ANTI-CANCEROUS METABOLITES AND EXTRACELLULAR ...

ANTI-CANCEROUS METABOLITES AND EXTRACELLULAR

ENZYME PRODUCTION BY ENDOPHYTIC PENICILLIUM AND

PAECILOMYCES STRAINS

Ph.D Thesis

By

Sajid Ali

CENTRE OF BIOTECHNOLOGY AND MICROBIOLOGY

UNIVERSITY OF PESHAWAR

2016

ANTI-CANCEROUS METABOLITES AND EXTRACELLULAR

ENZYME PRODUCTION BY ENDOPHYTIC PENICILLIUM AND

PAECILOMYCES STRAINS

By

Sajid Ali

A dissertation submitted to the University of Peshawar in partial

fulfilment of the requirements for the degree of

Doctor of Philosophy

In

Biotechnology and Microbiology

CENTRE OF BIOTECHNOLOGY AND MICROBIOLOGY

UNIVERSITY OF PESHAWAR

IN THE NAME OF ALLAH, THE BENEFICENT

THE MERCIFUL

Read! And thy Lord is Most Honorable and Most Benevolent, Who taught (to write) by pen, He taught man that which he knew not

(Surah Al-Alaq 30: 3-5) Al-Quran

Dedication

Dedicated to all humanity……

i

CONTENTS

Chapter Title Page No.

Contents i

List of Tables iii

List of Figures iv

Acknowledgement v

Abstract vi

1 INTRODUCTION AND LITRATURE REVIEW 1

1.1 Introduction to Endophytic Fungi 1

1.2 Role and Function of Endophytic Fungi 4

1.3 Essential Metabolites from Endophytic Fungi 5

1.3.1 Chemical Constituents from Endophytic Fungi 8

1.3.2 Phytohormones from Endophytic Fungi 11

1.3.3 Enzymes from Endophytic Fungi 11

1.4 Endophytic Biotechnology and Uses 13

1.5 Plant Species Selected 13

1.5.1 Ecology, Habitat and Traditional uses 14

1.6 Fungal Isolation, Identification and Diversity 15

1.7 Diversity of Endophytic Fungi by Using DGGE Analysis 20

1.8 Extracellular Enzymes, Indol Acetic Acid and ACC Deaminase

Production 21

1.9 Characterization of Bioactive Secondary Metabolites 24

1.10 Aims and Objectives of the Present Research Work 25

1.11 Study Benefits 26

2 MATERIALS AND METHODS 27

2.1 Plant Collection 27

2.2 Isolation of Fungal Endophytes 27

2.3 Morphological Characters and Molecular Identification of the

Fungal Endophytes

27

2.4 Genomic DNA Extraction 28

2.5 PCR Amplification 28

2.6 Nested PCR for DGGE analysis 29

2.7 Denaturing Gradient Gel Electrophoresis (DGGE) 30

2.7.1 Gel Casting Procedure 30

2.8 Phylogenetic Analysis 32

2.9 Diversity Analysis 33

2.10 ACC deaminase Activity of Endophytic Fungi 33

2.11 Quantification of Extracellular Enzymes 35

2.12 Reagents 36

2.12.1 Substrates 36

2.12.2 Buffer 37

2.12.3 Standards 37

2.12.4 Cultrul Filtrate 37

2.12.5 Microplate set-up 37

2.12.6 FluorescenceReadings 39

2.13 Indole Acetic Acid Quantification of Endophytic Fungi 40

2.14 Extraction and Purification of Bioactive Compounds 41

2.15 NMR Spectroscopy 41

ii

2.15.1 HSQC 41

2.15.2 HMBC 41

2.15.3 COSY 42

2.15.4 NOESY 42

2.15.5 Sample Preparation for NMR 42

2.16 Anticancer Activities 42

2.17 Statistical Analysis 43

3 RESULTS 44

3.1 Morphological Characteristics of Endophytic Fungi Colonies 44

3.1.1 Morphology of Endophytic Fungi Isolated from C. acutangula 44

3.1.2 Morphology of Endophytic Fungi Isolated from B. sacra 44

3.2 Diversity of Endophytic Fungi with Caralluma acutangula and

Boswellia sacra

45

3.3 Sequencing and Identification of Endophytes 47

3.4 Phylogenetic Analysis 48

3.5 ACC deaminase Activity of the Endophytic Fungi 52

3.6 Indole Acetic Acid Quantification of Endophytic Fungi 52

3.7 Extracellular Enzymes Production by Endophytes 53

3.7.1 α-glucosidase,Cellulases, Phospatases 55

3.8 Extraction and Purification of Compounds 57

3.9 Chromatographic and Spectroscopic Techniques 57

3.10 Characterization of Compounds 59

3.11 Enzyme Inhibitory Activities of Secondary Metabolites 65

3.12 MTT Assay 66

4 DISCUSSION 69

Section-1: Endophyte Diversity Assessment 69

Section-2: Potential Role of Endophytes 72

Section-3: Bioactive Metabolites from Endophytes 75

Section-4: Conclusion 81

5 REFRENCES 82

6 APPENDIXES 100

iii

LIST OF TABLES

Table No. Title Page No.

2.1 Constituents and their amounts in DGGE set up. 31

2.2 Ingredients of Stacking Gel 32

3.1 Endophytic fungi isolated from C. acutangula. 46

3.2 Endophytic fungi isolated from B. sacra 46

3.3 Sequence Similarities of Endophytic Fungi Isolated from

C.acutangula and Boswellia sacra

48

3.4 Gene Bank Numbers of Endophytic Fungi Isolated from C.

acutangula and B. sacra 49

3.5 Extracellular enzymes produced by endophytic fungi 54

iv

LIST OF FIGURES

Figure No. Title Page No.

1.1

Environmental continuum and endophytic interaction

with a plant during stress conditions (Adopted from

Khan et al., 2013).

4

1.2

Schematic diagrams of the research methods for

isolation and identification of endophyte community

(Adopted from; Sun and Guo, 2012). Xiang Sun and

Liang-Dong Guo (2012)

19

2.1 Schematic representation of a microplate set-up for the

study of kinetic parameters of β-glucosidase in two soils.

Adopted from (MC Marx, M Wood- 2001)

38

3.1 Evolutionary relationship endophytic fungal strains from

B. sacra

50

3.2 Evolutionary relationship endophytic fungal strains from

C. acutangula

51

3.3 ACC deaminase activity of the isolated endophytic fungi 52

3.4 Indole acetic acid production by endophytic fungi 53

3.5 Structures of Compounds 1-5 60

3.5a Demonstrate Compound 1; 11-Oxoursonic acid benzyl

ester

61

3.5b Demonstrate Compound 2; n-nonane 62

3.5c Demonstrate Compound 3;3-decene-1-ol 63

3.5d Demonstrate Compound 4; 2-Hydroxyphenyl acetic acid 64

3.5e Demonstrate Compound 5; Glochidacuminosides A 65

3.6 Enzyme inhibition activities of the secondary

metabolites isolated and characterized from the

endophytic fungi

66

3.7 Effect of cultural filtrates of endophytic fungi on the

viability of MCF-7 breast cancer cells in culture

67

3.8 Effect of pure compounds on the viability of MCF-7

breast cancer cells in culture

68

v

Acknowledgement

In the name of Allah, who has given me strength and courage to accomplish this PhD

work in the benefit of mankind.I bow my head on thanks and gratitude to Allah forhis

countless blessings.

My first debt of gratitude must go to my supervisor, Dr. Sumera Afzal. She patiently

provided the vision, encouragement and advice necessary for me to proceed through

the PhD program and completes my dissertation.

