00

00

0Ph

ylla

nthu

s m

ader

aspa

tens

is L

.0

01

00

00

00

00

00

00

00

00

00

00

00

00

00

00

03

00

00

00

00

0Ph

ylla

nthu

s niru

ri L.

03

00

00

02

00

02

00

00

00

00

00

00

00

00

00

00

20

00

00

00

00

Phyl

lant

hus

tene

llus

Roxb

.0

01

00

00

00

00

00

00

00

00

00

00

00

00

00

00

03

00

00

00

00

0Ph

ylla

nthu

s ur

inar

ia L

.0

10

20

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0Pi

cror

hiza

kur

roa

Roy

le e

x Be

nth.

03

00

00

02

00

02

00

00

00

00

00

00

00

00

00

00

00

00

02

00

00

Pipe

r cu

beba

Boj

er0

10

20

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0Pi

per

long

um L

.3

32

02

00

00

00

20

02

00

00

00

01

31

10

00

00

00

03

00

00

00

0Pi

per

nigr

um L

.3

01

00

00

00

00

00

01

00

00

00

01

31

00

00

00

00

00

00

00

00

0Pl

anta

go m

ajor

L.

00

10

00

00

00

00

00

00

00

00

00

00

00

30

00

00

00

00

00

00

00

Plan

tago

ova

ta F

orss

k.0

01

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

03

00

00

0Pl

umba

go ze

ylan

ica

L.

01

00

00

00

00

00

02

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Prem

na m

ollis

sima

Rot

h0

10

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

20

00

0Pr

unus

am

ygda

lus

Stok

es0

20

00

00

00

00

02

00

00

00

20

00

00

00

00

00

00

00

00

00

00

0Pt

eroc

arpu

s m

arsu

pium

Rox

b.0

20

02

00

00

00

00

02

00

00

00

00

00

00

00

00

00

00

00

00

00

0Pt

eroc

arpu

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ntal

inus

L.f.

01

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

02

00

00

00

00

Puer

aria

tube

rosa

(Will

d.) D

C.0

20

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

20

00

0Pu

nica

gra

natu

m L

.0

01

00

00

00

00

30

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0Ra

phan

us sa

tivus

L0

12

00

30

00

00

00

20

00

00

00

00

00

30

00

00

00

00

00

00

00

0Rh

eum

em

odi

L.0

10

00

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0Ro

sa ru

gosa

Thu

nb.

00

10

00

00

00

00

00

00

00

00

00

03

00

00

00

00

00

00

00

00

00

Rubi

a c

ordi

folia

L.

02

00

00

00

00

00

00

02

00

00

00

00

00

00

00

00

02

00

00

00

00

Rum

ex m

ariti

mus

L.

01

00

00

00

00

00

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

Sacc

haru

m b

enga

lens

e Re

tz.

01

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

02

00

00

Sacc

haru

m o

ffici

naru

m L

.0

10

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

20

00

0Sa

lvia

ple

beia

R.B

r.0

03

00

00

00

00

00

00

00

00

30

00

00

00

00

00

00

03

30

00

00

0Sa

ntal

um a

lbum

L.

01

00

00

00

00

00

00

00

00

00

00

02

00

00

00

00

00

00

00

00

00

Sara

ca a

soca

(Rox

b.) W

illd.

11

10

00

00

00

00

02

00

00

00

00

00

00

00

00

00

00

31

00

00

00

Saxi

fraga

gra

nula

ta L

.0

10

00

00

00

00

00

00

00

00

00

00

20

00

00

00

00

00

00

00

00

0Sa

xifra

ga li

gula

ta M

urra

y0

10

00

00

00

00

00

00

20

00

00

00

00

00

00

00

00

00

00

00

00

0Se

cale

cer

eale

L.

00

10

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

30

Sedu

m ja

poni

cum

Sie

bold

ex

Miq

.0

01

00

30

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0Se

nna

ale

xand

rina

Mill

.0

06

30

00

30

00

30

00

00

00

03

00

30

00

00

00

00

00

00

30

00

0Se

nna

aur

icul

ata

(L.)

Roxb

.0

01

00

00

00

00

00

00

00

00

00

00

30

00

00

00

00

00

00

00

00

0Se

nna

occ

iden

talis

L.

01

10

00

00

30

00

02

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Senn

a to

ra (L

.) Ro

xb.

00

20

00

00

00

03

00

00

00

00

00

00

00

00

00

00

00

00

00

00

30

Sesa

mum

indi

cum

L.

00

10

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

03

00

00

Sida

acu

ta B

urm

.f.0

03

00

00

00

00

00

00

00

00

00

03

03

00

03

00

00

00

00

00

00

0Si

da c

ordi

folia

L.

21

00

00

00

00

00

00

00

20

00

00

00

00

00

10

00

00

00

01

00

00

Supp

lem

enta

ry T

able

S5.

Det

ails

of su

m o

ccur

renc

es o

f a sp

ecie

s in

Ayu

rved

ic h

erba

l pro

duct

s use

d to

gen

erat

e he

atm

ap.

Sola

num

indi

cum

L.

01

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

02

00

00

Sola

num

l yco

pers

icum

L.

00

30

00

00

00

00

00

30

00

00

00

00

00

00

00

30

00

00

00

30

00

Sola

num

ni g

rum

L.

01

00

00

00

00

00

02

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Sola

num

sura

ttens

e Bu

rm. f

.0

10

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

20

00

0So

r ghu

m b

icol

or (L

.) M

oenc

h0

01

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

30

00

0S p

haer

anth

us in

dicu

s L.

02

00

00

00

00

00

00

20

00

00

00

02

00

00

00

00

00

00

00

00

00

Swer

tia c

hira

ta B

uch.

-Ham

. ex

Wal

l.0

20

00

00

00

00

00

02

00

00

00

00

00

20

00

00

00

00

00

00

00

0S y

zygi

um c

umin

i (L

.) Sk

eels

02

00

20

00

00

00

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

Tam

arix

gal

lica

L0

10

00

00

00

00

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

0Ta

raxa

cum

offi

cina

le (L

.) W

eber

ex

F.H

.Wig

g.0

01

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

30

00

00

0Te

phro

sia p

urpu

rea

(L.)

Pers

.0

10

00

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

0Te

rmin

alia

ar ju

na (R

oxb.

ex

DC.

) Wig

ht &

Arn

.1

50

00

00

00

00

02

20

00

00

00

00

01

02

00

02

00

00

00

00

00

2Te

rmin

alia

bel

liric

a (G

aertn

.) Ro

xb.

22

00

00

00

00

00

00

20

00

00

01

10

02

00

00

00

00

00

00

00

00

Term

inal

ia c

hebu

la R

etz.

07

00

00

00

00

00

22

20

20

00

02

20

02

00

00

00

00

00

00

00

00

Thys

anol

aena

latif

olia

(Rox

b. e

x H

orne

m.)

Hon

da0

01

00

00

00

00

00

00

00

03

00

00

00

00

00

00

00

00

00

00

00

0Ti

nos p

ora

cor

difo

lia (W

illd.

) Mie

rs

37

00

20

00

00

00

22

20

02

00

00

00

00

00

01

00

11

00

02

02

00

Trac

hysp

erm

um a

mm

i (L

.) Sp

ragu

e1

10

00

00

00

00

00

00

00

00

00

00

20

10

00

00

00

00

00

00

00

0Tr

iant

hem

a p

ortu

laca

strum

L.

00

30

00

00

00

00

00

00

00

00

00

00

00

00

30

03

00

03

00

00

00

Trib

ulus

terr

estri

s L.

04

60

00

00

00

03

00

23

02

22

00

03

33

00

30

00

00

00

00

00

00

Trig

astro

thec

a m

ollu

gine

a F

.Mue

ll.0

01

00

00

00

00

00

00

00

00

00

00

00

00

00

00

03

00

00

00

00

0Tr

igon

ella

foen

um-g

raec

um L

.2

11

01

30

02

00

00

00

00

00

00

00

00

10

00

00

00

00

00

00

00

0Tr

iticu

m a

estiv

um L

.0

01

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

03

0Va

leria

na o

ffici

nalis

L.

01

00

00

00

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Vale

riana

wal

lichi

i D

C.

01

00

00

00

00

00

20

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Vern

onia

cin

erea

(L.)

Less

.0

10

00

00

00

00

00

00

20

00

00

00

00

00

00

00

00

00

00

00

00

0Vi

gna

radi

ata

(L.)

R.W

ilcze

k0

01

00

00

00

00

00

00

00

00

00

00

03

00

00

00

00

00

00

00

00

0W

ithan

ia so

mni

fera

(L.)

Dun

al4

16

00

11

31

00

32

00

00

30

30

00

30

00

00

00

00

00

00

31

00

0Zi

ngib

er o

ffici

nale

Ros

coe

33

00

00

00

00

00

10

22

00

00

00

20

11

00

00

00

00

00

00

00

00

Tota

l (pr

esen

ce o

f MO

TU p

er c

ateg

ory)

2610

781

Tota

l (su

m o

f occ

uren

ces o

f MO

TU p

er c

ateg

ory)

4718

414

4

Supp

lem

enta

ry T

able

S6.

Det

ails

of g

enom

ic D

NA

con

cent

ratio

n, n

rITS

am

plic

on c

once

ntra

tion

and

succ

ess r

ate

of se

quen

ce y

ield

in h

erba

l pro

duct

s afte

r filt

erin

g th

e ra

w se

quen

ces b

etw

een

nrIT

S1 a

nd n

rITS

2, a

nd a

mon

g re

plic

ates

, and

num

ber o

f MO

TUs i

n nr

ITS1

and

nrI

TS a

fter N

CBI-

BLA

ST id

entif

icat

ion

nrIT

S1 a

mpl

icon

co

ncen

trat

ion

of

repl

icat

e 1

(ng/µl

)

nrIT

S1 a

mpl

icon

co

ncen

trat

ion

of

repl

icat

e 2

(ng/µl

)

nrIT

S1 a

mpl

icon

co

ncen

trat

ion

of r

eplic

ate

3 (n

g/µl

)

nrIT

S2 a

mpl

icon

co

ncen

trat

ion

of

repl

icat

e 1

(ng/µl

)

nrIT

S2 a

mpl

icon

co

ncen

trat

ion

of

repl

icat

e 2

(ng/µl

)

nrIT

S2 a

mpl

icon

co

ncen

trat

ion

of r

eplic

ate

3 (n

g/µl

)

No.

of r

eads

in

repl

icat

e1

(MO

TU

’s)

No.

of r

eads

in

repl

icat

e2 (M

OT

U’s

)

No.

of r

eads

in

repl

icat

e3

(MO

TU

’s)

No.

of r

eads

in

repl

icat

e1 (M

OT

U’s

)

No.

of r

eads

in

repl

icat

e2

(MO

TU

’s)

No.

of r

eads

in

repl

icat

e3

(MO

TU

’s)

No.

of M

OT

U's

in

rep

licat

e1

(MO

TU

's)

No.

of M

OT

U's

in

rep

licat

e2

(MO

TU

's)

No.

of M

OT

U's

in

rep

licat

e3

(MO

TU

's)

No.

of M

OT

U's

in

rep

licat

e1

(MO

TU

's)

No.

of M

OT

U's

in

rep

licat

e2

(MO

TU

's)

No.

of M

OT

U's

in

repl

icat

e3 (M

OT

U's

)

