Approaches towards Evaluation of Medicinal Plants prior to

Clinical Trials

Organized By

The Foundation for Medical Research 84-A, R. G. Thadani Marg, Worli, Mumbai – 400018, India

At

Yashwantrao Chavan Academy of Development Administration (YASHADA), Pune, India

November 8, 2006

Proceedings

Acknowledgements

The Foundation for Medical Research gratefully acknowledges the funding

support received for the workshop from the Pirojsha Godrej Foundation, Mumbai,

the Council of Scientific and Industrial Research, Government of India and The

Lotus Trust, Mumbai.

The publication of the workshop proceedings has been made possible through

the funding obtained from the Hinduja Foundation, Mumbai.

INDEX Workshop address

1

Session 1: Translation of ethnobotanical information obtained from a community for identification, subsequent testing and utilization of plant material Chairperson: Darshan Shanker

1.1 Field Tested Participatory Methodology for Rapid Assessment of the Community's Therapeutic Use of Medicinal Plants Darshan Shankar

6

1.2 Plant Based Traditional Knowledge for Improved Health Care Delivery System Raghu Bir S. Rawat

14

1.3 Ethnobotanical Scenario in Gujarat: Status and Prospects in Primary Health Care Minoo Parabia, Reddy M. N., Smita Pathak, and Falguni Sheth

24

1.4 Suggestions towards Improving the Quality of Ethnobotanical Surveys and Research Pundarikakshudu Tetali

34

Session 2: Principles of pre clinical evaluation of plants using appropriate bioassays Chairperson: Urmila M. Thatte

2.1 Bioassays in Traditional Medicine (Abstract only)

Urmila M. Thatte

42

2.2 Novel Imaging System for Determining Anti-Cancer Activity Dharmalingam Subramaniam, Rama P. Ramanujam, Joseph M. Betz, Panchapagesa M. Murali, Courtney Houchen and Shrikant Anant

44

2.3 Principles of pre-clinical evaluation of plants using appropriate bioassays (Abstract only) Jaswant Singh

57

2.4 Preclinical testing of medicinal plants: advantages and approaches Poonam G. Daswani, Brijesh S. and Tannaz J. Birdi

60

2.5 Challenges in Preclinical Testing of Traditional Medicines: In Search of Solutions (Abstract only)

Nirmala Rege

78

Session 3: Standardization of plant material Chairperson: Arvind A. Natu

3.1 Traditional Knowledge Guided Testing of Quality, Safety and Efficacy of Herbal Medicines Padma Venkatasubramanian

80

3.2 Principles of Quality Control, Standardization and Chemo profiling of Medicinal Plants and ISM Preparations R K. Khajuria and S. G. Agarwal

89

3.3 Approaches to Standardization of Medicinal Plant Preparations Brijesh S., Poonam G. Daswani and Tannaz J. Birdi

102

3.4 Medicinal Plant Compositional Consistency for Reliable Therapeutic Action Profiling: Key Issues and Concerns for Phytotherapeutics Rajender S. Sangwan

119

Abridged overview

129

List of participants

136

Workshop Address

Dr. Antia, ladies and gentlemen,

Good morning and welcome to this historic city of the Peshwas and to this

workshop organized by the Foundation for medical Research, Mumbai.

Established in 1974 as a public trust and an educational institute affiliated to the

University of Mumbai and located in a still beautiful area of Mumbai with a

glorious view of the Arabian Sea, this Foundation is 33 years young and is a

brainchild of Dr. Antia who we are honored to have on this dias with us. My

inheritance of piloting this Foundation, which began in September 2006 on Dr.

Antia’s retirement, is built on his mixed legacy of both innovative thought and

compassion both of which can see the light of day only through hard work and

motivation. I would like to take the opportunity of this occasion to pay tribute to

the originator of this precious inheritance, to my teacher and mentor, who has led

this Foundation for the past 33 years not only on the path of excellent and creative

science but also on a path of useful and compassionate science for the people of

this country.

Though small in size, the Foundation has developed a formidable reputation

in all the fields it has touched viz., the neurology and immunology of leprosy,

epidemiology and evolution of drug resistant tuberculosis and the preclinical

screening of medicinal for infectious diseases. The selection of these areas of the

Foundation’s work was guided firstly by the relevance quotient to the health

problems of the vast majority of this country, the availability of elegant but simple

technology to address these problems and the asking of the right questions that

could provide insights to larger and even universal issues.

To talk about the importance of herbal remedies or medicinal plants to this

audience would be impertinent. Nevertheless the work in the area of medicinal

plants was initiated by my senior colleague and long time friend, Tannaz Birdi, in

the late 90’s in collaboration with Vaidya Antarkar and our sister FRCH. The

initial documentation of herbal remedies for diarrhoeal diseases in children

whetted her appetite to ask more challenging questions. Cautious about jumping

straightaway into clinical trials, FMR chose to look at the fundamental area of

1

preclinical testing of antidiarrhoeal plants. This was a generic choice grounded in

several considerations, viz.,

• Geographical and environmental variation in medicinal plant properties

• Symbiotic and antagonistic interaction between plant constituents

• The identification of novel pathways of plant action that exert their action

through host parasite interaction rather than by direct action on pathogens

which unfortunately forms the bulk of screening assays employed currently.

Whilst the thrust in the work was to devise innovative assays that could

identify plant product actions, a rather major but ill expressed goal included a

desire to contribute to a resurgence of India’s traditional science and healing

heritage. The work initiated was meant to provide Ayurveda and modern science

and medicine articulated eloquently by Prof. RD. Lele in his seminal publication.

Ayurveda considers disease as disequilibria at the molecular level either

through excessive interaction between molecules (atiyoga) absence of interaction

(ayoga)) and erroneous interaction (mithyayoga). Restoration of that equilibrium

is the objective of medicine. The tridosh or the personalized concept is essentially

a concept also of molecular biology, which has expanded our understanding of not

only disease but also health through concepts of cell signaling and

communication, repair enzymes and molecular policemen and quiet cellular death

such as apoptosis.

Based on observation and experiential wisdom, Charaka, Sushruta and

Vagbhat described 700 herbal drugs with their properties and clinical effects.

Based on these they described 50 categories of drugs. It is our belief that by

eschewing the pathway of fishing expeditions that the modern pharmaceutical

industry often emplys, subjecting Ayurvedic drugs to mechanism based screening

will be very rewarding because their choice has already been backed by centuries

of experiential wisdom provided. We also consider the dynamism of evolution and

development in modification of plant properties over the centuries. As the R&D

process is long and demands large investments, research interests are lacking in

both industry and academia. Repeated attempts by the DST to arrange meetings

for arriving at preclinical screening norms had no takers and the meetings had to

be eventually cancelled. Pharmacopoeia standards and volumes on 326 single

drugs are published by the AYUSH department but there are no monographs for

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extracts that could reduce bulk drug utilization, help develop dosages, aid quality

assurance and reduce microbial / symptom burden.

This Workshop has attempted to devise a holistic programme that

encompasses ethnobotany and post assay downstreaming and standardization

besides biological screening in preclinical activities. The size of the Workshop has

been kept deliberately small so that the invited speakers and participants, all of

whom have been significant contributors in their field, can deliberate with focus

and in depth in the substantial time available after the presentations. The lead

discussants selected with same care as the participants have been pre-prepared

with presentation content so that the issues that they raise are critical. The

essential contributions that can be expected from this one day Workshop are

• Addressing of specific pitfalls that are likely to be encountered in the three

areas of ethnobotanical data collection, assay design and standardization

• Articulation of guiding principles for each of the three areas

• Articulation of inputs that pre-clinical screening can provide to clinical trials

• Building of a database for expertise available for each of the areas

These may please be borne in mind during the discussions so that definite

issues and recommendations are possible as outcomes.

It is not customary to thank in-house hands and heads who have helped in

shaping this Workshop. Nevertheless those who have supported this Workshop

with belief and finances deserve a mention:

• Mr. Jamshyd Godrej and the Pirjshah Godrej Foundation for their long-term

support to both FMR and this workshop

• The Hinduja Foundation for their contribution to the publication of the

Workshop proceedings

• The Council for Scientific and Industrial Research of the Govt. of India

• Dr. Bhushan Patwardhan of the University of Pune who lead us to many of

today’s participants and agreed to have this as a quiet satellite of the World

Ayurvedic Congress starting on 10th November at the University

• Our collaborators including the FRCH, Pune and the field investigators at its

rural project site of Parinche in Purandhar taluka; Dr. Tetali and his student

from The Naoroji Godrej Centre for Plant Research at Shirwal; Drs. Natu and

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Rojatkar from The National Chemical Labs, Pune; Drs. Minoo Parabia and

Kothari from South Gujarat University, Surat; Dr. Bhate from Analytical

Solutions, Mumbai. Our clinician friends include the late Vaidya Antarkar

and Dr. H.B. Singh, formerly of The Poddar Ayurvedic College Mumbai. We

hope we can make more friends and partners by the end of the day.

• The studies at FMR have been funded by Sir Dorabji Tata and Sir Ratan Tata

Trusts, Department of Science and Technology, Government of India and

now The Indian Council for Medical Research.

• Mrs. Jayalakshmi Iyer, FMR, for her administrative assistance throughout.

• Finally I would like to thank Mrs. Rajam John, Col. Phatak for the transport

team of FRCH for supporting us in their home territory. Also the staff of

Yashada for their extreme helpfulness and in ensuring all arrangements.

If the eminent physician Charaka was here instead of me giving you this

introduction then this is what he may have said;

I had charged you to learn the medical texts, but never to lose sight of reason

in the practice of medicine

Obsession with the written word is the habit of the mediocre; the wise go

beyond and inherit the experience of the world.

I urge you to learn from the sages and the shepherds: both are teachers in

their own way

I am not immutable or an icon. You made me one unaware that other nations

are sailing on.

You have taken to heart that where you proclaimed I was super-intelligent, I

have none; where you found plentiful cures in my texts, you now find none.

Distraught you have replaced me with new icons.

You have enthroned reason to the rejection of all other authority and justified

morality only in terms of reason and knowledge. It has escaped you that by setting

them apart you have enslaved morality to knowledge.

Does it worry you that the tree of knowledge without morality has given you a

harvest of bitter fruits?

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Do not expect miracles of me. I have left you a heritage which though all-

embracing exists within a world of unknown reserves of knowledge, experience

and faith. It behoves you to explore the trackless land as your forefathers did.

But far from seeking only knowledge, you should celebrate a good mind and

good conduct, free from extreme positions and errors of judgement.

In seeking to know my legacy, you but see the leaves of a universal tree too

vast for your eyes. May your sight grow and your quest never ends.

Thank you for your hearing and above all your presence and participation at

this Workshop.

Nerges Mistry, Ph. D. Director, The Foundation for Medical Research

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1.1 Field Tested Participatory Methodology for Rapid Assessment of the Community's Therapeutic Use of Medicinal Plants Darshan Shankar Foundation for Revitalisatoin of Local Health Traditions (FRLHT), Bangalore – 560064, India Email: darshan.shankar@frlht.org

1. Introduction

India has had a rich, vibrant and diverse cultural history. An important component

of this culture and tradition is that of health and healing. Thus there is a large

health and healing related knowledge base present in all ethnic communities

across the diverse ecosystems. However, over the last few centuries, this

knowledge base has been diluted with increased influences from the mainstream

culture, which is derisive of local health traditions. It is important to urgently put

in place effective documentation and assessment programs to revitalize local

health traditions otherwise this great people’s health culture will be irretrievably

lost.

There are two key Sanskrit words that describe the nature of the Indian

knowledge society. These are prakrit & sams-krit. The word prakrit refers to

ecosystem & ethnic community specific knowledge traditions. It is derived from

the term prakriti or nature, which means that dynamic phenomenon (kriti) that has

always existed (pra). The prakrit knowledge therefore refers to such emperical

traditions of knowledge that are directly derived from nature they include prakrit

language, arts, music, weaving, agriculture, architecture, and of course healthcare.

The word sams-krit refers to such dynamic phenomenon (kriti) that has been

refined or modified (samskar – from the prakrit state). This word thus refers in

Indian tradition to the various shastras or sciences like linguistics (vyakaran), the

fine and performing arts (shilpa, sangeet, nritya etc.,), agriculture (krishishastra),

architecture (vastu shastra) and healthcare (ayurveda).

In Indian society the samskrit traditions enjoy a symbiotic relationship with

the prakrit because samskriti (culture: in the broad sense of the term including all

arts and sciences) is derived from prakriti (nature). Thus ayurveda and folk

healthcare (lok swasthya parampar) are related and one should not visualize the

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future of Ayurevda, without envisaging the future of its prakrit counterpart which

lives amongst common folk in the society in both rural and urban areas.

The folk health traditions or lok swasthya paramparas are embedded in the

lifestyle, diet and health practices of thousands of local communities all over

India.

There are estimated to be around a million folk healers in the country. The

break up of these healers is shown in the chart below:

Carriers of village-based health traditions:

Traditional carrier Subjects Numbers Housewives Home remedies – Food & nutrition Millions Traditional Birth Attendants Normal deliveries 7 lakhs Herbal Healers Common ailments 3 lakhs Bone-setters Orthopaedics 60,000 Visha Vaidyas [Snake, Scorpion, Dog]

Natural poisons 60,000

Specialists Eyes Skin Respiratory Dental Arthritis Mental diseases Liver GIT Wounds Fistula/Piles

1000 in every State in rural communities

Figures based on extrapolations from micro-studies.

Apart from the distinct class of folk healers, there are also several millions of

mostly rural (and to a much lesser extent urban) knowledgeable households who

are also carriers of the community based oral health traditions of India. The

households have knowledge of home remedies about local food and their

nutritional value. The health traditions of households are based on the local eco-

system resources.

In the context of self-reliance of local communities in primary health care, it

is very important to strengthen and revitalize these community based health

traditions. Their revitalization holds the key to health security of the rural and

urban poor, even in the 21st century.

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1.1 Genesis of the Documentation of LHTs.

In the early nineties, the Foundation for the Revitalisation of Local Health

Traditions (FRLHT), started work with the mission for the revitalization of

India’s health traditions. FRLHT has been involved in Documentation and

Rapid Assessment (DRALHT) of the community's therapeutic use of

medicinal plants. Over the years, FRLHT has been able to consolidate its

work in the form of a fairly standardized documentation and assessment

process.

The DRALHT process consists of the following 3 steps:

• Ethnographic documentation and Identification of important local health

traditions

• Community validation of these identified traditions along with multi-

disciplinary assessment (using a comprehensive Ayurveda, Siddha,

Unani, and Pharmacology data-base) for encouraging the best practices,

adding to the incomplete and discouraging distorted ones.

• Promotion of the use of the validated health traditions at household and

community level.

1.2 Why Assess Local Health Traditions?

Assessment of community's therapeutic use of medicinal plants helps:

• To assess efficacy of local health traditions.

• To assess, differentiate and identify the sound practices from the

unsound.

• To understand the worldviews/ epistemologies of folk practitioners and

household health traditions.

2. The Method

The documentation of local health traditions is aimed at systematic recording of

Local Health Traditions and the resources used in it.

The next step after documentation is Rapid Assessment of Local Health

Traditions (RALHT). RALHT is aimed at selecting the best practices in household

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health traditions and folk health traditions, for their promotion in primary health

care, by means of a rapid assessment exercise. This form of assessment is termed

‘Rapid’ as it does not involve laboratory or clinical studies.

Community’s own assessment based on empirical data on efficacy

Desk Research consulting databases & Literature References from Ayurveda, Siddha, Unani and modern pharmacology Rapid Assessment of prioritized

Local Health Traditions

Identifications of Effective Remedies

Design of Promotional Programs led by Community Based Organizations and facilitated by NGOs

Community’s Prioritization of Health Conditions for assessment

Comprehensive ethnographic Documentation of Local Health Traditions

2.2 Data Sheets for ISM, Community & Pharmacological assessment

LHT DATA SHEET Local name of the prioritized condition Description (Cause/ stages) of the condition Ingredients name

Botanical Name

Part used Proportion used

Purification/ Remarks

Preparation of the medicine:

Dosage & Administration How much? How many times? How long? Pathya/ Apathya (Food & Regime advice) Any other remarks (contra indications, special precaution etc)

Reports of Successful Community Use Specify the number of people who reported using the remedy OR

Specify the number of villages, the remedy reported from OR

Specify the number of Talukas/ Districts remedy was reported from

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AYURVEDIC DATA SHEET (For individual resource references)

1 Local Name 2 Scientific Name 3 Rasa 4 Guna 5 Veerya 6 Vipakam 7 Dosha Karma 8 Prabhava 9 Rogagnatha

References from classical texts:

Disease/ Condition & stage

Text Chapter Sthana Mode of Administration

Other ingredients

Remark

SIDDHA DATA SHEET (For individual resource references)

No Plants/ Minerals/ Animal Names 1 Local Names 2 Scientific Name 3 Suvai 4 Gunam 5 Veeriyam 6 Vipakam 7 Others

References from classical texts: Disease Type

Condition Text Chapter Section

Mode of Administration

Other ingredients

Remarks

UNANI DATA SHEET

No Plants/ Minerals/ Animal – Unani Names 1 Local Names 2 Scientific Name & synonyms if any including family:

Part used

3 Mahiyath (Morphology): 4 Mizaj (Temperament):

10

5 Afaal (Actions): 6 Istamal (Uses): 7 Nafaekhas (Specific action): 8 Muzir (Contra Indication): 9 Muslah (Antidote): 10 Badal (Substitute): 11 Meqdar-e-kurak (Dosage):

PHARMACOLOGY DATA SHEET

Resource Name Botanical / Scientific name: (including family name) Part LHT uses Active constituents Biological activity Clinical reports Remarks Reference

2.3 Botanist

Herbarium records of the plants used by the community are sent to competent

local taxonomist for identification.

2.4 Desk Research Report

The report consists of assessment of the local health practices based on the

three Indian systems of medicine and the pharmacology report. An

ethnographically sensitive approach is used to draw correlation of the locally

described health problem with an ISM or modern health condition. This is a

pre-requisite to assess the local remedies. The details of the locally used

formulation such as the ingredients in local name, botanical names, part used,

proportions used, method of preparation, form of medicine and the dosage

used are conveyed to the evaluators.

3. RALHT Workshop

This is an essentially participatory method of assessment, which involves the

community, vaidyas, and medical practitioners from various systems of medicine,

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pharmacologists, botanists and the facilitators (NGOs, People’s Organisations

etc.)

3.1 Workshop Proceedings

The prioritized list of health conditions/illness and the causes and symptoms

are presented to the panel of practitioners present. The panel discusses with

the community (in the case of RALHT for household remedies) and the folk

healers (in the case of RALHT for folk remedies) and seeks clarifications if

required. The members of the panel then finalise their respective rapid

assessment proformas.

After the panel has filled in their data sheets, the following steps are

undertaken:

• Treatment or practice followed in the community with all the details as

given in the LHT data sheet.

• Presentation about understanding of health conditions and illness in the

community.

• Presentation of the assessment by writing on the blackboard or reading it

out.

• Initiating the discussion between the community members and

practitioners.

During the analysis, the following are noted:

• The total number of formula having consensus from the community and

all the experts of the Indian Systems of Medicine and modern medicine.

• The number of formulae having consensus from the community and one

of the ISM experts.

• Formulae suggested for modification or additions.

• Formulae to be discouraged as per consensus of the participants.

At the end of the exercise an ‘Analysis Chart For Assessment Result’ is

presented as given below:

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Community Folk healer Ayurveda Practitioner Siddha Practitioner Unani Practitioner Tibetan Practitioner Literature reference of ISM Literature: Modern Pharmacology Western Bio Medicine Practitioner Assessment (Promotion) Remarks

3.2 Promotion of the best traditional health practices

The positively assessed remedies are promoted among the

communities in the same region. This is taken up through promotional

workshops and awareness programmes for the households.

The end result of the program can be used to develop medicinal

plant kits for the home herbal gardens.

The data collected from the community is kept in a place as

decided by the community, where the community can access it. It

serves as a register of the health traditions of the area.

4. Conclusion

The key objective of the above methodology is to help revitalize household

remedies and folk healing practices that can contribute to the primary health

care of rural communities.

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1.2 Plant Based Traditional Knowledge for Improved Health Care Delivery System Raghu Bir S. Rawat Medicinal and Aromatic Plants Programme in Asia, International Centre for Integrated Mountain Development (ICIMOD), GPO Box 3226, Kathmandu, Nepal Email: rrawat@icimod.org Introduction:

The identification of plants useful to human beings from natural stands

commenced in prehistoric times. Experiments and trials were the two main ways

through which humans have learnt the various uses of plants. The use of plant

resources for medicinal and other purposes is one of a number of practices

developed by ancient people.

Plant derived medicines are used in all civilizations and cultures and, hence,

plants have always played a key role in health care systems worldwide. In most

developing countries, the indigenous modes of herbal treatment are a part of the

culture and the dominant method of healing therapy. These remedies, with a

considerable extent of effectiveness, are socially accepted, economically viable

and, mostly, are the only available source. Plants used in traditional medicine,

therefore, have a critical role in the maintenance of health all over the world.

Traditional medicine:

Initially all drugs were natural, such as vegetable, animal and mineral products in

their crude forms. Before the emergence of the twentieth century, all medical

practice was what we now call the traditional system. The World Health

Organization (WHO) has defined traditional medicine as “the sum total of all the

knowledge and practices, whether explicable or not, used in diagnosis, prevention

and elimination of physical, mental or social imbalance and relying exclusively on

practical experience and observation handed down from generation to generation,

whether verbally or in writing” (WHO, 1978). Traditional medicine is, therefore,

used mainly to distinguish the ancient and culture-bound health care practices,

which existed before the application of science to health matters in official

modern scientific medicine or allopathy. Some frequently used synonyms are

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indigenous, unorthodox, alternative, folk, ethno, fringe, and unofficial medicine

and healing.

All traditional medicines have their roots in folk medicines and household

remedies. WHO has listed 20,000 medicinal plants used in different parts of the

world. Other estimates indicate the number to range between 35,000 and 70,000

worldwide (Lewington, 1993; Bhattarai and Karki, 2004). Some of the earliest

remedies and prescriptions became widespread and were subjected to certain

refinements, revisions and improvements through practices by trained or

experienced medicine men and then got incorporated in organized or codified

systems of medicines. These organized or codified traditional medical systems

employ relatively few species, viz. 500-600 in traditional Chinese medicine, 1100

in Tibetan medicine (Sowangpa), 1500 in the Ayurveda, 450 in the Homoeopathy,

342 in the Unani, and 328 in the Siddha systems. Also, the various plants used in

these systems happen to be common both species-wise as well as disease-wise in

most of the cases. However, a major bulk of the plant species used as medicines

remained endemic to certain regions or people. Due to lack of communication,

intermingling and breeding of ideas, and varying ways of life, many of these

earlier remedies survived only by word of mouth from generation to generation.

This category of information and their uses still dominates the healing tradition in

the world.

WHO Concern:

Many countries, especially in Asia, Africa and East European countries, have

officially recognized the use of plant-based traditional medicine in their health

care delivery systems. WHO has estimated that 80% of the world’s populations

rely primarily on traditional medicine (WHO, 1978; Okerele, 1992). Considering

the coverage and effectiveness of various systems of traditional medicine

throughout the world, the Alma-Ata Declaration of the WHO in 1978 proposed

the theme “Health for all by the year 2000”. The examination of drugs used in

traditional medicine in the various parts of the world is, therefore, one of the

priority programs of the WHO (Pasquale, 1984).

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Characteristics of Traditional Medicines:

In almost every part of the world traditional healers are practicing means of health

care coverage that is culturally acceptable to the local population, dealing more or

less satisfactorily with many of their health problems. Animal sacrifices,

exorcism, religious ceremonies, preaching of supernatural powers and evil spirits,

psychotherapy, cauterization, prevention, surgery, use of animal and mineral

products, etc., are the important parts of most folk therapies. Occasionally, there

may be liberal use of musico-therapy, often associated with tribal dances and with

ritually induced trances or hypnotism. Herbal medicine is, however, the most

popular.

There are common features for herbal medicines:

• Herbal medicines are different from clinically defined medicines in their

character as well as in their medicinal value.

• They are based mostly on herbal products.

• Usually, they are multi-drug formulations including animal and mineral

products as essential components or additives.

• In herbal therapy, data on pre-clinical investigations are often incomplete

although in majority of cases the therapeutic experiences have been

accumulated over centuries.

• Some of them follow practices based on, for example, mistaken beliefs, faulty

experimentation, or inaccurate information that can be dangerous.

• They mostly include empirically defined doses and course of medication.

• The identity of plant species used is often controversial.

• Safety measures are poorly adopted, in most cases.

• Additives are frequently used; many of them also have therapeutic actions.

Use of plants in modern medicine

Many drugs of modern medicine have had their origin in traditional medicine.

Some common examples include the discovery of the alkaloid diosgenin in

Dioscorea deltoidea used as source for the partial synthesis of cortisone and

steroid hormones in the forties, the discovery of the hypotensive alkaloid reserpine

in Rauvolfia serpentina and the analgesic alkaloid aspirin in Filipendula ulmaria

in the fifties, the discovery of anti-asthmatic alkaloid ephedrine in Ephedra sinica

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and the anti-cancer alkaloid podophyllotoxin in Podophyllum hexandrum in the

sixties, to mention a few (Table 1).