My deepest regards to my co-supervisor Dr. Muhammad Hamayun, Department of

Botany, Abdul Wali Khan University Mardan. I am especially thankful to Dr. Abdul

Latif Khan, Dr. Liaqat Ali Malik and Dr. Tania Rizvi, University of Nizwa, Chair

of Oman's Medicinal Plants and Marine Natural Products, for providing me laboratory

facilities to conduct analysis of my study.Their love, encouraging behavior and

support were always there where things didn‟t seem to work.

I am thankful to Prof. Dr. Bashir Ahmad, Dean, Faculty of Life and Environmental

Sciences, and Prof. Dr. Ghousia Lutfullah, Director, Center of Biotehnology and

Microbiology, University of Peshawar for their cooperation and facilitation of my

Ph.D research project.

Perhaps, I would not be able to present this work in present form without co-operation

of Higher Education Commission (HEC) Pakistan by funding me through

indigenous PhD fellowship programme.

Thanks to all teachers, students, friends and staff members of Center of

Biotechnology and Microbiology, University of Peshawar for sharing expertise and

for providing a friendly environment. Thanks to everybody who had contributed

directly or indirectly for the completion of this study. Special thanks to my sweet

brothers Asad Ali and Nasir Khan for their moral support and encouragement

throughout the studies.

In the last I wish to thank my parents, wife and daughters (Sofia and Rubab), their

love provided me inspiration and was my driving force. I owe them everything and

wish I could show them just how much I love and appreciate them.

Sajid Ali

vi

Abstract

Fungal endophytes colonize an important niche within the plants through secretion of

secondary metabolites. The metabolites and extracellular enzymes produced by

endophytic fungi regulate the growth of the host plant and contribute in defence

mechanisms.The medicinal plants Caralluma acutangula and Boswellia sacra were used

for the isolation of endophytic fungi. The endophytic fungi were identified as Penicillium

citrinum, Paecilomyces variotii, Aspergillus nidulans, Fusarium oxysporum, Epicucum

nigram, Penicillium purpurogenum, Penicillium spinulosum, Aspergillus caespitosus,

Phoma and Alternaria sp. and were assessed for their potential to produce anti-cancerous

metabolites by performing MTT assay and extracellular enzymes such as cellulases,

phosphatases and glucosidases in growth media. P. variotii, P. citrinum and F. oxysporum

showed significantly higher amount of phosphatases and glucosidases as compared to

other strains. Additionally, P. variotii and F. oxysporum showed significantly higher

potential of indole acetic acid production (tryptophan-dependent and independent

pathways). ACC (1-Aminocyclopropane-1-carboxylate) deaminase results showed that

P.citrinum, P. purpurogenum and P. Variotii had shown prominent ACC deaminase

activity (300 nmol α- ketobutyrate mg-1

h-1

). Fluorescence-based MUB (4-methyl

umbelliferone) standards were used to analyze the presence of extracellular enzymes

glucosidase, phosphatase and cellulase. The bioactive secondary metabolites from

endophytic P. citrinum also revealed some prominent results by performing MTT assay

on breast cancer cell line (MCF-7). The current study concludes that these fungi are

producing bioactive constituents that could provide unique niche of ecological adaptation

by symbiosis and greatly contributing to the healthy life of their host plant. However,

some of the endophytic fungi offer a great potential to produce anti-cancerous metabolites

and extracellular enzymes.

Chapter 1 Introduction and Literature Review

1

INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction to Endophytic Fungi

In nature, almost all plants serve as a reservoir of asymptomatically occurring

microbial flora known as endophytes (Yang et al., 2014; Jain et al., 2015). The term

endophyte was first coined by De-Barry in 1866 (Verma et al., 2007). Hence

„endophyte‟ refers to „within the plant‟ and categorically used for microorganisms

which are inside the plant tissues (Mercado 2015). Microorganisms (bacteria/fungi)

occurring asymptomatically inside the plant tissues (leaves, stem, roots) without

causing any disease condition are classified as endophytes (Bacon and White, 2000;

Saikkonen et al., 2004; Schulz and Boyle, 2005). This definition excludes the

mycorrhizal fungi but does not imply that endophytic fungi are not cultivable on

synthetic media (Gallo et al., 2008). Most commonly these are the fungi that inhabit

in plant hosts for all or part of their life cycle, colonize the internal plant tissues

beneath the epidermal cell layers without causing any apparent harm or symptomatic

infection to their host and inhabit in all parts of the host plants (Selim et al., 2012).

The paleomycological evidences of endophytic symbioses with plants have been

estimated approximately 400 Million years old, thus placing it in the same geological

period as were mycorrhizal symbioses (Smith and Read, 1996; Sapp 2004; Krings et

al., 2007; Rodriguez and Redman, 2008; Heijdenet al., 2015). On the basis of

different parameters like evolution, classification, plant hosts and ecological niche

these endophytes are divided into two major groups i.e. (i) Calvicipitaceous (C) which

inhabit mostly in some grasses and (ii) Non-Calvicipitaceous (NC) which are

associated with tissues of nonvascular plants, ferns and allies, conifers and

angiosperms (Rodriguez et al., 2009; Harman 2011). C-endophytes are playing their


2

pivotal role mainly in stress condition and reported to extend benefits to the host

plants in biotic and abiotic stresses besides this also contributing in the increasing of

plant development (Bacon and White, 2000; Saikkonen et al., 2004; Faeth et al.,

2006; Rodriguez et al., 2009; Hamilton et al., 2010; Eaton et al., 2011; Harman

2011). These abilities of endophytic fungi are reliant on the species of host, its

genotype and environmental conditions (Faeth 2002; Redman et al., 2002; Waller et

al., 2005). NC-endophytes are more diverse in nature, may grow in both above and

below-ground tissues and can be recovered from almost every ecosystem of terrestrial

organisms. However, NC-endophytes represent at least three separate functional

groups (Class-I, II, III) which are based on their life style, features and their

ecological implication (Rodriguez et al., 2009). These endophytes asymptomatically

colonize and confer habitat-adapted fitness to plants (monocots and dicots/eudicots)

which have poor physiological capabilities to handle various environmental

conditions. Due to their adaptability between endophytic and free-living lifestyles,

they are explored with great interest (Selosse et al., 2004).

Since the last three decades of 20th

Century, most of the studies about endophytic

fungi were conducted on the population, habitat and classification. Endophytes are

isolated from variety of ecosystems ranging from hot deserts to tundra and temperate

forests (Hoffman and Arnold, 2008; Arnold and Lewis, 2005). Previously, some of

the studies estimated global diversity of fungi at 1.5 million species, drawing from a

ratio of six species of fungi per vascular plant species and only 7 % of the world‟s

fungi have so far been described. In the past century, many of the 0.1 million fungi

that have been described were those associated with various higher organisms as

either parasites or saprophyte on dead/ dying biological materials (Schulz et al.,2005;

Krings et al., 2007; Gallo et al., 2008). Thus, the question comes to our mind, where


3

are the remaining 0.9 million fungul species? Recently, Hibbett et al. (2011) and

Hawksworth (2001) suggested that this diversity could be more than 5.1 million

species. However, the global diversity of these endophytic fungi is limitless and still a

huge number of species and ecosystems need to be explored. In connection to this a

significant amount of work has also been carried out on the endophyte derived from

medicinal plants (Arnold and Lewis, 2005). The individual plant may serve as host to

more than one endophytes while plant and endophyte relationship is favoured by the

bioactive compounds, proposing that there may be many undiscovered endophytic

microbial flora (Strobel and Daisy, 2003; Wu et al., 2013). The identified strains of

endophytic microorganisms is very less in number which shows that there is

significant opportunities to explore the novel strains of endophytic microorganisms

from variety of ecosystems. Endophytic diversity and the symbiotic relationship of

host plant and endophyte greatly contribute to combat the adverse environmental

conditions and climatic change (Rodriguez et al., 2008; Khan et al., 2013; Yang et al.,

2014). This potential has also been considered for the ability of endophytes to produce

various kinds of biologically active metabolites and enzymes.