1T

able

ts2,

234,

16,

940,

659,

833,

2332

,53

012

3732

6231

260

3265

05

64

07

2T

able

ts3,

36,

15,

660,

856,

0210

,52

27,6

80

1198

9656

452

979

3619

04

21

11

3T

able

ts1,

240

10,7

963

,45

5,56

15,7

742

,95

062

910

330

2470

148

010

110

33

4T

able

ts2,

752,

970,

514,

24,

274,

9614

,41

00

00

352

180

00

03

15

Cap

sule

s0,

852,

150,

275,

923,

840,

0118

,68

00

00

1820

00

00

11

6T

able

ts1,

555,

131,

692,

453,

463,

9610

,115

0325

3313

700

680

1980

25

20

715

7C

apsu

les

24,6

5,35

4,39

3,3

3,63

8,55

5,87

2076

1997

1547

6291

750

7680

44

32

77

8C

apsu

les

0,7

4,66

5,04

6,11

1,35

00,

0112

319

00

00

11

00

00

9C

apsu

les

0,88

0,02

0,74

4,53

4,29

5,38

15,5

731

862

1001

719

070

281

914

11

10

27

10C

apsu

les

0,87

3,44

0,3

12,6

34,

684,

464,

5912

9715

4024

523

140

1630

7793

14

21

813

11C

apsu

les

0,82

00

00,

050

00

00

00

00

00

00

012

Tab

lets

1,95

1,7

0,21

52,1

81,

82,

564,

610

00

014

560

00

01

313

Tab

lets

1,9

1,42

0,4

6,15

9,87

4,06

7,76

014

9978

10

319

821

01

30

15

14T

able

ts2,

541,

990,

891,

686,

817,

2629

,03

1446

164

1434

720

00

04

32

00

015

Cap

sule

s1,

90,

860,

363,

094,

024,

010,

010

00

035

730

00

02

216

Tab

lets

1,27

4,84

3,18

1,16

3,87

0,52

0,69

1431

999

8939

820

00

22

60

00

17T

able

ts2,

451,

851,

1720

,55

2,66

3,36

11,1

682

7081

991

00

198

301

41

30

34

18T

able

ts6,

098

3,78

0,58

3,34

3,1

8,97

589

6054

1336

012

8467

82

32

04

219

Tab

lets

1,3

0,22

026

,16

2,16

6,87

4,52

347

00

031

0258

153

00

01

320

Tab

lets

0,84

00

00,

010

00

00

00

00

00

00

021

Tab

lets

1,89

5,5

1,81

0,17

15,1

93,

7821

,29

00

00

00

00

00

00

22T

able

ts1,

20,

010

03,

740,

651,

290

00

00

00

00

00

023

Tab

lets

3,04

3,07

2,84

3,03

9,45

4,05

15,7

658

6219

3410

20

9167

93

232

04

624

Ext

ract

s0,

173,

781,

61,

2715

,01

4,57

3,38

530

00

1491

00

00

12

25E

xtra

cts

0,13

3,27

1,46

4,16

34,6

78,

5918

,33

500

171

00

361

01

00

126

Ext

ract

s1,

13,

094,

051,

6711

,17

12,3

241

,32

088

921

420

093

01

20

01

27C

apsu

les

14,6

5,02

4,49

0,46

8,18

11,3

216

,75

4744

1934

1112

713

1065

234

2811

410

35

628

Cap

sule

s0,

166,

056,

260,

5630

,22

028

,14

00

00

00

00

00

00

29C

apsu

les

4,88

00

7,29

5,92

1,36

7,1

00

00

00

00

00

00

30C

apsu

les

0,27

3,85

0,33

22,

430,

8327

,87

2850

6146

1041

80

00

12

10

00

31T

able

ts4,

566,

790,

94,

041,

161,

332,

1568

923

5434

8515

20

247

57

10

132

Tab

lets

3,12

36,5

58,

81,

999,

284,

7947

,437

4429

5037

8132

4721

6636

024

54

315

1133

Tab

lets

5,4

7,8

8,23

0,36

52,2

17,1

475

,42

00

00

00

00

00

00

34T

able

ts18

,68,

4811

,15

1,43

28,6

23,

7312

,14

5764

1437

467

2455

4355

2169

108

46

814

35T

able

ts10

,74,

592,

650,

012,

3711

,62

38,1

00

00

00

00

00

00

36T

able

ts23

,37,

6710

,53

0,74

10,0

52,

566,

6548

2125

6810

6313

630

2970

76

51

11

37T

able

ts5,

196,

233,

783,

724,

943,

990

00

00

00

00

00

00

38T

able

ts3,

130

00

00

00

00

00

00

00

00

039

Tab

lets

6,07

3,59

3,07

0,07

11,4

913

,02

33,5

80

00

00

00

00

00

040

Cap

sule

s5,

332,

71,

371,

594,

594,

022,

419

321

6130

0796

711

053

22

46

941

Cap

sule

s15

,60,

031,

217,

371,

860,

420

00

00

00

00

00

00

42C

apsu

les

43,6

00

00,

140,

110,

90

00

00

00

00

00

043

Cap

sule

s0,

220

0,02

019

,62

4,11

16,0

50

00

00

00

00

00

044

Cap

sule

s31

,27,

5628

,99

1,64

8,04

22,8

953

,43

00

00

00

00

00

00

45C

apsu

les

67,

424,

370,

9631

,28

4,86

00

00

00

00

00

00

046

Cap

sule

s13

,90

00

00

00

00

00

00

00

00

047

Cap

sule

s31

,814

,68

4,47

0,51

28,8

47,

760

00

00

00

00

00

00

48Po

wde

rs19

,40

00

00

00

00

00

00

00

00

049

Pow

ders

19,4

00

12,7

21,

060,

330,

910

00

00

00

00

00

050

Pow

ders

4,83

00

02,

71,

280

00

00

00

00

00

00

51Po

wde

rs19

,229

,98

0,01

0,61

24,4

10

54,4

20

00

00

00

00

00

052

Pow

ders

42,5

7,92

0,59

2,95

3,52

3,28

9,7

539

012

1389

6616

7829

91

01

11

253

Pow

ders

190

7,27

1,96

1,11

6,59

1,93

5,26

00

00

00

00

00

00

54Po

wde

rs21

82,9

824

,36

0,2

39,0

845

,50

00

00

00

00

00

00

55Po

wde

rs21

014

,05

0,48

1,34

1,9

1,58

3,55

4115

1814

366

1994

1517

86

43

51

456

Pow

ders

3,31

32,6

12,

096,

760,

264,

5249

,64

00

00

00

00

00

00

57Po

wde

rs50

5,36

5,62

0,78

0,13

1,35

0,09

00

00

00

00

00

00

58Po

wde

rs22

,328

,88

0,48

0,35

0,14

0,32

0,06

423

4718

012

842

118

12

12

12

359

Cap

sule

s0,

144,

432,

951,

120,

150,

330,

140

00

00

00

00

00

060

Cap

sule

s0,

071,

4650

,22

48,4

50,

491,

640,

2413

440

00

046

151

10

00

12

61C

apsu

les

5,38

0,89

20,4

716

,42

2,07

3,2

0,83

3915

212

493

2892

510

288

511

157

86

12

562

Pow

ders

0,05

3,67

12,3

14,

60,

690,

10

00

00

00

00

00

00

63C

apsu

les

0,17

5,48

0,98

0,12

0,33

0,55

Not

App

licab

le11

1730

244

038

3547

41

20

67

64C

apsu

les

24,5

56,2

71,

580,

590,

352,

71,

80

00

00

00

00

00

065

Cap

sule

s11

,86,

356,

172,

692,

17,

422,

170

00

00

00

00

00

066

Cap

sule

s15

,60,

6515

,07

12,2

71,

367,

521,

3342

3262

119

812

809

22

21

34

67C

apsu

les

22,4

0,09

14,0

85,

472,

6214

,130

,70

00

00

00

00

00

068

Cap

sule

s3,

1920

,55

1,51

3,81

0,15

1,93

0,18

1157

6181

1306

626

367

015

494

54

110

769

Cap

sule

s3,

3510

,12

1,9

1,12

0,57

6,38

4,21

1681

5117

6291

9577

8674

1573

76

1215

43

570

Cap

sule

s3,

916

,12

1,38

3,29

0,52

1,57

0,36

2022

4082

373

348

778

013

581

43

40

371

Tab

lets

6,85

12,6

37,

882,

580,

161,

470,

130

00

00

00

00

00

072

Tab

lets

10,8

2,49

15,5

423

,59

1,44

2,4

2,03

00

00

00

00

00

00

73T

able

ts7,

392,

811,

513,

161,

142,

476,

7328

5930

3073

348

6511

4424

1241

1214

38

1718

74T

able

ts17

,53,

632,

881,

61,

193,

570,

1646

205

3320

511

9250

4842

682

15

14

675

Tab

lets

16,9

0,68

13,0

23,

491,

713,

121,

050

017

456

973

865

50

05

12

376

Pow

ders

547,

850,

881,

1168

,22

1,97

0,95

00

00

00

00

00

00

77Po

wde

rs6,

368

,41

0,52

0,54

0,45

0,49

Not

App

licab

le12

8327

9626

759

716

4682

92

33

15

878

Pow

ders

5312

,42

0,59

0,39

0,13

0,4

0,3

00

00

00

00

00

00

79Po

wde

rs0,

055,

684,

984,

710,

451

0,32

6010

6814

014

827

32

31

03

1N

egat

ive

Con

trol

10

00

00

00

00

00

00

00

00

00

Neg

ativ

e C

ontr

ol 2

00

00

00

00

00

00

00

00

00

0

Neg

ativ

e C

ontr

ol 3

00

00

00

00

00

00

00

00

00

0

Not

es. *

Val

ues

that

cor

resp

ond

to ‘0

’ in

ampl

icon

con

cent

ratio

n re

pres

ents

that

the

qPC

R m

easu

red

as ‘0

’ whi

ch im

plie

s th

at it

has

no

ampl

ifica

tion.

In s

uch

case

s, th

e co

mpl

ete

volu

me

from

PC

R w

ell w

ere

take

n fo

r nor

mal

izat

ion

and

sequ

enci

ng. T

he s

ampl

e re

pres

ente

d as

‘Not

App

licab

le’ i

s th

at q

PCR

resu

lts h

ave

not g

iven

any

val

ues

for t

hose

sam

ples

, in

such

cas

es G

el Im

ages

wer

e co

mpa

red

and

the

sam

ples

wer

e no

rmal

ized

bas

ed o

n th

e in

tens

ity o

f the

DN

A b

and

in th

e G

el.

nrIT

S2 -

Sequ

ence

yie

ld in

trip

licat

es a

fter

filte

ring

nrIT

S1 -

No.

of M

OT

U's

in tr

iplic

ates

afte

NC

BI-

BL

AST

iden

tific

atio

nnr

ITS2

- N

o. o

f MO

TU

's in

trip

licat

es a

fte N

CB

I-B

LA

ST

iden

tific

atio

nH

erba

l pr

oduc

ts c

ode

Her

bal

prod

uct

type

Tot

al g

enom

ic D

NA

co

ncen

trat

ion

m

easu

red

usin

g Q

ubit

2.0

Fluo

rom

eter

(ng/µl

)

nrIT

S1 -

Am

plic

on c

once

ntra

tion

of tr

iplic

ates

usi

ng q

PCR

* nr

ITS2

- A

mpl

icon

con

cent

ratio

n of

trip

licat

es u

sing

qPC

R*

nrIT

S1 -

Sequ

ence

yie

ld in

trip

licat

es a

fter

filte

ring

nrIT

S1nr

ITS2

Rep

licat

e 1

Rep

licat

e 2

Rep

licat

e 3

Rep

licat

e 1

Rep

licat

e 2

Rep

licat

e 3

Rep

licat

e 1

Rep

licat

e 2

Rep

licat

e 3

Rep

licat

e 1

Rep

licat

e 2

Rep

licat

e 3

Det

ectio

n in

re

plic

ates

1+

2+3

Det

ectio

n in

re

plic

ates

1+2

+3

Acac

ia a

rabi

ca (L

am.)

Will

d. (=

Aca

cia

nilo

tica

(L.)

Del

ile)

Yes

00

00

00

No

Achy

rant

hes

aspe

ra L

.Y

es0

00

00

0N

oBe

rgen

ia li

gula

ta E

ngl.

Yes

00

00

00

No

Boer

haav

ia d

iffus

a L

.Y

es0

00

00

0N

oCr

atev

a n

urva

la B

uch.

-Ham

Gen

us is

repr

esen

ted

00

00

00

No

Phyl

lant

hus

urin

aria

L.

Yes

00

00

00

No

Pipe

r cu

beba

Boj

erY

es0

00

00

0N

oCa

ssia

sp.

0

00

00

31

Cham

aecr

ista

ole

siphy

lla (V

ogel

) H.S

.Irw

in &

Bar

neby

00

011

70

131

Cham

aecr

ista

ole

siphy

lla (V

ogel

) H.S

.Irw

in &

Bar

neby

011

7+0+

131

Conv

olvu

lus

pros

tratu

s Fo

rssk

.0

2044

00

0Co

nvol

vulu

s pr

ostra

tus

Fors

sk.