Table 1: Important plant-derived drugs used in modern medicine

Drug Medicinal Use Plant species Family Ajmalicin Antihypertensive,

tranquilizer Apocynaceae

Ajmalin Heart arrhythmia Rauvolfia serpentina Apocynaceae Ajmaline Antihypertensive,

tranquilizer Rauvolfia serpentina Apocynaceae

Asprin Analgesic, anti-inflammation

Filipendula ulmaria Apocynaceae

Artemisine Antimalarial Artemisia annua Asteraceae Atropin Ophthalmology Atropa belladonna Solanaceae Benzoine Oral disinfectant Styrax tonkinensis Styracaceae Caffeine Stimulant Camellia sinensis Theaceae Camphor Rheumatic pain Cinnamomum

camphora Lauraceae

Cascara Purgative Rhamnus purshiana Rhamnaceae Cocaine Ophthalmologic

anaesthetic Erythroxylum coca Erythroxylaceae

Codeine Analgesic, antitussive

Papaver somniferum Papaveraceae

Colchicine Gout Colchicum autumnale Liliaceae Demecolcine Leukemia Colchicum autumnale Liliaceae Deserpidine Hypertension Rauvolfia canescens

Rauvolfia serpentina Apocynaceae Apocynaceae

Dicoumarol Thrombosis Mililotus officinale Fabaceae Digitoxin Atrial fibrillation Digitalis purpurea Scrophulariaceae Digoxin Atrial fibrillation Digitalis purpurea Scrophulariaceae Digoxin Cardiotonic Digitalis lanata Scrophulariaceae Diosgenin Induces

steralization Dioscorea deltoidea Dioscoreaceae

Emetine Antiamoebic Psychotria ipecacuanha Rubiaceae Emetine Amoebic

dysentery Cephaelis ipecachuanha

Rubiaceae

Ephedrine Bronchodilator Ephedra sinica Ephedraceae Eugenol Toothache Syzygium aromaticum Myrtaceae Gallotanins Hemorrhoid

suppository Hamamelis virginiana Hamamelidaceae

Gossypol Male contraceptive Gossypium herbaceum Malvaceae Hyoscyamine Anticholinergic Atropa belladonna

Datura stramonium Hyoscyamus muticus

Solanaceae

Hyoscyamine Anticholinergic Hyoscyamus niger Solanaceae Ipecac Emetic Cephaelis ipecacuanha Rubiaceae Ipratropium Bronchodilator Hyoscyamus niger Solanaceae

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Khellin Vascodilator Ammi visnaga Apiaceae L-DOPA Antiparkinsonian Mucuna pruriens Papilionaceae Marsilin Sedative,

anticonvulsant Marsilea minuta Marsileaceae

Morphine Analgesic Papaver somniferum Papaveraceae Noscapine Antitussive Papaver somniferum Papaveraceae Papain Attenuates mucus Carica papaya Caricaceae Papaverine Antispasmodic Papaver somniferum Papaveraceae Physotigmine Glaucoma Physostigma

venenosum Fabaceae

Picrotixon Barbiturate antidote

Anamirta cocculus Menispermaceae

Pilocarpine Glaucoma Pilocarpus jaborandi Rutaceae Podophyllotoxin Vermifuge, Cancer Podophyllum

hexandrum Podophyllum peltatum

Berberidaceae Berberidaceae

Proscillaridin Cardiac malfunction

Drimia maritime Liliaceae

Protoveratrine Hypertension Veratrum album Liliaceae Pseudoephedrine Central nervous

system stimulant Ephedra sinica Ephedraceae

Pseudoephedrine Rhinitis Ephedra sinica Ephedraceae Psoralen Vitiligo Psoralea corylifolia Fabaceae Quinidine Cardiac arrhythmia Cinchona pubescens Rubiaceae Quinine Malaria

prophylaxis Cinchona pubescens Rubiaceae

Rescinnamine Hypertension Rauvolfia canescens Rauvolfia serpentina

Apocynaceae Apocynaceae

Reserpine Hypertension Rauvolfia canescens Rauvolfia serpentina

Apocynaceae Apocynaceae

Rutine Decreases capillary fragility

Ruta graveolens Rutaceae

Sennoside-A Laxative Cassia angustifolia Caesalpiniaceae Sennoside-B Laxative Cassia angustifolia Caesalpiniaceae Scopolamine Motion sickness Datura stramonium Solanaceae Stigmasterol Steroidal precursor Physostigma

venenosum Fabaceae

Strophanthin Congestive heart failure

Strophanthus gratus Apocynaceae

Taxol Overian cancer, Breast cancer

Taxus brevifolia Taxus wallichiana

Taxaceae Taxaceae

Teniposide Bladder neoplasms Podophyllum hexandrum Podophyllum peltatum

Berberidaceae Berberidaceae

THC Antiemetic Cannabis sativa Cannabinaceae Theobromine Diuretic,

myocardial stimulant, vasodilator

Camellia sinensis Theaceae

Theophylline Cardiac stimulant, Camellia sinensis Theaceae

18

vasodilator, smooth muscle relaxant

Theophylline Diuretic, asthma Camellia sinensis Theaceae Toxiferine Surgery, relaxant Strychnos guianensis Loganiaceae Tubocurarine Muscle relaxant Chondrodendron

tomentosum Menispermaceae

Vinblastine Hodgkin’s disease Catharanthus roseus Apocynaceae Vincristine Pediatric leukemia Catharanthus roseus Apocynaceae Xanthotoxin Vitiligo Ammi majus Apiaceae

Today, there are 121 pure chemical substances extracted from about 130

species of higher plants used in the modern pharmacopoeias throughout the world.

Out of these, 89 plant derived drugs, currently used in modern medicine, were

originally discovered through the study of traditional cures and folk knowledge of

indigenous people (Choudhary and Atta-ur-Rahman, 2002; Bhattarai and Karki,

2004). In many countries modern medicines have replaced plants with many

synthetic products but almost 30% pharmaceutical preparations are still obtained

directly or indirectly from plants, majority of them based on ethnobotanical

information (Marino-Bettolo, 1980). There are 121 drugs in current use in the

USA derived from plant, with 95 species acting as sources, more than one drug

being obtained from some species. Approximately 25% of all prescriptions

dispensed in the United States contain plant extracts or active ingredients obtained

from, or patterned after, plant materials (Anonymous, 1994). Likewise, about 25%

of all prescription drugs in the Organization for Economic Cooperation and

Development (OECD) countries, and up to 60% of those in Eastern Europe,

consist of unmodified or slightly altered higher plant products (The Lancet, 1994).

Preference of herbal drugs in modern societies

Recent years have witnessed a renewed interest in plants as pharmaceuticals in the

western world. In the global context, herbal medicines flourish as the method of

therapy of choice in many parts of the world. In recent years, the increasing

demand for herbal medicines is being fueled by a growing consumer interest in

natural products. Now it is finding new popularity as an alternative conventional

medicine even in the industrialized countries and the adoption of crude extracts of

plants for self-medication by the general public is in the increase.

19

The landmark survey by Eisenberg et al. (1993) demonstrated a great demand

for alternative medical services in the United States. McPortland and Soons

(1997) reported that one-third of Americans used alternative medicine, and

approximately 10% of the Americans visited an alternative practitioner in the

calendar year 1990.

Safe and Efficacious Herbal Drugs

The overall goal in drug development is quality, safety and efficacy. All measures

in drug development are directed to this goal. The requirements of health

authorities on quality, safety and efficacy are standardized on a high level based

on the development procedure for the herbal as well as synthetic drugs. Health

authorities are reluctant to accept traditional drug preparations from other cultural

areas without well-documented data on quality, safety and efficacy. In many

developing countries, appropriate utilization of local resources to cover drug

needs is dependent on preliminary scientific study to determine the efficacy and

safety of the preparations based on plant drugs that are used on an empirical basis

in traditional medicine.

Despite many shortcomings, the number of users of herbal drugs is increasing

in the developing as well as the industrialized world. Traditional herbal medicines,

although currently serving the health care needs of majority of the world’s

population, can be further increased in coverage and broadened in terms of safety

and efficacy provided that some basic principles of drug preparation, evaluation

and uses are brought into practice.

The message is clear that phytotherapy acts as a bridge between traditional

medicine and modern medicine. The development of plant derive drugs have

always been a multi-step procedure starting with a crude extract followed by the

standardized extract and ending up with isolated constituents. Quite often

sufficient quality control and drug standardization is lacking for traditional

recipes. Ethnopharmacological leads have resulted in the introduction of new

single molecule drugs but have a greater role to play if crude extracts are accepted

for clinical use in the West.

20

Conclusions

Considering the status and trend of herbal medicine, and also considering the

shortcomings in the herbal mode of treatments, it is high time to suggest the

following measures:

• The herbal samples and raw materials should be genuine, well-identified and

harvested from the wild or organically cultivated sources and their subsequent

handling following the internationally recognized practices like those

suggested by WHO (2002, 2003) and other international bodies

(WHO/EDM/TRM, 2002; WHO/IUCN/WWF/TRAFFIC, 2004; Leaman and

Salvador, 2005).

• Herbal medicine for laboratory and clinical trials should be considered

together with its different components including substitutes and additives.

• The overall pharmaceutical practice including methods of drug preparation,

preparation of dosage forms, dose and course and route of drug

administration should be fully considered while conducting the laboratory and

clinical trials.

• Clinical trials and detailed case studies should be documented and well-

disseminated.

It has been realized that many of the modern tools used for the cure of

diseases are blunt, brutal, cumbersome and costly. What is needed is one that is

safe, effective and less expensive. Billions of people on earth depend mainly on

herbal medicines that have been significantly contributing to their primary health

care needs. Therefore, the role of medicinal plants in providing effective health

care services in most rural parts of the world is likely to continue far beyond the

recently emerged 21st century.

In the present state of rapidly expanding population, the rural health-related

challenges are enormous. The timely question, of course, is how the health

authorities are going to face these challenges. The most acceptable solution

would be to bring health care services to the rural people by helping them to apply

what are available in their environment and what is traditionally known to them.

But, otherwise, for the rural scenario to get better with more equality and evenly

shared health care facilities, the struggle ahead is sure to be long and difficult.

21

References

1. Anonymous, 1994. Interest increases in plant as medicine. Industrial

Uses/IUS 4: 17-19.

2. Bhattarai N, Karki, M. 2004. Medicinal and aromatic plants - Ethnobotany

and conservation status. In: J. Burley, J. Evans and J. Youngquist (Eds.).

Encyclopedia of Forest Sciences. Academic Press, London, UK. pp. 523-532.

3. Choudhary MI, Atta-ur-Rahman. 2002. Recent trends in medicinal plant

research. In: N. Bhattarai and M. Karki (Eds.). Sharing local and national

experience in conservation of medicinal and aromatic plants in South Asia.

Ministry of Forests and Soil Conservation, HMG/Nepal and International

Development Research Centre, SARO, New Delhi, India. pp. 62-66.

4. Eisenberg DM, Kessler RC, Foster C, Norlock FE, Calkins DR, Delbanco TL.

1993. Unconventional medicine in the United States – Prevalence, costs, and

patterns of use. N Engl J Med. 328: 245-252.

5. Leaman DJ, Salvador S. 2005. An international standard for sustainable wild

collection of medicinal and aromatic plants (ISSC-MAP): Principles, criteria,

indicators, and means of verification. Draft 2, April 2005. Steering group for

the development of practice standards and performance criteria for the

sustainable wild collection of medicinal and aromatic plants. 52p.

6. Lewington A. 1993. Medicinal plants and plant extracts: A review of their

importation into Europe. Traffic International, Cambridge, UK.

7. Marino-Bettolo, GB. 1980. Present aspect of the use of plants in traditional

medicine. J Ethnopharmacol. 2: 5-7.

8. McPartland JM, Soons KR. 1997. Alternative medicine in Vermont – A

census of practitioners: prevalence, patterns of use, and national projections.

The Journal of Alternative and Complementary Medicine 3(4): 337-342.

9. Okerele O. 1992. WHO Guidelines for the Assessment of Herbal Medicines.

Fitoterapia 63 (2): 99-110.

10. Pasquale AD. 1984. Pharmacognosy: The oldest modern science. J

Ethnopharmacol. 11: 1-16.

11. The Lancet, 1994. Pharmaceuticals from plants: great potential, few funds.

The Lancet 343: 1513-1515.

22

12. WHO. 1978. The Promotion and Development of Traditional Medicine.

WHO Technical Report Series, No. 622:8, Geneva, Switzerland.

13. WHO. 2002. WHO Traditional Medicine Strategy 2002-2005. World Health

Organization, Geneva, Switzerland. pp. 61.

14. WHO. 2003. WHO Guidelines on Good Agricultural and Collection Practices

(GACP) for Medicinal Plants. World Health Organization, Geneva,

Switzerland. pp. 72.

15. WHO/EDM/TRM. 2002. WHO Guideline on Good Sourcing Practices (GSP)

for Medicinal plants. World Health Organization, Geneva, Switzerland,

16. WHO/IUCN/WWF/TRAFFIC. 2004. Guidelines on the Conservation of

Medicinal Plants. World Health Organization, Geneva, Switzerland.

23

1.3 Ethnobotanical Scenario in Gujarat: Status and Prospects in Primary Health Care Minoo H. Parabia*, M. N. Reddy, Smita Pathak, and Falguni Sheth Shri Bapalal Vaidya Botanical Research Centre, Department of Biosciences, Veer Narmad South Gujarat University, Udhna-Magdalla Road, Surat – 395007, India Email: minoo_parabiain@yahoo.com * Corresponding Author Abstract

The state of Gujarat harbours approximately 2000 species of flowering plants. Out

of them about 1275 species are identified as medicinally useful. Different workers

at ten Universities of the state and few major institutions (Governmental and non

Governmental) are presently engaged in the studies. The ethnobotanical studies

began as gathering of supplementary information on the uses of plants. Gradually

the casual approach turned into a methodical and devoted study on ethnobotany.

The State has a document prepared by the concerted efforts of the workers of the

State and the support of the Government of Gujarat.

The article gives comprehensive results of the plants used for different

ailments hither to known as incurable by a modern medicine. The ailments

prevailing amongst tribes and their herbal treatments are suggested. Institutional

efforts for the revival of knowledge system are elucidated.

Introduction:

Ethnobotanical explorations in Gujarat dates back to early fifties. The important

ethnobotanical contributions are those of Bedi (1969), Bhatt (1975), Bhatt (1987),

Bhatt and Mitaliya (1999), Bhatt and Sabnis (1987), Chavan et al. (1963), Chavda

(2006), Chohan and Shah (1969), Gopal (1983), Gopal and Shah (1989), Hamir

(2001), Inamdar (1968), Jadeja (2006), Joshi et al. (1980), Joshi (1988), Karatela

(1974), Mitaliya (1998), More (1972), Murthy (1957), Nagar (2000), Oza (1961),

Oza (1991), Padte (1973), Patel (1971), Patel (2001), Patel (2002), Prajapati

(2002), Punjani (1997), Rao (1970), Shah (2006), Shah & Vyas (1973), Shah and

Yadav (1979), Suryanarayana (1968), Umadevi (1988), Umadevi et al. (1989).

24

The contributions could be classified into pre Shah (1978) and post Shah

(1978) publications. In addition to publications the sizeable amount of data is

buried in the Ph.D. thesis covering floristics of Gujarat. It just began as the

additional information on the economic uses of the components of flora of the

area.

With the advent of Jain’s series of publications viz. Medicinal Plants (1968),

Contribution to Indian Ethno botany (1981), Dictionary of Indian Folk Medicine

and Ethnobotany (1991), and with the establishment of Ethnobotanical Society in

1989, the ethnobotanical studies received the momentum. There was quite a good

flow of publications from the different corners of the state. Still, the publications

dealt only with the name of the taxa and the mention of their importance.

The monumental work than was created by Umadevi (1988), followed by a

comprehensive publication, by Umadevi et al. (1989).

In 2001 Gujarat Ecological Education and Research (GEER) Foundation

launched a state level coordinated project on the status of medicinal plants in

Gujarat. The sole purpose was to survey the identification, distribution and

relative abundance of medicinal plants in Gujarat. The project culminated in to the

publication of a document viz. Medicinal Plants of Gujarat (Pandey et al., 2005).

Shah (2006) carried out another survey viz. “Status of Ethnomedicine and

Grandma’s prescription in Valsad district (Gujarat), where data on 223 disorders

and 318 species were detailed out with recipes and posology.

In the mean time Shri Bapalal Vaidya Botanical Research Centre (BVBRC)

of Veer Narmad South Gujarat University took up work on the Primary Validation

of ethnic claims; such work is also underway at SRISTI, Ahmedabad as far as the

ethnoveterinary claims are concerned. The document viz. Ethnovet Heritage,

encompassing the details on the ethnoveterinary plants was prepared by Anjaria et

al. (2002)

The preliminary validation – a pilot studies - have so far been carried out for

the hypertension, oral disorders, dental hygiene, spondylosis, bronchial disorders,

osteoporosis and malaria at BVBRC, Surat.

BVBRC has also identified some simple recipes which could be taught to the

different tribes, underprivileged groups to help them in maintaining their primary

health. The recipes selected were aimed to help in dental care, burns, dandruff,

25

earache, constipation, cough-cold, general debility, cardio tonic, uterine fibroid,

asthma, diarrhoea, dysentery, menorrhagia, hyperacidity, malaria, joint pains, skin

ailments, iron deficiency etc.

Bird’s eye view of the ethno botanical research in Gujarat

The survey revealed that the numbers of papers published on Ethno botany of

Gujarat are about 49, the major floristic theses including notes on ethno botany are

32 and the theses devoted to the Ethno botany are 11.

The Universities/Institutions engaged in the ethnobotanical studies are Veer

Narmad South Gujarat University, Surat; The M.S. University, Baroda; Gujarat

University, Ahemedabad; H. North Gujarat University, Patan; S.P. University,

Vallabh Vidyanagar; Anand Agriculture University; Junagadh Agriculture

University; Bhavnagar Univeristy; Saurashtra University, Rajkot and Gujarat

Ayurved Univeristy, Jamnagar.

Most of the institutions are concentrating at the collection of data for the

purpose of documenting the information prevailing amongst tribes and the rural

communities. V.N.S.G.U. has also ventured into collecting recipes prevailing

amongst people at large in urban and rural communities. This has yielded a

beautiful work comprising information on 318 species used for 223 recipes (Shah,

2006). V.N.S.G.U. is also attempting to carryout validation of tribal claims. Pilot

studies completed so far covers ailments like Spondylosis, Psoriasis, Malaria,

hypertension, and dental hygiene.

V.N.S.G.U. and S.P.U. are presently handling collaborative D.S.T. funded

major research project entitled “Development and Standardization of herbal

antimalarial drug.”

Shri Bapalal Vaidya Botanical Research Centre of V.N.S.G.U. is solely

devoted to the studies in Medicinal Plants. The centre is presently holding leads

on diseases like Gangrene, Leukaemia, Neuralgia, Lupus nephritis, Spondylosis,

Panrcreatic tumor, Rabies, Pimples – acne, Obesity and Amoebiosis. It is hoped to

put them to critical scientific appraisal.

The tribes were consulted not only for obtaining information from them on

the medicinal cures but also to study their life style and the sufferings.

26

The major ailments noted are dental care, burns, dandruff, earache,

constipation, cough-cold, general debility, cardic disorders, iron deficiency,

diarrhoea, uterine fibroid, asthma, leucorrhoea, menorrhagia, hyperacidity, joint

pains, skin ailments, liver dysfunction, malaria etc.

It was also noted that the knowledge of medicine is not common for all the

tribesmen. Knowledge is largely scattered, each one having specialization in one

or few of the ailments. At times a wise man having knowledge would protect it

being highly reticent.

Authors, therefore, decided to disseminate the knowledge by way of

arranging training workshops.

The training workshops are normally arranged for 3 days. The first day, fore

noon will be reserved for introducing the plants growing around the camp area.

The participants were also being encouraged to bring more fresh samples for

discussions.

The participants were also told about the basic principles of hygiene and the

symptoms to identify the ailments. About 19 recipes were selected. They were

trained in the actual preparation of medicines. The prepared medicines were

distributed among the participants, so as they can carry the medicine home for

their personal use.

The recipes selected are given below.

• Burns & Dandruff: Creamy emulsion is prepared by emulsifying oils of

Azadirachta indica A. Juss. and Derris pinnata Lour. Oils were emulsified

with Ral (Shorea robusta Gaertn. f. resin). This is used as hair cream.

• Cardio tonic & Bone fractures: Powdered bark of Terminalia arjuna

(Roxb.) W. & A. is given orally, 5g twice a day either with water or as a

Kshir Pak to enhance the fracture healing. The Kshir Pak is prepared by

boiling 5g. of churna with 150 ml of water and 150 ml of milk, till the 150 ml

of water burns out. This is allowed to cool and given to drink.

• Joint pains: Make a paste of 100g Calotropis procera (Ait.) R. Br. leaves,

100g of Vitex negundo L. leaves, 100g of Tinospora cordifolia (Willd.)Miers

stem and boil with 250g of Sesame oil till water evaporates. Strain and store

the oil. Administer 5ml orally, twice a day.

27

• General Debility: Rasayan Churna (Tinospora cordifolia (Willd.) Miers,

Tribulus terrestris L., Emblica officinale Gaertn.) 5g twice a day with water.

• Cough – cold: Trikatu (Zingiber officinale Rosc., Piper nigrum Linn., P.

longum Linn.) 5g twice a day with water or milk.

• Constipation: Mix Harde (Terminalia chebula Retz.) and Sonamukhi

(Cassia angustifolia Vahl) in equal proportion. Give 5g at night with water.

• Diarrhoea: Roast a spoonful of Poppy (Papaver somniferum L.) seeds and

two fruits of Cardamom. Crush and give with a pinch of sugar. This recipe is

good for kids too.

• Hyperacidity: Poha (Rice flakes) and Saunf (Foeniculum vulgare Mill.) are

mixed in equal proportion and reduced to powder. 30g of this powder is

soaked in a litre of water overnight. Next day this liquid is to be taken during

the day whenever thirsty.

• Iron deficiency: Mix 5g of Trifala churna [Harde (Terminalia chebula

Retz.), Amla (Emblica officinale Gaertn), Baheda (T. bellirica (Gaertn.)

Roxb.)] with spoonful of jaggery and little water. Smear the mixture on the

wall of a small iron vessel at night. Next day in the morning add little warm

water and drink daily for about three weeks. The haemoglobin content is

usually restored, if not, continue for two more weeks.

• Earache: Crush 10g each of leaves of Annona squamosa L., Azadirachta

indica A. Juss., Ocimum basilicum L. and cloves of garlic (Allium sativum

L.). Boil with 100g of sesame oil till all water evaporates. Filter and store. Put

two – three drops twice a day in ears.

• Asthma Make a powder of following dried material:

Plant Species Part Used Qty. (g)

Justicia adhatoda L. leaves 500

Solanum surattense Burm. f. whole plant 500

Zizyphus jujuba Lam. bark 100

Terminalia bellirica (Gaertn.) Roxb. fruit rind 200

Datura metel Linn. leaves 50

Calotropis procera (Ait. )R..Br. leaves 50

Clerodendron serratum (Linn.) Moon wood & bark 50

28

Piper longum Linn. fruits 50

Cinnamomum zeylanica Breyn. bark 50

Glycirrhiza glabra Linn. roots 200

Mix them well and store. Give 5g four times a day. Patients are advised to

drink warm water only.

• Leucorrhoea & Menorrhagia: Soak 5g of gum of Sterculia urens Roxb. in

200ml of water or milk in the morning. The content will become a gel. Add

sugar and elaichi (Cardemon seeds) to taste and consume after lunch.

Continue till cured.

• Malaria: Make a powder of following material:

Plant Species Part Used Qty. (g)

Alstonia scholaris R.Br. bark 50

Calotropis procera (Ait.) R.Br. young apical bud 100

Enicostemma littorale Blume whole plant 100

Mix all and store. Take two gram thrice a day with warm water.

• Skin ailments: Paste of fresh rhizome of Curcuma domestica Valeton and

mature fresh leaves of Calotropis procera (Ait.) R. Br. is boiled in oil of

Brassica juncea (L.) Czern and Coss. till water part is evaporated. This

medicated oil is applied on affected part twice a day.

• Dental Care: Calcium powder is mixed with juice of fresh leaves of

Mimusops elengi L. to make a paste. Mix crystals of Menthol, Thymol and

Camphor. They will deliquesce. Add few drops of this mixture and bottle the

content after mixing thoroughly. Apply twice a day on teeth.

• Dysentery: Mix Belgarbha (Aegle marmelos (L.) Corr.), Indrajav

[Holarrhena antidysenterica (Heyne ex Roth) Wall.], Kutaj [Wrightia

tinctoria R. Br. (bark)] in equal proportion. Powder the mixture and store.

One spoonful three times a day is given with water.

• Uterine fibroid: Dried flowers of Woodfordia floribunda Salisb (Dhataki),

Cuminum cyminum L.(Jeera), Symplocos racemosa Roxb. (Lodra), in equal

proportion is taken and powdered. These powders are mixed with jaggery or

crushed raisins. Bolus are prepared. One bolus twice a day is prescribed.

• Liver Dysfunction (Jaundice): Swallow 10g of Aloe vera (L.) Webb. &

Berth. gel with water twice a day for two weeks.

29

• Aloe Health Drink: Scoop off the gel from the sheared leaves of Aloe. Mix

equal quantity of water. Add sugar (900g / L) and boil to attain a syrup of

good consistency. Add in a pack of flavours and preservative of a sherbet

maker available in the market. Filter and fill up the bottle. Daily dose

recommended is 30 ml with water.

Discussion:

There is a dire need of preparing comprehensive database on the ethnomedicinal

information. Each should have online database, inviting contribution from all

concerned. This must be a continuous process.

To re-establish the importance of local herbal resources the tribal training

programmes have shown some encouraging signs.

The follow up of tribal training programmes revealed that a very small

percentage of trainees took this up seriously and started using the knowledge for

monitoring their health. Some enterprising persons even sold their preparations.

We propose to develop a cadre of primary health workers trained in herbology. A

nation wide move in this regard could bring better results.

References:

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Ethnoveterinary Medicine: An overview, 1st edn. Pathik Enterprise,

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Panchmahals district in Gujarat State, Ph.D. thesis, S.P. University, Vallabh

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6. Bhatt RP, Sabnis SD. 1987. Contribution to the Ethnobotany of Khedbrahma

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30

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Plants. Prof.G.L. Shah Commemoration Volume. Pp. 89 – 96.

12. Hamir AM. 2001. Ethnobotanical studies of angiosperms of Arawally hills,

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31

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9(2): 125-142.

35. Shah GL. 1978. Flora of Gujarat State. Published by Sardar Patel University,

Vallabh Vidyanagar. Part I & II

32

36. Shah GL, Vyas KJ. 1973. Some interesting plants of Gujarat State. F Bombay

Nat Hist Soc. 69: 684-686.

37. Shah GL, Yadav SS. 1979. A contribution to the flora of Dangs forest in

Gujarat. Floristic composition, Floristic elements and Biological Spectrum.

Indian Journal of Forestry. 2(1): 13-19.

38. Shah BK. 2006. Status of Ethnomedicine and Grandma’s prescription in

Valsad district (Gujarat), Ph.D. thesis, Veer Narmad South Gujarat

University, Surat.

39. Suryanarayana B. 1968. A contribution to the flora of dang forest, Gujarat.

Ph.D. thesis, Sardar Patel University, Vallabh Vidyanagar.

40. Umadevi AJ. 1988. Identification and status survey of medicinal plants of

Gujarat. Ph.D. thesis, South Gujarat University, Surat.

41. Umadevi AJ, Parabia MH, Reddy MN. Medicinal plants of Gujarat. 1989. A

survey. In: Proceedings of All Indian Symposium on the Biology and Utility

of Wild plants. Prof. G.L. Shah Commemoration Volume. Department of

Biosciences, South Gujarat University, Surat.