In plant microbe interacton the rhizospheric band also offer a place of safety to the microbe

in transportation, reproduction and accessibility to nutrients via the plant roots. Once the

switch over is successful and mutualism is established, it lasts for generations throughout

the plant‟s life. The plant provides a safe haven to the endophyte while facilitating it with

food. In return, it extends diverse benefits to the host plant ranging from an influx of

nutrients to regulating the essential biochemicals after exposure to abiotic stresses. Thus, the

effects on the environmental continuum are ameliorated by promoting the metabolism of

the phyllosphere involving the rhizosphere. The '+', '-' and '-/+' in the phyllosphere show the

increase, decrease and altered activities of various processes during plant development,

respectively, under stress conditions and endophytic association (Khan et al., 2013).


4

Figure 1.1:- Environmental continuum and endophytic interaction with a plant

during stress conditions (Adopted from Khan et al., 2013).

1.2 Role and Function of Endophytic Fungi

Endophytic fungi display a great diversity and are known to have a considerable

effect on their host (Strobel and Daisy, 2003). The microbial flora of the plant and

their diversity present a pivotal role mainly in stress conditions (Schulz and Boyle,

2005; Krings et al., 2007). A variety of relationships can exist between endophytes

and their host plants, ranging from mutualism or symbiosis to antagonism or slight

pathogenesis (Schulz and Boyle, 2005; Arnold 2007; Waqas et al., 2012; Khan et

al., 2013). Generally, the host-endophytes relationships can be described in three

ways: host-specificity, host-recurrence and host-selectivity (Zhou et al., 2006;

Cohen 2006). In host-specificity, a microorganism is limited to a single host or a

single species (Strobel 2003, Strobel and Daisy, 2003) whereas host-recurrence


5

refers to the frequent or predominant occurrence of endophytes in a particular host

or a range of plant hosts, although the endophytes may also be found rarely in

other host plants in the same habitat (Zhou and Hyde, 2001). A single endophytic

species may form relationships with two or many related host plants, but where

there is a preference for one particular host the phenomenon is defined as host

selectivity (Cohen 2006). The term host-preference is most frequently used to

indicate a common occurrence or uniqueness of occurrence of endophytes in a

particular host, and is also used to indicate the difference in endophytic

community composition and relation frequencies from different host plants

(Suryanarayanan and Kumaresan, 2000). Endophytes are also able to colonize

multiple host species of the same plant family within the same habitat, and the

distribution of some endophytes can be similar in closely related plant species (Hu

et al., 2008). The differences in endophytes in their metabolic profile, and hence in

their biological activity, even if between isolates of a species, might be related to

the chemical difference of host plants. This raises the importance of studying host-

endophytes relationships, and the effect of host plants on endophytic metabolite

production. A number of endophytic fungi with their important metabolites are

reported from different medicinal plants.

1.3 Essential Metabolites from Endophytic Fungi

More attention is now being given to study the biodiversity of fungal endophytes, the

chemistry and bioactivity of metabolites produced by endophytic fungi and the

relation between endophytes and their host plants (Schulz et al., 2002; Khan et al.,

2013). Hence, Endophytic fungi represent a significant section of fungal biodiversity,

effect on plant community diversity and structure by dint of producing certain

essential metabolites (Sanders 2004; Gonthier et al.,2006; Krings et al., 2007).


6

Endophytic fungi are an important and novel resource of natural bioactive compounds

with their potential applications in agriculture, medicine and food industry (Morath et

al., 2012; Chen et al., 2014). A variety of interesting molecules have been isolated

from endophytes, including flavonoids, peptides, alkaloids, steroids, terpenoids,

lignans, and volatile organic compounds, many of them are biologically active (Zhang

et al., 2006; Gallo et al., 2008; Kusari et al., 2012). In fact, the study of Schulz et al.,

(2005) showed that about 51% of biologically active metabolites are derived from

endophytes. In the past two decades, a lot of valuable bioactive compounds with

antimicrobial, insecticidal, cytotoxic, and anticancer activities have been successfully

discovered from these tiny endophytic factories. Some endophytes have the ability to

produce the same or similar bioactive compounds as those originated from their host

plants (Kusari et al., 2012). The remarkable discovery of bioactive compound

revealed from the endophytic fungus Taxomyces andreanae in 1993 (Stierle et al.,

1993) and the production of world‟s first billion-dollar anticancer drug, paclitaxel

(Taxol) from endophytic fungi Pestalotiopsis microspore, colonizing in Himalayan

yew tree without causing any disease condition to its host plant (Strobel et al., 1996;

Gallo et al., 2008; Kusari et al., 2012) shifted the attention of many scientists and

started research on the fungal endophytes as potential source of novel and biologically

active compounds. Presently, the wild Taxus plants have been used for the production

of paclitaxel. However, the amount of paclitaxel found in the various parts of Taxus

plant was extremely low. To normalise supply and demand of the market, the

alternative resource and potential strategy should be developed. In the last four

decades, many efficient approaches such as field cultivation, cell and tissue culture,

chemical synthesize for paclitaxel production have been developed, and much

progress has been achieved (Zhou and Wu, 2006). Kumaran et al., 2014 screen the


7

fungus Phoma betae for taxol production which was isolated from Ginkgo biloba,

demonstrated the production of „Taxol‟. Similarly, the production of taxol from an

endophytic fungus, Lasiodiplodia theobromae isolated from the medicinal

plant Morinda-citrifolia showed its cytotoxicity against human breast cancer cell

lines. The endophytic fungal taxol was tested for its bioactivity against human cancer

cell line and the results showed that the bioactive compounds from endophytic fungi

Lasiodiplodia theobromae possess anticancer activity (Pandi et al., 2013).

These discoveries allowed the scientist to explore a variety of endophytes for the

production of valuable compounds such as paclitaxel. Since then, many scientists

have been increasing their interests in studying fungal endophytes as potential source

for the production of a number of valuable compounds. In the past two decades, many

valuable bioactive compounds i.e. alkaloids, terpenoids, steroids, phenols, lactones

and quinones have also been isolated from endophytic fungi and showed

antimicrobial, insecticidal, cytotoxic and anticancer activities (Zhang et al., 2006, Xu

et al., 2008; Zhao et al., 2011).

That‟s why Endophytic fungi are considered as one of the most active group of

microorganisms for the production of biologically active secondary metabolites that

plays important biological roles for human life. The symbiotic relationship and co-

evolution paved the way for a friendly relationship between endophyte and their host

plants. The host provides nutritional requirements and habitation for the survival of its

endophytic flora. While, the endophytes would produce a number of bioactive

compounds for helping the host plants to resist to any stress condition and promoting

the host growth in return (Rodriguez et al., 2009; Silvia et al., 2007). Therefore, to

produce same or similar bioactive compounds as those originated from the host plant


8

is of great importance to understand and utilize this sort of relation in host and

endophyte (Schulz et al., 2002; Zhao et al., 2011; Yang et al., 2014). This will greatly

contribute in the production of rare and important biologically active compounds

(Gunatilaka 2006, Zhou et al., 2009).