0+20

+44

0Co

riand

rum

sativ

um L

.0

190

00

58Cu

min

um c

ymin

um L

.0

00

335

065

Cum

inum

cym

inum

L.

033

5+0+

65Fo

enic

ulum

vul

gare

Mill

.0

015

00

0H

ibisc

us s y

riacu

s L.

065

110

00

Hib

iscus

syria

cus

L.0+

65+1

10

Ipom

oea

par

asiti

ca (K

unth

) G. D

on0

707

2972

963

014

46Ip

omoe

a p

aras

itica

(Kun

th) G

. Don

0+70

7+29

7296

3+0+

1446

Plan

tago

afra

L.

00

140

00

Senn

a a

lexa

ndrin

a M

ill.

042

620

617

110

1357

Senn

a a

lexa

ndrin

a M

ill.

0+42

6+20

617

11+0

+135

7W

ithan

ia so

mni

fera

(L.)

Dun

al0

00

00

177

Azad

irach

ta in

dica

A.Ju

ss.

Yes

00

00

00

No

Acac

ia a

rabi

ca (L

am.)

Will

d. (=

Aca

cia

nilo

tica

(L.)

Del

ile)

Yes

00

00

00

No

Curc

uma

long

a L

.Y

es0

00

00

0N

o

Enic

oste

mm

a lit

tora

le B

lum

eN

o (o

ther

gen

us a

re re

pres

ente

d)0

00

00

0N

o

Gym

nem

a sy

lves

tre (R

etz.

) R.B

r. ex

Sm

.Y

es0

00

00

0N

oM

omor

dica

cha

rant

ia L

.Y

es0

00

00

0N

oPi

per

long

um L

.Y

es0

00

00

0N

oPt

eroc

arpu

s m

arsu

pium

Rox

b.IT

S2 a

nd G

enus

is re

pres

ente

d0

00

00

0N

oSy

zygi

um c

umin

i (L

.) Sk

eels

Yes

00

00

00

No

Tino

s por

a c

ordi

folia

(Will

d.) M

iers

Y

es0

00

00

0N

oTr

i gon

ella

foen

um-g

raec

um L

.Y

es0

168

940

00

0Tr

igon

ella

foen

um-g

raec

um L

.0+

168+

940

0Y

esIp

omoe

a n

il (L

.) Ro

th0

390

00

0I p

omoe

a p

aras

itica

(Kun

th) G

. Don

0

980

8716

452

979

3619

Ipom

oea

par

asiti

ca (K

unth

) G. D

on

0+98

0+87

1645

2+97

9+36

19M

urra

ya k

oeni

gii

(L.)

Spre

ng.

011

00

00

Acac

ia a

rabi

ca (L

am.)

Will

d. (=

Aca

cia

nilo

tica

(L.)

Del

ile)

Yes

00

00

00

No

Aspa

ragu

s ra

cem

osus

Will

d.Y

es0

00

00

0N

oCh

loro

phyt

um a

rund

inac

eum

Bak

erY

es0

00

00

0N

oW

ithan

ia so

mni

fera

(L.)

Dun

alY

es0

00

022

9424

With

ania

som

nife

ra (L

.) D

unal

00+

2294

+24

Yes

Azad

irach

ta in

dica

A.Ju

ss.

051

00

00

Bras

sica

nig

ra (L

.) K

.Koc

h0

017

00

0Ci

chor

ium

int y

bus

L.0

188

157

015

691

Cich

oriu

m in

tybu

s L.

0+18

8+15

70+

156+

91Cu

llen

cor

ylifo

lium

(L.)

Med

ik.

023

110

00

Culle

n c

oryl

ifoliu

m (L

.) M

edik

.0+

23+1

10

Foen

icul

um v

ulga

re M

ill.

014

430

00

Foen

icul

um v

ulga

re M

ill.

0+14

+43

0H

ygro

phila

schu

lli M

.R.A

lmei

da &

S.M

.Alm

eida

00

820

00

Ipom

oea

par

asiti

ca (K

unth

) G. D

on0

183

214

020

33Ip

omoe

a p

aras

itica

(Kun

th) G

. Don

0+18

3+21

40+

20+3

3M

omor

dica

cha

rant

ia L

.0

5071

00

0M

omor

dica

cha

rant

ia L

.0+

50+7

10

Plan

tago

ova

ta F

orss

k.0

120

00

0Ra

phan

us sa

tivus

L0

3319

00

0Ra

phan

us sa

tivus

L0+

33+1

90

Sedu

m ja

poni

cum

Sie

bold

ex

Miq

.0

1023

00

0Se

dum

japo

nicu

m S

iebo

ld e

x M

iq.

0+10

+23

0Tr

igon

ella

foen

um-g

raec

um L

.0

6538

60

00

Trig

onel

la fo

enum

-gra

ecum

L.

0+65

+386

0Ze

a m

ays L

.0

010

00

05

Caps

ules

1W

ithan

ia so

mni

fera

(L.)

Dun

alY

es0

00

018

240

00

00

1W

ithan

ia so

mni

fera

(L.)

Dun

al0

0+18

+24

Yes

11

100

%Al

oe b

arba

dens

is M

ill. (

= Al

oe v

era

(L.)

Burm

.f.)*

Yes

00

00

00

No

Boer

haav

ia d

iffus

a L

.*Y

es0

00

00

0N

oCa

ppar

is sp

inos

a L

.*Y

es0

00

00

0N

oCi

chor

ium

inty

bus

L.*

Yes

00

00

199

620

Cich

oriu

m in

tybu

s L.

0

0+19

9+62

0Y

esEc

lipta

alb

a (L

.) H

assk

. (=

Ecl

ipta

pro

strat

a (L

.) L.

)Y

es0

00

00

0N

oPh

ylla

nthu

s niru

ri L.

Yes

00

00

00

No

Picr

orhi

za k

urro

a R

oyle

ex

Bent

h.*

Yes

00

00

00

No

Rheu

m e

mod

i L.

*Y

es0

00

00

0N

oTe

phro

sia p

urpu

rea

(L.)

Pers

.*Y

es0

00

00

0N

oCi

nnam

omum

iner

s Re

inw

. ex

Blum

e0

00

00

59Co

riand

rum

sativ

um L

.0

00

029

58Co

riand

rum

sativ

um L

.0

0+29

+58

Cum

inum

cym

inum

L.

00

00

1841

Cum

inum

cym

inum

L.

00+

18+4

1Ec

hium

vul

gare

L.

00

00

025

Embe

lia ts

jeria

m-c

otta

m (R

oem

. & S

chul

t.) A

.DC.

037

00

00

Foen

icul

um v

ulga

re M

ill.

022

6538

00

00

Foen

icul

um v

ulga

re M

ill.

0+22

65+3

800

Ipom

oea

par

asiti

ca (K

unth

) G. D

on0

00

00

43Ja

troph

a c

urca

s L.

00

00

033

Mom

ordi

ca c

hara

ntia

L.

00

00

053

Mus

a la

terit

a C

hees

man

500

00

00

Parth

eniu

m h

yste

roph

orus

L.

00

00

049

Puni

ca g

rana

tum

L.

00

00

051

Senn

a a

lexa

ndrin

a M

ill.

1453

9499

00

184

247

Senn

a a

lexa

ndrin

a M

ill.

1453

+94+

990

0+18

4+24

7Tr

ach y

sper

mum

am

mi

(L.)

Spra

gue

012

50

00

50Tr

ibul

us te

rres

tris

L.0

00

015

173

Trib

ulus

terr

estri

s L.

00+

15+1

73Tr

igon

ella

foen

um-g

raec

um L

.0

120

00

0Tr

iticu

m a

estiv

um L

. 0

00

010

0W

ithan

ia so

mni

fera

(L.)

Dun

al0

00

022

542

2W

ithan

ia so

mni

fera

(L.)

Dun

al0

0+22

5+42

2Ze

a m

ays

L.0

00

00

56Al

lium

sativ

um L

.Y

es0

00

00

0N

oAs

para

gus

race

mos

us W

illd.

Yes

00

00

1310

Aspa

ragu

s ra

cem

osus

Will

d.0

0+13

+10

Yes

Lept

aden

ia re

ticul

ata

(Ret

z.) W

ight

& A

rn.

Yes

00

00

00

No

Puer

aria

tube

rosa

(Will

d.) D

C.Y

es0

00

00

0N

oTr

igon

ella

foen

um-g

raec

um L

.Y

es0

00

00

0N

oW

ithan

ia so

mni

fera

(L.)

Dun

alY

es0

014

050

261

With

ania

som

nife

ra (L

.) D

unal

00+

50+2

61Y

esCa

lluna

vul

garis

(L.)

Hul

l91

128

284

00

0Ca

lluna

vul

garis

(L.)

Hul

l91

+128

+284

0Ci

chor

ium

int y

bus

L.0

00

00

37Co

ccin

ia se

ssili

folia

(Son

d.) C

ogn.

011

00

00

I pom

oea

par

asiti

ca (K

unth

) G. D

on

386

350

021

90

14Ip

omoe

a p

aras

itica

(Kun

th) G

. Don

38

6+35

0+0

219+

0+14

Pluk

enet

ia lo

rete

nsis

Ule

340

00

00

Senn

a o

ccid

enta

lis (L

.) Li

nk15

6515

0812

4960

7268

773

14Se

nna

occ

iden

talis

(L.)

Link

1565

+150

8+12

4960

72+6

87+7

314

Sola

num

ni g

rum

L.

00

00

032

Trac

hysp

erm

um a

mm

i (L

.) Sp

ragu

e0

00

00

12Al

bizia

lebb

eck

(L.)

Bent

h.G

enus

is re

pres

ente

d0

00

00

0N

oCu

rcum

a lo

nga

L.

Yes

00

00

00

No

Peril

la se

toye

nsis

G.H

onda

123

190

00

0Pe

rilla

seto

yens

is G

.Hon

da12

3+19

+00

Hum

ulus

lupu

lus

L.Y

es0

00

00

0N

oVa

leria

na o

ffici

nalis

L.

Yes

00

00

00

No

Cann

abis

sativ

a L

.31

862

1001

719

070

00

Cann

abis

sativ

a L

.18

62+1

0017

+190

0Ca

ssia

sp.

00

00

018

Cich

oriu

m in

tybu

s L.

00

00

7440

2Ci

chor

ium

inty

bus

L.0

0+74

+402

Cucu

mis

sativ

us L

.0

00

00

13Ec

hina

cea

pur

pure

a (L

.) M

oenc

h0

00

00

83O

cim

um b

asili

cum

L.

00

00

207

164

Oci

mum

bas

ilicu

m L

.0

0+20

7+16

4O

cim

um te

nui fl

orum

L.

00

00

013

3Pl

anta

go o

vata

For

ssk.

00

00

010

1Ba

copa

mon

nier

i (L

.) W

ettst

.Y

es0

00

00

0N

oBo

erha

avia

di ff

usa

L.

Yes

00

00

00

No

Cam

ellia

sine

nsis

(L.)

Kun

tze

Yes

00

00

00

No

Eclip

ta a

lba

(L.)

Has

sk.

(= E

clip

ta p

rostr

ata

(L.)

L.)

Yes

00

00

00

No

Phyl

lant

hus n

iruri

L.Y

es0

00

00

0N

oPi

cror

hiza

kur

roa

Roy

le e

x Be

nth.

Yes

00

00

00

No

Pipe

r lo

ngum

L.

Yes

00

00

00

No

Anch

usa

ital

ica

Ret

z.0

00

066

164

Anch

usa

ital

ica

Ret

z.0

0+66

+164

Aspa

ragu

s ra

cem

osus

Will

d.0

00

00

32Ci

nnam

omum

iner

s Re

inw

. ex

Blum

e0

00

00

46Co

nvol

vulu

s m

icro

phyl

lus

Sieb

er e

x Sp

reng

.0

00

00

1612

Conv

olvu

lus

pros

tratu

s Fo

rssk

.0

024

127

056

0Cu

min

um c

ymin

um L

.0

00

00

23Fo

enic

ulum

vul

gare

Mill

.0

221

00

00

Gre

enio

psis

sibu

yane

nsis

Elm

er0

210

00

0La

ctuc

a sa

tiva

L.