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1.4 Suggestions towards Improving the Quality of Ethnobotanical Surveys and Research Pundarikakshudu Tetali Naoroji Godrej Centre for Plant Research, Gat No. 431, Shindewadi, Shirwal – 421801, Dist. Satara, Maharashtra, India Email: ngcpr@lawkimindia.com “I am tired of sending rejection letters to authors of Ethnobotanical research.” – McClatchey (2006).

Abstract

Design and development of appropriate health care delivery system is one of the

most critical factors that determine the success of health related programmes all

over the world. The role of ethnobotany in health care and health care delivery

systems is vital, particularly for traditional societies who are not accessible to

modern health care facilities. The World Health Organization (WHO) and the

United Nations Educational, Scientific & Cultural Organization (UNESCO) have

rightly recognised the importance of Traditional botanical knowledge, its

utilization and practice as a heritage. This heritage needs to be properly

documented, protected and sustained. Documentation of plants that are used by

traditional societies for health purpose is an important branch of ethnobotany,

which is multidisciplinary and needs expertise in plant taxonomy, pharmacology

and medicine. Mc Clatchey recognized clear cut pattern of problems that cause

poor quality ethnobotany manuscripts i.e two categories – a) poor quality

presentation of science and b) poor quality science. We on the other side believe

that the problem lies more in the training and curriculum since documentation of

ethnobotanical information is generally carried out by plant taxonomists who are

not generally familiar with the terminology and science of pharmacology and

medicine. As a result, the documented and deciphered information becomes either

diluted to process further or filled with misleading terms. In either case the

research is useless. We therefore, feel the need to integrate the science of

ethnobotany with other subjects such as pharmacology and medicine. This is

possible through the introduction of integrated syllabus/ courses or preparation of

34

special work-manuals to train ethno botanists. This would help to create a more

meaningful and technically appropriate database that can be scientifically

screened, tested, utilized and finally can be integrated into health care delivery

systems.

Introduction

Health systems designed by nations all over the world aim to improve, maintain or

restore citizen’s health. Nations, therefore, design and implement their own

appropriate health care systems that suit to the needs of people. India, like many

other biodiversity rich countries rightly opted to promote the so-called traditional

or alternate systems of medicine. Among these, Ethno medicine is one such

branch of science which tries to decipher hitherto unknown traditional medicinal

knowledge that was transmitted orally from generation to generation is gained

considerable importance in recent years. Ethno medical information when

properly documented, evaluated and analyzed can help to attain the highest

possible level of health to millions of people. Moreover, sustenance and

revitalization of this system is crucial for the survival of many traditional societies

who are not generally accessible to modern systems of medicine.

On the other hand, as a nation we boast about a great heritage of well

established anicent medical systems. India is a Centre sub-centre of origin for

many traditional systems of medicine such as Ayurveda, Unani, Siddha, Tibetian

yoga etc. In addition, we are number one interims of documented ethnomedical

information. The published data is enormous. About 40% of the known 17,500

flowering plants are recorded to have some or other medicinal use. It is claimed

that Jeevaka an ancient Ayurvedic specialist in cranial surgery, who also reputed

to have cured Buddha, claimed to have said that "He found no plant that is not

medicinal". Despite all the positive points and support of our systems of medicine,

there is an increasing feeling among people that our public health care systems are

very poorly organized to cater the needs of the people. Child deaths due to

epidemics and other diseases are still very high. The available statistics casts

shadow on our claims. We know now that it is not just economic success alone

that puts us in the list of developed nations but the general health of citizens which

35

is a determining factor that decides the successful and developed nations. If it is

so, then are we in the right direction? What is the role of alternate systems of

medicines and how far they are really successful in delivering at least primary

health care to the people? Why is it that we are still a small player in global

medical plant business? Where did we fail to convert our ethnomedical knowledge

into producing competitive formulations? How far we are successful to integrate

ethnomedicines of our systems into modern systems of medicine? There are many

such questions which I don't want to dwell. Never the less I have deliberately

highlighted these questions before the respectable scientific community so as to

show that they are somewhat linked to the type of ethnomedical research

information that we are generating.

McClatchey identified two types of basic problems in ethnobotanical research

– a) Poor quality presentation of science and b) Poor quality science. I am not

tired but worried in a different context. My worries are concerned with ethno

medical surveys and conversion of the published data into useful products.

If we are able to see things in a proper perspective, we can recognize that

hypothesis, accuracy of information and proper interpretation are important points

that decide the quality of a research paper. In case of ethnomedical research

communications tactful questioning, careful interpretation of answers, deciphering

of oral communication and giving clues to other researchers to explore the

problem further becomes the hall mark. Such research based knowledge will help

other researchers to analyze further and come out with better products/

medicines/solutions. In the present paper, I would like to discuss some of these

points that cripple processing of ethno medical research. These can be broadly

classified as 1) Vague data 2) Insufficient data, 3) Ignorance in other related fields

of science, and 4) Wrong deciphering of information.

Methodology

Ethno medical information presented in each example is collected from journals

and books published in India. References have been deliberately not cited so as to

avoid controversies.

1. Vague data

E.g. 1: Parts sold: Leaves

36

Botanical name: Andrographis paniculata Nees.

Vernacular name: Nilavaembu

Family: Acanthaceae

Properties/cures: Antidote

• Antidote is a noun, means a medicine etc. taken or given to counteract

poison. Here, recording just “antidote” does not signify anything.

• The author should specify clearly for what poison it is used as an

antidote.

E.g. 2: Saussurea albescens (DC) Sch. (Asteraceae). ‘Pushkar’ 950. Powder

of dried roots mixed with mustard oil on skin diseases.

• Skin is the largest organ of the body.

• Although many skin diseases are isolated, some are manifestations of

internal disease. Wikipedia, the free encyclopedia lists about 93 diseases

of skin. Ranging from simple Acne to Skin cancer

(http://en.wikipedia.org/w/index.php, List of skin diseases = 119633219).

• Some skin diseases are occupational in nature.

• It is imperative to discuss, finalize and publish such information with a

professional Dermatologist (Skin specialist).

E.g. 3: Scabiosa speciosa Royle (Dipsacaceae). ‘Munik, 775’. Extract of

leaves for abdominal pain.

• Abdomen is the part of the body containing the stomach, bowels,

reproductive organs, etc. The information is impossible to interpret. The

pain could be from any above mentioned parts.

2. Insufficient data

E.g. 1: Albizzia amara Boiv. (Mimosaceae) Narlangi- Leaf paste in goat’s

milk is applied externally over fractures 4-5 times for fast healing.

Leaf paste: Leaves could be fresh or dried; young or mature.

Goat’s milk: Milk could be fresh or boiled/ hot or cold

Fractures: Specific location, Fracture/ hair split/ injury

4-5 times: Per day/ per week/ during the whole healing process.

How is it applied? ; Is it possible to apply the paste when fractured portion is

bandaged?

37

• Authors in the present case have collected insufficient information.

• A few more questions during the interview would have served the

purpose better.

• Giving details about mode of application and time of application is also

necessary.

E.g. 2: Salvia campanulata Wall. (Lamiaceae) ‘Kokai’, ‘Sholar’ 1089.

Infusion of herb applied to scalp supposed to darken hair and stimulate their

growth. Decoction of tender herb in water for tooth ache.

• Infusion: To extract the content in liquid

• The information needs to be elaborated further by adding preparation,

dosage & application procedures.

3. Where knowledge from other fields of science is necessary

Ex: 1: Plumbago zeylanica Linn. (Plumbaginaceae) Chitramulam – The root

paste is applied over Snake bite and Scorpion sting for relief.

• Snakes are classified as poisonous and non poisonous.

• Venom is a poisonous fluid secreted by snakes and scorpions.

• There are around 2,700 snake species. India has about 261 species of

snakes. Most of them are non poisonous. Snake venom is classified into

two types – 1) Neurotoxic - affects the nervous system (Cobra, Krait &

Sea snakes) 2) Haemotoxic – affects the cardiac system (Russel’s viper,

Saw scaled viper) (Khaire, 1996).

• All scorpions possess venomous sting. However, majority of the

scorpions are harmless but the sting is extremely painful and requires

treatment. Globally about 20-25 species of scorpions are known to be

dangerous and their sting can cause death. About 84 species of scorpions

are reported from India. The sting of Buthus tamulus can be fatal.

Scorpions’ venomos are a mixture of neurotoxins.

• While presenting ethnomedical information about antidotes to snake bites

and scorpion stings it is important to present some data relevant to the

Zoological or vernacular names of snakes or scorpions of the study area.

38

If it is not possible, mention at least poisonous or non poisonous snakes

by asking more relevant questions.

• Relief from pain or poison or from both.

E.g. 2: Cardioprotective medicinal plants; Antiplatelet plants.

Name of plant & its family: Zingiber officinale

Vernacular name: Ginger, Adrak

Part used: Rhizome

Active constituents: 6-gingerol; 6, 10-Dehydrogi-mgerdione and 6, 10-

gingerdione

• The medical information related to usage and active constituents

provided by the authors might not be original, but appears to be copied

from other literature (No reference is given). Its usage described is

accurate but with many typing and other errors.

• The author is not familiar with the rules of botanical nomenclature. He

has not mentioned the family name as well as the authority of the taxon.

• The author is also not familiar with agricultural knowledge. Zinger is a

cultivated species. A number of cultivars and varieties are available in its

centres of origin and distribution. The author would have bothered to

check and incorporate the vernacular name of the cultivars that have been

tested for the purpose.

• Cardiac drugs are different from other drugs as they work directly on

heart. Also cardiac drugs are in general are alkaloids they are potent in

dosage. It is therefore essential for the authors to mention the dosage

regime. Otherwise it can lead to dangerous complications.

E.g. 3: List of Trees and Climbers that can be promoted for Primary Health

Care.

Emblica officinalis: Gooseberry, Amla (Hindi); Eyes, promotes growth of

hair (fruit juice).

• Information provided is insufficient.

• The Indian Gooseberry or Anola is indigenous to Tropical South East

Asia. The author must know that the tree is found in wild as well as

cultivated conditions. More than 5 named cultivars are grown

commercially (Banarsi, Chakaiya, Francis etc.) in different parts of India.

39

• The author must specify in the first case whether he is talking about wild

or cultivated varieties. Secondly if they are cultivated they should

disclose the name of varieties or cultivars. The help of Agriculture

botanists is essential in such situations.

4. Misinterpretation of oral information

E.g. 2: Ricinus communis Linn. (Euphorbiaceae) V: Arandi; Andaua. Seeds

are used as purgative, scorpion sting and in impotency.

• The Castor plant is native to North-east Africa and Middle East. It is

cultivated in many parts of the world. India A number of varieties are

available all over the world and

• The author made a dangerous misinterpretation. It is the seed oil that is

used as purgative not the seeds.

• The seeds of castor contain a powerful cytotoxin called ricin and RCA

(Ricinus communis agglutinin). Ricin is a protein and causes weak

agglutination of Red Blood Cells (RBC), while RCA is a powerful

hemagglutinin. It is said that Ricin content of just one seed is toxic

enough to kill a child. Poisioning in humans is only due to ricin ( Knight,

1979; www.ansci.cornell.edu/plants/toxicagents/ricin.html

The research data cited above did not include common minimum details

such as formulation preparation, dosage regiment, contraindications,

frequency of quotes etc. Without which the data becomes obsolete.

Discussion

Designing and implementation of appropriate health system is one of the critical

indicators that determine the progress and development of a nation. Common man

recognizes only two types of medical systems – the one that works for him and the

one that don’t work. . It is therefore necessary for planners to understand some of

the ground realities and to adopt the health systems. Ethnomedical research is

believed to offer new vistas for planning and implementing appropriate health

care systems in India. Nonetheless, lot of money is already pumped into this

discipline to recognize and identify some solutions to some of the health problems

of our country. Despite the generation of a huge ethnomedical database, we have

40

failed to take the knowledge a few steps further and convert it into useful

medicines for the society. It is suggested here that it is not just poor quality

presentation of science or poor science as McClatchey highlighted in his editorial,

but the abilities and training of the surveyor in a) questioning, b) collecting

complete information, c) its interpretation, d) knowledge in other disciplines

accounts to the quality and usefulness of data. Hence, the author suggests that

ethno botanists must be trained in other related fields of science such as medicine,

pharmacy, agriculture, intellectual prosperity rights etc. or make it mandatory to

form research teams with specialists from different disciplines to generate

authentic and useful ethnomedical information. The other option, may be little

difficult to implement, but essential keeping the intellectual prosperity rights in

mind that is to create a platform for tribals to train and document their own

information with the coordinated efforts of ethnobotanists and other experts. The

research information generated through such organized efforts will help other

researchers to evaluate and convert into useful formulations/drugs. This can

ultimately help to design and develop appropriate health care systems.

Acknowledgements: The author thanks Mr. V.M. Crishna, Director, Naoroji

Godrej Centre for Plant Research, for encouragement and Mr. Ravindra Ghule for

useful discussions.

References

1. Khaire N. 1996. Indian snakes. Indian Herpetological Society, Usant, Pune.

2. Knight B. 1979. Ricin – a potential homicidal poison. Br Med J. 278. 350-

351.

3. McClatchey. 2006. Improving quality of international ethnobotany research

and publications. www.ethnobotanyjournal.org/vol3/i1547-3465-04-001.pdf

41

2.1 Bioassays in Traditional Medicine Urmila Thatte Department of Clinical Pharmacology, TN Medical College & BYL Nair Charitable Hospital, Mumbai – 400008, India. Email: clinpharm@hathway.com, urmilathatte@gmail.com Abstract

Drug development poses great challenges in traditional medicine. From the choice

and standardisation of test material to the choice of which bioassay to use, the

path is long and treacherous – perhaps more so than when working with new

chemical entities. This paper examines some of these challenges.

In vitro assays are very useful to identify putative drug actions while working

with pure compounds. However, with herbal materials several issues come up. For

example, what dose to use, what extract to use and what cut-offs to use? Doing

viability assays (using Trypan Blue exclusion or MTT assays) help in deciding

upper cut-offs, however, it is not fail-safe to lead to potentially active

concentrations. Many herbal extracts are not water soluble and therefore a solvent

that is not toxic to cells have to be selected and this has to be used as a control.

Extrapolation from an in vivo dose helps in identifying the possibly effective dose

– but with crude extracts acceptable cut-offs must be predetermined. If the extract

is coloured, then interpretation of results when being estimated colourimetrically

has to be done cautiously. Incorporation of appropriate controls is also essential

to confirm robustness of the assay system. If the substance is not soluble and we

use suspensions, this can seriously interfere with assays e.g. when we use herbo-

mineral preparations. Bioassays are expensive and need sophisticated

instrumentation and expert personnel. Additionally, de-differentiation and

instability of cell lines adds to some uncertainties. Additional variables that affect

analysis include variability in media composition, temperature, viscosity,

osmolarity, and buffering. Naturally, therefore, in vitro bioassays cannot totally

replace in vivo studies

However, the advantages of in vitro studies are many. Thus, we can study cell

interactions, cell-environment interactions, intracellular activity, cell products, site

and mechanism of action and genetic studies with the herbal medicines are

42

possible. The technology to use stem cells has opened new vistas. Apart from the

convenience, the lack of ethical dilemmas (unlike in human or animal studies), a

control of the experimental environment, the ability to characterize the sample and

maintenance of homogeneity in the procedure are other advantages. Up-scaling

and mechanization are also major advantages, allowing high-throughput screens.

It must be emphasized here that in vivo bioassays are also a powerful tool used in

pharmacology for studying the effects of medicines, and are also of some use in

herbal drug development. Dose relationships and mechanistic studies are some of

the classical examples. However, the challenges with in vivo animal studies are

also many, including what dose, route and for how long? Extrapolation of data

from animals to humans and vice versa is difficult.

Using examples of research from our group, these issues will be discussed to

map out a path for drug development in traditional medicine.

Full text not received

43

2.2 Novel Imaging System for Determining Anti-Cancer Activity Dharmalingam Subramaniam1, Rama P. Ramanujam2, Joseph M. Betz3, Panchapagesa M. Murali4, Courtney Houchen1 and Shrikant Anant1, 5,*

1Department of Medicine, Digestive Diseases and Nutrition, and 5Department of Cell Biology, University of Oklahoma Health Sciences Center, 920 Stanton L. Young Blvd WP1360, Oklahoma City, OK 73190, USA 2Swaasth, Inc., 800 Research Pkwy Ste 350, Oklahoma City, OK 73104, USA 3Dietary Supplement Methods and Reference Materials Program, Office of Dietary Supplements, National Institutes of Health, 6100 Executive Blvd, Bethesda, MD 20892, USA 4Dalmia Centre for Research and Development, B-133, Siruvani Main Rd, Kalampalayam, Coimbatore 641010, India

* Corresponding Author

Abstract

The lack of effectiveness with modern medicines combined with increased

consumer desire for better health has resulted in a dramatic increase in the use of

dietary supplements to improve ones general health by reducing pain and

inflammation, improving mental alertness and enhancing energy levels. Our

studies and those of others have indicated that dietary curcumin, an active

ingredient in the spice turmeric, potently inhibits intestinal tumorigenesis and

inflammation. We have now characterized the mechanisms by which curcumin

modulates this function. Curcumin and turmeric have been used extensively in

the Ayurvedic system of medicine for treatment of many disorders including

inflammation and cardiovascular disease. Inflammation is a common consequence

to injury in general. In particular, diet-induced injury to gastrointestinal tract can

occur due to varying constituents in the ingested materials, resulting in a robust

inflammatory response. The ubiquitous transcription factor nuclear factor kappa B

(NF-κB) differentially regulates cyclooxygenase-2 (COX-2) expression in colon

cancer cells. NF-κB directs high-level transcription of many cytokines, adhesion

molecules, and other pro-inflammatory genes in tissue cultures; however, the

extent to which NF-κB controls specific biological processes in vivo is unknown.

NF-κB is regulated at the posttranscriptional level by ubiquitination-mediated

proteosomal degradation of the inhibitor I-κB protein. My presentation will

highlight some of our recent studies on developing a rapid high throughput system

44

using the IkappaB degradation activity for identifying anti-cancer medicinal

activity.

Background and Aims: Dietary supplements are being increasingly consumed by

the local population in United States of America. However, there is no specific

assay available to determine batch-to-batch variations and between those of

different companies of any given product. This study aimed to develop a high

throughput assay to identify the activity of dietary supplements. Methods: We

generated an HCT-116 colon cancer cell line stably expressing a chimeric I-κB-

luciferase fusion protein. In vitro assay was performed to demonstrate that

curcumin and turmeric extracts inhibit the proliferation of these cells in the

presence and absence of prostaglandin E2 (PGE2). Western blot was performed to

demonstrate the phosphorylation and degradation of endogenous I-κB. Luciferase

activity measurements were performed to determine the levels of the I-κB-

luciferase fusion protein. Results: HCT-116 cells proliferation was significantly

inhibited by both curcumin and turmeric extracts in a dose dependent manner even

in the presence of a PGE2, a potent apoptotic inhibitor. Western blot analyses

demonstrated that TNF-α induced degradation of I-κB in the cells, which was

inhibited by turmeric extracts. Luciferase assay measurements also demonstrated

that curcumin and turmeric extracts inhibited TNF-α-mediated degradation of I-

κB-luciferase fusion protein in the cells. Conclusion: This assay may be utilized

to demonstrate the level of activity in turmeric extracts based on its ability to

inhibit TNF-α-mediated degradation of the I-κB-luciferase fusion protein.

Introduction

In recent years, due to the increasing dissatisfaction with modern medicines and

increased consumer desire in healthy living, there has been a dramatic increase in

the consumption of natural foods and the use of dietary supplements. However,

although some natural products have been reported to have clinically proven

health benefits, they have been used in all cultures from ancient times without any

functional quality-assurance testing. In addition, it is evident that many natural

foods may contain potentially toxic substances and cause adverse interactions with

45

modern drugs. This problem is apparent with functional foods such as fruits,

vegetables and nuts containing beneficial amounts of polyphenols and flavanoids

(Halsted, 2003).

The major issues for the use of natural products for public benefits or risks,

relate to the content of active constituents and bioavailability of such active

ingredients Currently, High Pressure Liquid Chromatography (HPLC) appear to

detect the presence of chemical constituents in foods and dietary supplements

(Reddy, et al., 1982, Robins, 1994). But, there is a serious lack of adequate

scientific tools and techniques to analyze the bioavailability of active ingredients

in ingested foods and dietary supplements.

Epidemiologic studies and laboratory tests of animals have indicated that

consumption of spices, fruits, vegetables and whole grains can reduce the risk of

cancer and inflammation (Corpet and Tache, 2002; Reddy, 1992, Slaga and

Gimenez-Conti, 1992). However, traditional dietary recommendations lack

physiological specification to determine the appropriate beneficial dietary dosage

to be consumed from spices, fruits and vegetables. Accordingly, in this report, we

demonstrate a gene expression measurement (GEMTM) assay using the

degradation of I-κB to measure activity of antiproliferative and anti-inflammatory

compounds. As a proof-of-principle, we have used extracts of turmeric (Curcuma

longa Linn), a commonly used spice that is used worldwide as a seasoning and is

an essential ingredient of curry. In addition, we have tested the effects of

curcumin, an active ingredient in turmeric (Aggarwal et al., 2003; Sharma et al.,

2005, Sinha et al., 2003). In the Indian subcontinent and in Southeast Asia,

curcumin has been traditionally used in the treatment of throat ulcers,

inflammation, skin wounds and cancer (Sinha et al., 2003).

Materials and Methods

Preparation of turmeric extracts and chemical reagents: The turmeric rhizome

was obtained from a farm in South India. Hard-dry turmeric rhizomes were

pulverized to a fine powder with the aid of a kitchen mixer, and ethanolic extracts

were prepared. Curcumin was purchased from LKT Laboratories, Inc, St. Paul,

Minnesota, USA. All routine molecular biology reagents were purchased from

Sigma-Aldrich, St. Louis, MO, USA.

46

Cell culture: HCT-116 human colon cancer cells were grown in Dulbecco’s

modified Eagle medium (DMEM) containing 10% heat inactivated fetal bovine

serum (Sigma Chemical Co, St. Louis, MO), standard antibiotics in a carbon

dioxide incubator with 5% CO2 at 37 0 C. The cells were stably transfected with

PGL3- I-κB firefly luciferase (I-κB FLuc) plasmid as previously described (Gross

and Piwnica-Worms, 2005).

Proliferation assay: HCT116 cells were seeded on a 96 well plates at a density of

1x 103 cells/ well and allowed to adhere and grow overnight. The cells were then

treated with increasing concentration (0-50 µg/ml) curcumin or turmeric extracts

in DMEM-10% FBS. Where indicated, the cells were also treated with

Prostaglandin E2 (1 μM). Proliferation activity was determined by enzymatic assay

as previously described (Landegren, 1984). Results were further confirmed by

manual cell counts.

Western Blot Analysis: HCT 116 cells were treated with curcumin (10 µg/ml) or

turmeric extracts (10 µg/ml) for 90 min, followed by PGE2 (1 µM) or TNF-α (10

ng/ml) for 30 min. Cell lysates were prepared and subjected to polyacrylamide gel

electrophoresis and blotted onto Immobilon polyvinylidene difluoride membranes

(Millipore, Bedford, MA). Antibodies were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA). Specific proteins were detected by the enhanced

chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ).

Determination of the effect of turmeric extracts on I-kappaB-luciferase

expression: Briefly, HCT-116 cells stably expressing I-κB-FLuciferase were

plated in a 6-well dishes at a concentration of 5 x 105 cell/well and allowed and

grow for 24 h. The cells were treated with increasing concentrations of curcumin

or turmeric extracts (0-10 μg/ml) for 90 min, followed by TNF-α (10 ng/ml final

concentration) for 20 min. The cells were lyzed with a lysis buffer (Promega,

Madison, USA), and luminescence was measured by using a luciferase assay

reagent (Promega, Madison, USA) in the Synergy-HT 96 well plate reader

(BioTek, USA).

47

Results and Discussion

Curcumin and turmeric extracts inhibit HCT-116 cell proliferation. Curcumin is

an active ingredient in turmeric, and the amount of curcumin varies depending on

the source of turmeric. Hence, the first step in the evaluation of the turmeric

activity to determine the level of curcumin. Fresh turmeric was obtained from

Erode in Tamil Nadu, and an ethanolic extract was prepared and subject to HPLC

analysis. The curcumin content in the extract was determined to be 2.2% (data not

shown). As a comparison for the subsequent studies, curcumin was commercially

obtained and determined to be 98% pure using a HPLC assay (data not shown).

Minor amounts of bisdemethoxycurcumin and demethoxycurcumin were present

in the preparation.

Previous studies have demonstrated that curcumin is a potent inhibitor of

colon cancer cell proliferation (Aggarwal et al., 2003). To determine whether the

turmeric extract that we generated is also active in inhibiting proliferation of colon

cancer cells, we performed an in vitro proliferation assay. Cells were treated with

increasing concentrations of pure curcumin or the turmeric extract for 24 h and its

effect on cell proliferation was determined. Both curcumin and turmeric inhibited

the proliferation of HCT-116 cells in a dose dependent manner (Fig 1). PGE2 has

been shown to stimulate colon cancer cell growth through its heterotrimeric

guanine nucleotide-binding protein (G protein)-coupled receptor, EP2 (Castellone

et al., 2005). To determine whether curcumin and turmeric can inhibit colon

cancer cell proliferation in the presence of PGE2, we performed the experiments in

the presence of exogenous PGE2. Both, curcumin and turmeric extract inhibited

proliferation of the cells even in the presence of PGE2 (Fig 1), suggesting that

curcumin and turmeric were able to override the PGE2-mediated growth induction

activity.

48

Fig. 1: Curcumin and turmeric inhibits the proliferation of HCT 116 Cells. HCT 116 cells treated with increasing doses of curcumin or turmeric (0-50 µg/ml) and presence or absence of PGE2 (1 µM) for 24 h were analyzed for proliferation based using a hexosaminidase enzyme activity. Curcumin (Panel A) and turmeric (Panel B) treatment resulted in dose dependant decrease in cell number in HCT116 cells. The presence of PGE2 did naffect the proliferation inhibition activity of either curcumin or the turmeric extracts.

ot

Curcumin and turmeric extracts were further investigated at various

concentrations to determine their inhibition Concentration (IC50) values for the

inhibition of proliferation. Curcumin and turmeric extracts, a 50% reduction in

proliferation was observed at a dose of 5 and 6 µg/ml, respectively (Fig 2). Based

on the molecular weight (368), the calculated concentration of curcumin that

demonstrated a 50% inhibition was 1.5 µM. Since, turmeric only contains 2.2%

curcumin, the concentration of curcumin in the turmeric extracts was calculated to

be 132 nM. These data suggest that additional active ingredients are present in the

extract, which either act by themselves or enhance curcumin function.