The isolation and characterization of different therapeutic drugs from endophytic

fungi have shifted the focus of drug discovery from plants to these endophytes

(Tolulope et al., 2015). The recent advancement in biotechnological research paved

the way for the utilization of endophytes as a promising source of bioactive

compounds and has greatly contributed to preserve the endangered species of

medicinally valuable plants by confirming their activities in a short period of time

(Kharwar et al., 2011). Various endophytic fungi have been identified to yield several

modern medicines with main health cures.As Yang et al., (2003) reported six

endophytic fungi obtained from Sinopodophyllum hexandrum, Diphylleia sinensis and

Dysosma veitchii that have the ability to produce podophyllotoxin inevitable for warts

disease. In a simple way, more than 20% of the identified antibiotics and other drugs

have been produced by fungal and bacterial sources (Strobel and Daisy, 2003). While

the growing demand of human population for food, drug and effective agriculture

paved the way for the exploration of natural synthesizers of bioactive compounds.

1.3.1 Chemical Constituents from Endophytic Fungi

The biological diversity of endophytic fungi coupled with their capability to

biosynthesize bioactive secondary metabolites has provided the stimulus for a number

of investigations on endophytes and their chemical constituents. The endophytic

fungus Entrophosporain frequens obtained from Nothapodytes foetida has the ability

to produce camptothecin (CPT) (Amna et al., 2006). CPT and its analogue10-


9

hydroxy-camptothecin have been regarded as the most effective antineoplastic agents

and in clinical use against ovarian, small lung and refractory ovarian cancers

prevalently all over the world (Sirikantaramas et al., 2007). Camptothecine (CPT) is a

quinoline alkaloid and also known as a potent inhibitor of eukaryotic topoisomerase-I.

CPT is also produced by plant species belonging to the asteroid-clade and recently

efforts have been made to isolate endophytic fungi from some of these plants as

possible alternative sources of CPT. Shweta et al., (2013) documented the isolation of

endophytic fungi from fruit and seeds of Miqueliadentata that produce CPT, 9-

methoxy CPT and 10-hydroxy CPT from endophytic fungi Fomitopsis, Alternaria

alternate and Phomposis species (Shweta et al., 2013).

Similarly, xylarenic acid and xylarenones A and B were isolated from the endophytic

fungus Xylaria species NCY2. These compounds were evaluated for antitumor and

antimicrobial assays in which they showed antitumor activities against HeLa cells (Hu

et al., 2008). The ethyl acetate extract of endophytic fungus Periconiaspecies F-31

isolated from Annona muricata were evaluated for anti-tumor activities. Two new

terpenes named (3S, 6S, 7R, 8S)-periconone A and (1R, 4R, 6S, 7S)-2-caren-4,8-olide

were identified and compounds were pharmacologically evaluated for cytotoxic effect

against different human tumor cell lines. However, they exhibited low cytotoxic effect

(Han-Lin et al., 2011). According to Wu et al., (2013) the endophytic fungus of

Phomopsis species isolated from Aconitum carmichaeli showed very good results for

the production of steroids, more than four different types of steroids were isolated

from the culture broth and their inhibitory activities were evaluated against different

pathogenic fungi (Wu et al., 2013). Ying et al., 2014 used Huperzia serrate (toothed

clubmoss) for endophytic fungal isolation; they isolated and identified the novel types

of metabolites like norcyclocitrinol A, erythro-11α-hydroxy-neocyclocitrinol, and

pesudocyclocitrinol A, from fungal endophyte Penicillium chrysogenum P1X. These


10

compounds were identified by spectroscopic methods and revealed that it share the

C25steroid skeleton. In particular, norcyclocitrinol A, represents the first example of a

C25 steroid. All compounds were evaluated for their cytotoxic activities against HeLa

and HepG2 cell lines while showed no significant results (Ying et al., 2014).

Some of the earlier investigations have proved that Fusariumis a rich source of

biologically active secondary metabolites, including the anti-fungal agents

oxysporidinone and 6-epioxysporidinone which is an antimicrobial agents beauvericin

and bikaverin, fungal toxins fumonisin and sambutoxin, phosphatidylinositide 3-kinase

inhibitor wortmannin, and immunosuppressive agent cyclosporine A. In some of the

study, F. oxysporum culture led to the isolation of two new compounds, a new

oxysporidinone analogue and a new 3-hydroxyl-2-piperidinone derivative. The utilization

of endophytic fungi as a source agent in food industry, drug discovery and controlling

farm pests and pathogens is enormous. As the synthetic compounds showed high level of

toxicity toward environmental conditions the endophytes are the chemical synthesizers

inside plants could be greatly utilized in the synthesis of natural bioactive compounds

(Owen and Hundley 2004, Morath et al., 2012; Chen et al., 2014). Host plant itself has

functioned as a selection system for endophytic microbial flora and developed a

symbiotic association. There are number of examples of endophytes which produce the

same types of bioactive compounds as produce by their host plant. The endophytic fungus

Phomabetae isolated from Ginkgo biloba the endophytic fungus Lasiodiplodia

theobromae isolated from the medicinal plant Morinda citrifolia, the endophytic fungal

strain QJ18 from host plant Gentianama crophylla encodes forgentiopicrin like its host

plant G. macrophylla (Yin et al., 2009; Pandi et al., 2013; Kumaran et al., 2014). Thus,

the endangered species of medicinally important plants and other natural resources can be

protected by utilizing the endophytic microbial flora to satisfy the requirement of drugs

via production of plant-derived pharmaceutical leads by fermentation.


11

1.3.2 Phytohormones from Endophytic Fungi

Waqas et al., (2012) and Khan et al., (2013) suggested that endophytic fungi can

produce physiologically active phytohormones such as gibberellins and auxin, where

the ameliorative effects of endophytes is comparable with that of commercially

available plant growth regulators (Gibberellic Acid) during stressful conditions (Khan

et al., 2014; 2015). Initially, gibberellin was discovered in 1930s from the culture

filtrates of Gibberella fujikuroi (Ogas J, 2000). While, recently different types of GAs

have been recognised and a number of fungal species associated with plants has been

reported as GA producers (Kawaide H, 2006). Host-plants without endophyte-fungal

association are devastated by the waves of extreme temperature, drought, salinity and

pathogen attack (Saikkonen et al., 2010). Hence, the productivity is frequently

compromised in such situations. These endophytes get higher macro- and micro-

nutrients like phosphorus, sulfur, calcium, magnesium and potassium. This capability

has often been considered due to the potential of these endophytes to produce various

biologically active metabolites and enzymes (Yuan et al., 2010). Among metabolites,

plant hormones like GAs and auxin production is a new phenomenon in the endophytic

fungi. Both GAs and auxin have been reported to play an important role in plant

growth, reproduction, metabolism and respond to various environmental signals. In last

decade or so, it has been a known factor that these endophytic fungi, residing inside

host confer abiotic stress tolerance (Yuan et al., 2010; Arnold et al., 2007).

1.3.3 Enzymes from Endophytic Fungi

In addition to the role of endophytic fungi in the production of anticancer and

antimicrobial metabolites, the endophytic fungi have been also known for their potential

to produce different types of extracellular enzymes. The extracellular enzymes are

produced by endophytic fungi for penetrating the host cell wall, and their production

supplements the direct uptake of nutrients by microorganisms and is linked to nutrient

availability and environmental conditions (Latif Khan et al., 2016).