00

00

011

Men

tha

can

aden

sis L

.0

00

00

33Pu

nica

gra

natu

m L

.12

370

00

2632

8Pu

nica

gra

natu

m L

.0

0+26

+328

Senn

a a

lexa

ndrin

a M

ill.

00

396

084

936

42Se

nna

ale

xand

rina

Mill

.0

0+84

9+36

42Se

nna

tora

(L.)

Roxb

.0

00

047

301

Senn

a to

ra (L

.) Ro

xb.

00+

47+3

01So

lanu

m ly

cope

rsic

um L

.0

300

00

0St

r ych

nos

nux-

vom

ica

L.

00

00

150

Trac

hysp

erm

um a

mm

i (L

.) Sp

ragu

e0

00

00

62Tr

ibul

us te

rres

tris

L.0

00

014

321

Trib

ulus

terr

estri

s L.

00+

143+

21W

ithan

ia so

mni

fera

(L.)

Dun

al0

1268

014

042

815

18W

ithan

ia so

mni

fera

(L.)

Dun

al0

140+

428+

1518

Acor

us c

alam

us L

.*Y

es0

00

00

0N

oAn

ethu

m so

wa R

oxb.

ex

Flem

ing

Gen

us is

repr

esen

ted

00

00

00

No

Baco

pa m

onni

eri

(L.)

Wet

tst.*

Yes

00

00

00

No

Baco

pa m

onni

eri

(L.)

Wet

tst.

Yes

00

00

00

No

Cela

strus

pan

icul

atus

Will

d.*

Yes

00

00

00

No

Cent

ella

asia

tica

(L.)

Urb

. *Y

es0

00

00

0N

oEl

etta

ria c

arda

mom

um (L

.) M

aton

Yes

00

00

00

No

Embe

lia ri

bes

Burm

.f.*

Yes

00

00

00

No

Embl

ica

offic

inal

is G

aertn

. (=

Phyl

lant

hus e

mbl

ica

L.)*

Yes

00

00

00

No

Evol

vulu

s al

sinoi

des

(L.)

L.*

Yes

00

00

00

No

Ipom

oea

digi

tata

L. (

= Ip

omoe

a ch

eiro

phyl

la O

'Don

ell)

Gen

us is

repr

esen

ted

00

00

00

No

Muc

una

pru

riens

(L.)

DC.

Yes

00

00

00

No

Nard

osta

chys

jata

man

si*Y

es0

00

00

0N

oO

rox y

lum

indi

cum

(L.)

Kur

z i*

Yes

00

00

00

No

Prun

us a

myg

dalu

s St

okes

*Y

es0

00

00

0N

oTe

rmin

alia

arju

na (R

oxb.

ex

DC.

) Wig

ht &

Arn

.Y

es0

00

00

0N

oTe

rmin

alia

che

bula

Ret

z.*

Yes

00

00

00

No

Tino

spor

a c

ordi

folia

(Will

d.) M

iers

*Y

es0

00

00

0N

oVa

leria

na w

allic

hii D

C. (=

Val

eria

na ja

tam

ansi

Jone

s)*

Yes

00

00

00

No

With

ania

som

nife

ra (L

.) D

unal

*Y

es0

00

00

0N

oW

ithan

ia so

mni

fera

(L.)

Dun

alY

es0

00

00

0N

oZi

ngib

er o

ffici

nale

Ros

coe

Yes

00

00

1415

Zing

iber

offi

cina

le R

osco

e0

0+14

+15

Yes

Trib

ulus

terr

estri

s L.

00

00

016

Triti

cum

aes

tivum

L.

00

00

025

Achi

llea

mill

e fol

ium

L.*

Yes

00

00

00

No

Boer

haav

ia d

iffus

a L.

Yes

00

00

00

No

Capp

aris

spin

osa

L.

Yes

00

00

00

No

Cass

ia o

ccid

enta

lis L

. (=

Senn

a oc

cide

ntal

is (L

.) Li

nk)*

Yes

00

00

00

No

Cich

oriu

m in

tybu

s L.

Y

es0

1499

760

319

440

Cich

oriu

m in

tybu

s L.

0+

1499

+76

0+31

9+44

0Y

esEm

belia

ribe

s Bu

rm.f.

Yes

00

00

00

No

Num

ber

of M

OTU

's (n

rITS

2)

Spec

ies i

dent

ified

in th

e pr

oduc

t by

DN

A m

etab

arco

ding

(Cri

tieri

a of

the

iden

tiy: a

sp

ecie

s was

con

sider

ed a

nd v

alid

aded

as b

eing

pre

sent

with

in th

e pr

oduc

t onl

y if

it w

as

dete

cted

in a

t lea

st 2

out

of t

he 3

rep

licat

es)#

Doe

s nrI

TS se

quen

ce o

f the

pla

nt sp

ecie

s in

the

prod

uct l

abel

foun

d b

y D

NA

met

abar

codi

ng?

Tot

al n

umbe

r of

pla

nt sp

ecie

s de

tect

ed w

ithin

the

prod

uct b

y D

NA

met

abar

codi

ng

Tota

l num

ber

of sp

ecie

s list

ed

on th

e pr

oduc

t lab

el d

etec

ted

by D

NA

met

abar

codi

ng

Fida

lity

rate

per

he

rbal

pro

duct

Det

ecte

d sp

ecie

s by

app

lyin

g ou

r qu

ality

cri

teri

a se

lect

ion#

Supp

lem

enta

ry T

able

S7.

Res

ults

of th

e D

NA

met

abar

code

ana

lysis

of h

erba

l pro

duct

s

Her

bal

prod

uct

num

ber

Prod

uct t

ype

Num

ber

of

spec

ies a

s per

la

bel

Scie

ntifi

c na

mes

of t

he p

lant

ingr

edie

nts a

s ind

icat

ed o

n th

e pr

oduc

t lab

elA

vaila

bilit

y of

nrI

TS se

quen

ces

in G

enBa

nkSp

ecie

s det

ecte

d by

DN

A m

etab

arco

ding

Num

ber

of r

eads

(nrI

TS1)

Num

ber

of r

eads

(nrI

TS2)

Num

ber

of M

OTU

's (n

rITS

1)

40

76

00

%1

Tabl

ets

70

56

11

12

19

%2

Tabl

ets

110

42

03

39

125

%3

Tabl

ets

40

1011

07

157

111

%6

Tabl

ets

92

52

23

75

233

%7

Caps

ules

64

43

00

01

00

%8

Caps

ules

21

10

02

73

00

%9

Caps

ules

21

11

18

136

00

%10

Caps

ules

71

42

01

31

113

%12

Tabl

ets

220

00

Embl

ica

offic

inal

is G

aertn

. (=

Phyl

lant

hus e

mbl

ica

L.)

Yes

00

00

00

No

Fum

aria

offi

cina

lis L

.Y

es0

00

00

0N

oPh

ylla

nthu

s am

arus

Sch

umac

h. &

Tho

nn.

Yes

00

00

00

No

Plum

bago

zeyl

anic

a L

.Y

es0

00

00

0N

oRa

phan

us sa

tivus

L. (

= Ra

phan

us ra

phan

istru

m su

bsp.

sativ

us (L

.) D

omin

)Y

es0

00

00

0N

oSo

lanu

m n

igru

m L

.*Y

es0

00

00

0N

oTa

mar

ix g

allic

a L

*Y

es0

00

00

0N

oTe

rmin

alia

arju

na (R

oxb.

ex

DC.

) Wig

ht &

Arn

.*Y

es0

00

00

0N

oTe

rmin

alia

che

bula

Ret

z.Y

es0

00

00

0N

oTi

nos p

ora

cord

ifolia

(Will

d.) M

iers

Yes

00

00

00

No

Conv

olvu

lus

pros

tratu

s Fo

rssk

.0

067

50

00

Echi

nace

a p

urpu

rea

(L.)

Moe

nch

00

00

015

0Ec

li pta

pro

strat

a (L

.) L.

00

00

011

0La

ctuc

a sa

tiva

L.

00

00

062

Phyl

lant

hus

mad

eras

pate

nsis

L.

00

300

059

Abut

ilon

indi

cum

(L.)

Swee

tY

es0

00

00

0N

oAl

oe b

arba

dens

is M

ill. (

= Al

oe v

era

(L.)

Burm

.f.)*

Yes

00

00

00

No

Aspa

ragu

s ra

cem

osus

Will

d.*

Yes

00

00

00

No

Berb

eris

aris

tata

DC.

*Y

es0

00

00

0N

oBo

erha

avia

di ff

usa

L.*

Yes

00

00

00

No

Case

aria

esc

ulen

ta R

oxb.

*G

enus

is re

pres

ente

d0

00

00

0N

oCu

rcum

a lo

nga

L.

Yes

00

00

00

No

Embl

ica

offic

inal

is G

aertn

. (=

Phyl

lant

hus e

mbl

ica

L.)

Yes

00

00

00

No

Euge

nia

jam

bola

na L

am. (

= Sy

zygi

um c

umin

i (L.

) Ske

els)

*Y

es0

00

00

0N

oG

lycy

rrhi

za g

labr

a L

.*Y

es0

00

00

0N

oG

mel

ina

arb

orea

Rox

b.*

Yes

00

00

00

No

Gos

sypi

um h

erba

ceum

L.*

Yes

00

00

00

No

Gym

nem

a sy

lves

tre (R

etz.

) R.B

r. ex

Sm

.*Y

es0

00

00

0N

oM

omor

dica

cha

rant

ia L

.Y

es0

00

00

0N

oO

cim

um te

nui fl

orum

L.

Yes

00

00

00

No

Phyl

lant

hus

amar

us S

chum

ach.

& T

honn

.*Y

es0

00

00

0N

oPi

per

long

um L

.Y

es0

00

00

0N

oPi

per

nigr

um L

.Y

es57

4549

1321

465

00

0Pi

per

nigr

um L

.57

45+4

913+

2146

50

Yes

Pter

ocar

pus

mar

supi

um R

oxb.

*Y

es0

00

00

0N

oRu

mex

mar

itim

us L

.G

enus

is re

pres

ente

d0

00

00

0N

oSp

haer

anth

us in

dicu

s L.

*Y

es0

00

00

0N

oSw

ertia

chi

rata

Buc

h.-H

am. e

x W

all.*

Yes

00

00

00

No

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Roxb

.Y

es0

00

00

0N

oTe

rmin

alia

che

bula

Ret

z.Y

es0

00

00

0N

oTi

nos p

ora

cor

difo

lia (W

illd.

) Mie

rs*

Yes

00

00

00

No

Trib

ulus

terr

estri

s L.

*Y

es0

00

00

0N

oZi

n gib

er o

ffici

nale

Ros

coe*

Yes

00

00

00

No

Bras

sica

ole

race

a L

. 80

930

1318

50

00

Bras

sica

ole

race

a L

. 80

93+0

+131

850

Coria

ndru

m sa

tivum

L.

504

00

00

0M

edic

a go

sativ

a L

.0

00

00

0So

lanu

m ly

cope

rsic

um L

.11

90

700

00

Sola

num

lyco

pers

icum

L.

119+

0+70

0Ve

roni

ca c

ham

aedr

ys L

.0

1501

00

00

Ach y

rant

hes

aspe

ra L

.Y

es0

00

00

0N

oC y

peru

s sc

ario

sus

R.Br

.G

enus

is re

pres

ente

d0

00

00

0N

oD

idym

ocar

pus p

edic

ella

ta (

Ait.

) Ait.

F.

Gen

us is

repr

esen

ted

00

00

00

No

Ono

sma

brac

teat

um W

all

Gen

us is

repr

esen

ted

00

00

00

No

Rubi

a c

ordi

folia

L.

Yes

00

00

00

No

Saxi

fraga

ligu

lata

Mur

ray

(= S

axifr

aga

stolo

nife

ra C

urtis

)Y

es0

00

00

0N

oVe

rnon

ia c

iner

ea (L

.) Le

ss. (

= Cy

anth

illiu

m c

iner

eum

(L.)

H.R

ob.)