49

Fig. 2: Proliferation inhibitory constant for proliferation inhibition. The inhibitory constant (IC50 value) for curcumin- and turmeric emediated suppression of PGE

xtracts-

on in 2

induced cell proliferatiHCT 116 cells was determined after plotting the percent inhibition. The IC50 value for curcumin (Panel A) and turmeric (Panel B) was determined to be 5 µg/ml and 6 µg/ml, respectively.

Curcumin and turmeric extracts inhibit I-κB activity: NF-κB is a transcription

factor that consists of 2 subunits: a 50 kDA (p50) and a 65 kDa subunit (p65)

subunit (Figure 3A) (Karin, 2006). It is rapidly induced following stimulation of

the cells and controls the expression of a wide range of genes especially in

immunological processes. A primary level of control for NF-κB is through

interactions with an inhibitor protein called I-κB (Karin, 2006). Under normal

conditions, NF-κB is sequestered in the cytoplasm by I-κB. However, when cells

are exposed to LPS or inflammatory cytokines such as TNF-α, I-κB is

phosphorylated through the activity of I-κB kinase resulting in release of the NF-

κB. In addition, the phosphorylated I-κB is degraded by the ubiquitin-degradation

pathway (Fig 3 A) (Karin, 2006).

50

Fig. 3: Curcumin and turmeric extracts inhibit I-κB degradation. A. Schematic representation of NF-κB pathway. The diagram shows the signaling cascade that flows from the signaling complex to the activation of NF-κB. Both curcumin and turmeric extracts inhibit the I-κB phosphorylation. B. Turmeric extracts protect endogenous I-κB from TNF-α-mediated degradation. Total lysates from HCT-116 cells were subjected to western blot analyses for phosphorylated I-κB. Cells were pretreated with either curcumin or turmeric (10 µg/ml) followed by treatment with PGE2 or TNF-α Turmeric inhibited both the phosphorylation and degradation of I-κB *p<0.001.

Curcumin has been previously shown to inhibit NF-κB activation by

inhibiting the degradation of I-κB (Aggarwal and Shishodia, 2006). To confirm

that curcumin, and determine whether the turmeric extracts that we prepared also

inhibited I-κB degradation, we performed a western blot analysis. We incubated

cells with TNF-α, a cytokine that binds to one of to surface membrane receptors,

tumor necrosis factor receptor (TNFR) 1 and 2, and triggers a signal cascade that

results in the activation of I-κB kinase (Fig 3 A) (Clark, et al., 2005). Treatment of

cells with TNF-α resulted in significant decrease in total I-κB levels in cells (Fig 3

B). Furthermore, pretreatment of the cells with 10 µg/ml curcumin did not affect

the levels of total I-κB in the TNF-α treated cells. On the other hand, pretreatment

of the cells with 10 µg/ml turmeric significantly increased the levels of total I-κB

in the cells suggesting that it inhibited the TNF-α-mediated degradation of I-κB

51

(Fig 3 B). When the cells were treated with PGE2, no such effect was observed in

the time frame of the experiment. However, incubation of the cells for longer

times with PGE2 resulted in a similar degradation activity (data not shown). Since

degradation requires the initial step of phosphorylation, we also determined the

effect of the extracts on TNF-α-mediated I-κB phosphorylation. Increased

phosphorylation over baseline was not observed following TNF-α treatment (Fig

3B). However, we believe this is because the protein is rapidly shunted to the

ubiquitin-proteosome degradation pathway following when it is phosphorylated.

Hence the steady state levels of phosphorylated I-κB may not be significantly

changed. On the other pretreatment of the cells with turmeric significantly

suppressed the phosphorylation of I-κB at baseline in the TNF-α-treated cells.

Again, curcumin at the dose used did not demonstrate this activity, but also

significantly suppressed I-κB phosphorylation when used at higher levels (data not

shown). Taken together, these data suggests that the extracts inhibit I-κB

degradation in the cells and hence was used to develop a rapid in vitro assay

presented below.

Development of an in vitro assay using I-kB degradation activity. As a first

step in the development of a high throughput detection mechanism for I-κB in

colon cancer cells, we generated HCT-116 cells that were stably transfected with a

plasmid that encodes I-κB as a fusion protein with the firefly luciferase under of

the cytomegalovirus immediate early gene promoter-enhancer. Presence of I-κB

was monitored by luminescence activity. As a first step, we determined if the

fusion protein is subject to similar levels of degradation in response to a TNF-α

similar to that observed above with the native I-κB. Total cell lysates from TNF-α

treated cells demonstrated significant decrease in the luciferase levels, suggesting

that the protein underwent TNF-alpha-mediated degradation (Fig 4 A). To

determine whether curcumin and turmeric extracts protected the protein from

TNF-α induced degradation, the cells were pretreated with the compounds. Both

curcumin and turmeric extracts suppressed TNF-α-mediated degradation of the I-

κB-luciferase fusion protein (Fig 4 B). We have performed similar studies is a

second cell line, HeLa cells, a cervical carcinoma cell line and obtained similar

results (data not shown). These data demonstrate that the I-κB-luciferase fusion

52

protein responds to external stimuli in manner similar to that observed with

endogenous I-κB.

NF-

ed

e

1

. The

-10

ic

Fig. 4: Curcumin and turmeric extracts protect I-κB from Tinduced degradation. A. TNF-α induces degradation. HCT-116 cells stably expressing the I-κB-Fluc fusion protein were treatwith TNF-α. Total cell lysates were prepared and the luciferasactivity was measured. TNF-α induced the degradation of the IκB-luciferase protein. *P<0.0B. Curcumin and turmeric extracts protect I-κB-Fluc fromTNF-α-induced degradationcells were pretreated with increasing concentrations (0mg/ml) of curcumin or turmeric extracts for 90 min, followed by the treatment with 10 ng/ml TNF-α. Both curcumin and turmerextracts demonstrated a dose dependent protection against TNF-α induced I-κB degradation. *P<0.01

We further determined the inhibitory constant for curcumin and the turmeric

extracts in inhibting the TNF-α-mediated degradation of the I-κB-luciferase by

plotting the percent inhibtion of the degradation against the amount of the

compound/mixture used (Fig 5 B). The calculated IC50 value for curcumin was

0.4µg/ml and 0.25 µg/ml for curcumin and turmeric respectively. For curcumin,

the IC50 value was further calculated to be 75 nM. In addition, based on the

amount of curcumin that is present in the turmeric extract, it is apparent that other

ingredients in the extract, which could include other curcuminoids, also have

activity in inhibiting the degradation of the protein. This is further proof that the

purified molecule in the extract is not better than the whole extract in regulating

anti-inflammatory function through inhibiting I-κB degradation.

53

Fig. 5: Inhibitory constant value for I-κB degradation. The inhibitory constant (IC50 value) for curcumin- and turmeric extracts-mediated suppression of TNF-α-mediated I-κB degradation in the HCT-116-I-κB-FLuc cells was determined after plotting the percent inhibition. The IC50 value for curcumin (Panel A) and turmeric (Panel B) was determined to be 0.4 µg/ml and 0.25 µg/ml, respectively.

Finally, these data demonstrate the feasibility of using this approach to

determine the activity of phytochemical compounds that regulate NF-κB activity

in cells. The utility of this system is in the development of a high throughput assay

to determine the activity of the various turmeric extracts including different lots of

the same material or different mixtures. Collectively, these observations suggest

that turmeric and curcumin may qualify for use as anti-proliferation and anti-

inflammatory compound(s) for in vitro studies and the development of high

throughput IC50 unit measurement system to quality assure turmeric and other

phytochemicals as chemo-preventive agents.

Acknowledgements

This work is partly funded by NIH grant CA109269 (to SA) and a NIH-ODS MD-

611266 (to RPR). We gratefully acknowledge the gift of the luciferase plasmid

from Dr. David Piwnica-Worms of Washington School of Medicine.

54

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preclinical and clinical studies. Anticancer Res. 23: 363-98.

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prevention and therapy of cancer. Biochem Pharmacol. 71: 1397-421.

3. Castellone MD, Teramoto H, Williams BO, Druey KM, Gutkind JS. 2005.

Prostaglandin E2 promotes colon cancer cell growth through a Gs-axin-beta-

catenin signaling axis. Science 310:1504-10.

4. Clark J, Vagenas P, Panesar M, Cope AP. 2005. What does tumour necrosis

factor excess do to the immune system long term? Ann Rheum Dis. 64 (S4):

70-6.

5. Corpet DE, Tache S. 2002. Most effective colon cancer chemopreventive

agents in rats: a systematic review of aberrant crypt foci and tumor data,

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6. Gross S, Piwnica-Worms D. 2005. Real-time imaging of ligand-induced IKK

activation in intact cells and in living mice. Nat Methods. 2:607-14.

7. Halsted CH. 2003. Dietary supplements and functional foods: 2 sides of a

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fermented foods: their preparation and nutritional quality. Crit Rev Food Sci

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14. Sinha R, Anderson DE, McDonald SS, Greenwald P. 2003. Cancer risk and

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Cancer Inst Monogr. 13: 55-60.

56

2.3 Principles of pre-clinical evaluation of plants using appropriate bioassays Jaswant Singh Division of Pharmacology, Indian Institute of Integrative Medicine (formerly Regional Research Laboratory), Canal Road, Jammu-Tawi – 180001, India Email: jsishar1@yahoo.com Abstract

The molecular and cellular mechanisms that underlie the pathophysiology of

various diseases vary in proportions to the changes in their genes expression and

therefore of phenotypes of the cells. Understanding of the molecular basis of the

disease is therefore important for rational approach to drug design and

development. Newer knowledge in every distinct disease adds to the existing

information to define and refine molecular targets so as to develop active leads to

convert into safer therapeutics. In some cases a single molecular target may be

important for disease modification and presumably its prevention, but to manage a

disease process effectively and provide a quality life, several targets nevertheless

are to be considered. This undoubtedly asks for a battery of bioassays to apply in

screening herbal products against disease phenomena. Ancient knowledge in the

traditional use of herbal plants has provided us a treasure, which continues to

bolster therapeutics, leads and template molecules to be developed into new

chemical entities and investigative new drugs. A brief discussion shall highlight

some current emerging targets with respect to diabetes, liver dysfunctions, drug

bioavailability enhancement, inflammation/ arthritis neurodegenerative diseases,

etc. while our current efforts into the development of therapeutic leads from plants

shall be discussed in detail.

It is of paramount importance first to standardize plant extract to establish a

chemical profile using modern chemical instrumentation. The extract is subjected

to fractionation for which again chemical signature is established. Pure molecules

are isolated from bioactivity-guided fractions while bioactivity depends upon the

bioassay employed. Several types of in vitro and in vivo systems are employed to

screen and validate the bioactivity of the test extract/fraction/molecules. Use of

mammalian cell cultures mimicking in vivo functions is important for rapid

57

screening of large number of plant extracts /isolates. This would minimize

dependency on indiscriminate killing of large number of animals. With known 3D

structures of proteins as molecular targets, drug like molecules can be designed

using in silico biology approaches to get the best Hits to be taken for in vivo

validation.

My group is actively engaged in the pre-clinical development of anticancer

plant leads employing mechanism driven approaches using human cancer cell

lines. We know that dysregulation of apoptosis (programmed cell death) is the

hall-mark of many cancer cells. It plays an essential role in the control of cell

number while apoptosis is impaired in many human tumors suggesting that

disruption of apoptosis contributes substantially in the transformation of normal

cell into tumor cell. Thus, induction of apoptosis in cancer cells provides

pragmatic means to develop early anti-cancer therapeutic leads. Thus apoptosis

has become a focal point in chemotherapy-induced tumor cell killing. Apoptosis is

a complex phenomenon where the switching on and off of apoptosis is

determined by the ratio of pro-apoptotic and anti-apoptotic proteins. Therefore

many plant products may induce apoptosis by triggering the core components of

the cell death machinery, which involves chromatin condensation, cell blebbing

and DNA fragmentation into apoptotic bodies. More than 50 % of anti-cancer

drugs in clinical practice have come from plants, which are also rich source of

simple to complex molecules. We treat human cancer cell lines with the test

compounds and measure the extent of apoptosis employing several end-points

assays using flow cytometry, agarose gel electrophoresis, fluorescence

microscopy; the bioassays offer rapid screening of plant products as pro-apoptotic

agents. In cancer cell several genes are mutated and the next interest would be to

find the cell signaling pathways involved in cancer cell death by the test plant

material and that how the test material induces apoptosis.

Once the plant products produce apoptosis in cancer cells, other studies into

the mechanism of action would help in optimizing leads in suitable in vitro and in

vivo tumor models. Moreover, such leads may also form important adjuncts in

traditional chemotherapy besides protecting normal human cells from the

chemotherapeutic drugs. Thus investigations into the inter-relationship between

pro-apoptotic compounds and anti-cancer activity will be a promising field to

58

understand and elucidate the possible mechanisms for the functionality of

medicinal plants for cancer prevention and treatment. The lead molecules are

subjected to PK studies, acute and sub-acute toxicity as a part of pre-clinical

profile. Some highlights of our current studies shall be discussed.

Full text not received

59

2.4

Preclinical testing of medicinal plants: advantages and approaches Poonam G. Daswani, Brijesh S. and Tannaz J. Birdi*

The Foundation for Medical Research, 84A, RG Thadani Marg, Worli, Mumbai - 400018, Maharashtra, India. Email: fmrbom@hathway.com * Corresponding Author Abstract

According to WHO, 80% of the population of developing countries rely on

traditional medicines, mostly plant drugs, for their primary health care needs.

Also, modern pharmacopoeias still contain at least 25% drugs derived from plants.

With the growing importance and popularity of plants, several attempts are being

made to provide validation to the use of medicinal plants. Approaches towards

validation include both preclinical and clinical testing. However in view of the

limitations of directly utilizing clinical trials on the basis of information obtained

from databases, cited literature and/or ethnobotanical studies, preclinical testing

becomes an important prerequisite.

The paper will underline the various advantages of preclinical testing of medicinal

plants justifying its niche. Shortcomings of some of the commonly used

preclinical assays especially with respect to screening for antimicrobial activity

and generalized immunological parameters will also be discussed. The various

approaches to preclinical testing with emphasis on using bioassays representative

of disease process as exemplified by data obtained at FMR on plants with

antidiarrhoeal activity will also be discussed.

Introduction

There is no doubt that modern medicine offers tremendous advantages and its

application is wide spread. Despite this, traditional medicine has maintained its

popularity all over the developing world and its use is rapidly spreading. The

history of usage of plants is probably as ancient as the human civilization; modern

pharmacopoeias contain at least 25% drugs derived from plants. Even today it is

60

estimated that 80% of the population of developing countries relies on traditional

medicines, mostly plant drugs, for their primary health care needs. However use

of these medicines is mainly in raw and semi-standardized form and often based

on empirical evidence. The lack of pharmacological and clinical data on the

majority of herbal medicinal products is a major impediment to the integration of

herbal medicines into conventional medical practice. The present paper will

discuss some of the relevant issues associated with the preclinical testing of plants

related to the approaches, the advantages and the limitations.

Before covering the pre clinical testing, the various approaches to plant

selection and some issues related to adverse effect profile, clinical testing of

medicinal plants are briefly discussed.

Approaches to Medicinal Plant Selection

Several reviews have described approaches that can be used for selecting plants of

potential therapeutic interest (Verpoorte, 2000; Phillipson and Anderson, 1989;

Kinghorn, 1994; Vlietinck and Vanden Berghe, 1991; Farnsworth, 1996;

Farnsworth and Bingel, 1977).

The search can follow three main routes: random, ethno (including

ethnobotanical, ethnomedical and ethnopharmacological) and ecological search

(Fabricant and Farnsworth, 2001). The ethnobotanical and ethnopharmacological

approach uses information obtained from traditional medical practitioners and

other people such as village elders and local women who are traditional users of

medicinal plants. Ethnomedical information is available from ancient texts of

different systems of medicines such as Ayurveda, Unani, Kampo, and traditional

Chinese medicine. Plants with medicinal properties can also be selected using the

ecological approach. The absence of predation in areas infested with herbivores,

for example, can indicate the presence of toxic compounds. Selection can also be

based on an approach called zoopharmacognosy, a variation to the ecological

approach, which proposes the selection of plant species regularly ingested by

animals, mostly primates for reducing pain, microbial or worm infestations (Berry

et al., 1995).

61

Evaluation of Medicinal Plants

Artuso (1997) has outlined the entire process which include formulating an

appropriate strategy, obtaining biologic extracts, screening those extracts,

isolating active compounds, conducting preclinical tests and chemical

modification, etc. This approach is very demanding since there is an estimated

250,000 species of higher plants present on this earth (Ayensu and DeFilipps,

1978). Of the 6% plants that have been screened for biologic activity, only 15%

have been evaluated phytochemically (Verpoorte, 2000). However, this scenario

would change due to use of the advanced screening methods that are available

today. Reverse pharmacology based on the documented therapeutic effects of

plants in ancient texts can prove to be a more productive and cost effective

approach in development of safe, effective and acceptable therapeutic agents

(Vaidya, 2006).

Before a plant can be used and popularized, depending on the intended usage,

it is necessary to establish its efficacy through biological assays and obtain its

adverse effect profile through literature or from toxicological studies (both short

term and long term) followed by controlled clinical trials.

Clinical Studies:

Well-established, randomized controlled clinical studies lead to the better

acceptance of herbal medicines. Clinical studies are necessary to confirm the

pharmacological effects of medicinal plants before they can be integrated into

conventional medical practice. This would be especially true in case of some

unrelated effects of therapy contributing to efficacy that may be difficult to

measure pre-clinically. Well recorded case reports can contribute towards useful

information at such times and put forward new hypothesis and stimulate further

study (Morris, 1989). However, double blind clinical trials may not be required

when an extensive and detailed database of case studies is available. Such a

database is especially important when a particular treatment is individualized.

The methods and guidelines used for clinical validation of modern medicines

must be applied to herbal products even though the latter has a holistic approach

to treatment. However, conventional concepts of clinical research design may be

difficult to apply when using clinical research to evaluate various systems and

62

practices of traditional medicine (WHO, 2000). This could be due to the fact that

herbal remedies are individualized (each person has certain predispositions to

disease and susceptible to factors like environment, genetics, dietary and lifestyle)

therapies.

The number of patients required for undertaking clinical trial of medicinal

plants is large not only since the study design needs to be adequate and

statistically appropriate but also to cater to the control, confounders and placebo

groups to provide sufficient evidence for judging efficacy of the plant under study.

The increase in patient number also increases the time commitment and the

expenses involved. Moreover, it may not always be possible to include all the

groups in a single study for e.g., use of a placebo may not be possible when the

plant preparation has a strong smell or taste as is the case of certain essential oils.

In addition, patients who have been treated previously with the herbal medicine

under investigation that has a characteristic organoleptic property cannot be

randomized into control groups (WHO, 2000).

Therefore only a limited number of plants can be subjected to clinical

trials. Hence, it is essential to undertake appropriate preclinical testing to

short list plants for clinical evaluation. The main goals of the preclinical

studies are to determine a drugs pharmacodynamics, pharmacokinetics and

toxicity through animal studies. This data allows researchers to estimate a

safe starting dose of drug for clinical trials in humans.

Preclinical testing

Preclinical testing is an integral part of the modern drug discovery process which

helps in collection of important efficacy and safety data before clinical trials can

be carried out. It is a vital step towards sources of new, effective and safe drugs.

The preclinical evaluation of medicinal plants involves documentation and

testing of their biological efficacy, studies of toxicology and chemical profiling.

These have been covered in the subsequent sections.

Phytochemical studies:

Medicinal plant preparations are chemically complex and may contain one or

many structurally related active compounds that produce a combined effect.

63

Phytochemical studies help in standardizing the herbal preparations so as to get

the optimal concentrations of these active constituents, as well as in preserving

their activities. The aim of phytochemical studies is to identify the bioactive

constituents in the plants, devise suitable methods for their extraction, help in

standardization and quality control. For a detailed discussion see (Brijesh et al.,

this issue).

Toxicity studies:

Toxicological studies include acute, subchronic and special toxicology such as

immunotoxicity, genotoxicity, carcinogenicity and reproductive toxicity (Remirez,

2006). These test help in the identification of possible target organs involved and

the toxic symptoms. Studies of special toxicology such as carcinogenesis are very

important if the plants contain compounds with known mutagenic or carcinogenic

activities (Chanabra et al., 2003). It is recommended that a minimum of 2 or 3

mammalian species be used for the in vivo screen. Rodents like mice, rats, or

guinea pigs are used in the initial screen to be used later in combination with other

species such as dogs and monkeys.

Adverse effect profile: It is often argued that prolonged and apparently uneventful

use usually is testimony of safety of medicinal plants. However, a history of

traditional usage is not always a reliable guarantee of safety since it is difficult for

traditional practitioners to detect or monitor delayed effects (e.g. mutagenicity),

rare adverse effects, and adverse effects arising from long-term use (Ernst, 1998),

such as for food supplements and nutraceuticals. Absence of any such

documentation does not automatically rule out the possibility of toxicity. It is

possible that that the plant preparation taken up for clinical trial may lead to some

unanticipated / unknown / unrelated side effect that may vary from person to

person.

The use of herbal preparations may also lead to hypersensitivity reactions

ranging from transient dermatitis to anaphylactic shock (Ernst, 1998). Many

widely used medicinal plants have been implicated as possible causes of long-

term disease manifestations such as liver and kidney diseases. The widespread use

of Scenecio, Crotalaria and Cynoglossum has been implicated in the occurrence

64

of liver lesions and tumours, lung and kidney diseases in certain areas of Ethiopia

(Addae-Mensah, 1992). Another example is of Psoralea corylifolia Linn. which is

used for treating conditions like psoriasis, leucoderma, and non-healing ulcers and

wounds is known to cause hepatosplenomegaly in experimental animals

(CHEMEXCIL, 1992).

There is increasing information available on adverse side effect such as

undesirable outcome on excessive or prolonged usage e.g., Glycyrrhiza glabra

which is used for conditions like bronchitis and peptic ulcers causes not only

hypertension, weight gain and hypokalaemia but also lower levels of aldosterone

and anti diuretic hormone on excessive or prolonged usage (Newall et al., 1996;

toxic effects, e.g., Kava-kava, a well-established hypnotic drug, was reported to

show hepatotoxicity and had to be eventually banned in most countries worldwide

(Wheatley, 2005); allergic reactions, e.g., allergic reactions such as

rhinoconjunctivitis and contact dermatitis have been reported with Allium cepa

(Valdivieso et al., 1994)., interactions with drugs and other herbs, e.g., it has

been advised not to use Silybum marianum with a protease inhibitor during

antiretroviral therapy (Rogers, 2004).

Hence it becomes necessary to carry out toxicological studies, both short

term and long term before initiation of clinical trials, and the risk-benefit

ratio of the herbal drugs also need to be evaluated (Seth and Sharma, 2004).

Biological testing:

Biological screening is a necessary approach to provide a scientific basis for the

continued use of the plants, thereby validating their traditional utilization. It is

necessary not only to establish the therapeutic potential of medicinal plants but

also for identifying and comparing various plant preparations for potency.

Additionally, these studies aid to correlate the activity with some component in

the plant. Thus biological screening along with chemical profiling aids in

standardization of plant material.

Biological screening involves

1. In Vitro Screening: wherein isolated cells, tissues and organs provide an in

vitro model system to initially determine the biological activity of the drug.

65

2. In Vivo Screening: wherein the in vitro biological activity is assessed in

suitable animal models. Animals are also used when a suitable in vitro model

is not available for a particular condition. Besides testing of plant in powder

form or products active on in vivo breakdown, which cannot be tested in vitro

system, have to be tested in animals. Animal testing lays the foundation for

the later clinical trials by aiding in determining several important parameters

like – route of administration, dosage, duration of treatment, fate of the drug

within the host, etc.

Designing of screening assays: The design of a screening assay is an array of

multiple choices, all of which have significant impacts on the outcome of the

overall drug discovery process. The selected assay should be able to mimic the in

vivo dynamics as far as possible with high sensitivity and specificity in examining

the target activity. The basis for designing a screening assay is the identification of

valid target. An estimated 30-40% of experimental drugs fail due to an

inappropriate target (Butcher, 2003) and hence it is important to develop new

screening assays with newer and more appropriate targets. It is crucial to establish

the role of the target in question in the cause or symptoms of a disease (Williams,

2003). Pharmacological manipulation of the target should consistently lead to

desired phenotypic changes. The desired changes must also be reproducible in at

least one relevant animal model (Drews, 2003). To meet these demands, a great

deal of research is required in areas such as target selection and in the

development of improved methodologies for detection and cell based screens.

Emphasis has to be placed on assessment of assay quality and validation of the

parameters being used.

Assay formats employed in screening can be either cell-based or biochemical.

Though the logistics of cell-based assays are more challenging than with

biochemical assays due to requirement of significant investments in cell culture

infrastructure (Moore and Rees, 2001), the current trend in drug discovery is

clearly shifting towards cell-based assays. Cell-based screening has multiple

advantages. It can provide biologically more relevant information on the nature of

the activity (Moore and Rees, 2001; Johnston and Johnston, 2002). In addition,

66

information regarding cellular membrane permeability and cytotoxicity can also

be obtained.

Several researchers have worked on medicinal plants with activity against

different ailments. However, a large proportion of medicinal plant research is

focused on nutraceuticals, chronic and metabolic disorders (diabetes,

cardiovascular etc.) and other diseases like HIV/AIDS, malaria etc. Common

infectious diseases, especially in resource poor communities, such as diarrhoeal

diseases and acute respiratory tract infections are often not addressed or testing is

limited to microbicidal assays.

The general approaches that are commonly used for studying the

pharmacological effects of medicinal plants are: use of single bioassay for

screening multiple plants and use of multiple bioassays for studying single plant.

The latter approach has been used widely for metabolic diseases; bioassays often

represent different steps of a disease cascade with individual bioassay representing

one or more steps in the disease process e.g screening of Magnifera indica for

inhibitory effect on tumor necrosis factor (TNF) and nitric oxide (NO)

representing antioxidative activity (Remirez, 2006).

Unfortunately when screening plants for infectious diseases the assay system

is often limited to testing for antimicrobial activity. However this approach is not

always appropriate. Plants can exhibit their efficacy against infectious diseases by

mechanisms other than antimicrobial activity. When screening plants for immuno-

enhancing properties, often synthetic antigens and immunological assays are used

which do not have any biological relevance to disease(s) in question. The

importance of using relevant and where necessary multiple bioassays for

screening medicinal plants for infectious diseases is highlighted in the studies

conducted by Foundation for Medical Research on antidiarrhoeal (infectious

diarrhoea) medicinal plants. In these studies, medicinal plants were studied for

their effect on different stages of diarrhoeal pathogenesis and not restricted to

antimicrobial profile / gastrointestinal motility as reported by others (Akah et al.,

1999; Kambu et al., 1990; Tona et al., 1999). To the best of our knowledge the

effect of medicinal plants on infectious diarrhoeal pathogenesis has not been

studied.