12

The enzymes derived from fungi and bacteria are often more stable than other sources

and are used in food, medicine, beverages, sweets, textiles and leather industries to

process raw materials (Sunitha et al., 2013; Castro et al., 2014). Fungal Kingdom

investigates the roles and importance of fungi play in the biosphere. Recently, only

five genera i.e. Aspergillus, Humicola, Penicillium, Rhizopus and Trichoderma account

for more than 50% fungal enzymes used in the different processes (Ostergaard and

Olsen, 2010).

Chimata et al., 2013 used the fungal strain Aspergillus sp. MK07 for the production of

extracellular amylases enzyme by utilizing wheat-bran as a substrate. Amylases are

important enzymes, mainly used in the starch processing industries to hydrolyse

polysaccharides into simple sugars, about 30% of the world‟s enzymes production is

based on amylases, being used in different processing like confectionary, baking, paper,

textile, detergent and pharmaceutical (Chimata et al., 2013).

Most of the amylases have been screened from soil fungi such as Aspergillus, Penicillum

and Rhizopus (Pandey et al., 2000) while the study of Zaferanloo et al., 2014 showed

that endophytic fungus also encodes for the production of amylases i.e. Preussia minima

can be isolated from Eremophilia longifolia, which can greatly contribute in the

production of α-amylase. The two strains of endophytic fungi Philaophora

finlandia and Philaophora fortinii isolated from the roots of alpine plant communities

were able to breakdown the polymeric forms of carbon, nitrogen and phosphorus found in

the plants (Caldwell et al., 2000). Similarly, Marlida et al., (2000) reported starch

degrading enzyme from endophytic fungi Gibberella pulicaris, Acremonium species

and Nodilusporium species. While, amylase production by few endophytic isolates from

mangrove angiosperm Acanthus ilicifolius and mangrove fern, Acrostichum aureum was

reported by Maria et al., in 2005.


13

1.4 Endophytic Biotechnology and Uses

Biotechnology has motivated the utilization of a distinctive group of plant-associated

microorganisms, known as endophytes. However, the biological and ecological roles of

fungal endophytes still totally unexplored. The European Cooperation in Science and

Technology intended to utilize endophytes in biotechnology and agriculture. There are

four working group activities started research on Ecology of endophytes, Identification of

new competent endophytes and new industrial products in life sciences.

The use of endophytic biotechnology to control plant-pathogenic bacteria and fungi is

receiving increasing attention as a sustainable alternative to synthetic pesticides and

antibiotics. Furthermore, these endophytic microorganisms are likely to be adapted to

the presence and metabolism of complex organic molecules and therefore, show

useful biodegradation activities. In order to reduce the input of pesticides and

fertilizers bring to eco-friendly agriculture, it will be important to develop inocula of

biofertilizers, stress protection and biocontrol agents.

The use of endophytic biotechnology to provide solutions for the economically and

ecologically compatible exploitation of endophytes will greatly contribute to our

present state of knowledge.

1.5 Plant Species Selected

In the present research work the endophytic fungi were isolated from Caralluma

acutangula and Boswellia sacra. Medicinally valuable plant, C. acutangula is found

in the northern and FATA region of Khyber Pakhtunkhwa, Pakistan. Boswellia sacra

a primary tree in the genus Boswellia from which frankincense, a resinous dried sap,

is harvested were collected from salalah region of Oman.


14

1.5.1 Ecology, Habitat and Traditional uses

Caralluma acutangula is a flowering plant belongs to family Asclepiadaceae, mostly

occurring in the northern and FATA region of Khyber Pakhtunkhwa, Pakistan. It is a

compactly branched, cactus-like, perennial stem succulent and having a height of 40-

100 cm with purple-black flowers. Caralluma acutangula is used in some of the

traditional medicine, while very little is known about the endophytic microorganism

from this plant.

Boswellia sacra a primary tree in the genus Boswellia from which frankincense is

harvested. Frankincense is an aromatic resin as an exuded gums obtained from trees of

the Burseraceae family. The resin has been used in incense and fumigants, as well as a

fixative in perfumes. The prominent chemical components in frankincense that provide

anti-inflammatory activities have been documented as boswellic acids. The traditional

applications of frankincense are very diverse - ranging from dental disease to skin

conditions, to respiratory complaints and digestive troubles. The resin was chewed to

stimulate the gums and treat dental infections and sore gums and to generally strengthen

the teeth. Buds and fruit provided a cleansing tonic for the digestive system. Modern

research has focused on frankincense anti-inflammatory properties, particularly in the

treatment of rheumatoid arthritis and soft tissue rheumatism for which it appears to be

extremely useful. Today, frankincense essential oil is used as a fixative and precious oil

not only in the perfume industry, but also lends its scent to soaps, detergents and

numerous cosmetic articles. Considerable amount of work has been attempted to

identify chemical composition of the plant, utilization of its component in various

medicines and isolation of different rhizospheric and endophytic microorganisms

associated with Boswellia sp. Boswellic acids have been identified as a major chemical

component in Boswellia sp. extracts that provide the anti-inflammatory activity.


15

In present research work the endophytic fungi were isolated from medicinally valuable

plants Caralluma acutangula and Boswellia sacra.

1.6 Fungal Isolation, Identification and Diversity

Endophytic fungi that asymptomatically reside in the internal tissues of plants beneath the

epidermal cell layer and contribute in the production of bioactive secondary metabolites

compounds, in addition provide defence to their host plant by producing a plethora of the

substances (D. Wilson, 2000; Strobel G, 2012; Kusari et al., 2014; Mercado-Blanco

2015). The occurrence of endophytic fungi inside plant tissues has been known since the

end of 19th century (Guerin, 1898). Their biological diversity is enormous in a variety of

ecosystems world-wide. The fungi are hosted in nearly 300,000 land plant species, with

each plant hosting one or more of these fungi. Endophytic strains have been isolated from

different parts of diverse plants species including trees, vegetables, fruits and other crops

(M. Rosenblueth and E. Martinez-Romero, 2006; Schulz et al., 2006).

Fungal endophytes have been isolated from different tissues of the plants and

representatives of all major lineages of land plants, that has been inspected (Carroll

and Petrini 1983; Schulz et al., 1999; Strobel G, 2012, Kusari and Spiteller, 2012b).

Previously, two major groups of endophytic fungi have been recognized which shows

dissimilarities on the basis of evolutionary relatedness, taxonomy, plant hosts and

ecological functions the clavicipitaceous endophytes (C-endophytes) and the

nonclavicipitaceous endophytes (NC-endophytes) (Clay & Schardl, 2002; Rodriguez

et al., 2009 ). To date, the best studied groups of fungal endophytes are species in the

family clavicipitaceae that form associations with some cool and warm season grass

species. These species are vertically transmitted, abundant in host tissue, and can

provide a wealth of fitness benefits to the host, for example, herbivore limitation


16

(Clay et al. 1985; De Battistaet al., 1990; Clay and Schardl, 2002). NC-endophytes

are representing a highly diverse assemblage of fungi and have been recovered from

every major lineage of land plants, most commonly from terrestrial ecosystems

(Arnold and Lutzoni, 2007). On the basis of host colonization patterns, mechanisms

of transmission and ecological function the NC-endophytes are classified into three

functional classes (Schardl et al., 2004; Rudgers and Clay 2007; Rodriguez et al.,

2009).