Yes

00

00

00

No

Zing

iber

offi

cina

le R

osco

eY

es0

00

00

0N

oAc

anth

ospe

rmum

hisp

idum

DC.

00

205

00

0Be

rgen

ia li

gula

ta E

ngl.

175

472

2010

00

0Be

rgen

ia li

gula

ta E

ngl.

175+

472+

2010

0Cu

cum

is m

elo

L.

00

455

00

0O

enot

hera

dru

mm

ondi

i H

ook.

00

180

00

Oen

othe

ra la

cini

ata

Hill

00

570

00

0Tr

ibul

us te

rres

tris

L.14

144

9517

724

00

0Tr

ibul

us te

rres

tris

L.14

144+

9571

+724

0Sa

raca

indi

ca L

.Y

es0

00

00

0N

oAs

para

gus

race

mos

us W

illd.

*Y

es0

00

00

0N

oCe

ntel

la a

siatic

a (L

.) U

rb.

Yes

00

00

2012

Cent

ella

asia

tica

(L.)

Urb

. 0

0+20

+12

Yes

Gly

cyrr

hiza

gla

bra

L.

Yes

00

00

00

No

Sida

cor

difo

lia L

.Y

es0

00

00

0N

oTe

rmin

alia

che

bula

Ret

z.Y

es0

00

00

0N

o0

00

00

0Al

thae

a o

ffici

nalis

L.

2632

00

00

0D

raba

niv

alis

Lil j

.50

600

00

00

Dr y

moc

allis

rupe

stris

(L.)

Soj k

8081

90

00

0D

rym

ocal

lis ru

pestr

is (L

.) So

j k80

+819

+00

Luffa

ech

inat

a R

oxb.

00

00

132

0Ne

lum

bo n

uci fe

ra G

aertn

.0

047

10

460

Pipe

r lo

ngum

L.

00

126

00

0Se

nna

ale

xand

rina

Mill

.0

00

00

157

Tria

nthe

ma

por

tula

castr

um L

.0

031

30

010

5Tr

ibul

us te

rres

tris

L.0

00

00

27Tr

igon

ella

foen

um-g

raec

um L

.49

80

00

00

Bosw

ellia

serr

ata

Rox

b. e

x Co

lebr

.*G

enus

is re

pres

ente

d0

00

00

0N

oAl

pini

a g

alan

ga (L

.) W

illd.

*Y

es0

00

00

0N

oCo

mm

i pho

ra w

ight

ii (A

rn.)

Bhan

dari*

Yes

00

00

00

No

Gl y

cyrr

hiza

gla

bra

L.*

Yes

00

00

00

No

Tino

spor

a c

ordi

folia

(Will

d.) M

iers

Y

es0

00

00

0N

oTr

ibul

us te

rres

tris

L.Y

es0

00

00

0N

oAl

thae

a o

ffici

nalis

L.

577

1392

715

00

0Al

thae

a o

ffici

nalis

L.

577+

1392

+715

0Cu

cum

is sa

tivus

L.

00

00

754

0C y

nodo

n d

acty

lon

(L.)

Pers

.0

00

068

0D

raba

niv

alis

Lil j

.12

4579

00

00

Dra

ba n

ival

is L

ilj.

12+4

579+

00

Frax

inus

pen

nsyl

vani

ca M

arsh

all

00

00

412

0M

eliss

a o

ffici

nalis

L.

00

621

00

0Pe

dicu

laris

flam

mea

L.

083

00

00

Sola

num

l yco

pers

icum

L.

00

00

015

4W

ithan

ia so

mni

fera

(L.)

Dun

al0

00

050

524

With

ania

som

nife

ra (L

.) D

unal

00+

50+5

24A

kik

pish

ti$0

00

00

0As

para

gus

race

mos

us W

illd.

Yes

00

00

00

No

Caes

alpi

nia

bond

ucel

la (L

.) Fl

emin

g (=

of C

aesa

lpin

ia b

ondu

c (L

.) Ro

xb.)

Yes

00

00

00

No

Crat

eva

nur

vala

Buc

h.-H

amG

enus

is re

pres

ente

d0

00

00

0N

oTr

ibul

us te

rres

tris L

.Y

es0

00

00

0N

oAb

utilo

n in

dicu

m (L

.) Sw

eet

117

00

00

0D

r ym

ocal

lis ru

pestr

is (L

.) So

j k13

50

00

00

Festu

ca g

igan

tea

(L.)

Vill

.0

00

00

62M

omor

dica

cha

rant

ia L

.95

00

00

0Sa

lvia

ple

beia

R.B

r.0

00

00

50Th

ysan

olae

na la

tifol

ia (R

oxb.

ex

Hor

nem

.) H

onda

00

00

3102

5703

Thys

anol

aena

latif

olia

(Rox

b. e

x H

orne

m.)

Hon

da0

0+31

02+5

703

Aste

raca

ntha

long

ifolia

Nee

s (=

of H

ygro

phila

aur

icul

ata

(Sch

umac

h.) H

eine

)Y

es0

00

012

42As

tera

cant

ha lo

ngifo

lia N

ees (

= of

Hyg

roph

ila

auric

ulat

a (S

chum

ach.

) Hei

ne)

00+

12+4

2Y

es

Croc

us sa

tivus

L.

Yes

00

00

00

No

Prun

us a

myg

dalu

s Bat

sch

(= P

runu

s dul

cis (

Mill

.) D

.A.W

ebb)

Yes

00

00

00

No

Trib

ulus

terr

estri

s L.

Yes

00

00

00

No

Cent

aure

a c

yanu

s L.

330

280

00

Cent

aure

a c

yanu

s L.

33+0

+28

0Cu

cum

is sa

tivus

L.

00

740

020

Frax

inus

alb

ican

s Bu

ckle

y0

00

054

372

Frax

inus

alb

ican

s Bu

ckle

y0

0+54

+372

Hyo

scya

mus

nig

er L

.37

245

00

00

Hyo

scya

mus

nig

er L

.37

+245

+00

Mel

issa

offi

cina

lis L

.57

9216

890

00

0M

eliss

a o

ffici

nalis

L.

5792

+168

9+0

0Sa

lvia

ple

beia

R.B

r.0

00

010

44Sa

lvia

ple

beia

R.B

r.0

0+10

+14

Senn

a a

lexa

ndrin

a M

ill.

00

00

085

With

ania

som

nife

ra (L

.) D

unal

00

00

1511

6W

ithan

ia so

mni

fera

(L.)

Dun

al0

0+15

+16

Gym

nem

a sy

lves

tre (R

etz.

) R.B

r. ex

Sm

.Y

es64

012

00

0G

ymne

ma

sylv

estre

(Ret

z.) R

.Br.

ex S

m.

64+0

+12

0Y

esAm

mi

trifo

liatu

m (H

.C.W

atso

n) T

rel.

110

00

00

Boer

havi

a d

i ffus

a L

.0

017

00

0Br

assic

a o

lera

cea

L.

330

100

00

Bras

sica

ole

race

a L

.33

+0+1

00

Cent

ella

asia

tica

(L.)

Urb

.0

00

038

0Ch

aero

phyl

lum

refle

xum

Aitc

h.31

018

00

0Ch

aero

phyl

lum

refle

xum

Aitc

h.31

+0+1

80

Cham

aecr

ista

ole

siphy

lla (V

ogel

) H.S

.Irw

in &

Bar

neby

00

00

640

Cocc

inia

sess

ilifo

lia (S

ond.

) Cog

n.52

170

00

0Co

ccin

ia se

ssili

folia

(Son

d.) C

ogn.

52+1

9+0

0Co

nvol

vulu

s pr

ostra

tus

Fors

sk.

00

100

00

Coria

ndru

m sa

tivum

L.

410

00

090

Cum

inum

cym

inum

L.

00

00

3918

1Cu

min

um c

ymin

um L

.0

0+39

+181

Cyna

nchu

m st

aubi

i Bo

sser

00

00

056

Fago

pyru

m e

scul

entu

m M

oenc

h25

00

00

0Fo

enic

ulum

vul

gare

Mill

.14

317

790

00

0Fo

enic

ulum

vul

gare

Mill

.14

3+17

7+90

0Ip

omoe

a p

aras

itica

(Kun

th) G

. Don

3695

572

1036

733

243

918

85Ip

omoe

a p

aras

itica

(Kun

th) G

. Don

3695

+572

+103

6732

2+43

9+18

85M

omor

dica

cha

rant

ia L

.15

00

00

0Ph

ylla

nthu

s em

blic

a L

.0

018

00

0Se

nna

ale

xand

rina

Mill

.63

411

6857

383

172

1206

Senn

a a

lexa

ndrin

a M

ill.

634+

1168

+573

831+

72+1

206

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Roxb

.0

012

00

0Tr

ach y

sper

mum

am

mi

(L.)

Spra

gue

00

00

010

With

ania

som

nife

ra (L

.) D

unal

00

014

70

0Em

blic

a o f

ficin

alis

Gae

rtn. (

= of

Phy

llant

hus e

mbl

ica

L.)

Yes

00

00

00

No

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Roxb

.Y

es25

8016

590

00

0Te

rmin

alia

bel

liric

a (G

aertn

.) Ro

xb.

2580

+165

9+0

0Y

esTe

rmin

alia

che

bula

Ret

z.Y

es0

00

00

0N

oAl

thae

a of

ficin

alis

00

1041

80

00

Bras

sica

oler

acea

0

4487

00

00

Oci

mum

tenu

iflor

um L

.Y

es0

00

152

024

Oci

mum

tenu

iflor

um L

.0

152+

0+24

Yes

Cinn

amom

um ze

ylan

icum

Blu

me

(= C

inna

mom

um v

erum

J.P

resl)

Yes

00

00

00

No

Curc

uma

lon g

a L

.Y

es0

00

00

0N

oEm

blic

a o f

ficin

alis

Gae

rtn. (

= of

Phy

llant

hus e

mbl

ica

L.)

Yes

00

00

00

No

Gly

cyrr

hiza

gla

bra

L.

Yes

3937

128

00

0G

lycy

rrhi

za g

labr

a L

.39

+37+

128

0Y

esPi

per

long

um L

.Y

es35

813

3621

850

00

Pipe

r lo

ngum

L.

358+

1336

+218

50

Yes

Pipe

r ni

grum

L.

Yes

117

015

00

0Pi

per

nigr

um L

.11

7+0+

150

Yes

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Roxb

.Y

es13

390

711

210

00

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Roxb

.13

3+90

7+11

210

Yes

Term

inal

ia c

hebu

la R

etz.

Yes

00

00

00

No

Zing

iber

offi

cina

le R

osco

eY

es0

00

00

0N

oCo

nvol

vulu

s pr

ostra

tus

Fors

sk.

180

00

00

Peril

la se

toye

nsis

G.H

onda

1348

100

00

Peril

la se

toye

nsis

G.H

onda

13+4

8+10

0Si

da a

cuta

Bur

m.f.

1126

00

00

Sida

acu

ta B

urm

.f.11

+26+

00

Term

inal

ia a

rjuna

(Rox

b. e

x D

C.) W

ight

& A

rn.

00

110

00

Trib

ulus

terr

estri

s L.

00

150

00

Caru

m c

optic

um (L

.) Be

nth.

& H

ook.

f. ex

C.B

.Cla

rke

(= T

rach

yspe

rmum

am

mi (

L.) S

prag

Yes

00

00

00

No

Cora

llium

rubr

um#

00

00

00

No

Curc

uma

lon g

a L

.Y

es0

00

00

0N

oH

emid

esm

us in

dicu

s (L

.) R.

Br.

ex S

chul

t.Y

es0

00

00

0N

oSa

ntal

um a

lbum

L.

Yes

00

00

00

No

Saxi

fraga

gra

nula

ta L

.Y

es0

00

00

0N

oS p

haer

anth

us in

dicu

s L.

Yes

00

00

00

No

Alce

a ro

sea

L.

00

00

274

0Be

rber

is a

siatic

a R

oxb.

ex

DC.

00

00

850

Cent

ella

asia

tica

(L.)

Urb

.0

00

066

626

Cent

ella

asia

tica

(L.)

Urb

.0

0+66

6+26

Conv

olvu

lus

mic

roph

yllu

s Si

eber

ex

Spre

ng.