67

Based on the pathogenesis, adherence & invasion (as markers of colonization)

to HEp-2 epithelial cells and production & action of bacterial enterotoxins [E. coli

heat labile toxin (LT) and cholera toxin (CT); and E. coli heat stable toxin (ST)] in

addition to the antimicrobial activity were used at FMR to test the efficacy of

medicinal plant for antidiarrhoeal activity and thus define the possible

mechanism(s) of action in infectious diarrhoea. The decoctions of two plants viz.

Cyperus rotundus (unpublished data) and Pongamia pinnata (Brijesh et al., 2006)

tested using the multiple bioassays have been used in this paper as examples.

C. rotundus decoction had no antibacterial to six strains of bacteria (Fig. 1a)

or antirotaviral activity (data not shown), but had a static action against G. lamblia

(Fig. 2a). It inhibited production of CT but not its action (Fig. 3a). On the other

hand it inhibited action of LT but not its production (Fig. 4a). It had no effect on

ST (Fig. 5a). The decoction reduced the bacterial adherence to and invasion of

HEp-2 cells (Fig. 6a and 7a respectively). In comparison, P. pinnata had no

antimicrobial activity (Fig. 1b and 2b); reduced only production of CT (Fig. 3b)

and bacterial invasion (Fig. 7b). It did not reduce bacterial adherence (Fig. 6b) and

had no effect on action of CT (Fig. 3b) or production & action of LT (Fig. 4b) and

ST (Fig. 5b).

A B

Fig. 1: Antibacterial Activity: (A) Cyperus rotundus; (B) Pongamia pinnata.

68

Fig. 2: Antigiardial activity: (A) Cyperus rotundus; (B) Pongamia pinnata.

A B

Fig 3: Effect on action and production of cholera toxin: (A) Cyperus rotundus; (B)

A B

Pongamia pinnata.

A B

Fig. 4: Effect on action and production of E. coli labile toxin: (A) Cyperus rotundus; (B) Pongamia pinnata.

69

Fig. 5: Effect on production and action of E. coli stable toxin: (A) Cyperus rotundus; (B) Pongamia pinnata.

A B

A B

Fig. 6: Effect on adherence of E. coli B170 to HEp-2 cells: (A) Cyperus rotundus; (B) Pongamia pinnata.

A B

Fig. 7: Effect on invasiveness of E. coli E134 and Shigella flexneri to HEp-2 cells: (A) Cyperus rotundus; (B) Pongamia pinnata.

70

In summary, C. rotundus has a wider efficacy and would be effective in most

forms of diarrhoea as compared to P. pinnata which has limited efficacy and

would be effective only against invading organisms (and hence bloody diarrhoea)

and V. cholerae. Thus though both the plants did not have marked antimicrobial

action; they are effective antidiarrhoeal agents with different mechanism(s) of

action. Had the study included only screening for antimicrobial activity, both C.

rotundus and P. pinnata would have been considered not to be efficacious. Thus

the study highlighted the necessity of looking at different parameters and not just

concentrating on singular assays like antimicrobial activity for determining the

biological efficacy of plants. In addition based on the results one can arrive at a

rational plant combination for combating different forms of diarrhoea. Ingredients

of this combination can be used to formulate an appropriate ‘herbal package’ for

diarrhoea, which could be effective against all or most of diarrhoeal agents. Such

a package can then be popularized in rural communities where it is difficult for the

people to differentiate between causative agents. This can then lead to generation

of self-help at grassroots.

Another approach used especially when plants are screened for their

immunomodulatory is studying their effects using general immunological

parameters to defined antigens such as lymphoproliferation, nitric oxide

production, antibody production, monocyte phagocytosis etc. However this

approach is not always appropriate as generalized immunological assays can give

misleading results. For example lymphoproliferation can sometimes measure

DTH, or antibodies may not always be protective against a disease, or increase in

phagocytic activity can lead to paralysis of phagocytes.

1. The studies lead to the determining of the therapeutic effect of the plant in

question and also elucidate the efficacy and / or the mechanism of action of

medicinal plants including cell interactions, cell-environment interactions,

intracellular activity, and genetic studies. Plants with novel and / or multiple

mechanism(s) of action can be identified.

2. One of the major advantages of preclinical studies is that one can easily study

and compare the efficacy of different plants in a cost effective manner in a

71

short period of time. The identification of the most efficacious plant part and

the type of extract to be used can also be achieved simultaneously.

3. Development of a rational drug combination can be achieved. This includes

using multiple bioassays designed on the disease process and / or screening

for activity against a spectra of microorganisms (in case of infectious

diseases) to get an insight of varied and diverse mechanism(s) of action of a

plant under study. An example of this has already been discussed dealing with

FMR data on antidiarrhoeal medicinal plants.

4. Isolation of phytoconstituents in the crude extract will be time consuming and

costly. Bioassay guided fractionization can lead to the identification of active

principles and reduces an overwhelming task to isolate all major compounds

from a crude extract.

5. Important information pertaining to the rate and extent to which the

therapeutic moiety is absorbed; is made available to the site of drug action

and the kinetics of drug absorption, its distribution and elimination can be

achieved through studies on bioavailability and pharmacokinetics of the plant

in question.

6. Shelf life of plant material is usually ignored due to the general belief that the

plant materials do not have an expiry date. However this is not always true.

Biological screening can help monitor the degradation of plant material and

decide the appropriate storage period.

7. Preclinical pharmacological safety data obtained from both in vitro (cell-

based or biochemical) and in vivo studies on animals can be used as indicators

of potential toxicity.

8. Unlike the clinical trials, the burden of ethics in the preclinical testing is

minimal.

Limitations of Preclinical testing:

1. Suitable pharmacological models have not yet been developed for many

common diseases with unknown, or multifactorial origins (Hamburger and

Hostettmann, 1991)

2. The lack of a positive result in a screening assay does not always mean the

absence of bioactive constituents. This may occur under two circumstances-

72

a) the active principle (s) may be present in insufficient quantities in the crude

extracts to show activity in the dose levels employed, b) the assay system

employed may not be the appropriate one. Alternatively, if the active

principle is present in high enough quantities, there could be other

constituents exerting antagonistic effects or negating the positive effects of

the active principles during the assay (Farnsworth, 1993).

3. The in vitro bioassays used for preclinical testing often have certain

limitations for e.g. Phenols can affect enzyme-based targets while saponins

may disrupt membranes in cellular targets or dislodge substrates absorbed

onto assay wells (Taylor et al., 2001). Another limitation would be the

interference of pigments in colorimetric or quenched assays (Taylor et al.,

2001).

4. In vitro assays cannot screen for active principles that are generated as

metabolized products in vivo (Farnsworth, 1993)

5. The pharmacological investigation of drug interactions in multi /compound

preparations is difficult due to the presence of constituents from several plants

where some plants may show less specific activity and some plants may have

been added to reduce the toxicity of the more therapeutically effective plants

(Taylor et al., 2001).

6. In case of bioassay-guided fractionation, there is a possibility of loss of

activity as the compound in question may be sensitive to temperature, light,

acidity, basicity or the extracting solvent and consequently it is progressively

degraded during fractionation. Alternatively, some compounds are inactive in

situ but act synergistically with other constituents of the extract. Separation

into different fractions during purification will thus result in a decrease or

total loss in activity in all the fractions e.g. loss of activity of Cirriformia

tentaculata upon its hexane fractionization (Kicklighter et al., 2003).

7. All human ailments do not have animal models.

8. Some of the most common side effects are difficult to recognize in animal

models e.g. nausea, nervousness, lethargy, heartburn, headache, depression,

stiffness, etc.

9. Extrapolation of in vitro dose to in vivo animal models and humans is

difficult.

73

10. Difficult to test organic extracts and dry powders not soluble in water in vitro.

Conclusions:

The area of medicinal plant research is fast developing. Both pre clinical and

clinical testing are integral components of medicinal plant research. Preclinical

testing of plants for medicinal properties is of vital importance, not only to

provide a scientific basis for their usage but also validate their historical utilization

by traditional healers and herbalists, and thus provides the society with sources of

new, effective and safe drugs. With the opening of newer vistas in the field of

medicine including the modern molecular biology tools, high output automated

bioassays and newer technologies for rapid structure determination in the area of

medicinal plant research, the field of preclinical testing seems to have a bright

future. Despite the limitations, the various advantages the pre clinical testing

offers, justify it as an essential prerequisite of a drug discovery process. It is

possible that with the advancement of technology, current tests could prove

successful where negative results may have been obtained 20 years ago (Prance,

1994). Thus preclinical testing can serve as an important link between a plant

selection and its subsequent mass usage following proper clinical testing.

Acknowledgements

The financial assistance of Sir Dorabji Tata Trust, Sir Ratan Tata Trust and

Department of Science and Technology, Ministry of Science and Technology,

Government of India (grant number 91283) is acknowledged.

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2.5 Challenges in Preclinical Testing of Traditional Medicines: In Search of Solutions Nirmala Rege Department of Pharmacology and Therapeutics, Seth GS Medical College &KEM Hospital, Mumbai - 400012, India. Email: nimarege@gmail.com Abstract

There is no doubt that pre-clinical development of plant drugs from traditional

systems of medicines should be based on scientific principles. However, while

working with drugs from traditional medicines several problems are posed

pertaining to agent itself, assay methods and analysis of data. These are discussed

by the speakers of this session giving examples of various assays they have used

in their laboratories.

What is additionally important is the review of literature, both from

traditional system of medicine and modern science. Critical appraisal and

interpretation of the text of traditional system helps one to select appropriate

extraction and fractionation procedure and also provide guidance for selection of

relevant model.

Ideal conditions for pre-clinical testing are described in the literature and it is

necessary for the researcher to use standardized plant material for the study and

adhere to the GLP. At the same time, to meet these ideal criteria may not always

be possible. It is therefore essential to define bare minimum or acceptable limits.

Selection of appropriate concentration for in vitro assay and its correlation to the

dose for in vivo model remains the problem for traditional drugs. However, it is

essential to clarify whether the concentrations/doses are expressed in terms of

extract concentration or crude drug concentration. In case of former, percentage

yield from the crude drug needs to be mentioned. Without proper clarification, if a

cut-off for concentration is decided then, a clinically used drug may get rejected in

bioassay. In vitro effects may not correlate with in vivo effects and it is essential to

use at least 2-3 in vivo models to confirm the results.

The combined results of in vitro and in vitro models assist in extrapolating the

findings to human in better way.

78

Some other points which need to be discussed are unique mode of action of a

plant drug, especially herbo-mineral formulation which may be totally different

from the known drugs in that therapeutic area; widely used screening models may

not be suitable for detecting therapeutically relevant pharmacological properties of

plant drug; and in conventional in vitro models effect of metabolites are not

detected.

The discussion of the present session will focus on addressing these

challenges and deriving the best possible solutions.

Full text not received

79

3.1 Traditional Knowledge Guided Testing of Quality, Safety and Efficacy of Herbal Medicines Padma Venkatasubramanian Joint Director, Foundation for Revitalization of Local Health Traditions (FRLHT), B74/2, Jarakbande Kaval, Post Attur, Via Yelahanka, Bangalore – 560064, India. Email: padma.venkat@frlht.org Abstract

The world is witnessing an unprecedented growth in the usage of herbal products

at national as well as international levels. The issue of quality is becoming more

and more pertinent since the herbal medicines available in a country may have its

origins in an alien country/culture, thereby making regulation and quality control

that much more difficult. Unlike in olden times where traditional physicians

prepared and tested the quality of herbal medicines, the problems faced today are

those of economics of industrial scale production, shelf-life, and distribution to

long distances. These have necessitated development of modern and objective

standards for evaluating the quality, safety and efficacy of these medicines. The

current standards, parameters and protocols available to test the quality of herbal

medicines were originally developed for allopathic drugs and can at best

authenticate the identity of plant materials (maybe purity to some extent) not their

safety or efficacy.

Herbal medicines are natural products and their phytoconstitutents vary

depending on time and region, processing and storage. Variations in the

collection, processing or storage of an herb could impact its efficacy profile. Since

prior knowledge regarding appropriate collection and usage of most medicinal

plants exists in tradition, it can be used as a guide to quality standardisation. The

parameters of testing of the quality of materials (dravya) in traditional medicines,

such as rasa (taste), guna (properties), virya (potency), vipaka (post-digestion

effect) and karma (action), are very different from the western methods. These

traditional parameters reflect not only the quality but also efficacy. Having said

which, there are no direct written protocols available in traditional medicine either

for collection or for testing the action. The methods of testing are lost today and

need revivification.

80

The issues for deliberation in the present paper would be:

1. medicinal plant quality and factors affecting the same

2. the relevance of western & traditional parameters used to identify the quality

of a herbal product

3. R & D efforts at FRLHT, Bangalore

The objective of research at FRLHT is two-fold with respect to quality

standardization of medicinal plants: (i) development of modern scientific

standards for medicinal plants taking into consideration the traditional advice on

medicinal plant identity, collection, processing and storage and (ii) development

of contemporary scientific methods to test traditional parameters such as rasa.

The strategy adopted has been to firstly document the advice and protocols on

medicinal plants usage from texts and from living traditions of medical

practitioners, conduct preliminary laboratory tests and then to identify the unique

chemical/biological markers. The laboratory techniques such as phytochemistry,

chromatography (HPLC, HPTLC), molecular biology and bioassays are used to

study the quality. Biological activities are subsequently verified through

appropriate animal testing.

In a pioneering attempt, modern sensory evaluation has been used in a

systematic manner, as one of the quality testing tools for evaluation of quality of

herbal medicines. Sensory evaluation methods and standards are being developed

for herbal medicines.

Why do we need new strategies?

The standards used for checking the quality of medicinal plants are broadly based

on modern botanic description (macroscopy), anatomy, chemistry and

microbiology. On scrutiny (Table 1) one can understand that the values indicated

in a modern Certificate of Analysis (CoA) for any raw drug either reflects its

identity, purity or strength but not its biological activity, when the whole purpose

of using the raw drug is for its medicinal properties. The specific conditions

(time/ region) of collection of the plant are also not specified. These are precisely

the drawbacks in the current approaches to standardization of medicinal plants.

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Table 1: Modern Certificate of Analysis for Ginger (Mukherjee, 2002)

Botanical Name: Zingiber officinale Roscos Part Used: Rhizome Organoleptic characters: Yellowish brown, in colour, aromatic, pungent

Test Limits Protocol used Foreign Organic Matter <1 % Sand & silica Absent WHO Insects Nil Rodents Nil Ash Content <8 % w/w Acid Insoluble Ash <1.0 % w/w Moisture Content <12 % w/w WHO Volatile Oil Content 1-2.6 % w/w Water Soluble Extractive >14% w/w Alcohol soluble extractive >6% w/w Markers/Active Gingerol, Shogaol Phytoconstituents TLC Fingerprint HPLC Fingerprint

Traditional Indian Medicine such as Ayurveda has a different way of

classification of quality of medicinal plants which combines at once the

pharmcognosy (properties) and pharmacology (action). The parameters of quality

in Ayurveda (Table 2) are rasa (taste), guna (properties), virya (potency), vipaka

(post-digestive effect) and karma (action).

Table 2: Ayurvedic standards of ginger (Anonymous, 2001)

Parameter Standard Rasa (Taste) Katu (pungent) Guna (Properties) Laghu (light), Snigdha (unctuous) Virya (Potency) Usna (hot) Vipaka (Post-digestive effect)

Madhura (sweet)

Karma (Action) Deepaka (stimulates digestive), Pachana (digestive), Hrdya (good for heart), Anulomana (creates movement) etc.

It can be noted that the pharmacognosy and pharmacology of the raw drug are

inseparably evaluated in Ayurveda. This was done by trained scholars called

aptas, through human perception. As to know when these qualities are fully

expressed there were general (Table 3) as well as specific recommendations either

available in texts or are well known to the traditional healers and physicians.

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Table 3: Ayurvedic recommendations for collection of medicinal plant parts

Plant Part Collection Season Branches & leaves Rainy & Spring Roots Summer & Late winter Bark, Rhizome, Sap Autumn Heartwood Early winter Flowers & Fruits As per season

However, the major hurdle in this is the access to information and protocols

of assessment of Ayurvedic properties. It is not methodically documented and

available as a ready reckoner. Significant proportion of the information may also

be lost over the centuries. Due to this, modern parameters and standards are being

used to screen/test Ayurvedic medicines, which do not truly reflect its safety or

efficacy. These include phytochemical, anatomical or molecular standards.

Unlike in Ayurveda, which used human body and perception to study the

properties and action of a drug, there is no single instrument in modern S & T that

can check at once the pharmacognosy and pharmacology of the drug. Human

sensory evaluation is of particular value in the Food & Beverage industry (such as

wine and tea) but not in the modern pharma sector. Sensory evaluation is still

used by the traditional Ayurvedic drug industries for raw drug identification and

preparation of formulations, but protocols are not documented and therefore

appear subjective. With the onset of industrialisation of Ayurvedic products, finer

details such as best time and place of collection etc are being ignored.

The team at FRLHT has taken a traditional knowledge guided approach to

standardize the quality of medicinal plants. It is two-pronged approach: (i) the

method of reverse pharmacognosy is being employed, where, the plant material

taken for standardization is according to traditional knowledge, (ii) Traditional

Quality Parameters, such as rasa are being researched both from Ayurvedic view

point as well as through modern scientific tools.

Strategy

The following strategy has been adopted for research into Traditional Quality

Standards (TQS) at FRLHT:

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1. Documentation: Since there are no ready-reckoners available in traditional

knowledge on quality standards, advice on collection etc. of medicinal plants,

it is important to firstly document the same from traditional texts such as

Caraka Samhita as well as from living traditions. Through a limited study

funded by National Geographic Society, over 20000 verses pertaining to

quality aspects of 2000 medicinal plant names have been compiled from

12 classical Ayurvedic texts by FRLHT (Venkatasubramanian and

Geeta, 2004). Important information on traditional methods of processing

and purification of 10 toxic plants such as Strychnos nux-vomica, Croton

tiglium, and Abrus precatorius were also documented from traditional elderly,

but practicing physicians during this study.

2. Informatics: In order to store, retrieve and analyse the wealth of information

compiled, a prototype CD, has been developed with information on the

purification methods on 10 toxic plants. This contains facility to query, save,

print and browse not only text but also crucial images and video-clippings

collected during the documentation (FRLHT, 2004).

3. Prioritisation of Specific Recommendations: A prioritized list of 30

recommendations was prepared based on the specificity and practicality of

the traditional advice. A few examples are provided in Table 4. These

recommendations were taken up for conducting further studies.

Table 4: Examples of specific traditional advice on medicinal plants

Identity Vidanga seeds (Embelia ribes) are also called chitra tandula (patterns on the seeds) and resemble Kapala (bowl shaped structure)

White Seeds of Abrus precatorius are better than red ones for medicine preparation

Collection Mature vidari (Ipomea mauritiana) should be used in formulations Haridra (Curcuma longa) is best collected at night

Bhallataka (Semecarpus anacardium) fruits should be collected when it resembles ripened ‘Jamboo’ (Syzygium cumini) fruits

Processing Pippali (Piper longum) should be prepared as a milk decoction Commiphora mukul (guggul) should only be used after shodhana (purifiction)

Vidanga (Embelia ribes) seeds should be used after storing for one year

Storage

84

4. Inter-cultural studies: Understanding the context and purpose of traditional

advice requires an inter-cultural approach that in turn requires a dialogue

between different disciplines such as trade and distribution information on the

raw drug, Ayurvedic information from text and practice, collection of

botanically correct specimens for testing and standardization using modern

phytochemistry/biological methods.

Study 1: Inter-cultural study on Vidanga

FRLHT has conducted an inter-cultural study to identify the authentic species

that can be called as vidanga as per Ayurveda. Vidanga is one of the 10 most

traded plants in volume and there are at least 3-4 botanical species being

traded as Vidanga. Prior correlations have indicated that Embelia ribes to be

the authentic species. However, our market and distribution study pointed out

that > 95% of the traded species are E. tsjeriam-cottam. There were also other

Myrsinacea members such as Myrsine africana, My. capitellata and Maesa

indica that were being used as vidanga. On analysis of Ayurvedic

terminologies pertaining to vidanga, such as chitratandula (patterns on

seeds), kapaala (bowl-shaped structure), kshudra tandula (small seeds), some

of the candidates such as M. indica and My.africana were eliminated. The

closest matches were E. ribes and E. tsjeriam-cottam (Fig 1).

Fig. 1: Characteristics of vidanga seed.

Bowl shaped

Patterns on seed

Upon learning that both E. ribes and E. tsjeriam-cottam are good

candidates for vidanga as per Ayurveda, further work on microscopy, HPTLC

and HPLC were used to distinguish the authentic from the adulterants.

85

Molecular tools such as RAPD-PCR have been used to distinguish between

the species at the DNA level (Fig 2).

Fig 2: Distinction of species traded as vidanga RAPD-PCR technique.

Study 2: Traditional knowledge guided Quality standards for Ipomea

mauritiana tubers

Tuber of Vidari (I. mauritiana) is a galactagogue and an immunomodulator as

per Ayurveda. However, the immature tubers are discarded while selecting

the mature ones during preparation of medicine. The main parameter based

on which the distinction is made by physicians is through the size. Mature

ones are larger than the immature ones (Fig 3).

Our aim was to (i) determine if the traditional ways of culling had a

bearing on the activity of the raw drug (ii) identify the chemicals that would

reflect its activity not just the identity. The culling of the tubers was done by

a qualified and experienced physician on which grading was noted as per size

and categorized as totally immature and totally mature. The bioactivities of

the extracts of the tubers were compared to reflect their overall difference

through Brine Shrimp Bioassay (Meyer et al., 1982). The bioactivities of the

mature tubers were twice as active as that of the immature ones. The HPLC

profiles of the tubers indicated that the ratio of peaks at ~11 min and 15 min

increased significantly with maturity (Fig 4). Fig. 3: Morphology of mature and immature tubers of I. mauritiana.

86

Preliminary studies on immunomodulatory effect on rats (Indian ink,

oral) have shown that the phagocytic index of mature extracts is twice that of

immature. The compounds of interest appear to be β-sitosterol as studied

through LC-MS. Further investigations are ongoing to identify maturity and

bioactivity related marker.

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Detector A - 1 (254nm)L0512009 3stage (2.5 mgpml)L0512008 3stage (2mg-1ull)

Detector A - 1 (254nm)L0512016 10 stage (2.5 mgpml)L0512016 10 stage (2mg-1ul)

Retention Time -- Mature Fig 4: Comparative HPLC of mature and immature tubers of Ipomea mauritiana.

-- Immature

Study 3: Sensory Evaluation as a Quality Control tool

Even today, sensory evaluation is used by the managers in traditional

medicine industry (especially small scale manufacturing units) to check the

quality of raw ingredients as well of end products. It also is an effective on-

line process control tool. Sensory evaluation is an accepted scientific quality

control tool in the food and beverage industry as well as the perfume industry.

It requires rigorous protocol development for each product, testing by trained

panel and statistical analysis. At FRLHT, protocols have been developed

(combining traditional and modern sensory parameters) for 3 products and 10

raw drugs including trikatu choorna, nisha amlaki and bala taila. Easy-to-use

formats and instructions have been developed for use by the SSUs.

Conclusion

Standards for medicinal plants have to reflect not only the identity but also their

efficacy and safety. Working out the safety and efficacy profiles through animal

studies and clinical trials for the enormous number of plants would not only be

expensive but also time-consuming. Traditional knowledge about collection and

usage of medicinal plants has the advantage of experience that can be tapped. Just

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as these days, reverse pharmacology is being used to identify bioactive molecules

from sound formulations of traditional medicine, reverse pharmacognosy can be

employed to identify quality determining standards as per traditional knowledge.

The paper has illustrated with examples the strategy that needs to be followed in a

traditional knowledge guided approach to build relevant standards.

The hurdles that need to be crossed are of various kinds, such as:

• Policy and fund allocation for research in Traditional Medicine

• Mind set of modern scientists & journals in accepting the importance of

cross-cultural research

• Documentation of TQS from texts and living traditions

• Access of plants and information to researchers

• Identification of appropriate scientific tools to study the TQS

• Networking and sharing of research findings/standards within and outside the

country, especially the users such as small scale manufacturing units,

physicians, farmers.

References

1. Anonymous. 2001. The Ayurvedic Pharmacopoeia of India, Vol 1 Part 1

(First Edition), pp 103

2. FRLHT’s Traditional Quality Standards—Purification of 10 Toxic Plants. A

Prototype CD. 2004, FRLHT, Bangalore.

3. Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin

JL. 1982. Brine Shrimp: A Convenient General Bioassay for Active Plant

Constituents. Planta Medica 45: 31-34.

4. Mukherjee P. 2002. Quality Control of Herbal Drugs, Pharmaceutical

Publishers.

5. Venkatasubramanian P, Geeta UG. 2004. Documentation of traditional Indian

Methods of Medicinal Plants Collection, Processing and Storage- A Report

submitted to National Geographic Society, FRLHT, Bangalore.

88

3.2 Principles of Quality Control, Standardization and Chemo profiling of Medicinal Plants and ISM Preparations Ravi K. Khajuria* and S. G. Agarwal Indian Institute of Integrative Medicine (formerly Regional Research Laboratory), Canal Road, Jammu Tawi – 180001, India * Corresponding Autior Overview:

The recent years have witnessed resurgence of interest in herbal medicines as

more and more people throughout world are turning to use medicinal plant

products in healthcare system. The sales for herbal medicine products have

plateaued to such an extent that these products have become available to

consumers as positive healthcare just like vitamins. They are now found in

supermarkets, pharmacies and numerous other main stream retail outlets as over-

the-counter drug products.

Botanical medicines, similar to all pharmacologically active substances have

the potential to contribute positively, neutrally, or negatively to the health status of

the people. World wide need of alternative medicine has resulted in growth of

natural product markets and interest in traditional systems of medicine. The use of

plants for health or medicinal purposes has been a part of every culture or region

but now this science of herbal drugs is passing through the age of renaissance.

Through out the world, the major problem that hinders the prospects of herbal

products in becoming the main stay of any treatment modality is the difficulty in

standardizing a formulation. System of Ayurveda is well respected globally and

many of the herbals used in Ayurveda, are covered in WHO monographs on

selected medicinal plants, thus confirming the efficacy of the Ayurvedic, Siddha

and Unani (ASU) System.