According to Xiang Sun and Liang-Dong Guo, (2012) endophytic fungal diversity can

be identified by two basic techniques i.e. direct observation (microscopy) and

cultivation-dependent methods. In the direct observation method, endophytic fungal

structures within living plant tissues are directly examined under a light and electron

microscope, which can show all endophytic mycobiota within the plant tissue,

particularly biotrophic fungi that cannot be cultured on standard growth media (Deckert

et al., 2001; Lucero et al., 2011). However, most endophytic fungi within plant tissue

have only a hyphal structure, and therefore cannot be identified to any taxonomic

category according to morphology due to lack of spore-producing structures and sexual

or asexual spores. In addition, endophytic isolates cannot be obtained as microbial

resources for further use with the direct observation method. Therefore, this is not

commonly used in endophyte diversity studies (Deckert et al., 2001).

In contrast to direct observation methods, cultivation dependent techniques have been

routinely employed in endophyte diversity studies (Petrini et al., 1982; Rodrigues and

Samuels 1990; Sun et al., 2011; Vieira et al., 2011). It is important to isolate

endophytic fungi for further detailed studies into their characterization, population

dynamics, species diversity to improve plant growth and health, or screening for novel

biologically active secondary metabolites (Zhang et al., 2010; Tejesvi et al., 2011).


17

With cultivation-dependent techniques, the isolation procedure is a critical and

important step in working with endophytic fungi. The living plant tissues are

subjected to a serial process of surface sterilization to remove all organisms from the

surface of the plant. Only internal fungi are isolated by means of incubation of the

plant samples onto nutrient plates.

Cultivation-dependent techniques generally include the following steps;

1) To remove adhering soil particles, debris and major epiphytes, the plant tissues

are washed under tap water.

2) The surface sterilization of plant tissue is important to remove any

epiphyticmicroorganisms on the host surface; a number of protocols are required

for different tissue types.

3) Isolation of endophytic fungi growing out from samples placed on nutrient agar

for further screening and analysis.

4) Purification and sporulation of endophytic isolates under various incubation

conditions.

5) Identification of the isolated endophytic fungi based on morphological

characteristics in the cultures. (Wei et al., 2007; Guo et al., 2008; Sun et al.,

2011; Sun and Guo, 2012).

The development of new techniques in molecular biology brings a novel perspective to

the study of endophyte diversity. Application of molecular techniques, such as DNA

fingerprinting and sequencing methods has the potential to overcome the obstacles in

traditional cultivation-dependent methods. Molecular methods are required for the

identification and understanding of the diversity of endophytic fungi. In a survey of

endophytic fungi from L. chinensis in Hong Kong, a large number of isolates (16.5% of


18

total) did not sporulate, remaining as Mycelia sterilia (Guo et al., 2012). These non-

sporulating isolates were grouped into 19 morphotypes based on their cultural

morphology and identified to different genera (Diaporthe, Mycosphaerella and Xylaria),

families (Pleosporaceae and Clypeosphaeriaceae), and order (Xylariales) based on ITS

sequence analyses. Sun et al. (2011) grouped 221 non-sporulating endophyte strains into

56 morphotypes, and placed these morphotypes into 37 taxa based on ITS sequence

similarity and phylogenetic analyses.

In our present work we did isolation, identification, extracellular enzymes assay and

anticancerous metabolites screening for endophytic fungi isolated from Caralluma

acutangula and Boswellia sacra.


19

Figure 1.2: Schematic diagrams of the research methods for isolation and

identification of endophyte community (Adopted fromGuo et al., 2012).


20

1.7 Diversity of Endophytic Fungi by Using Denaturing Gradient

Gel Electrophoresis (DGGE) Technique

The techniques used in molecular biology offer new opportunities for the analysis of the

structure and species composition of microbial communities. In particular, sequence

variation in rRNA has been exploited for deducing phylogenetic relationships among

variety of microbes and for designing specific nucleotide probes for the detection of

individual microbial taxa in natural environment (Woese 1987; Giovannoni et al.,

1988). All these techniques have also been applied to determine the genetic diversity of

microbial communities and to identifying a number of uncultured microbes. They

constitute the cloning of ribosomal copy DNA or polymerase chain reaction (PCR)-

amplified ribosomal DNA (rDNA) followed by sequence analysis of the resulting

clones (Giovannoni et al., 1990). Denaturing Dradient Gel Electrophoresis is an

approach for directly determining the genetic diversity of complex microbial

populations. The procedure is based on electrophoresis of PCR-amplified 18S rDNA

fragments in polyacrylamide gels containing a linearly increasing gradient of

denaturants. In denaturing gradient gel electrophoresis (DGGE), DNA fragments of the

same length but with different base-pair sequences can be separated. The separation in

DGGE is based on the electrophoretic mobility of a partially melted DNA molecule in

polyacrylamide gels, which is decreased, compared with that of the completely helical

form of the molecule. The melting of fragments proceeds in discrete so-called melting

domains: stretches of base pairs with an identical melting temperature.Once the melting

domain with the lowest melting temperaturereaches its melting temperature at a

particular positionin the DGGE gel, a transition of helical to partially meltedmolecules

occurs, and migration of the molecule will practicallyhalt. Sequence variation within

such domains causes their melting temperatures to differ. Sequence variants ofparticular


21

fragments will therefore stop migrating at different positions in the denaturing gradient

and hence can be separated effectively by DGGE (Lerman et al. 1984; Ward et al.,

1990). This technique has been successfully applied to identifying sequence variations

in a number of genes from several different organisms. PCR can beused to selectively

amplify the sequence of interest before DGGE is used. In a modification of the latter

method, GC-rich sequences can be incorporated into one of the primers to modify the

melting behaviour of the fragment ofinterest to the extent to which close to 100% of all

possible sequence variations can be detected. This procedure allows one for the first

time to directly identify the presence and relative abundance of different species and

thus, to profile microbial populations both qualitatively and semi-quantitatively.

1.8 Extracellular Enzymes, Indole Acetic Acid and ACC Deaminase

Production

Endophytic fungi are relatively unexplored producers of metabolites useful to

pharmaceutical and agricultural industries. A single endophyte produces several

bioactive metabolites. As a result, the role of endophytes in production of various

natural products with greater bioactivity has received increased attention over the last

three decades. Endophytes can influence soil stability directly by their mycelia in the

soil as well as indirectly altering roots and physical conditions of the host plants

(Patil, M. G et al., 2015).An endophytic fungus shows a complex interaction with host

plants and has been extensively studied over the last three decades as a productive

source of novel bioactive natural products. The enzymes derived from fungi and

bacteria are often more stable than other sources and are used in food, medicine,

beverages, sweets, textiles and leather industries to process raw materials (Sunitha et

al., 2013; Castro et al., 2014). The advantageous special effects of endophytic fungi

have been regarded for their potential to produce biologically active secondary


22

metabolites and other substances which can contribute in the defense, growth and

development of their host. Similarly, a variety of substances and enzymes have been

isolated from these endophytes (Khan, A. L et al., 2014).

The enzymes are produced by endophytic fungi in the host and their production

supplements the direct uptake of nutrients by microorganisms and is linked to nutrient

availability and environmental conditions (Khan, A.L et al., 2016). In connection to

this, extracellular enzymes are also produced by some endophytic fungi for

penetrating the host cell wall, as well as to contribute in biocontrol. The enzymes like

glucosidases and cellulases have been assessed from different endophytic microbial

flora. Some of the endophytes have also been known to produce various classes of

secondary metabolites and most of the work has been done on endophytic microbial

flora for screening of biologically active secondary metabolites. In case of bioactive

metabolites, phytoharmones are the natural substances mainly produced by plants to

regulate its growth.