00

00

017

Conv

olvu

lus

pros

tratu

s Fo

rssk

.0

00

039

0Co

rcho

rus

olito

rius

L.0

00

025

14Co

rcho

rus

olito

rius

L.0

0+25

+14

Coria

ndru

m sa

tivum

L.

00

00

3921

Coria

ndru

m sa

tivum

L.

00+

39+2

1

01

51

19

%13

Tabl

ets

160

13

00

03

110

%14

Tabl

ets

274

32

00

02

00

%16

Tabl

ets

82

26

03

42

120

%17

Tabl

ets

64

13

04

23

00

%18

Tabl

ets

62

32

01

31

00

%19

Tabl

ets

43

00

04

67

125

%23

Tabl

ets

43

22

35

68

110

0 %

27Ca

psul

es1

114

10

00

01

133

%30

Caps

ules

31

21

10

17

550

%31

Tabl

ets

107

57

315

1112

00

%32

Tabl

ets

64

54

Cum

inum

cym

inum

L.

00

00

148

901

Cum

inum

cym

inum

L.

00+

148+

901

Nard

osta

chys

jata

man

si (D

.Don

) DC.

00

00

160

Nelu

mbo

nuc

i fera

Gae

rtn.

1588

2344

1126

00

0Ne

lum

bo n

ucife

ra G

aertn

.15

88+2

344+

1126

0Pi

per

long

um L

.21

2043

326

030

00

Pipe

r lo

ngum

L.

2120

+433

+260

30

Pipe

r ni

grum

L.

2637

120

00

Pipe

r ni

grum

L.

26+3

7+12

0Py

rus

calle

ryan

a D

ecne

.0

00

029

0Ro

sa ru

gosa

Thu

nb.

00

00

3912

Rosa

rugo

sa T

hunb

.0

0+39

+12

Senn

a a

lexa

ndrin

a M

ill.

016

4027

276

18Se

nna

ale

xand

rina

Mill

.0+

16+4

027

2+76

+18

Senn

a a

uric

ulat

a (L

.) Ro

xb.

00

00

246

313

Senn

a a

uric

ulat

a (L

.) Ro

xb.

00+

246+

313

Sesa

mum

indi

cum

L.

00

00

025

Trib

ulus

terr

estri

s L.

100

027

9044

020

59Tr

ibul

us te

rres

tris

L.0

2790

+440

+205

9Ve

roni

ca p

lebe

ia R

. Br.

012

00

00

0W

ithan

ia so

mni

fera

(L.)

Dun

al0

00

185

4419

6W

ithan

ia so

mni

fera

(L.)

Dun

al0

185+

44+1

96Am

omum

subu

latu

m R

oxb.

Yes

00

00

00

No

Cinn

amom

um ze

ylan

icum

Blu

me

(= C

inna

mom

um v

erum

J.P

resl)

Yes

00

00

00

No

Gly

cyrr

hiza

gla

bra

L.

Yes

358

164

143

00

0G

lycy

rrhi

za g

labr

a L

.35

8+16

4+14

30

Yes

Iris

ger

man

ica

L.

ITS2

and

Gen

us is

repr

esen

ted

00

00

00

No

Pipe

r lo

ngum

L.

Yes

3017

1083

189

00

0Pi

per

long

um L

.30

17+1

083+

189

0Y

esPi

per

nigr

um L

.Y

es10

950

800

00

Pipe

r ni

grum

L.

1095

+0+8

00

Yes

Term

inal

ia a

rjuna

(Rox

b. e

x D

C.) W

ight

& A

rn.

Yes

389

3455

00

0Te

rmin

alia

arju

na (R

oxb.

ex

DC.

) Wig

ht &

Arn

.38

9+34

+55

0Y

esZi

ngib

er o

ffici

nale

Ros

coe

Yes

6210

00

00

Zing

iber

offi

cina

le R

osco

e62

+10+

00

Yes

Boer

havi

a d

iffus

a L

.0

00

133

2864

Boer

havi

a d

iffus

a L

.0

133+

28+6

4Ci

nnam

omum

cas

sia (L

.) J.P

resl

00

011

023

0011

5Ci

nnam

omum

cas

sia (L

.) J.P

resl

011

0+23

00+1

15Co

nvol

vulu

s m

icro

phyl

lus

Sieb

er e

x Sp

reng

.0

00

052

324

8Co

nvol

vulu

s m

icro

phyl

lus

Sieb

er e

x Sp

reng

.0

0+52

3+24

8Co

nvol

vulu

s pr

ostra

tus

Fors

sk.

3710

065

715

0Co

nvol

vulu

s pr

ostra

tus

Fors

sk.

37+1

0+0

657+

15+0

Conv

olvu

lus

subh

irsut

us R

egel

& S

chm

alh.

00

00

029

2Co

riand

rum

sativ

um L

.0

00

00

28Cu

min

um c

ymin

um L

.0

00

017

26Cu

min

um c

ymin

um L

.0

0+17

+26

Inul

a ra

cem

osa

Hoo

k.f.

00

017

60

114

Inul

a ra

cem

osa

Hoo

k.f.

017

6+0+

114

Justi

cia

adh

atod

a L

.0

00

00

185

Men

tha

s pic

ata

L.

00

010

40

0Ny

ctan

thes

arb

or-tr

istis

L.

00

00

6567

Nyct

anth

es a

rbor

-trist

is L

.0

0+65

+67

Pista

cia

atla

ntic

a D

esf.

120

00

00

Plan

tago

maj

or L

.0

00

00

107

Rosa

rugo

sa T

hunb

.0

00

00

10Se

nna

occ

iden

talis

(L.)

Link

00

00

044

Sesa

mum

indi

cum

L.

00

00

069

Sida

acu

ta B

urm

.f.10

116

00

00

Sida

acu

ta B

urm

.f.10

1+16

+00

Trib

ulus

terr

estri

s L.

100

170

1275

814

800

Trib

ulus

terr

estri

s L.

100+

17+0

1275

+814

+800

Vign

a ra

diat

a (L

.) R.

Wilc

zek

593

103

00

00

Vign

a ra

diat

a (L

.) R.

Wilc

zek

593+

103+

00

With

ania

som

nife

ra (L

.) D

unal

00

00

593

0An

dro g

raph

is p

anic

ulat

a (B

urm

.f.) N

ees

Yes

00

00

00

No

Bosw

ellia

serr

ata

Rox

b. e

x Co

lebr

.Y

es0

00

00

0N

oCa

rum

co p

ticum

(L.)

Bent

h. &

Hoo

k.f.

ex C

.B.C

lark

e (=

Tra

chys

perm

um a

mm

i (L.

) Spr

agY

es0

00

00

0Tr

achy

sper

mum

am

mi

(L.)

Spra

gue

013

630+

29+7

0Y

esCu

rcum

a lo

nga

L.

Yes

00

00

00

No

C ypr

aea

mon

eta$

00

00

00

No

Embl

ica

offic

inal

is G

aertn

. (=

Phyl

lant

hus e

mbl

ica

L.)

Yes

00

00

00

No

Lotu

s ar

abic

us L

. Y

es0

00

00

0N

oM

yrist

ica

frag

rans

Hou

tt.Y

es0

00

00

0N

oPi

per

long

um L

.Y

es81

320

0130

30

00

Pipe

r lo

ngum

L.

813+

2001

+303

0Y

esSw

ertia

chi

rata

Buc

h.-H

am. e

x W

all.

Yes

00

00

00

No

Term

inal

ia b

ellir

ica

(Gae

rtn.)

Roxb

.Y

es0

00

00

0N

oTe

rmin

alia

che

bula

Ret

z.Y

es0

00

00

0N

oTr

igon

ella

foen

um-g

raec

um L

.Y

es36

2342

568

50

00

Trig

onel

la fo

enum

-gra

ecum

L.

3623

+425

+685

0Y

esZi

ngib

er o

ffici

nale

Ros

coe

Yes

1416

120

00

Zing

iber

offi

cina

le R

osco

e14

+16+

120

Yes

Ole

a e

urop

aea

L.

315

8753

00

0O

lea

eur

opae

a L

.31

5+87

+53

0Pi

per

nigr

um L

.14

00

00

0Ra

phan

us sa

tivus

L.

240

100

00

Raph

anus

sativ

us L

.24

+0+1

00

Trac

hysp

erm

um a

mm

i (L

.) Sp

ragu

e0

290

1363

029

70Tr

ibul

us te

rres

tris

L.18

100

00

0Tr

ibul

us te

rres

tris

L.18

+10+

00

Ciss

us q

uadr

angu

laris

L.

Yes

00

00

5113

Ciss

us q

uadr

angu

laris

L.

00+

51+1

3Y

esO

cim

um g

ratis

simum

L.

Yes

00

00

00

No

Phyl

lant

hus

embl

ica

L.

Yes

00

00

00

No

Term

inal

ia a

r juna

(Rox

b. e

x D

C.) W

ight

& A

rn.

Yes

00

00

00

No

Boer

havi

a d

i ffus

a L

.0

1019

103

037

Boer

havi

a d

iffus

a L

.0+

10+1

910

3+0+

37D

amro

ngia

cya

nant

ha T

ribou

n0

00

018

724

4D

amro

ngia

cya

nant

ha T

ribou

n0

0+18

7+24

4D

rype

tes

hoae

nsis

Gag

nep.

00

019

950

020

5D

rype

tes

hoae

nsis

Gag

nep.

019

9+50

0+20

5Lo

xoca

rpus

ala

ta R

.Br.

00

00

019

Oci

mum

bas

ilicu

m L

.90

00

2466

138

239

Oci

mum

bas

ilicu

m L

.0

2466

+138

+239

Oci

mum

tenu

i flor

um L

.0

00

239

7028

3O

cim

um te

nuifl

orum

L.

023

9+70

+283

Peril

la se

toye

nsis

G.H

onda

8911

420

00

Peril

la se

toye

nsis

G.H

onda

89+1

1+42

0Pl

anta

go m

ajor

L.

00

00

2126

Plan

tago

maj

or L

.0

0+21

+26

Tino

spor

a co

rdifo

lia (W

illd.

) Mie

rs

00

00

039

Vite

x ag

nus-

castu

s L.

140

00

00

Eclip

ta a

lba

(L.)

Has

sk.

(= E

clip

ta p

rostr

ata

(L.)

L.)

Yes

539

012

1389

6616

7829

7Ec

lipta

alb

a (L

.) H

assk

. (=

Ecl

ipta

pro

strat

a (L

.) L.

)53

9+0+

1213

89+6

616+

7829

7Y

esAm

aran

thus

viri

dis

L.0

00

00

12Si

da c

ordi

folia

L.

Yes

027

200

00

Sida

cor

difo

lia L

.0+

27+2

00

Yes

Justi

cia

adh

atod

a L

.0

120

00

11M

eliss

a o

ffici

nalis

L.

100

00

00

Mom

ordi

ca c

hara

ntia

L.

220

018

00

0Po

l ypo

gon

mon

spel

iens

is (L

.) D

esf.

00

013

20

0Si

da a

cuta

Bur

m.f.

3834

1621

318

00

16Si

da a

cuta

Bur

m.f.

3834

+162

1+31

80

Sola

num

vir g

inia

num

L.

00

012

40

0Te

rmin

alia

che

bula

Ret

z.10

00

00

0Tr

iant

hem

a p

ortu

laca

strum

L.

310

034

215

34Tr

iant

hem

a p

ortu

laca

strum

L.

034

2+15

+34

Trib

ulus

terr

estri

s L.

208

154

012

160

117

Trib

ulus

terr

estri

s L.

439+

222+

073

75+1

6+11

7Vi

gna

mun

go (L

.) H

eppe

r0

028

00

0Ti

nos p

ora

cor

difo

lia (W

illd.

) Mie

rs

Yes

390

4713

70

3939

Tino

spor

a c

ordi

folia

(Will

d.) M

iers

39

0+47

+137

0+39

+39

Yes

Cocc

inia

ado

ensis

(Hoc

hst.

ex A

. Ric

h.) C

ogn.

00

430

00

Cocc

inia

gra

ndis

(L.)