There are about 10,000 plant species which are being used in Indian System

of Medicines (ISM) /traditional medicines in Indian subcontinent. Out of these,

450-500 species are mostly utilized in over 85% of the Ayurvedic, Unani and

Siddha formulations and about 40 plant species are used in modern drugs. Twenty

five of these are cultivated while other plant species are collected by

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proper/destructive harvesting. Important parameters for collection of the plant

materials are proper botanical identification, proper chemo-type/ecotype, desired

state of maturity, post-harvest processing technology and proper storage

conditions. These herbals account for a large chunk of global drug market,

therefore assurance of their quality, safety and efficacy needs additional attention.

Safety Parameters:

Standardization of herbal drugs is gaining momentum in India and as a result, it is

also proposed to include the safety parameters as per International norms. So far

as drugs are concerned one must remember that “Safety comes first and quality

afterwards”. Therefore, In addition to proper botanical identification and physico-

chemical parameters, test for heavy metals (Mercury, Lead, Cadmium, Arsenic),

microbial contaminants (Total viable aerobic count, Total Enterobacteriaceae,

Total fungal count), specific pathogens (E. coli, Salmonella spp., S. aureus,

Pseudomonas aeruginosa), pesticide residue (organochlorine, organophosphorus,

pyrethroids & others), aflatoxin (B1, B2, G1, G2) should also be an important part

of any Quality control protocol. State-of-art facilities have been installed at

Quality Control Laboratory of RRL, Jammu to test such parameters.

Botanical concerns: (Identification of true ASU herbal drugs, Taxonomic

characterization, Collection, Post-Harvest Processing and Storage)

Many a time similar names have been very frequently used for several related

umbelliferous fruits, mint leaves and various other plant materials and thus,

different plant species are sold in the market under the same name in different

parts of Indian sub-continent. We would like to mention a few to draw attention.

“Ajamoda” (Trachspermum roxbrghianum), a reputed drug of the Ayurvedic and

Unani systems of medicine but in the market Apium graveolens, Apium

leptophyllum, Trachspermum roxbrghianum and T. ammi are sold under one trade

name i.e. Ajamoda (Table1) in different regions (Dutt, 1974). The major chemical

constituents of all these four-plant spp. are different and can easily be

characterized on the basis of chemical markers (S. G. Agarwal, unpublished data).

Many species of Mentha such as M. spicata, M. virides, M. longifolia, M.

piperita, M. arvensis, M. aquaicta and M. pulegium are sold under one name i.e.

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Pudina in various parts of the country but all the spp. contain different

monoterpenes. In some cases constituting monoterpenes are same; they are

present in different amounts in different species (Agnihotri et al., 2005). Three

plant species such as Carum carvi, Bunium persicum and Bunium cylindricum are

sold under one name i.e. Kalazeera and all the three contain entirely different

constituents. Fruit of another plant i.e. Cuminum cyminum is sold under the name

of Safed zeera has constituents similar to one of Kalazeera i.e. Bunium persicum

[Thappa et al., 1991; Agarwal et al., 1974; Agarwal et al., 1979 (Table 3)].

Similarly different ecotypes yield different constituents with various proportions

(Table 4)

On the other hand, many a time different plant species contain similar

chemical markers and same is true with Mucuna puriens, M. utilis and M.

cochchinensis, which all contain L-dopa as marker compound, although these are

very much different in size and appearance. Our own studies (Ahmed et al.,

2006) on three Tinospora spp which are sold in the market under the name of

“Guduchi” have revealed that there is lot of variation in the chemical constituents

of three spp of the plant i.e. Tinospora cordifolia, T. malabarica and T. crispa.

Further, chemical composition of many plant species is directly related to the

phenological stages of plant growth and changes with season. The best example is

commonly used vasaka (Adhatoda vasica) for cough & cold. At vegetative stage,

the total alkaloid content are highest with vasicine (90%) and vasicinone (5%); but

at full bloom stage alkaloid content falls with simultaneous variations in

quantitative composition with vasicine (45%) and of vasicinone (25%) of the

total alkaloids along with several other water soluble glycosylated, oxides and

other derivatives of vasicine/ vasicinone (Pandita et al., 1993). The post harvest

processing of Crocus sativus stigma by traditional methods which takes 3-5 days,

the amount of total crocins content is 7-9% and if the same stigma is processed at

45oC ± 5oC with tray load of 1M3 which requires 6-7 hours of drying, the amount

of crocins (Table 5) from the plant increases to more than 14% (Raina et al.,

1996).

Thus, there is a need to remove ambiguities related to proper herbals to be

used in ASU System of medicine followed by proper taxonomical identification,

harvesting time, post-harvest processing, storage conditions etc. as these are of

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utmost importance in maintaining the quality of an herbal product. Therefore,

“Standard Operating Procedures” should be adopted for collection, processing

storage and sampling of herbs and herbal products. Further, ASU drugs should be

categorized separately, from the EU Herbal drugs, as these have evidence based

documented records and are widely used in Indian sub-continent for centuries.

Additionally, ASU drugs belong to organized system of healthcare and practiced

by experts in the field.

Chemical Standardization and Chemo-profiling of ASU drugs:

There are always chances of wide variations with respect of their chemical

contents in crude drugs/ raw materials of plant origin due to varied reasons such as

climatic conditions, geographical distribution, source and season of collection and

lack of scientific methods of post-harvest processing, storage and preservation.

Keeping these facts in view for acceptance or rejection criteria, minimum-

maximum limits for marker compounds, based on chemical analysis of large

number of samples for each drug/ herbal collected from different agro-climatic

zones with passport data has to be worked out, to have reproducible results.

Therefore, the only solution to place ASU System of medicines on global

market, is to chromatographically standardize the herbal products and wherever

possible quantitate them for active/or phytochemical marker(s). Thin Layer

chromatography is a powerful and simple analytical tool, used for this purpose;

there were situations where this tool of analysis did not give satisfactory results

because of its own limitations (Singh et al., 2003). Quantitative and qualitative

HPLC, HPLC/MS, HPLC/MS/MS, GC, GC/MS, GC/MS/MS, HPLC-UV–DAD

and HPTLC are well-suited analytical methods of choice to control the quality of

phyto-pharmaceuticals.

In modern times the issues of quality, safety and efficacy of medicines are

interrelated. The quality of drug is of paramount importance as it can affect the

issues of efficacy and safety. Long history of use and better patient tolerance as

well as acceptance, renewable source, cultivation and processing, environmental

friendly, local availability especially in the third world and several important

recent break-through are the major factors responsible for the resurgence of

interest in plant based drugs. The people all over the world have realized that vast

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plant wealth has much to offer in the shape of new remedies which are

efficacious, safe and accessible to masses. By laying guidelines for the assessment

of herbal medicines under the programme on traditional medicines, WHO has

recognized the role of traditional system of medicines in healthcare programmers

all over the world. We in India therefore, have to gear up to the challenge

especially when we possess wealth of natural drugs second to none in the world.

According to world statistics developing one new drug needs screening of

10,000 molecules with 10-15 years of time and an expenditure of more than $ 900

million. But if we screen plant drugs from known natural sources with assured

therapeutic efficacy, the percentage of hits be increased, thus saving time and

money. WHO currently encourages, recommends and promotes traditional herbal

remedies in national healthcare programmes because such drugs are available at

reasonable price, comparatively safe and people have faith in such remedies. At

the same time, WHO in its number of resolutions have emphasized the need to

ensure quality.

In the present global market scenario related to traditional and herbal drugs,

People Republic of China is leading with 50% market share followed by Japan

(20%) and Russia (16%).Unfortunately India’s share is only 2.5% although the

internal market has crossed over Rs.4000 crore per year. The reason is very simple

and to ensure fair share in world market the quality control and scientific

evaluation data for Indian products has to be generated / presented to global

community. Development of ASU drugs with proper efficacy has been undertaken

by Department of Ayush, ICMR and CSIR through Golden Triangle Programme

utilizing traditional knowledge, latest safety, chemo-profiling and reverse

pharmacology route to arrive at standardized formulation (preferably with 3 to 5

ingredients) with IPR, for placing at global market.

Why standardization?

Reproducible assays of the plant preparation generate confidence in the mind of

the user and prescriber. To ensure consistent quality of the preparation, the

qualitative and quantitative chromatographic fingerprint on the basis of

characteristic substance(s) for raw materials and finished products should be

provided. Thus, the standardization of the herbal drugs and preparation thereof is

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not just an analytical operation that ends with the identification and assay of an

active principle; rather it embodies total information (passport data of raw

material, quality parameters, process control) and controls that are necessary to

guarantee consistency of composition. The question of quality acquires greater

relevance today than ever before. Regulatory agencies and consumer groups on

one hand and increasingly stringent quality norms for global positioning of the

products on the other hand, demand that new materials and herbal products there

from, used for medicinal, nutritional and cosmetic purposes should be of

consistent reproducibility; scientifically (chemically and biologically) validated

and supplemented by data on documented evidence of efficacy and safety through

limited human clinical trials.

However, with rapid advancement in the field of instrumentation where limits

of detection (LOD) and limits of quantification (LOQ) have routinely been

upgraded to femto & atto levels, the chemical standardization of herbal products

by hyphenated techniques (HPTLC, HPLC, LC/MS and LC/MS, GC/MS) has

become more credible than biological evaluation of data.

Although liquid and gas chromatography are powerful tools for chemical

profiling of the herbals, but these in combination with mass spectrometric

detection has become more effective and convenient. The techniques are very

useful for the characterization and quantification of the individual constituents in

the plant extracts. LC/MS and GC/MS based multi component hyphenated

methods allow the determination of the components in the mixture. These

techniques are sensitive, selective, fast with inexpensive clean up for sample

preparation; where simultaneous separation as well as identification of the

components in a mixture is possible. LC/GC perform the function of separation

whereas MS performs the function of identification of the components in the

mixture on the basis of molecular mass and fragmentation pattern. It gives us two

dimensional information, the information from the UV/VIS or diode array

detector in LC and FID/ECD/NPD detectors in GC is the a first dimensional

information in the form of retention time whereas the second dimension of

information comes from the mass detector in the form of molecular mass and

fragmentation pattern. Techniques are very sensitive, selective and specific and

allow the detection of the compounds even in picogram amounts.

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LC/MS has been playing a more significant role in plant medicine research

(Caig et al., 2002) because the technique is capable of characterizing active

components ranging from small molecules to macromolecules and recent

scientific results and publications show that application of LC/MS has been

rapidly expanding into the area of structure elucidation and characterization of

active components, in addition to valuable quantitative analysis. The LC/GC-MS

instruments are of two types, Ion trap and linear quadrupoles. Linear quadrupole

instruments are very sensitive and are used for quantitative analysis whereas Ion

trap technology is multi step fragmentation technology and the instruments based

on this technology are used for characterization of the molecule. Under CSIR’s

NMITLI Project, our studies on quantification of four chemical markers in

different cultivars (cultivated/ wild accessions) of Withania somnifera (Khajuria et

al., 2004) have provided us a very valuable and significant data. These studies

have revealed that an accession cultivated at Regional Research Laboratory

(CSIR), Jammu and coded, as AGB002 can be the best source for withaferin-A.

The PCR based RAPD and AFLP studies on this accession have revealed that this

accession has entirely different DNA sequence when compared with other

accessions. Gradual improvements in the LC/MS methods led to the development

of methods for quantification on the basis of five (Fig.1), seven (Fig.2) and finally

eight withanolides and withanosides (Fig.3).

LC/MS methods are very useful in determining active components and their

metabolites in pre-clinical studies. Studies on the metabolites of piperine, an

alkaloid constituent of Piper nigrum and Piper longum led to the characterization

(Bajad et al., 2003; Bajad et al., 2003) of two metabolites in rat urine. These

metabolites were characterized on the basis of LC/MS/MS and LC/NMR/MS/MS

data.

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Fig. 1: HPLC Chromatogram of five markers of Withania somnifera

Fig. 2: HPLC Chromatogram of seven markers of Withania somnifera

Fig. 3: HPLC Chromatogram of eight markers of Withania somnifera

Over recent decade, a number of lead compounds and new natural products

derived from medicinal herbs have been successfully isolated and identified.

96

Chemical analysis plays a central role in development and modernization of

herbal or herbal-based medicines and mass spectrometry coupled to LC is

emerging as the technique of choice in the identification of active ingredients,

compositional analysis and chemical finger printing studies. Tandem mass

spectrometry (MS/MS) is a powerful technique for detecting a target compound in

complex matrices, including plant extracts and herbal drugs. The strength of this

technique lays in the selectivity, high sensitivity and fast screening capabilities

compared with many other separation and identification techniques. We have

developed a rapid LC/MS/MS screening method for detection of camptothecin in

an endophytic fungus Entrophosphere infrquens, which resides in the plant

Nothapodytes foetida (Touseef et al., 2006). For preparation of marker grade

hyperforin from Hypericum perforatum a preparative HPLC method (Anand et al.,

2003) has been developed by our group and similarly a very sensitive LC-MS

method was developed to check the purity and stability of the same. LC/MS

methods for the detection of different compounds like ß, ß-dimethylacrylshikonin

in tissue cultures of Onosoma echiodes etc. have been developed (Lattoo et al.,

2005). These methods are also used to detect the same compounds in the plant

extracts.

Conclusions

Proper botanical identification, proper chemo type/ecotype, proper/desired stage

of maturity, post harvest processing, storage, isolation of marker compounds

(preferably the therapeutic ones), validated analytical (GC, GCMS, LC, LC-MS

and LC-MS/MS) methodologies for the quantification of the extracts/fractions

are the important milestones in the herbal drug standardization. Ever increasing

discovery costs and increased failures at the end of the discovery line makes

medicines unaffordable to the developing countries. This makes new approaches

such as system biology and reverse pharmacology more attractive that provide

innovation opportunities that are based on experiential wisdom and holistic view

point of ‘Traditional Medicine’. It is known to all of us that drug discovery

pipeline in modern drug discovery is getting dry and modern world is looking

towards the herbal world with great expectations.

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Table 1: Different plants sold under the name Ajmoda

Ajmoda Species constituent Volatile Oil Important 1. Apium graveolens

(Celery) 1-2% Limonene (~85%), pentyl benzene, 3-

n-butyl phthalide & other phthalides 2. Apium leptophyllum 1-2% Monoterpenes, coumarins 3. T. roxberghianum 4-5% Limonene (~15%), Cadinener (~ 24%),

β-cyclolavandulala/ acid is 15-25%, Seselin (~12-15%)

4. T. ammi 2-5% p-cymene, γ-terpinene, thymol Table 2 Different species of Mentha sold under the same name as Pudina

Pudina Species Volatile Oil

O

O

O

OH

O

O

Important constituents M. spicata l-limonene, l-carvone

M. virides Piperitenone oxide

M. longifolia l-limonene, l-carvone

M. piperita l-menthol (~ 50%), isomenthone others

M. arvensis l-menthol (~75%), menthone, others

M. aquatica Isopinocamphone O

O

M. pulegium Pulegone (~80%), piperitone, menthone

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Table 3 : Different plants sold under the name Kalazeera and Safed Zeera

Zeera Species Volatile Oil Important constituents Kala Zeera Carum Carvi 2.5-4% d-limonene, d-carvone Bunium persicum 4-12% p-cymene, γ-terpinene, cuminaldehyde, p-

mentha-1,3-dienals, p-mentha-1,4-dienals Bunium cylindricum 1-2% Myristicin, Dillapiole, elemenes Nigella sativa - Carvone, limonene, p-cymene, thymol,

nigellone Safed Zeera Cumin cyminum 1.5-3% p-cymene, γ-terpinene, Cuminaldehyde, p-

mentha-1,3-dienals, p- mentha-1,4-dienals Table 4: Different ecotypes giving different constituents in different ratios

Ecotype Ajwain (Trachyspermum ammi)

Oil Thymol p-cymene + γ-terpinene

Moti Ajwain 1-3% 85-90% 10-15 % Choti Black 3-5% 35-45% 40-60% Khus (Vetiveria zizaniodes)

North Indian oil l -Vetiverol 63-78%

South Indian oil d-Khusinol (major) Table 5: Yields of crocins by using different drying techniques Crocus sativus

Post Harvest Processing Crocins pigment content Traditional processing 3-5 days 7-9% Processing at 45oC + 5oC (6 hrs drying) 14-17%

References

1. Agarwal SG, Thappa RK, Dhar KL, Atal CK. 1979. Essential oils of the

seeds of Bunium bulbocastanum Linn. Carum gracile Lindle and Cuminum

cyminum Linn. Indian Perfumer. 23: 34-37.

2. Agarwal SG, Vashist VN, Thappa RK, Singh K, Ghosh SC, Atal CK. 1974.

Terpenes and other components from Bunium cylindricum seeds.

Phytochemistry, 13: 2024-2025.

3. Agnihotri VK, Agarwal SG, Dhar PL, Thappa RK, Meena B, Kapahi BK,

Saxena RK, Qazi GN. 2005. Essential oil composition of Mentha pulegium L.

Flavour& Fragrance Journal. 20: 607-610.

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4. Ahmed SM, Manhas LR, Verma V, Khajuria RK. 2006. Quantitative

Determination of four Constituents of Tinospora sps. by Reverse Phase

HPLC- UV(DAD) method: Broad based studies revealing variation in content

of four secondary metabolites in the plant in different eco-geographical

regions in Indian J Chromatographic Sci. 44: 504.

5. Anand A, Puri SC, Verma N, Handa G, Khajuria RK, Gupta VK, Suri OP,

Qazi, GN. 2003. A simple and reliable semi preparative HPLC technique for

the isolation of marker grade hyperforin from Hypericum Perforatum L. J

Chromatographic Sci. 41(8): 444-446.

6. Bajad S, Coumar M, Khajuria RK, Bedi KL, Suri OP. 2003. Characterization

of a new major metabolite 3,4-methylenedioxy phenyl-2E.4E-pentadienoic

acid - N-(3yl-propionic acid) amide of piperine , an ominipresent food

component by LC-NMR- Positive ESI-MS. European J Pharmaceutical Sci.

19(5): 413-421.

7. Bajad S, Khajuria RK, Bedi KL, Suri OP, Singla AK. 2003. Characterisation

of a new minor urinary metabolite by LC-MS/MS. J Separation Sci. 26 (9-

10): 943-946.

8. Caig Z, Lee FSC, Wang XR, Yu WJ. 2002. A capsule review of recent

studies on the application of mass spectrometry in the analysis of Chinese

medicinal herbs. J Mass Spectrometry. 37: 1013-1024.

9. Dutt AK. 1974. The Botanical Identity of Ajmoda. J Res Ind Med. 9: 96-98.

10. Lattoo, SK, Kaul S, Dhar MK, Khajuria, RK, Gupta DK, Qazi GN. 2005.

Production of β-dimethylacryl shikonin in callus cultures of Onosoma

hispidum, Var. hispidum. J Plant Biochem Biotechnol. 14: 193-196.

11. Khajuria RK, Suri KA, Gupta RK, Satti NK, Amina M, Suri OP, Qazi GN.

2004. Separation, identification, and quantification of selected withanolides in

plant extracts of Withania somnifera by HPLC-UV (DAD) - Positive ion

electrospray ionisation-mass spectrometry. J Separation Sci. 27(7-8): 541-

546.

12. Pandita K, Bhatia MS, Thappa RK, Agarwal SG, Dhar KL, Atal CK. 1983.

Seasonal variation of the alkaloids of Adhatoda vasica and the detection of

glycosides and N-oxides of vasicine and vasicinone. Planta Medica. 48: 81-

82.

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13. Raina BL, Agarwal SG, Bhatia AK, Gaur GS. 1996. Changes in pigments

and volatiles of saffron (Crocus sativus L) during processing and storage. J

Sci Food Agric.71: 27-32.

14. Singh S, Pandey SC, Srivastava S, Gupta VS, Paetro B, Ghosh AC. 2003.

Chemistry and medicinal properties of Tinospora cordifolia (GUDUCHI)

Indian J Pharmacol. 35: 83-91.

15. Thappa RK, Agarwal SG, Singh K, Ghosh S. 1991. Comparative studies on

the major volatiles of Kalazira (Bunium persicum Seed) of wild and cultivated

sources. Food Chemistry. 41: 129-134.

16. Touseef A, Khajuria RK, Puri SC, Verma V, Qazi GN. 2006. Determination

and quantification of camptothecin in an endophytic fungus by liquid

chromatography - Positive mode electrospray ionisation tandem mass

spectrometry. Curr Sci. 91(2): 208-212.

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3.3 Approaches to Standardization of Medicinal Plant Preparations Brijesh S., Poonam G. Daswani and Tannaz J. Birdi*

The Foundation for Medical Research, 84A, RG Thadani Marg, Worli, Mumbai - 400018, Maharashtra, India. Email: fmrbom@hathway.com * Corresponding Author Introduction

In the not too distant past disease treatment was entirely managed by traditional

forms of medicine, what is now considered in the developed world as alternative

medicine, especially of herbal origin. The interest in traditional medicine started

declining in late 19th century after the discoveries of antibiotics and other medical

technologies. However, currently it is estimated that about 80% of the world

population residing in the vast rural areas of the developing and under developed

countries still rely mainly on medicinal plants since it is the only affordable and

accessible source of primary health care especially in the absence of access to

modern medical facilities (WHO, 2002). Herbal medicinal preparations are

becoming popular in some developed countries also such as Germany, France,

Italy and the United States (Calixto, 2000). Significant economic gains have been

attained by the global and national markets as the demand for herbal medicines

have increased rapidly over the last two decades. According to the Secretariat of

the Convention on Biological Diversity, global sales of herbal products were an

estimated US $ 60000 million in 2000.

The increased use of herbal medicines in developed countries is mainly due to

the failure of modern medicine in providing effective treatment for chronic

diseases and emergence of multi-drug resistant bacteria and parasites. These

include various new diseases such as cancer, HIV/AIDS, diabetes, hepatitis,

allergies and mental disorders. The adverse effects of chemical drugs, questioning

of the approaches and assumptions of allopathic medicine, their increasing costs

and greater public access to information on safety and efficacy of medicinal plants

has also led to an increased interest in medicinal plants (WHO, 2002).

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With the tremendous increase in their global use there have been several

concerns regarding the safety and quality of the herbal medicines from health

authorities and the public alike (WHO, 2002). There have been reports of

adverse/suboptimal effects of herbal medicines which have been attributed to

several factors such as inadvertent use of the wrong plant species, adulteration,

contamination, over dosage, inappropriate use by health-care providers or

consumers, and interaction with other medicines, resulting in an adverse drug

interaction.

The poor quality of herbal medicines can be attributed to the use of

substandard raw medicinal plant materials. The factors that can affect the safety

and quality of the raw medicinal plant materials and finished products can be

either intrinsic e.g. genetic or extrinsic such as environment, collection methods,

cultivation, harvest, post-harvest processing, transport and storage practices.

Microbial or chemical contamination during processing can also compromise the

safety and quality. Mistakes in proper identification of the plant species,

accidental contamination or intentional adulteration by other species or plant parts

can also lead to poor quality of the end product.

It is necessary to standardize the safety and quality assurance measures so as

to ensure a steady, affordable and sustainable supply of medicinal plant materials

of good quality. The pharmaceutical industry has shown interest in development

of standardized plant preparations with proven safety and efficacy. However, their

focus has been on isolating newer active principles from plants unlike the rural

communities that use fresh/dried plant material or their crude extracts. Moreover,

it is generally believed that standardization of the plant material is not required

when used by rural communities for their primary health care. But, regardless of

whether the medicinal plant is to be used by local communities or by

industry, a systematic approach is required for the validation of efficacy and

safety of the medicinal plants.

To ensure safety and quality of the medicinal plants it is necessary to focus on

all aspects of the medicinal plant research: from ethno-pharmacology, utilization,

isolation and identification of active constituents to efficacy evaluation, safety,

formulation and clinical evaluation. Quality control of the medicinal plants starts

right at the source of the plant material. The phytochemical composition of the

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plant material and the resulting quality can vary due to several factors including a

number of environmental factors such as geographical location, soil quality,

temperature and rainfall etc. Taxonomy, the time of collection, method of

collection, cultivation, harvesting, drying and storage conditions, preparation and

other processing methods can also affect composition. Contamination by

microbes, chemical agents such as pesticides and heavy metals, as well as by

insects and animals during any of these stages can also lead to poor quality of the

finished product. Standardization of all these factors is necessary to meet the

current standards of quality, safety, and efficacy.

Source of Plant Material

Wild harvesting is still the prominent mode of obtaining medicinal plants and

most of the requirement of medicinal plants for the industries is still met through

wild collection (Lange, 1998). Though many medicinal plants are commonly

available in the wild and can be freely harvested, collection and sale of large

quantities of plant material from the forest can lead to destruction of many forest

plants especially the endemic species that have a restricted geographical

distribution. For example, medicinal plants like Curcuma caesia, Rauwolfia

serpentina were reported to occur abundantly (IUCN, 1994) in central India.

However, due to their growing economic importance and rampant harvesting,

these plants have now been categorized as critically endangered (Prasad and

Patnaik, 1998). The present deteriorating condition of medicinal plants in forests

needs immediate attention not only for conservation but also for propagation.

Countries can protect their biodiversity in medicinal plants by working with

industry towards monitoring and maintaining controlled non-destructive

harvesting with habitat management.

Cultivation of medicinal plants would seem as a commercially attractive

option to companies because they have greater control over supply of the plant

material and it is easier to control post-harvest treatment. Moreover, cultivation

can reduce the dependence on collection of plants from wild and thus have the

potential to save wild populations and conserve their genetic diversity.

The feasibility of such an approach would, however, depend on a number of

factors such as the ability of the species to thrive under mono culturing, while its

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economic viability will depend on the demand and market prices. Moreover,

cultivation of medicinal plants requires intensive care and management and the

conditions and duration required can vary depending on the quality of the

medicinal plant material required. Risks of contamination from pollution by

hazardous chemicals should be avoided. Introduction of non-indigenous plant

species into cultivation can lead to detrimental consequences on the ecological

balance of the region (Sharma et al., 2005). However, collection from the wild

may be unavoidable for those medicinal plants that grow slowly, are difficult to

domesticate or for which only small quantities are needed.

A point that needs specific consideration is that cultivated plants are

sometimes considered qualitatively inferior to the wild collections. The medicinal

properties in plants are due to the combinations of secondary products. Different

plants often have taxonomically distinct combinations of these secondary

metabolites resulting in unique medicinal properties in individual plants (Wink,

1999). Secondary metabolites that are generally produced for defense against

predators, pathogens or competitors or for protection/adaptation to environmental

stress related to changes in soil conditions, temperature, water status, light levels,

UV exposure, and mineral nutrients in their natural habitats; and are responsible

for most of the biological activities, perhaps would not be expressed when

cultivated under optimum conditions to obtain better vegetative yields. For

example, the wild ginseng roots are 5-10 times more valuable than cultivated roots

because the cultivated roots lack the characteristic shape of wild roots (Robbins,

1998.). These beliefs were also reflected in the conclusion reached through

research on Arnica montana by the herbal company Weleda (Ellenberger, 1998).