The production of plant like hormones such as Indole acetic acid (IAA) and

Gibberellins is recently documented from different types of endophytes (Waqas et al.,

2012; Kusari et al., 2013; Khan, A. L et al., 2016). IAA plays a very important role in

plant development, growth and combating environmental stimuli while most of the

fungal endophytes are documented for encoding of IAA (Waqas et al., 2012; Khan,

A.L et al., 2016). Consequently, different biosynthetic pathways have been proposed

for IAA production: Tryptophan dependent and Tryptophan independent while the

proposed Trp-dependent pathways are of four types (indole-3-acetamide pathway

(IAM), Indole-3-acetaldoxime pathway (IAOX), Tryptamine pathway (TAM), and

Indole-3-pyruvic acid pathway (IPA)). In this way, a single endophytic strain


23

sometimes exploits more than one biosynthetic pathway by expressing the genes

present on plasmid and chromosome to contribute in the development of their host

(Idris et al., 2007).

In 1978, for the first time Honma and Shimomura reported ACC from Pseudomonas

(Honma and Shimomura, 1978).The enzyme 1-Aminocyclopropane-1-carboxylate

(ACC) deaminases have the capability to hydrolyze ACC which is the immediate

precursor of ethylene in plant. The lowering down of ethylene levels by ACC

deaminase is considered one of the major mechanisms employed by microbial flora of

the plant to facilitate plant growth. ACC deaminase has been found in various

bacteria, yeasts, and fungi, and it can convert ACC into α-ketobutyrate and ammonia

(Abeles, F. B et al., 2012; Yim et al., 2013).

The microbial flora of the plants are containing ACC deaminase and can take up and

degrade ACC from the plant and therefore decrease ethylene synthesis in the plants

(Glick, 2014). ACC deaminases are normally related to free-living soil

bacteria/rhizobacteria and some of the mycorrhizal. However, a very few endophytic

microbes have been known to produce ACC deaminase. The exuded ACC is of plant is

metabolized by fungi and bacteria possessing ability to produce ACC deaminase. This

stimulates plant ACC efflux, decrease the root‟s ACC and ethylene concentration, thus

increase root growth and development (Glick, 2014). The relationship of ACC and IAA

affect each other, the endogenous content of ACC increased the rate of ethylene

production in the presence of IAA, but failed to increase the ACC content in the

absence of IAA (Yoshii and Imaseki 1981). Therefore, it is suggested that the combine

effect of ACC deaminase and IAA are responsible for the pragmatic plant growth

promotion and development.


24

The utilization of endophytes as a potential source of industrially relevant enzymes is

in queue. Hence, they occupy a relatively unexplored site and can represent a new

source in obtaining different enzymes. The present research was carried out to explore

new sources of valuable bioactive compounds and extracellular enzymes from

endophytic fungi of Caralluma acutangulaandBoswelliasacra also to understand their

functional role with the host.

1.9 Characterization of Bioactive Secondary Metabolites

There are more than 20,000 bioactive metabolites of microbial origin (Berdy 2005). In

eukaryotic organisms fungi are among the most important group that are well known

for producing many novel metabolites while many of endophytic fungi were recently

reported to produce bioactive metabolites such flavonoids, peptides, alkaloids,

steroids, phenolics, terpenoids and lignans with antimicrobial, anticancer and antiviral

potentiality. The discovery of taxol producing fungi increased the importance of

endophytes and shifted natural products research from plant to endophytic fungi

(Schulz et al., 2002; Chinet al., 2006).

Schulz et al., (2006) revealed that more than 50% of biologically active metabolites

originate from endophytes (Schulz et al., 2006) and many novel bioactive compounds

with antimicrobial, insecticidal, cytotoxic, and anticancer properties have been

successfully isolated and characterized from endophytic fungi (Berdy 2005; Khan et

al., 2011; Xiao et al., 2014).

The potential for the production of anticancer drug „taxol‟ (paclitaxel) from

Pestalotiopsis microspore and many other endophytic fungi such as Fusarium solani

isolated from Taxus chinensis, Pestalotiopsis guepini and Periconiaspecies encoding


25

for taxol (Strobel et al., 1997) Alternaria and Aspergillus isolated from Ginkgo biloba

and Podocarpus species respectively, has set the stage for increasing interest in fungal

endophytes (Liu et al., 2009).

In view of these potentialities of endophytic fungi, the present research work was

designed to isolate the endophytic fungi from Caralluma acutangula and Boswellia

sacra for the production of bioactive metabolites. The endophytic wealth is yet to be

explored from a variety of plants. The metabolomics produced at extracellular level

during the growth of these endophytic fungi were assessed using advanced

chromatographic and NMR spectroscopic techniques.

1.10 Aim and Objectives of the present research work

The aim of this research work is to explore biologically active secondary metabolites

from endophytic fungi isolated from medicinally valuable plant. New methodologies

and their utilization are important for the discovery of drugs from endophytes. As

most of free living fungi are pathogenic to human and causing a variety of diseases.

The self-medication, false practices and repeated use of antibiotics has led to the

increase resistant species of some existing available drugs in our country. Therefore, it

is very important to explore and identify biologically active secondary metabolites

from endophytic sources and preserve the endangered species of medicinally

important plants.

To isolate endophytic fungi from medicinally important plant Caralluma and

Boswellia species.

Screening and identification of isolated endophytic fungi by morphological

and molecular analysis.

Isolation and purification of secondary metabolites.


26

Structural elucidation of the purified metabolites.

Anti-cancerous and Enzyme inhibitory activities of isolated compounds.

1.11 Study Benefits

Plants and fungi are very important in health-care. Worldwide more than 80% of the

population relies on traditional medicine, much of which is based on plant remedies.

But recent trends and development in endophytic biotechnology focus on the isolation

of endophytic fungi and there bioactive compounds from medicinally important plants.

This will strengthen the importance of biotechnology and its products in our

country.

This will help in the utilization of bioactive compounds isolated from

endophytic fungi. Which may leads to result in the development of new

valuable pharmaceutical compounds.

This will greatly contribute in the study of extracellular enzymes from

endophytic fungi.

This can put up an association among the researchers and pharmaceutical

industries.

This will provide job opportunities in the field of biotechnology and

pharmaceutical industries.

Plants and microorganisms are used in a variety of medicine which is beneficial in

one way or the other. The isolation, identification and utilization of bioactive

compounds from endophytic fungi can greatly contribute to the research and

industries in a country like Pakistan.

Chapter 2 Materials and Methods

27

MATERIALS AND METHODS

2.1 Plant Collection

Different samples of Caralluma acutangula and Boswellia sacra were collected

from northern areas of Pakistan and salalah region of Oman, respectively. The

samples were subjected for the isolation of endophytic fungi and were brought to

laboratory in a sterilized zip bags (121°C for 20 min) in ice box (4°C). The

samples were labelled and stored till further processing.

2.2 Isolation of Fungal Endophytes

The samples were surface sterilized with sodium hypochlorite (2.5%; 30 min in a

shaking incubator at 120 rpm) and repeatedly washed with autoclaved distilled

water (DDW) to remove any epiphytic microbes and ecto-mycorrhizae (Bayman et

al., 1997). Isolation of fungi from bark/leaf were carried out on Hagem minimal

medium, containing 0.5% glucose, 0.05% KH2PO4, 0.05% MgSO4.7H2O, 0.05%

NH4Cl, 0.1% FeCl3, 100ppm streptomycin and 1.5% agar (pH 5.8±0.2). The newly

emerged fungal spots were separated and further grown and stored on potato

dextrose agar (PDA, 50 ppm). The efficiency of sterilization was monitored by

imprinting the tissues on Hagem and PDA plates. Upon contaminant growth, the

tree samples were again sterilized. The morphologically different (Arnold et al.,

2007) endophytic fungal strains were grown in Czapek broth medium (1%

Glucose, 1% Peptone, 0.05% KCl, 0.05% MgSO4.7H2O, and 0.001% FeSO4.7H2O;

pH 7.3±0.2) and incubated on shaking incubator (28ºC with 150 rpm for 8 days).