Voi

gt0

00

128

382

78Co

ccin

ia g

rand

is (L

.) V

oigt

012

8+38

2+78

Vign

a ra

diat

a (L

.) R.

Wilc

zek

330

00

00

With

ania

som

nife

ra (L

.) D

unal

00

00

064

Term

inal

ia a

rjuna

(Rox

b. e

x D

C.) W

ight

& A

rn.

Yes

00

00

00

No

Eclip

ta p

rostr

ata

(L.)

L.0

00

00

77H

umul

us lu

pulu

s L.

1344

00

00

00

Sola

num

lyco

pers

icum

L.

00

00

4674

Sola

num

lyco

pers

icum

L.

00+

46+7

4Bo

erha

avia

diff

usa

L.

Yes

195

1249

00

00

Boer

haav

ia d

iffus

a L

.19

5+12

49+0

0Y

esAl

thae

a o

ffici

nalis

L.

00

00

011

1Ba

ccha

roid

es a

nthe

lmin

tica

(L.)

Moe

nch

00

2769

00

0Cu

cum

is sa

tivus

L.

1731

60

00

00

Cum

inum

cym

inum

L.

00

00

6424

Cum

inum

cym

inum

L.

00+

64+2

4D

onio

phyt

on w

edde

llii

Kat

inas

& S

tues

sy26

887

80

00

0D

onio

phyt

on w

edde

llii

Kat

inas

& S

tues

sy26

8+87

8+0

0D

onio

phyt

on w

edde

llii

Kat

inas

& S

tues

sy0

071

90

00

Dra

ba n

ival

is L

ilj.

043

290

00

0Ec

li pta

pro

strat

a (L

.) L.

1461

70

3654

00

133

Eclip

ta p

rostr

ata

(L.)

L.14

617+

0+36

540

Euod

ia h

orte

nsis

J.R.

For

st. &

G. F

orst.

014

070

00

0H

ibisc

us sy

riacu

s L.

103

00

00

0H

iero

nym

a a

lcho

rneo

ides

Alle

mão

012

10

00

0M

omor

dica

cha

rant

ia L

.0

343

00

00

Oci

mum

bas

ilicu

m L

.0

170

00

0Pl

uken

etia

mad

agas

carie

nsis

Lea

ndri

00

110

00

Salv

ia ro

smar

inus

Sch

leid

.0

00

00

143

Senn

a a

lexa

ndrin

a M

ill.

6633

00

00

0Si

da a

cuta

Bur

m.f.

00

394

00

0Tr

iant

hem

a p

ortu

laca

strum

L.

2041

4921

378

102

821

705

Tria

nthe

ma

por

tula

castr

um L

.20

+414

9+21

378

102+

821+

705

Phyl

lant

hus

embl

ica

L.

Yes

00

00

00

No

Phyl

lant

hus n

iruri

L.Y

es0

00

00

0N

oTi

nos p

ora

cor

difo

lia (W

illd.

) Mie

rs

Yes

127

036

096

39Ti

nosp

ora

cor

difo

lia (W

illd.

) Mie

rs

127+

0+36

0+96

+39

Yes

00

00

00

Cocc

inia

gra

ndis

(L.)

Voi

gt0

00

030

36Co

ccin

ia g

rand

is (L

.) V

oigt

00+

30+3

6Ec

hino

chlo

a c

olon

a (L

.) Li

nk68

330

208

013

379

Echi

noch

loa

col

ona

(L.)

Link

683+

30+2

080+

13+3

79Ec

hino

chlo

a p

yram

idal

is (L

am.)

Hitc

hc. &

Cha

se72

00

00

0O

cim

um te

nui fl

orum

L.

00

00

034

Phyl

lant

hus

mad

eras

pate

nsis

L.

235

00

021

132

6Ph

ylla

nthu

s m

ader

aspa

tens

is L

.0

0+21

1+32

6Ph

ylla

nthu

s te

nellu

s Ro

xb.

00

00

3427

08Ph

ylla

nthu

s te

nellu

s Ro

xb.

00+

34+2

708

Trig

astro

thec

a m

ollu

gine

a F

.Mue

ll.0

00

014

25Tr

igas

troth

eca

mol

lugi

nea

F.M

uell.

00+

14+2

5Az

adira

chta

indi

ca A

.Juss

.Y

es29

1752

00

22Az

adira

chta

indi

ca A

.Jus

s.29

+17+

520

Yes

Curc

uma

long

a L

.Y

es0

00

00

0N

oO

cim

um te

nuifl

orum

L.

Yes

00

011

927

146

Oci

mum

tenu

iflor

um L

.0

119+

27+1

46Y

esPt

eroc

arpu

s sa

ntal

inus

L.f.

Yes

00

00

00

No

Rubi

a c

ordi

folia

L.

Yes

00

00

00

No

Tino

spor

a co

rdifo

lia (W

illd.

) Mie

rs

Yes

00

00

1511

9Ti

nosp

ora

cord

ifolia

(Will

d.) M

iers

0

0+15

+119

Yes

Oci

mum

bas

ilicu

m L

.0

00

077

052

2O

cim

um b

asili

cum

L.

00+

770+

522

Peril

la se

toye

nsis

G.H

onda

130

100

00

Peril

la se

toye

nsis

G.H

onda

13+0

+10

0Sp

ondi

as tu

bero

sa A

rrud

a0

150

00

0G

lycy

rrhi

za g

labr

a L

.Y

es0

00

00

0N

oAl

thae

a o

ffici

nalis

L.

00

00

3235

2Al

thae

a o

ffici

nalis

L.

00+

32+3

52Ba

ccha

roid

es a

nthe

lmin

tica

(L.)

Moe

nch

00

00

1616

Bacc

haro

ides

ant

helm

intic

a (L

.) M

oenc

h0

0+16

+16

Cich

oriu

m in

tybu

s L.

00

00

890

Coria

ndru

m sa

tivum

L.

00

00

022

5D

ysch

orist

e sip

hona

ntha

(Nee

s) K

untz

e70

110

106

00

0D

ysch

orist

e sip

hona

ntha

(Nee

s) K

untz

e70

+110

+106

0Ec

lipta

pro

strat

a (L

.) L.

00

00

160

Gym

nem

a sy

lves

tre (R

etz.

) R.B

r. ex

Sm

.0

00

012

0Ip

omoe

a p

aras

itica

(Kun

th) G

. Don

0

00

00

188

Justi

cia

adh

atod

a L

.12

611

1230

610

00

Justi

cia

adh

atod

a L

.12

6+11

12+3

061

0O

cim

um b

asili

cum

L.

00

026

317

044

Oci

mum

bas

ilicu

m L

.0

263+

170+

44O

ddon

iode

ndro

n n

orm

andi

i A

ubre

v.0

137

00

00

Pipe

r lo

ngum

L.

6125

180

00

Pipe

r lo

ngum

L.

61+2

5+18

0Sa

lvia

ple

beia

R.B

r.0

00

017

237

1Sa

lvia

ple

beia

R.B

r.0

0+17

2+37

1Sa

raca

aso

ca (R

oxb.

) Will

d.90

047

9798

810

00

Sara

ca a

soca

(Rox

b.) W

illd.

900+

4797

+988

10

Sola

num

lyco

pers

icum

L.

00

00

320

Tria

nthe

ma

por

tula

castr

um L

.0

00

00

353

Trib

ulus

terr

estri

s L.

00

00

570

With

ania

som

nife

ra (L

.) D

unal

00

00

740

Sara

ca a

soca

(Rox

b.) W

illd.

Yes

6971

60

00

0Sa

raca

aso

ca (R

oxb.

) Will

d.69

+716

+00

Yes

Allo

phyl

us b

ojer

ianu

s (C

ambe

ss.)

Blum

e0

044

00

0Al

thae

a o

ffici

nalis

L.

044

922

160

00

Alth

aea

offi

cina

lis L

.0+

449+

2216

0Ba

ccha

roid

es a

nthe

lmin

tica

(L.)

Moe

nch

026

9820

50

00

Bacc

haro

ides

ant

helm

intic

a (L

.) M

oenc

h0+

2698

+205

0Ci

chor

ium

int y

bus

L.10

50

00

00

Coria

ndru

m sa

tivum

L.

045

1513

016

Coria

ndru

m sa

tivum

L.

0+45

+15

13+0

+16

Eclip

ta p

rostr

ata

(L.)

L.0

015

00

00

Evol

vulu

s al

sinoi

des

(L.)

L.62

877

560

00

0Ev

olvu

lus

alsin

oide

s (L

.) L.

628+

77+5

600

Foen

icul

um v

ulga

re M

ill.

554

653

790

00

Foen

icul

um v

ulga

re M

ill.

554+

653+

790

Gos

sypi

um h

irsut

um L

.0

120

00

0H

ibisc

us sa

bdar

i ffa

L.

00

017

40

314

Hib

iscus

sabd

ariff

a L

.0

174+

0+31

4I p

omoe

a p

aras

itica

(Kun

th) G

. Don

9110

213

40

00

Ipom

oea

par

asiti

ca (K

unth

) G. D

on91

+102

+134

0Ja

troph

a c

urca

s L.

00

300

00

Justi

cia

adh

atod

a L

.0

00

9218

8457

1537

1Ju

stici

a a

dhat

oda

L.

092

18+8

457+

1537

1M

enth

a a

rven

sis L

.0

00

00

21

315

1112

00

%32

Tabl

ets

64

54

68

1415

563

%34

Tabl

ets

810

84

11

17

431

%36

Tabl

ets

137

65

46

98

125

%40

Caps

ules

43

22

11

21

110

0 %

52Po

wde

r1

11

1

51

44

110

0 %

55Po

wde

r1

64

3

12

32

110

0 %

58Po

wde

r1

21

2

01

21

10

%60

Caps

ules

11

00

12

55

110

0 %

61Ca

psul

es1

78

6

06

76

133

%63

Caps

ules

34

12

13

45

350

%66

Caps

ules

62

22

110

78

00

%68

Caps

ules

14

54

43

513

110

0 %

69Ca

psul

es1

612

15

Oci

mum

bas

ilicu

m L

.0

00

061

0O

cim

um te

nuifl

orum

L.

00

017

215

60

Oci

mum

tenu

iflor

um L

.0

172+

156+

0Pe

rilla

seto

yens

is G

.Hon

da0

140

00

0Sa

lvia

ple

beia

R.B

r.0

1768

00

0Sa

lvia

ple

beia

R.B

r.0+

17+6

80

Senn

a a

lexa

ndrin

a M

ill.

00

500

00

Swer

tia c

hira

yita

(Rox

b.) B

uch.

-Ham

. ex

C.B.

Clar

ke0

011

00

0Ta

raxa

cum

offi

cina

le (L

.) W

eber

ex

F.H

.Wig

g.0

129

2672

00

0Ta

raxa

cum

offi

cina

le (L

.) W

eber

ex

F.H

.Wig

g.0+

129+

2672

0Ti

nosp

ora

cor

difo

lia (W

illd.

) Mie

rs0

010

00

0Tr

iant

hem

a p

ortu

laca

strum

L.

234

205

470

00

Tria

nthe

ma

por

tula

castr

um L

.23

4+20

5+47

0Tr

iticu

m a

estiv

um L

.0

00

00

15Ae

gle

mar

mel

os (L

.) Co

rrêa

Yes

00

00

00

No

Bacc

haro

ides

ant

helm

intic

a (L

.) M

oenc

h0

3954

269

783

460

010

Bacc

haro

ides

ant

helm

intic

a (L

.) M

oenc

h0+

3954

2+69

783

460+

0+10

Boer

havi

a c

occi

nea

Mill

.0

00

203

00

Cucu

mis

sativ

us L

.0

00

100

00

Eleu

ther

ococ

cus

sess

iliflo

rus

(Rup

r. &

Max

im.)

S.Y

.Hu

020

50

00

0H

iera

cium

fusc

um V

ill.

2022

00

00

0H

ypoc

haer

is m

acul

ata

L.

00

107

00

0La

ctuc

a sa

tiva

L.

00

3458

00

0Pl

anta

go o

vata

For

ssk.

00

015

021

3Pl

anta

go o

vata

For

ssk.

015

+0+2

13Sw

ertia

chi

rayi

ta (R

oxb.

) Buc

h.-H

am. e

x C.