Analysis of the biochemical properties of the cultivated plants showed differences

when compared with wild plants that grow in poor meadows with acidic soils in

mountainous areas of Europe. The rhizomes of the cultivated stocks had lost much

of Arnica’s characteristics, reducing its commercial potential.

Selection of Plants

As per WHO guidelines (WHO, 2003), the plant selected for collection should be

taxonomically same as recommended by the national pharmacopoeia or other

related documents. If a new plant is being selected for collection then it should be

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properly identified and documented. The voucher specimens of the plant material

should be submitted to regional or national herbaria for authentication. Complete

taxonomical identification including varieties of the medicinal plants is an

important factor during selection as taxonomy of the plant species can play an

important role in their biological activity. This was observed in our study with two

varieties of Zingiber officinale wherein one variety showed immune enhancing

properties while the other did not (unpublished data).

Several reviews have described approaches that can be used for selecting

plants of potential therapeutic interest (Verpoorte, 2000; Phillipson and Anderson,

1989; Kinghorn, 1994; Vlietinck and Vanden Berghe, 1991; Farnsworth, 1996;

Farnsworth and Bingel, 1977). In general, the search for the medicinal plants can

follow three main routes: random, ecological (Fabricant and Farnsworth, 2001)

and ethno (including ethnobotanical, ethnomedical and ethnopharmacological)

based search. Random search is extremely laborious and the success rate could be

very low (Basso et al., 2005). The ecological approach uses information such as

absence of predation in a particular area infested with herbivores, which would

indicate the presence of toxic compounds.

The ethnobotanical, ethnomedical or ethnopharmacological approach is based

on information obtained from ethnobotanical survey. Undertaking of the survey

should be by a team of local botanists, traditional healers and medical

practitioners. While the traditional healers would identify medicinal plants for

treatment of different diseases, the botanist can carry out appropriate taxonomical

and botanical characterization of these medicinal plants, whereas the medical

practitioners would help in proper identification of the disease conditions (e.g.

differentiate between muscle pain and pain due to arthritis) and help in

understanding whether a treatment is curative or is alleviating the symptoms only

or whether it is a placebo effect.

It has to be specifically understood that there are certain differences in

approaches when selecting plants for an industrial or a rural application. The rural

community requires medicinal plants for their primary health care and hence

focuses more on selection of plants for treatment of common diseases such as

diarrhoea, malaria, pneumonia, wound infections etc. On the other hand

pharmaceutical industry requires medicinal plants for formulation of herbal drugs

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for commercial gain and hence focuses more on urban problems such as metabolic

disorders, chronic diseases, and multi-drug resistance among infectious pathogens.

Whether for rural community or for industrial application the selection of plant

should be based on its therapeutic efficacy in terms of its effect on the causative

agent or on the host. From the rural perspective, since the understanding of

disease in terms of causative agents is not possible in the community, it is

important that the plant formulation should address the common causative agents

resulting in a given symptom e.g. diarrhea which is caused by various infectious

agents including bacteria, viruses and protozoa. The plants selected for utilization

by rural communities should be able to control the respective diseases or else at

least act as a stop gap until further medical aid becomes available. Moreover,

these plants should be easily available so that the users of these medications can

become self reliant.

Collection of Medicinal Plants

Good collection practices are necessary for the long term survival of wild

populations and their habitats. WHO guidelines (WHO, 2003) can be followed

while collecting medicinal plant materials. Prior to initiating collection, essential

information regarding the target plant species should be obtained. The botanical

identity, scientific name including genus, species, subspecies or variety and family

of the plant should be recorded. If available, the local name should also be

verified. Information regarding environmental conditions, such as topography,

geology, soil, climate and vegetation at the collection site, should be obtained.

Information such as the geographical distribution of the plant, its abundance,

whether it is threatened or endangered, shrub/fast growing tree etc should also be

obtained. It is of immense importance that a voucher specimen be deposited in a

national or regional herbarium for identification and further consultation by other

researchers.

Medicinal plant materials should be collected in the proper season so as to

ensure the best possible quality of both the starting material as well as the finished

product. Seasonal variations can affect the chemical composition of the plants and

thus its biological activity. This was demonstrated in one of our studies where the

decoctions of Psidium guajava leaves collected in two different seasons showed

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variable antibacterial activity against six bacterial strains, the November collection

being more active than the March collection (data unpublished). In most cases,

maximum accumulation of chemical constituents occurs at the time of flowering

which then declines at the beginning of the fruiting stage (Mendonca-Filho, 2006).

The time of harvest should also depend on the plant part to be used since it is well

known that depending on the plant species the level of biologically active

constituents can vary in different parts at different stages of the plant growth and

development. For example, Kursar et al. (1999) found that younger leaves of

tropical rainforest plants contained secondary metabolites that were either present

in very little quantities or totally absent in matured leaves. The extracts from these

younger leaves showed better biological activity when tested for anticancer

activity or activity against Bacillus subtilis and Artemia salina (brine shrimp). It

also applies to other components in the plant material such as the toxic

components. Climatic conditions, e.g. light, rainfall, and temperature (including

daytime and nighttime temperature differences) also influence the physical,

chemical and biological qualities of medicinal plants. The water and temperature

stress related increase in the content of active constituents such as the total

phenolic compunds was shown by Nacif de Abreu and Mazzafera (2005) in

Hypericum brasilience. Hence the best time of collection should be determined

according to the levels of the biologically active constituents rather than the

vegetative yield.

Information such as the correct plant parts that are used (roots, leaves, fruits

etc.) and whether these parts are seasonal or replenishable should be obtained. The

collection levels and the collection practices should also be known before

initiating collection. It is necessary that the collection practices employed should

be non-destructive. For example, while collecting roots, the main root should not

be cut or dug up or while collecting bark, the tree should not be girdled or

completed stripped of its bark. Parts that are not required or are decomposed and

any foreign matter such as soil or toxic weeds should be removed during

collection.

Collection of medicinal plants should not be done from places that are prone

to or close to sources of contamination such as areas where high levels of

pesticides or other possible contaminants are used or found e.g. roadsides,

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drainages, mine tailings, garbage dumps and industrial facilities which may

produce toxic chemicals or active pastures that may lead to microbial

contamination. Quality control ensures that the plant material is not contaminated

with microbes, pesticides, heavy metals or other toxic agents (Mendonça-Filho,

2006) and that the final product is of consistent high standard.

Rapid and safe transportation of the collected plant materials should be

arranged in advance. Handling of the plant material such as cleaning, drying and

storage, should be carried out by trained personnel.

Processing of Plant Materials and their Preparation

Preliminary processing of the plant material that can be done include elimination

of undesirable materials and contaminants, washing to remove soil, sorting and

cutting. It would be advisable to dry the plant materials prior to transportation if

the processing facilities are located away from the collection sites. Cross

contamination of the different collected plants or plant parts should be avoided

during transportation. The plant materials should be protected from conditions that

may cause deterioration such as rain, moistures etc during or after transportation

till the processing begins. The plant materials that need to be used fresh should be

delivered as quickly as possible to the processing facility to prevent microbial

fermentation or thermal degradation.

Specific processing methods are often required, to reduce drying time, to

detoxify the inherent toxic constituents, to reduce side effects or to enhance

therapeutic effects. For example, the methods and temperatures used for drying

may have a considerable impact on the quality of the resulting medicinal plant

materials. Shade drying is the preferred method for drying plant material since it

can maintain or minimize loss of color of leaves and flowers; and the lower

temperatures can prevent the loss of volatile substances in the plant materials

(Ibanez et al., 2003; Bartram, 1995). However, plants can be dried in a number of

other ways: in drying ovens/rooms and solar dryers; by indirect fire; baking;

lyophilization; microwave; or infrared devices. Pre-selection, peeling the skins of

roots and rhizomes, boiling in water, steaming, soaking, pickling, distillation,

fumigation, roasting, natural fermentation, treatment with lime and chopping are

some of the common processing practices. All processed medicinal plant materials

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should be protected from contamination and decomposition as well as from

insects, rodents, birds and other pests, and from livestock and domestic animals.

Medicinal plant preparations can be prepared in several ways that usually

vary based upon the plant being used, and sometimes, the condition for which it is

being used. These preparations can be in the form of infusions, decoctions,

tinctures, macerations, fresh juices etc. Some other methods include hot baths,

powdered plants, steam inhalation and even aromatherapy. Adherence to the

method of preparation as mentioned in the ancient texts or by traditional

practitioner is necessary depending on the form of preparation or the plant used as

they may hold important information for obtaining an effective herbal preparation.

A juice of a plant may be recommended instead of decoction/powder if the active

ingredients are volatile or thermo labile e.g. fresh leaf juice of Adhatoda vasica is

used for reducing blood glucose level of diabetic patients (Ahmad, 2007).

Sometimes, it is possible that due to the difficulty in preparation of the extracts

and the time required, whole fresh material (e.g. leaves) or dried powder is used

instead of the required extract for treatment. This may lead to potential toxicity

which would otherwise not be observed due to the elimination of the toxic

constituent during extraction. In this context, an example that can be cited from

our study is the extraction of negligible amounts of the toxic component karanjin

from the leaves of Pongamia pinnata in the aqueous decoction (Brijesh et al.,

2006).

The medicinal property of plants is closely related to the different classes of

phytoconstituents (such as essential oils, alkaloids, acids, steroids, tannins,

saponins etc.) present in the plant, each of which would have a preferred effective

method of extraction, facilitating maximum yield in preparation. E.g. preparing a

decoction might extract a group of anti-inflammatory plant steroids to treat

arthritis and yet when the same plant is prepared in alcohol different antibacterial

alkaloids are extracted instead (http://www.rain-tree.com/prepmethod.htm)

Storage

Storage can also influence the physical appearance and chemical quality of plant

materials and hence it is necessary to maintain appropriate storage conditions so

as to increase their shelf life. It is customary to store the plant material in dried

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form since preparations like decoctions/infusions can only be stored for a few

days. Dried plant materials can be stored in whole, crushed or powdered forms in

storage conditions that include use of cloth bags, clear glass bottles and plastic.

Plant materials that are used fresh should be stored under refrigeration, in jars or

sandboxes, or using enzymatic or other appropriate conservation methods.

However, they should be used as quickly as possible to avoid microbial

contamination. Shelf life of plant material is usually ignored due to the general

belief that the plant materials do not have an expiry date, however, dried plant

materials usually retain their activity for about six months only. It is observed that

the powdered plant material degrades faster than the whole or crushed plant

material (unpublished data). Different types of plastics can be used which prevent

absorption of moisture and oxidation of the plant material by preventing the

exchange of gasses to increase the shelf life of the plant material.

Phytochemical Studies

Medicinal plant preparations are chemically complex and may contain one or

many structurally related active compounds that produce a combined effect.

Phytochemical studies help in standardizing the herbal preparations so as to get

the optimal concentrations of these active constituents, as well as in preserving

their activities. The aim of phytochemical studies is to identify the bioactive

constituents in the plants, devise best methods for their extraction, understand

their side effects and calculate appropriate dosages.

Standardization can be carried out by obtaining a chemical fingerprint/profile

or through bioactivity guided fractionation. Chemical fingerprints through

chromatographic techniques are more commonly used for standardization and are

obtained in terms of one or more marker compounds. It would be ideal to use the

active constituent in the plant as the marker compound, however in cases where

active constituents are not known, the marker compound can be independent of

the therapeutic activity. Furthermore, the plant extracts can also be standardized to

class of compounds e.g. ginsenosides in ginseng, kava lactones in kava, or

oxindole alkaloids in cat’s claw. Such an approach would be suited to situations

where though the active constituents are not known but are expected to belong to a

particular class of compounds.

111

According to European Medicines Agency guidelines (EMEA, 2005),

quantification of substances with known therapeutic activity or markers is

obligatory. As per the European Pharmacopoeia, marker compounds should be

characteristic or unique for the herbal material or herbal preparation; have an

established chemical structure; be present in the starting material as well as the

finished product in sufficient amounts; be accessible to quantification with

common analytical methods such as high-performance liquid chromatography

(HPLC) or high-performance thin layer chromatography (HPTLC); be sufficiently

stable; and be commercially available or able to be isolated by the company in its

own laboratory.

Thin layer chromatography (TLC) and HPLC are the most commonly used

methods for obtaining chemical fingerprints and identification of the crude plant

extracts. However, there are several possibilities that may arise while using these

techniques for standardizing the crude extracts. It is possible that the plant

materials collected from the same plant in two different seasons can show

different phytochemical fingerprint and therefore different biological activity or

two plants with identical taxonomy collected under same environmental

conditions can show different phytochemical fingerprint but similar biological

activity. In such situations comparisons of the phytochemical profiles as an

indicator of important constituents can act as a shortcut for identifying

biologically active constituents. Another possibility that may arise is when two

different plants showing similar phytochemical fingerprints show different

biological activity. In such situations bioassay guided fractionation or any other

suitable method is the only option in identifying the biologically active

constituents.

DNA fingerprinting is another technique, which though still in its early years,

seems to be of immense potential in identification of medicinal plants, particularly

when profiling the genotypic differences (Vasudevan, 2004). Apart from

identifying these genetic variations, it can also aid in identification of germplasms

of important or endangered plants for future cultivation or conservation.

Use of isolated compounds can result in better biological activity due to

higher concentrations, but it can also lead to potential side effects e.g. the active

constituent conessine isolated from Holarrhena antidysenterica, a plant

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commonly used by Ayurvedic practitioners in treatment of diarrhea, was found to

be toxic to the central nervous system (CHEMEXCIL, 1992). More recent studies

have also indicated at reduced biological activity with isolated active constituents

compared to crude extracts (Kicklighter et al., 2003).

When using crude extract, a factor that can affect the outcome in terms of the

biological activity, as observed above, is the synergism between the different

active constituents that may be present in the extract. Synergism can lead to better

activity as well as decrease in potential toxicity of some individual constituents.

Synergism can be due to the individual action of different constituents present in

the extract at multiple target sites/parameters. This was observed in a study

conducted by FMR on the antidiarrhoeal activity of P. guajava (unpublished

data). It was observed that the decoction of the dried leaves of P. guajava showed

antidiarrhoeal activity by showing antimicrobial activity against five out of the six

bacterial strains tested, Giardia lamblia and rotavirus. It inhibited adherence to

and invasion of the bacterial pathogens to the epithelial cells. It also inhibited

production and action of enterotoxins such as Escherichia coli labile toxin and

cholera toxin. These results suggested that the different constituents present in the

decoction could be individually responsible for the different activities observed

against these parameters. Another mechanism by which these constituents can

show synergism is by having an additive effect against a single target

site/parameter. It was observed that the decoction of P. guajava leaves were

consistently more active at a dilution of 1% than at 5% against bacterial adherence

to epithelial cells. This effect could be due to the fact that the ratio of constituents

achieved at 1% was more optimal for activity than at 5%.

The modern analytical and isolation methods that are used for screening and

isolation of plant constituents are the chromatographic and spectroscopic

techniques such as TLC, thin layer electrophoresis, HPLC, nuclear magnetic

resonance, HPTLC etc. These techniques have proved very useful in isolation

and proper identification of the active constituents in the plant extracts.

These techniques, however, are not applicable at the grassroots, as they

cannot be used by the community. Hence it is necessary to devise simple

techniques for standardization that can be used by the community for identifying

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plants with good biological activity. Use of a class of compounds, as mentioned

earlier, as a surrogate marker is a potential approach to identifying plants with

good biological activity at the grassroots. For example, instead of a single

polyphenol, tannins can be used as surrogate marker, which the schoolchildren

can estimate easily in their laboratories. This approach has been attempted by us

in collaboration with the Foundation for Research in Community Health, in their

field project at Parinche, Maharashtra.. We estimated levels of tannins in

decoctions (prepared as per standard Ayurvedic texts) of five different

collections of P. guajava leaves and compared them with their respective

activities against action of cholera toxin (data unpublished). It was observed that

the decoctions with >10.5 mg/ml tannins showed good activity with no

significant difference in their activities. However, below this level the activity

was significantly poorer. Hence, 10.5mg/ml tannin level may be taken as a cut-

off value for differentiating a P. guajava plant with good activity from that with

poor activity.

Biological Screening

Biological screening along with phytochemical standardization forms an integral

part of preclinical studies that is essential for generating important efficacy and

safety data to validate the claimed therapeutic potential of the plant before it is

tested in clinical trials. It is helpful in understanding various factors affecting the

biological activity of a plant right from the selection to the formulation of the

herbal drug and their standardization. This point, however, will not be discussed

in detail in this paper.

Conclusions

With the tremendous increase in the global use of medicinal plants, several

concerns regarding the safety and quality of the herbal medicines have also been

observed. Hence it has become necessary to standardize the safety and quality

assurance measures so as to ensure supply of medicinal plant materials of good

quality. Though the pharmaceutical industry has been focusing on standardization

of plant materials when manufacturing herbal drugs, it is generally believed that

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standardization is not required when used by rural community for their primary

health care. However, irrespective of whether the plant is being used by the

industry or by the rural community standardization of plant material is required.

The difference comes when using isolated active constituents that are required by

the pharmaceutical industry for manufacturing herbal drugs whereas rural

communities may use standardized extracts. The decision on whether to collect

plants from the wild or to cultivate it would depend on the feasibility of the

approach for that particular species. After proper botanical identification, WHO

guidelines should be followed for collecting plant material in terms of proper

season and climatic conditions, correct plant part, practices that are non-

destructive and would prevent contamination from soil, toxic weeds or microbes.

Post collection, appropriate processing and storage conditions are required to

reduce drying time, detoxification to reduce side effects and to enhance

therapeutic value of the plant material and to improve its shelf life. Phytochemical

standardization for identification of the plant material can be carried out by

obtaining chemical fingerprint through chromatographic techniques in terms of a

known marker compound or through bioassay guided fractionation and/or DNA

fingerprinting techniques. Chromatographic and spectroscopic techniques have

proved very useful in isolation and proper identification of active constituents in

the plant extracts. Biological screening plays a vital role in generating important

efficacy and safety data to validate the claimed therapeutic potential of the plant

before clinical trials are carried out. Hence, ‘standardization’ involves the quality

control of various factors affecting the therapeutic activity of a plant right from

selection of the plant species to the formulation of the herbal drug so that it

minimized batch-to-batch variation and meets standards of quality, safety, and

efficacy.

Acknowledgements The financial assistance of Sir Dorabji Tata Trust, Sir Ratan Tata Trust and

Department of Science and Technology, Ministry of Science and Technology,

Government of India (grant number 91283) is acknowledged.

115

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3.4 Medicinal Plant Compositional Consistency for Reliable Therapeutic Action Profiling: Key Issues and Concerns for Phytotherapeutics Rajender S. Sangwan Central Institute of Medicinal and Aromatic Plants, PO CIMAP, Lucknow-226 015, India Email: sangwan.lab@gmail.com The putative efficacy of medicinal herbs relies on empirical or anecdotal data and

on traditions of use that frequently fail to satisfy the requirements of evidence

based medicine. Consequently, establishing sound pharmacological basis of

therapeutic action remains a constant challenge and that necessitates consistency

of the quality of the test herbal material or therapeutic product thereof. The quality

of a phytomedicine is defined by several parameters in sequence like quality of

herbal resource and herb, manufacturing of the drug preparations from the herb,

properties of the finished product etc., thus, demanding special quality attention

on each individual herbal species in accordance with Good Manufacturing

Practice (GMP) standards at each step.

The new era of popularity with medicinal plants stems from at least two

distinct fields of activism: (i) current-model discovery of therapeutic activity

associated with one or more phytochemicals isolated from the herbs, (ii)

increasing trust in ancient systems of medicines to deal with new era ailments like

stress, geriatric and oxidative damages, focus on ageing healthily, aspirations of

longer life span etc. The former began either as an inspiration from traditional

pharmacies (like Ayurveda, TCM, Unani, Kampo etc.) or as an ab initio

pharmacological research driven on novel/characteristic phytochemical isolates to

scientifically evolve out the individual plant molecule or its derivative (e.g.

artemisinin, taxol, podophyllotoxin, morphanes, ergot alkaloids, vinblatine,

vincristine etc.) as drugs with molecular mechanisms of action. However, the later

is largely a trendy holistic phytotherapy approach primarily based on beliefs on

ancient knowledge insufficiently reinforced with consistent

clinical/epidemiological data (e.g. Echinacea, Kava Kava, Sal Palmetto,

Hypericum etc.). Thus, for convenience of addressing the issues, the applicable

quality pointers in the former case may be called as Type-One Quality Standards

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(TOQS) while those for the later case may be referred to as Type-Two Quality

Standards (TTQS).

The type one quality parameters applicable to the pure phyto-molecules with

established, validated drug action and cleared full sequence of clinical trails

(clinical action, bioavailability, toxicology, contra-indications etc.) are more or

less similar to those relevant to their synthetic counterparts in allopathic

medicines. Nevertheless, specifically stating they include important aspects like

herbal resources of minimized variation in phytochemical composition as any

significant deviation would result in hurdles in assurance of pure phytochemical

molecules during the manufacturing process, disease-free herbal phyto-resources

as use of diseased botanical parts of the plant may entail contamination of

microbial toxins, some of which could be fatal at even at levels of contamination

usually considered insignificant for the other contaminant so the drug.

The type two quality parameters applicable for the herbs or herbal preparation

(except the pure phyto-molecules isolated there from) bear issues and concerns

that are a brand different and need stringent and special attention, if their case in

clinics is to be advanced. Facets of their standardization are far more diverse and

complicated due to several reasons. Some of the concerns that need to be a priori

considered are while focusing on the applicable quality parameters needed to be

recruited and assessed:

• although, some of the plants of the herbs under group-II (TTQS) are well

documented for their unique secondary metabolome but scientifically strong

correspondence between the phytocemicals and clinical affects is yet be

established, thus, phytochemical markers to be applicable and tested can not

be unambiguously selected.

• clinical action, if any, residing with chemical(s) contained, the phytochemical

data need to be more comprehensive to promote their status from botanical

markers to therapeutic markers.

• data from different laboratories/research groups on a herb, formulation of the

herb and even on different batches of the same formulation can not be

compared unless the existing huge compositional variability in the herb

(mainly due to its random resourcing or wild crafting) is minimized.

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• some recent revelations in this regard on specific cases like Ashwagandha

(Sangwan et al., 2004), Echinacea, Kava Kava (Krochmal et al., 2004) etc.

bring to light the issue of quality standards in herbals vis-à-vis therapeutic

affects fluctuating between avowal and denial.

• recognition of confounding factors of compositional heterogeneity calls for

generating method and protocols to ensure consistency for useful

therapeutical data.

• even in absence of enough association between the botanical markers and

therapeutic actions of the plant, former can serve at least as ‘surrogate

markers’ to ensure reproducibility and quality of the preparatory process.

• the general premise that traditional use of these medicinal products for

generations establishes their safety does not necessarily attest to their safety

and efficacy. Indeed,

• adverse effects of long-term herbal use, adulteration with toxic compounds

and contamination by toxicogenic microbes or natural toxins like mycotoxins

have been sometimes reported for herbal products and medicinal plants due to

lack of proper quality pre-screens of the herbs traded or tested for the clinical

action.

• herbal multi-component (polyherbal) preparation used in multi-target

therapy, pharmaceutical prerequisites have to be ensured for all components

and for their combinations.

Besides the intensified application of regulatory issues, certain developmental

elements are essential for better perception of their therapeutics in modern

settings. The recent developments in sensitive and high resolution analytical

technologies in combination with advances in disease development and pre-

clinical/clinical research have led to several paths for creating strong knowledge

base via Good Agricultural Practices (GAP) of cultivation, monitoring

composition, ensuring consistency across preparations/samples and on generating

data-based pharmacological inferences on a specific herb. Characteristic

compounds of the extracts/herbs need to be identified and different analytical

methods such as HPTLC, HPLC, CE etc. with low coefficients of variation should

be developed to analyze each of the standardized extracts and the finished

product. Detailed understanding of the constituents and their chemical behavior in

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the extracts in case of multi-component products is essential as also identification

of phytochemical components co-responsible for efficacy (co-effectors) or as

matrices creators.

The issue of quality standards in herbal medicines fulfilling requirements of

current clinical studies is too vast to be stated briefly. However, thematically, it

may be attempted to be addressed as Seven Phase Process (SPP), each having

several facets of research and application as below:

Selection: The selection of the herb should be made more herb action

information-centric after their critical analyses and each herb need to be defined in

terms of chemotaxonomic status and clustering with other congeners of the

phytomolecules per se or as class/variants. The selection of herbs for exploratory

therapeutics should focus on those health needs that are unmet and appear to be

important to be dealt.

Structure: Anything apart, identification of active and/or characteristic secondary

metabolites or other phytochemical constituents of the herb is necessary not only

to from a firm knowledge to the herb and its action but also for the drug

development in the foreseeable future. The drugs from such phytochemcial and

pharmacological studies in tune with traditional knowledge are highly

motivational like artemisinin from Artemiasia annua, quinine from Cinchona,

camptothecin from Camptotheca, vinblastine and vincristine fron Catharanthus

roseus etc. At the same time, the cohort studies in this line can lead to the

development of herb-specific phytochemical marker library as well as

identification or development of specific chemotypes for the herbs. Such a

development forms the grit to the development of high resolution, sensitive and

validated analytical methods that could serve as standard operating protocols

(SOPs) for the quality evaluations of the herbs proposed to be subjected to the

clinical trials.

Source: The source of the medicinal herb is not merely the authentic identity of

the herb but includes comprehension of its chemotaxonomy and phytochemical

analysis of the larger number of individuals from the populations from diverse

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geographic locations to discern the different chemotypes. The source of the

therapeutic herb might begin with the taxonomically identification but it never

ends before its specific description as a chemotype and its chemotypic position

within the family of chemotypes known/existing for the plant within the

geographically region sampled but elsewhere. While the present world of

intellectual property rights restricts the movement of live herbs or cultivable part

thereof, globalized and free trade options of the day assure unrestricted movement

of herbs across the glob for manufacturing. Since a good account of chemotypic

position of different herbs in different countries is lacking, it need to be developed

through intense research. Keeping the extens of variability within a chemotype, it

is important to shift the herbal trade description from authenticity of identification

of the herb to its next level, the chemotype. Thus, extending focus from

chemotaxonomy to chemotypes to ensure better quality in terms of compositional

uniformity across generations and regions, development of good agricultural

practices (GAPs), mathematically better defined compositional variation in multi-

agri-environments, standard operating procedures for post harvest management

and processing chemical technologies.

Standardization: Standardization of herbal drugs is most critical parameter for

assurance of quality and compositional consistency but also as a diagnostic tool. A

fully characteristic profile for each herb is not feasible due to the lack of complete

information about the complete composition of the secondary metabolome of each

herb. Nevertheless, several approaches have been proposed to reach a significant

level of reliability of the phytochemical diagnostics for the herbs. These include

marker approach, multi-component approach, pattern approach and multi-pattern

approach chemical fingerprinting, development of chemometric databases, fractal

fingerprints and wavelet analysis, PTR-MS, pharmacological profiling.

The marker approach and multi-component approach both are component

based approaches (Xie et al., 2006). These approaches explicitly focus on content

and concentrations of the specified chemical components present to characterize

the herb, extract or preparation. This system relies on identification through one or

two markers or active compounds and is the simplest kind of marker system.

However, this marker system is far from adequate as the dimensions of variations

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and chemo-variants of medicinal herbs are much more than the test dimensions in

the system. Therefore, multi-component approaches are being increasingly applied

for the better quality control in herbals. This is a natural extension of the marker

approach and uses quantitative parameters of the all analytes (known or unknown)

in the sample/chromatogram in terms of relative concentration (peak area).

The concept of pattern approach is basically considering whole spectrum as

the feature. Though, seemingly there may be no discernible relationship between

the pattern and chemical composition but under identical conditions the pattern

has its origin in the chemical composition. Such patterns are generated using any

of the spectroscopic techniques like Fourier Transform-Infrared spectroscopy (FT-

IR), Near Infrared Reflectance (NIR), Nuclear magnetic Resonance (NMR), Mass

Spectrometry (MS) etc. Using conceptually such an approach, Wang et al. (2003)

have used both objective and robust NMR-based metabolomics combining high

resolution 1H-NMR spectroscopy with chemometric analysis in chamomile.

Recently, an objective method for the determination of a herb extract’s quality

based headspace measurements by proton-transfer-reaction mass spectrometry

(PTR-MS) that enabled excellent quality assurance and product source diagnosis

of herbals (Jaksch et al., 2004). The advantage of the chromatogram as a whole

rather than peak-centric approach is that it better covers the components of the

herb. Such an approach when used with hyphenated instrumentation, couples the

power of separation with the power of identification. Thus, when the identities of

the components present are not fully known and chromatogram peaks can not be

precisely labeled, similarity of the chromatogram is used as an index of identity

and comparison of herbs. Such component fingerprints for quality verification of

herbs are acceptable in several countries like USA, China, Britain and India and

form part of even World Health Organization guidelines for manufacture of herbal

medicinal products (Mok and Chau, 2006).

Thus, instead of fingerprints constructed from a single chromatogram,

multiple chromatographic fingerprinting (consisting of more than one

chromatographic fingerprinting) is much more adequate to profile the composition

of complex botanicals like multi-herb drug products as the later represents nearly

whole characteristics of chemical composition of complex medicines. Amongst

them binary chromatographic fingerprinting are more common and are

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significantly more reliable than their single chromatogram counterparts (Fan et al.,

2006). The pattern approach fingerprints are quite often used by the

pharmaceutical industry to examine the source of the drug substance and the

method of chemical preparation. Even such a system can be advanced to provide a

“phytochemcal logo” by augmentation by qualified chemical pattern from safe

and edible herb in the product. However, this approach similar to the component

approaches still bears the limitation of revealing only one set of patterns displayed

by single technique and thus may exclude several other patterns pertaining to

secondary metabolites of other classes. In other words, TLC, HPLC or GC alone

may not be sufficient to reflect all that is contained in the herb. Thus, use of multi-

dimensional data patterns derived from different techniques of resolution form

multi-patterns and use of different detectors along with could add value to such

multi-patterns. Chemometrics assisted application of mathematical and statistical

techniques can greatly improve the quality of the fingerprints (Alonso-Salces et

al., 2004). A major problem with the direct use of chromatograms for comparison

is that retention times on the basis which the comparisons are made often shift

from run to run. Such shifts can be large in HPLC due to gradual degradation of

the stationary phase, environmental fluctuations (temperature, instrumental

reproducibility of the experimental conditions including flow rates, composition

of mobiles phase and gradient elution, shifts in detector responses and interaction

of analytes. Of course, several retention alignment algorithms are available to

estimate the shifts and align chromatograms and these methods work fine for

samples with reasonably closed chemical compositions but less to herbals where

the variations in chemical compositions could be large.

Surveillance: Surveillance of the phytochemical resources that is scheduled to be

procured for the manufacturing processes is essential although the crop cultivation

process to ensure compliance of standard agronomic practices including post

harvest handling, pre-processing storage, residue monitoring, microbial load

assessment, chemical process contamination, pharmaco-surveillance with respect

to use, efficacy, toxicity/ secondary effect(s). The augmentation of the supply

through wild crafted herb (for any reason) is one of the most common practice

and most predominant factor contributing to the variations in the herbal drugs. An

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appropriate and real-time statistics of cultivated source of herb production and

herbal drug production would provide an estimate of such augmentations in the

industry and trade as a whole. In legal perspectives, it should be mandatory to

codify the phyt-resource of the manufactured drug that could be traceable in the

manufacturing premises although from procurement to processing in quantifiable

terms.

Safety: Doubts on safety of the herbal drugs is one of the most convenient weapon

that comes into play in any event of fall out of ill effects of the herbal drugs, even

when it could be due to misuse or unauthorized use/prescription. Citation of

historical or traditional use is often cited as the basis of safety. However, in

modern perspectives such a reference can only be of collateral corroboration and

appears to lack any significance standalone. Rationally, the safety aspects have

been less rigorously attended and emphasized in the literature. The therapeutic

references on the herb have over-shadowed any effect as the therapeutic action

appears to be the key focus and toxicity appears to have gone barley beyond

tolerating (via co-administration of another herb) or specific linkage of the herb

with food or drinks. Thus, a growing library of the epidemiological, toxicity data,

co-effects, secondary effects etc. need to be appended to the information on

historical/traditional use of the herb. Also, such growing database information

would keep the potential consumers well educated and informed about the herb

and forms an important platform for academic and investigative discussion.

Particularly, sharing, documentation and publication of the negative (adverse

effect) or null data is most important for rational conclusions to be drawn for the

therapeutic action of the herbs. Similarly, a good placebo system needs to be

evolved for herbs and herbal products for corroboration of the claimed benefits. In

my opinion, use of another herb (preferably of the same family or species of the

same genus) that lacks (or has little of) the potential drug action raison de etre

phytochemicals could serve as most convincing secondary controls.

Substantiation: Substantiation of the all claims on analysis, processing and

therapeutic action need to be dynamically substantiated in a diversified and non-

end fashion. Frequent reviewing of the pre-existing data including methodology

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(as one can not get right data with wrong method), assessment of the cohortness

and robustness of study parameters, sample size, full-scale multi-parallel controls

are key not only to keep the importance of the drug imply but to develop a

knowledge force to drive its importance upstream, en route diminuting the pitfalls.

All these parameters need to be defined for each phytomedicine by SOPs and

all notions need to be subjected to full-scale tests through upstream science to

facilitate creation of a strong, trustable and sustainable herbal medicine regime

sans current caveat emptor. Research into new analytical methods for stricter

standardization of phytomedicines would continue to be a necessity to improve

the accuracy and consistency of phytomedicine preparations. Such a thrust is also

believed to continue from regulatory authorities for speedy and innovative

developments in the area. Very recently, Obradovic et al. (2007) have developed a

new method for the authentication of plant samples by analyzing fingerprint

chromatograms. Instead of conventional procedure of integrating the

chromatograms and using peak heights or areas of several peaks in a supervised

pattern recognition method to confirm the authenticity of the product, these

workers have proposed a section approach of analyzing chromatograms wherein

(i) the chromatograms are split into sections (ii) each section is described by four

variables (number of peaks in the section, average retention time of the peaks in

the section, total area of peaks in the section and average area of peaks in the

section, (iii) these variables have then been used in the statistical analysis. The

approach has been proposed to be especially useful when the peaks on the

chromatogram are not well separated and linking and corresponding peaks across

chromatograms is not easy. The more objective results and ease of interpretation

of data from the approach have been shown for the chromatograms of willow-herb

extracts resolved through capillary electrophoresis. The correct combination of

variables is crucial for such discriminate analysis.

Acknowledgements

I am thankful to The Foundation for Medical Research (FMR) and Dr. Tannaz J.

Birdi and Dr. Nerges Mistry, the Organizers of the conference “Approaches

Towards Evaluation of Medicinal Plants Prior to Clinical Trails” for giving me an

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opportunity to deliver a lecture at the conference. This paper is the full review of

the subject matter thematically presented and discussed at the conference.

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An Abridged Overview

It is estimated that about 80% of the world population of the developing and

under developed countries still rely mainly on medicinal plants. It has become

clear that the use of medicinal plants is not only a valuable resource and a

necessity but also provides an affordable and accessible alternative for primary

health care. Thus medicinal plants have begun to get the attention they deserve.

Conventionally, however, understanding the mode of plant action is often

trivialized with overt emphasis on clinical trials; the pre-clinical testing of

medicinal plants being often neglected. The Foundation for Medical Research

being involved in the medicinal plant research for over a decade, felt a necessity

to have a comprehensive discussion for various approaches towards the preclinical

evaluation of medicinal plants. With this aim, the Foundation organized a one day

workshop with the following three themes:

Translation of ethnobotanical information obtained from a community for

identification, subsequent testing and utilization of plant material.

Principles of pre-clinical evaluation of plants using appropriate bioassays.

Standardization of plant material

Session I:

Session I focused on the importance of ethnobotanical surveys and the

documentation of the information obtained as well as collection, processing of

plant material, that need to be considered prior to validation. The presentations

and the following discussion emphasized that the information obtained would help

in not only preserving and popularizing the local health traditions but also

following of standard protocols for collection and processing would ensure quality

of the medicinal plant preparation.

It was noted that there is an urgent need for effective documentation and

assessment of local health traditions (LHT). Dr. Darshan Shankar pointed out that

the once rich and diverse cultural & traditional heritages of India are getting

diluted due to the increased influences from the mainstream culture. He discussed

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the work undertaken by FRLHT in the past towards the revitalization of health

traditions and demonstrated the methodology towards revitalizing household

remedies and folk healing practices that can contribute to primary health care of

rural communities. He emphasized that apart from documentation and

identification of important LHT, community validation of these traditions and

their promotion at household and community levels is necessary.

Information obtained through the ethnobotanical survey and previously

documented data can help in development of safe, effective and acceptable

therapeutic agents. WHO guidelines should be followed for sustainable cultivation

and collection of medicinal plant materials to ensure safety and quality. The

necessity of good collection practices for the long term survival of wild plant

populations and their habitats was emphasized by Dr. RS. Rawat. He also

emphasized on the continuous need for finding sensitive and selective analytical

methods towards assessing the safety and quality of not only the raw plant

material but also the finished products.

The importance of ethnobotanical studies was reemphasized by Dr. M.

Parabia and Dr. P. Tetali. Dr. Parabia presented the studies undertaken by Shri

Bapalal Vaidya Botanical research Centre (BVBRC) of Veer Narmad South

Gujarat University, Surat, towards the development of simple recipes for common

ailments that could be taught to tribals and underprivileged groups for their

primary health care. He also gave a brief historic description of several

ethnobotanical surveys in Gujarat and described the work of BVBRC on the

preliminary validation of these ethnic claims.

Dr. Tetali added that one of the main issues was the need to integrate the

science of ethnobotany with other subjects such as pharmacology and medicine.

He opined that the undertaking of ethnobotanical surveys should be by a team

consisting of a local botanist, traditional healer and medical practitioner and

suggested the introduction of an integrated syllabus/ courses or preparation of

special work-manuals to train ethnobotanists to help create a more meaningful and

technically appropriate database that can be scientifically screened, tested, utilized

and finally integrated into health care delivery systems.

As demand for medicinal plants is growing, the necessity for cultivation of

medicinal plants was discussed. In this context, issues related to pesticide residue

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were highlighted and organic farming suggested. Alternatively, cultivation using

tissue culture could be considered. With respect to the usage, especially within a

community, the need for protection of flora, traditional rights and IPR were

stressed. From the taxonomical point of view, the possibility of developing and

using of molecular tools for classification was suggested. Participation of industry

was highlighted with respect to emphasis on quality of plant material and its

processing. There was an active discussion on whether validation is really

required or can plants directly undergo a Phase III clinical trial which was the

subject matter for the next session.

Session II:

Session II focused on the importance of conducting preclinical screening

assays. It was emphasized that preclinical studies are necessary for validation of

medicinal plants and preparation of rational polyherbals. Each speaker in this

session talked extensively on the principles, the advantages, limitations and the

approaches of conducting preclinical studies on medicinal plants. These included

testing of plants for their pharmacological efficacy in vitro and in vivo systems

and studies of toxicology, specificity, biopharmaceutical properties and drug

interactions.

A number of participants stressed the need for toxicity testing of plants, both

short and long term, depending on the intended usage. It is possible that the plant

treatment taken up for the clinical trials may lead to some

unanticipated/unknown/unrelated side effects. Dr. S. Anant commented upon the

easy purchase on herbal products without a proper prescription and raised the need

to regularize how the traditional medicines are prescribed.

Clinical trials though required for the ultimate validation of medicinal plants,

have several limitations in terms of the number of patients required, the time

commitment and the expenses involved. Several advantages that the preclinical

testing offers, justifies it as an important prerequisite for short listing plants that

can be subjected to clinical trials.

The importance of preclinical testing was reemphasized by Dr. N. Rege who

said that the testing of traditional medicines should be based on scientific

principles. Towards this both the national and international guidelines need to be

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followed. She opined that research on medicinal plants should be of a

collaborative nature with active participation of academicians, students,

researchers and industry.

Dr. U. Thatte and Dr. Anant stressed on the importance of selecting

appropriate bioassays for studying the pharmacological efficacy of medicinal

plants. Dr. Thatte used the example of Tinospora cordifloria to illustrate this. She

also stated that T. cordifloria when tested for antimicrobial activity failed to show

efficacy but when tested for immunomodulatory activity proved to be an excellent

immunomodulator.

Dr. Anant, on the other hand, discussed high through-put assays. He

elaborated that the basis for designing a high through-put screening assay is the

identification of a valid target; choosing an appropriate testing model and the right

type of extract. He demonstrated their utilization giving the example from his own

laboratory where the high through-put system was used to screen the anti-cancer

activity of curcumin.

The other speakers in this session also stressed on the need for selecting

appropriate bioassays. It was pointed out by Ms. P. Daswani that since the disease

process involves multiple steps several targets need to be considered which

undoubtedly would require a battery of bioassays which may include both in vitro

and in vivo systems. She mentioned that unfortunately when screening plants for

infectious diseases the assay system is often limited to testing for antimicrobial

activity. However plants can exhibit their efficacy against infectious diseases by

mechanisms other than antimicrobial activity. Ms. Daswani highlighted this

approach by presenting data of her work carried out at FMR on antidiarrhoeal

medicinal plants.

Dr. J. Singh mentioned that the use of mammalian cell cultures which mimic

in vivo functions can not only minimize dependency on large number of animals

but also allow for rapid screening of large number of plant extracts/isolates. He

added that modern approaches like silico biology can be used to shortlist best hits

to be taken for in vivo validation.

Dr. Thatte, Dr. Rege and Ms. Daswani all drew attention to the limitations of

the preclinical testing. It was pointed out that the extrapolation of dosage from in

vitro studies to in vivo systems may not always be possible. Secondly some

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extracts may only show in vivo activity due to the metabolism of inactive

compounds into active forms or conversely, some compounds which show good

activity in vitro may be metabolized in vivo into inactive forms. Besides these, Dr.

Rege pointed out that availability of standard plant material, limited infrastructure

especially in academic institutes and difficulty in obtaining standard markers for

fingerprinting pose a challenge.

In her concluding remarks, Dr. Thatte said, that in order to get Ayurveda

accepted, we need right attitude, motivation, passion and curiosity especially from

young medical students.

Session III:

Session III focused on importance of phytochemical standardization of the

plant preparations. Various aspects and issues regarding standardization of plant

material were discussed. It was noted that stringent norms for standardization of

efficacy and safety measures is necessary to ensure consistent supply of medicinal

plant materials with good quality. All speakers in the session gave a brief

description of procedures that can be followed for standardization.

Dr. R. Sangwan stated that issues related to standardization did not exist

earlier, since herbal healers collected the plants by themselves. However, in the

current scenario of self medication and / or reliance on plant material suppliers

there is an urgent need for standardization. Using Withania somnifera as an

example, he demonstrated the need for standard plant material. With respect to

quality, Dr. Sangwan emphasized on a “seven phase quality process” which

included selection of herbs, structural features of phytochemicals, source of raw

materials, standardization for pre process quality assurance, surveillance of

variability factors, safety assurance and substantiation of effects through stringent

controls.

Dr. P. Venkatasubramanian pointed out that the parameters of testing the

quality of materials (dravya) in traditional medicines, such as rasa (taste), guna

(properties), virya (potency), vipaka (post-digestion effect) and karma (action),

reflect not only the quality but also efficacy. However, as there are no ready,

documented protocols available in traditional medicine either for collection or for

testing the action, there is a need for reviving and verifying these traditional

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protocols. Towards this, she discussed the two-fold objective of research at

FRLHT with respect to quality standardization of medicinal plants: (i) the method

of reverse pharmacognosy is being employed, where, the plant material taken for

standardization is according to traditional knowledge, (ii) Traditional Quality

Parameters, such as rasa are being researched both from Ayurvedic view point as

well as through modern scientific tools.

Mr. Brijesh reemphasized the need for standardization. In addition he

discussed several factors including collection, processing, storage, that can affect

phytochemical profiles and stressed on the approaches for correlating these to

biological assays. He presented examples from his own studies with antidiarrhoeal

medicinal plants to substantiate this.

Dr. R. Khajuria emphasized on the need for stringent quality norms for global

positioning of herbal products. He discussed some of the procedures that are used

for standardization of medicinal plants and described in detail the rapid strides that

have been made in the field of instrumentation and their applications in isolation

of marker compounds necessary for chemical profiling.

It was suggested that a suitable mode of administration for the plant

formulations like chewing gum / tablet may be considered which would not only

meet the demand of the industries but also be acceptable to the masses.

It was agreed by the participants during the course of discussion that the

standardization of herbal preparation is not just an analytical assay of an active

principle; rather it embodies total information and controls that are necessary to

guarantee consistency of composition. Hence, a systematic approach is required

for a plant identified from traditional medicine, as is followed in modern

medicine.

Citing the example of the variations in W. somnifera as demonstrated by Dr.

Sangwan, Dr. Singh lamented upon “the mess we are in” due to large variations in

plant material. He said that we need to change our present attitude to bring

Ayurveda into international scenario. According to him, instead of tackling

variations, we should concentrate on one plant variety based on traditional

knowledge, apply all the modern tools towards appropriate biological and

phytochemical standardization, and bring out an efficacious formulation in the

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market. This approach would lead to a “national plant” with clear cut efficacy

based on our rich heritage.

In conclusion, the workshop comprehensively touched on all aspects of

preclinical medicinal plant research, from selection to formulation. Besides re-

emphasizing the importance of medicinal plants, the workshop stressed on

participation/partnership amongst academicians, researchers and the industry to

integrate utilization of medicinal plants into modern medicine.

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List of Participants

Name Address Anant S.

Associate Professor of Medicine and Cell Biology & Director of Gastroenterology Research, University of Oklahoma Health Sciences Centre, 920 Stanton L. Young Boulevard, WP 1360, Oklahoma City, OK 73190, USA.

Tel: 001-1-405-271-2175, ext. 3, Fax: 001-1-405-271-5450

Email: Shrikant-anant@ouhsc.edu

Antia N. H. Trustee, Foundation for Medical Research & Foundation for Research in Community Health, 6, Koregaon Park, Pune – 411001, India.

Tel: 020-26121875

Email: nhantia@vsnl.net

Bhate V. R.

Director, Analytical Solutions, Plot no. R-610, TTC, MIDC, Rabale, Navi Mumbai – 400701, India.

Tel: 022-27601122, Fax: 022-27602483

Email: anasolutions@rediffmail.com

Birdi T. J.

Deputy Director, Foundation for Medical Research, 84A, RG Thadani Marg, Worli, Mumbai – 4000018, India.

Tel: 022-24934989 / 24938601, Fax:022-24932876

Email: fmrbom@hathway.com

Brijesh S. Research Student, Foundation for Medical Research, 84A, RG Thadani Marg, Worli, Mumbai – 4000018, India.

Tel: 022-24934989 / 24938601, Fax: 022-24932876

Email: fmrbom@hathway.com

Chaturvedi S. Junior Research Officer, Foundation for Research in Community Health, 3&4, Trimiti B Apts., 85, Anand Park, Aundh - Pune 411007, Maharashtra, India.

Tel: 020-25887020, Fax: 020-25881308

Email: frchpune@giaspn01.vsnl.net.in

Charegaonkar D. Managing Director, Anchrom Enterprises (I) Pvt. Ltd, 101, Shree Aniket Apartments, Navghar Road, Mulund (E), Mumbai – 400081. India.

Tel: +919870326638

Email: info@anchrom.com

Darshan Shankar

Executive Director, Foundation for Revitalisation of Local Health Traditions (FRLHT), 74/2, Jarakbande Kaval, Post Attur, Via Yelahanka, Bangalore – 560064, India.

Tel: 080 28565708, Fax: 080 28567926

Email: darshan.shankar@frlht.org

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Daswani P. G.

Research Assistant, Foundation for Medical Research, 84A, RG Thadani Marg, Worli, Mumbai – 4000018, India.

Tel: 022-24934989 / 24938601, Fax: 022-24932876

Email: fmrbom@hathway.com

Joshi S. P. Division of Organic Chemistry: Technology, National Chemical Laboratory, Homi Bhabha Road, Pune- 411008, India.

Tel: 020-25902327, Fax: 020-25893614

Email: sp.joshi@ncl.res.in

Khajuria R. K.

Head, LC-MS Lab and Bioanalytical Division, Indian Institute of Integrative Medicine (formerly Regional Research Laboratory), Canal Road, Jammu Tawi – 180001, India.

Tel: (0191) 2569000, Fax: (0191) 2569333

Email: khajuriak@yahoo.com

Kulkarni D. K.

Senior Scientist, Department of Botany, Agarkar Research Institute, G.G.Agarkar Road, Pune – 411004, India.

Tel: 020-25654357, Fax: 020- 25651542

Email: dkkulkarni@indiatimes.com

Mistry N. F.

Director, Foundation for Medical Research, 84A, RG Thadani Marg, Worli, Mumbai – 400018, India.

Tel: 022-24934989 / 24938601, Fax: 022-24932876

Email: fmrbom@hathway.com

Natu A. A.

Indian Institute of Science Education and Research, Near National Chemical Laboratory, Homi Bhabha Road, Pune- 411008, India.

Tel: 020-25536212

Email: aa.natu@iiserpune.ac.in

Parabia M. H. Professor of Biosciences, Veer Narmad South Gujarat University, Udhna –Magadalla Road, Surat – 395007, India.

Tel: +9198251-34422 (M)

Email: minoo_parabiain@yahoo.com

Pendse N. M.

Professor, Kayachikitsa & Panchkarma, Tilak Ayurveda Mahavidyalaya, Deenanath Mangeshwar Hospital, Mahalaxmi Nagar, Bibwewadi, Pune – 411037, India.

Tel: 020-2420 4103/ 020-2432-9353

Email: drpendse@vsnl.net

Ranadive B. Junior Research Officer, The Foundation for Research in Community Health, 3&4, Trimiti B Apts., 85, Anand Park, Aundh - Pune 411007, Maharashtra, India.

Tel: 020-25887020, Fax: 020-25881308

Email: frchpune@giaspn01.vsnl.net.in

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Rawat R. B.

Regional Program Co-ordinator, Medicinal and Aromatic Plants Programme in Asia (MAPPA), International Centre for Integrated Mountain Development (ICIMOD), Dhapakel, Lalitpur, PO Box 3226, Kathmandu, Nepal.

Tel: 977-1-5525313, Fax: 977-1-5524509

Email: Rrawat@icimod.org

Rege N. N.

Associate Professor, Pharmacology & Therapeutics, Seth GS College & KEM Hospital, Parel, Mumbai – 400012, India.

Tel: 022-24181744, Fax: 022- 24121711

Email: kemarc@vsnl.com, nimarege@gmail.com

Samathanam G.J. Director, Department of Science & Technology, Ministry of Science & Technology, Technology Bhavan, New Mehrauli Road, New Delhi - 11 0016. India.

Tel: 011-26862512 (telefax)

Email: samathan@nic.in

Sangwan R. S.

Senior Scientist, Department of Biochemistry, Central Institute of Medicinal and Aromatic Plants (CSIR), PO CIMAP, Lucknow – 226016, India.

Tel: 0522-2717434, +919451246764

Email: sangwans@sify.com, sangwan.lab@gmail.com

Sharma P. P.

Lecturer, Post Graduate Department of Botany, Deogiri College, Station Road, Aurangabad – 431005, India.

Tel: +919422519569, 0240/2334577, Fax: 0240/2334577

Email: dr_ppsharma@yahoo.co.uk

Sharma S. Ayurvedic Physician, C/O, Jan Swasthya Sahyog, Village and PO Ganiyari, Bilaspur – 495112, Chhattisgarh, India.

Tel: 07753-244819, 244819, Fax: 07752 - 247966

Email: janswasthya@gmail.com, jss_Ganiyari@rediffmail.com

Singh J.

Deputy Director, Division of Pharmacology, Indian Institute of Integrative Medicine (formerly Regional Research Laboratory), Canal Road, Jammu Tawi – 180001, India.

Tel: 0191-2572002, 2549051, Fax: 0191-2547850

Email: Jsishar1@yahoo.com

Tetali P. Senior Scientist, Naoroji Godrej Centre for Plant Research, Lawkin Ltd. Campus, Shindewadi, Shirwal, Satara – 412801, India.

Tel: 02169-244401

Email: ngcpr@lawkimindia.com

Thatte U.

Professor & Head, Department of Clinical Pharmacology, T N Medical College and BYL Nair Charitable Hospital, Mumbai Central, Mumbai – 4000008, India.

Tel: 022-23014713, Fax: 022-23050347

Email: clinpharm@vsnl.net

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Venkatasubramanian P.

Joint Director, Foundation for Revitalisation of Local Health Traditions (FRLHT), 74/2, Jarakbande Kaval, Post Attur, Via Yelahanka, Bangalore – 560064, India.

Tel: 080-28565618/080 28568000 ext. 142, Fax: 080-28567926

Email: Padma.venkat@frlht.org

Wele A. A.

Professor and Head, Dept of Rasashastra and Bhaishajyakalpanavigyan (Ayurveda Pharmacology), BVDU College of Ayurveda, Dhankawadi Campus, Pune – 411043, India.

Tel: +919823059970

Email: wele@vsnl.com

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