B.Cl

arke

030

40

00

0Ta

raxa

cum

offi

cina

le (L

.) W

eber

ex

F.H

.Wig

g.0

772

00

00

Tria

nthe

ma

por

tula

castr

um L

.0

00

00

1135

Aspa

ragu

s ra

cem

osus

Will

d.Y

es0

00

00

0N

oCa

ssia

fistu

la L

.Y

es0

00

00

0N

oEc

hino

ps e

chin

atus

Rox

b.Y

es0

00

00

0N

oH

orde

um v

ulga

re L

.Y

es0

00

032

27H

orde

um v

ulga

re L

.0

0+32

+27

Yes

Ory

za sa

tiva

L.

Yes

169

100

00

0O

ryza

sativ

a L

.16

9+10

+00

Yes

Picr

orhi

za k

urro

a R

o yle

ex

Bent

h.Y

es0

00

00

0N

oPr

emna

muc

rona

ta R

oxb.

(= P

rem

na m

ollis

sima

Roth

)Y

es0

00

00

0N

oPu

erar

ia tu

bero

sa (W

illd.

) DC.

Yes

00

00

00

No

Sacc

haru

m m

unja

Rox

b. (=

Sac

char

um b

enga

lens

e Re

tz.)

Yes

00

00

00

No

Sacc

haru

m o

ffici

naru

m L

.Y

es0

00

00

0N

oSi

da c

ordi

folia

L.

Yes

00

00

4410

6Si

da c

ordi

folia

L.

00+

44+1

06Y

esSo

lanu

m in

dicu

m L

.Y

es0

00

00

0N

oSo

lanu

m su

ratte

nse

Burm

. f.

Yes

00

00

00

No

Tino

spor

a c

ordi

folia

(Will

d.) M

iers

Y

es0

00

00

0N

oAb

elm

osch

us e

scul

entu

s (L

.) M

oenc

h0

410

059

26Ab

elm

osch

us e

scul

entu

s (L

.) M

oenc

h0

0+59

+26

Alth

aea

offi

cina

lis L

.22

00

00

45Ar

achi

s co

rren

tina

(Bur

kart)

Kra

pov.

& W

.C.G

reg.

120

00

00

Bacc

haro

ides

ant

helm

intic

a (L

.) M

oenc

h0

069

783

00

0Bo

erha

via

di ff

usa

L.

032

00

00

0Ce

nchr

us a

mer

ican

us (L

.) M

orro

ne0

190

4420

1544

246

Cenc

hrus

am

eric

anus

(L.)

Mor

rone

044

20+1

544+

246

Citru

llus

lana

tus

(Thu

nb.)

Mat

sum

. & N

akai

180

00

00

Com

bret

um n

i gric

ans

Lepr

. ex

Gui

ll. &

Per

r.0

148

00

00

Conv

olvu

lus

pros

tratu

s Fo

rssk

.0

241

010

510

674

Conv

olvu

lus

pros

tratu

s Fo

rssk

.0

105+

106+

74Co

riand

rum

sativ

um L

.0

00

00

26Cu

llen

cor

ylifo

lium

(L.)

Med

ik.

012

00

00

Foen

icul

um v

ulga

re M

ill.

352

00

00

0G

oss y

pium

hirs

utum

L.

2312

024

252

72G

ossy

pium

hirs

utum

L.

23+1

2+0

242+

52+7

2H

elwi

ngia

chi

nens

is B

atal

in38

00

00

00

Hib

iscus

sabd

ari ff

a L

.0

00

014

117

1H

ibisc

us sa

bdar

iffa

L.

00+

141+

171

Hom

aliu

m c

eyla

nicu

m (G

ardn

er) B

enth

.0

00

012

0H

ypoc

haer

is m

acul

ata

L.

00

107

00

0I p

omoe

a p

aras

itica

(Kun

th) G

. Don

075

60

301

1357

28Ip

omoe

a p

aras

itica

(Kun

th) G

. Don

030

1+13

57+2

8Ja

troph

a c

urca

s L.

00

00

037

Lact

uca

sativ

a L

.0

034

580

00

Men

tha

can

aden

sis L

.0

00

00

59M

icro

berli

nia

bra

zzav

illen

sis A

.Che

v.14

00

00

0Nu

xia

opp

ositi

folia

(Hoc

hst.)

Ben

th.

520

00

00

0O

cim

um b

asili

cum

L.

00

00

040

Oci

mum

tenu

i flor

um L

.0

00

192

120

Oci

mum

tenu

iflor

um L

.0

192+

12Pl

euro

peta

lum

dar

wini

i H

ook.

f.0

00

012

0Ps

eudo

sela

go se

rrat

a (P

.J. B

ergi

us) H

illia

rd

138

00

00

0Ra

uvol

fia se

rpen

tina

(L.)

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h. e

x K

urz

00

00

084

Senn

a a

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a M

ill.

027

20

959

210

129

Senn

a a

lexa

ndrin

a M

ill.

095

9+21

0+12

9Se

nna

tora

(L.)

Roxb

.0

00

162

00

Sesa

mum

indi

cum

L.

00

00

5240

Sesa

mum

indi

cum

L.

00+

52+4

0Si

lene

ura

lens

is (R

upr.)

Boc

quet

130

00

00

Sor g

hum

bic

olor

(L.)

Moe

nch

1198

988

013

014

12So

rghu

m b

icol

or (L

.) M

oenc

h11

98+9

88+0

130+

14+1

2Ta

mar

indu

s in

dica

L.

021

00

00

Thilo

a g

lauc

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pa (M

art.)

Eic

hler

017

70

00

0Vi

gna

ung

uicu

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(L.)

Wal

p.0

130

00

0W

ithan

ia so

mni

fera

(L.)

Dun

al0

00

067

419

With

ania

som

nife

ra (L

.) D

unal

00+

674+

19Xe

noste

gia

trid

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ta (L

.) D

.F. A

ustin

& S

tapl

es i

00

00

550

Zea

may

s L.

00

00

480

Acor

us c

alam

us L

.*Y

es0

00

00

0N

oBa

copa

mon

nier

i (L

.) W

ettst

.*Y

es0

00

00

0N

oCe

lastr

us p

anic

ulat

us W

illd.

*Y

es20

011

00

2014

Cela

strus

pan

icul

atus

Will

d.20

+0+1

100+

20+1

4Y

es

Conv

olvu

lus p

luric

aulis

Cho

isy (=

Con

volv

ulus

pro

strat

us F

orss

k.)*

Yes

00

3300

511

9248

0839

37Co

nvol

vulu

s plu

ricau

lis C

hoisy

(= C

onvo

lvul

us

pros

tratu

s For

ssk.

)0

1192

+480

8+39

37Y

es

Cora

llium

rubr

um$

00

00

00

Foen

icul

um v

ulga

re M

ill.

Yes

00

00

00

No

Lava

ndul

a st

oech

as L

.Y

es0

00

00

0N

oM

ytill

us m

arga

retif

erou

s$0

00

00

0N

oNa

rdos

tach

ys ja

tam

ansi

(D.D

on) D

C.Y

es0

00

00

0N

oO

nosm

a br

acte

atum

Wal

lG

enus

is re

pres

ente

d0

00

00

0N

oW

ithan

ia so

mni

fera

(L.

) Dun

alY

es0

00

011

611

With

ania

som

nife

ra (

L.) D

unal

00+

116+

11Y

esBo

erha

via

diff

usa

L.

00

00

021

Cent

ella

asia

tica

(L.)

Urb

.0

00

010

425

8Ce

ntel

la a

siatic

a (L

.) U

rb.

00+

104+

258

Cucu

mis

mel

o L

.0

016

00

27El

euth

eroc

occu

s se

ssili

floru

s (R

upr.

& M

axim

.) S.

Y.H

u0

062

00

0Ph

ylla

nthu

s m

ader

aspa

tens

is L

.0

012

00

0So

lanu

m l y

cope

rsic

um L

.26

205

00

00

Sola

num

lyco

pers

icum

L.

26+2

05+0

0Az

adira

chta

indi

ca A

.Juss

.Y

es0

049

073

33Az

adira

chta

indi

ca A

.Juss

.0

0+73

+33

Yes

Oci

mum

tenu

iflor

um L

.Y

es0

00

066

512

8O

cim

um te

nuifl

orum

L.

00+

665+

128

Yes

Tino

spor

a c

ordi

folia

(Will

d.) M

iers

Y

es0

00

00

0N

oO

cim

um b

asili

cum

L.

00

056

90

494

Oci

mum

bas

ilicu

m L

.0

569+

0+49

4Pe

rilla

seto

yens

is G

.Hon

da0

087

00

0Si

da a

cuta

Bur

m.f.

00

160

00

Spon

dias

tube

rosa

Arr

uda

00

100

00

Vign

a m

ungo

(L.)

Hep

per

00

120

00

Aspa

ragu

s ra

cem

osus

Will

d.Y

es0

00

597

1507

353

Aspa

ragu

s rac

emos

us W

illd.

059

7+15

07+3

53Y

esBe

hnia

retic

ulat

a (T

hunb

.) D

idr.

1239

2765

00

00

Behn

ia re

ticul

ata

(Thu

nb.)

Did

r.12

39+2

765+

00

Conv

olvu

lus

pros

tratu

s Fo

rssk

.0

00

00

12La

wson

ia in

erm

is L

.0

1630

00

26La

wson

ia in

erm

is L

.0+

16+3

00

Men

tha

spic

ata

L.

00

00

023

Oci

mum

bas

ilicu

m L

.0

00

015

59O

cim

um b

asili

cum

L.

00+

15+5

9Ps

eudo

sela

go se

rrat

a (P

.J. B

ergi

us) H

illia

rd0

150

00

0Se

cale

cer

eale

L.

440

150

330

Seca

le c

erea

le L

.44

+0+1

50

Senn

a to

ra (L

.) Ro

xb.

00

00

6123

0Se

nna

tora

(L.)

Roxb

.0

0+61

+230

Sida

acu

ta B

urm

.f.0

022

20

00

Triti

cum

aes

tivum

L.

00

00

3010

9Tr

iticu

m a

estiv

um L

.0

0+30

+109

With

ania

som

nife

ra (L

.) D

unal

00

00

017

Term

inal

ia a

rjuna

(Rox

b. e

x D

C.) W

ight

& A

rn.

Yes

00

00

00

No

Alth

aea

offi

cina

lis L

.0

727

00

730

Cyam

opsis

tetra

gono

loba

(L.)

Taub

.41

00

00

0Lo

phoc

ereu

s m

argi

natu

s (D

C.) S

.Aria

s & T

erra

zas

057

00

00

Oci

mum

bas

ilicu

m L

.0

00

044

273

Oci

mum

bas

ilicu

m L

.0

0+44

+273

Pisu

m sa

tivum

L.

00

140

00

Sten

ocer

eus

chry

soca

rpus

Sán

chez

-Mej

.19

00

00

0Tr

ibul

us te

rres

tris

L.0

00

031

0Xe

rone

ma

cal

liste

mon

W.R

.B.O

liv.

028

40

00

0

40

32

00

%70

Caps

ules

11

43

817

1814

321

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Tabl

ets

1412

143

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Tabl

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92

15

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267

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Tabl

ets

30

05

Not

es. "

#Our

qua

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crite

ria s

elec

tion

for p

lat s

peci

es d

etec

tion

usin

g D

NA

met

abar

codi

ng: a

spe

cies

was

con

side

red

and

valid

aded

as

bein

g pr

esen

t with

in th

e pr

oduc

t onl

y if

it w

as d

etec

ted

in a

t lea

st 2

out

of t

he 3

repl

icat

es. $

Non

-pla

nt in

gred

ient

s. *

Scie

ntifi

c na

mes

that

indi

cate

the

use

of v

ario

us re

fined

/sta

ndar

dize

d he

rbal

sub

stan

ces

with

in th

e pr

oduc

t; w

here

as, a

ll ot

hers

indi

cate

the

use

of n

ot e

xtra

cted

pla

nt m

ater

ial,

incl

udin

g si

mpl

y pr

oces

sed

and

com

min

uted

pla

nt m

ater

ial,

with

in th

e pr

oduc

t."

15

87

110

0 %

77Po

wde

r1

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3

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der

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