2.3 Morphological Charaters and Molecular Identification of the

Fungal Endophytes

The endophytic microbes were grouped into different groups on the basis of colony

shape, thickness, colour of aerial hyphae, colony reverse colour, growth rate and


28

pattern, margin characteristics, surface texture, and growth depth into medium

(Arnold et al., 2007). The endophytes were identified by genomic DNA extraction,

PCR techniques, nucleotide sequencing, and phylogenetic analysis as described by

Khan et al., (2011).

2.4 Genomic DNA Extraction

In DNA extraction, two methods were applied in this study.gDNA was isolated

according to manufacturing instructions from fresh mycelial mates with a Solgent

Genomic DNA preparation kit and another efficient method was developed for the

isolation of genomic DNA from endophytic fungi, because usual CTAB extraction

method and mycelial grinding was causing DNA shearing. Rich mycelial culture was

obtained by growing fungus in Czapek culture broth (supplemented with 1% glucose

and peptone) for 7 days on rotary shaking incubator (120 rpm and 28°C), and

lyophilized for 24 hrs. A 0.5 g of lyophilized sample was broken carefully in 2 ml

eppendorf, with the blunt end spatula or with a glass rod. Double volume of lysis

buffer (20 mM Tris-HCL, pH8.0; 10 mM EDTA; 1% SDS) containing 1% of 2-

mercaptoethanol was added. The mixture was vortexed briefly (30 sec) to obtain

homogeneity and left to incubate for 2 hr in water bath set at 55°C. 250 μl/ml of pre-

heated 4% CTAB extraction buffer was added to lysed cells mixture and incubated

further at 65°C for 1 hr. Chloroform extraction followed by iso-propanol precipitation

yielded condensed strand of nucleic acid, which was cleaned from RNA using 10 µl

of RNase A for 2 hr of incubation at 37°C.The isolated DNA was suspended in 50µl

of autoclave deionized distilled water and tested for purity (Hamayun et al., 2009).

2.5 PCR Amplification

The fungal isolate was identified through sequence analysis of the internal transcribed

region (ITS) of 18S rDNA, using universal primers ITS-1 (5´-TCC GTA GGT GAA


29

CCT GCG G-3´) and ITS-4 (5´-TCC TCC GCT TAT TGA TAT GC-3´) (Taylor and

Bruns 1999). A 25 µl of PCR mixture contained 2.5 µl of dNTPs and Ex-Taq buffer,

2 µl of each primer, 0.5 µl of DNA sample, and 0.2 µl of Ex-Taq polymerase. The

remaining volume was adjusted with 15.3 µl of autoclaved deionized distilled water.

For the amplification of ITS1 and ITS4 regions of 18S rDNA, the reaction cycle

consisted of initial denaturation (95°C) for 2 min, 35 cycles of denaturation (95°C) for

30 s, annealing (55°C) for 60 s, extension (72°C) for 30 s and a final extension time

for 5 min (72°C). The resultant products were gene cleaned using a Nucleogen gene

clean kit, ligated in T-vector using Takara Perfect T-cloning kit, and then inserted into

E. coli competent cells (RBC) by overnight incubation (37°C). Transformed cells

were selected, grown overnight (37°C) in LB broth and their plasmids were extracted

using SolGent Plasmid mini-prep kit, which were later sequenced.

2.6 Nested PCR for DGGE Analysis

Fungal 28S rDNA fragments fromsamples were amplified by nested PCR using specific

primers. In first round the primer set P1 (5P-ATCAATAAGCGGAGGAAAAG-3P) and

P2 (5P- CTCTGGCCTTCACCCCTATTC-3P) were used, yielding a PCR product of

approximately 800 bp. The 25-Wl PCR assays contained 2 W1 template, 5U PCR buffer

(335 mM Tris (pH 8.8), 83 mM (NH4)2SO4, 3.75 mM EGTA, 25% glycerol, 0.1%

Tween 20), 2.5 mM MgCl2, 200 WM of each dNTP, 10 pmol of each primer and 3 mg

ml31 BSA. After initial denaturation at 94°C for 4 min and cooling to 80°C, 2 U Tth-

Polymerase (Hybaid) were added. Thirty five cycles were performed by using 94°Cfor 1

min, 40°Cfor 1 min, 72°C for 2 min, followed by 72°C for 10 min. Negative controls

produced no PCR products. Nested PCR was performed using the U1 and U2 primers

with an additional 37-bp GC-clamp at the 5P-end of the primer U1. Forty PCR cycles

were performed as described above, except for the annealing temperature, which was set


30

to 50³C. All amplifications were performed as hot start reactions in a PCR Express cycler

(Hybaid). DNA extracts of the fungal strains inoculated on the mortar blocks were used

as positive controls in the second PCR round following DGGE analysis. All PCR

products were analysed by electrophoresis in 1.5% agarose gels for 60 min at 100 V

before DGGE was carried out.

2.7 Denaturing Gradient Gel Electrophoresis (DGGE)

PCR products of fungal isolates were analysed by the DCode System (Bio-Rad) using

10% (w/v) acrylamide (37.5:1acrylamide: bisacrylamide) gels. Detailed procedure is

as follow.

2.7.1 Gel Casting Procedure

Before, gel casting, the gel casting plates were cleaned thoroughly, either with a

commercial solution for this purpose, or with detergent, DI water, and ethanol. The gel

casting plates were dried with a lab wipe. The blue gaskets were fixed to the glass plate

with curved corners and gaskets were sealed. To keep separate the glass plated we

inserted separators on the inside of the gasket‐covered glass. The plates were kept

together with the stronger casting clamps with two on each side and two on the bottom.

Frozen aliquot of 10% APS was used and 1000ul DI water with 0.1 g ammonium

persulfate. The following reagents and their respective amount were utilized.


31

Table 2.1:- Constituents and their amounts in DGGE set up.

Reagents Chamber 1L Chamber 2R

Milli‐Q Water 10.832 mL

3.102 mL

Loading dye 500 uL 40% Acrylimide, 1.06 %

Bis‐Acrylimide 3.075 mL 3.075 mL 7 M urea, 40% formamide

6.183 mL 13.413 mL

50x TAE 410 uL 410 uL APS 100 uL 120 uL TEMED 6 uL 7.5 uL


3.102 mL



6.183 mL 13.413 mL



3.102 mL



6.183 mL 13.413 mL



3.102 mL

Loading dye ‐‐‐ 500 uL 40% Acrylimide, 1.06 %

The addition of APS and TEMED resulted into polymerization. Immediately after the

addition of APS and TEMED and stirring, the valve was get opened on the mixing

chamber, as well as the in‐line valve on the tubing. The pump was switched on and

outlet needle was placed on one side of the gel casting rig. When the gel was 4‐5 cm

from the top of the casting plates the pump was turned off. The gel polymerized in

about 2 hours.


32

The tank was filled with TAE buffer: 200 mL 50x TAE and 20 L DI water and heated

to 60°C. After the gel had polymerized water was poured off on top of the gel.

Stacking gel was prepared in a 15 mL conical tube as follow.

Table 2.2:- Ingredients of Stacking Gel

Reagents Volume

40% Acrylimide: 1.06 % Bis‐Acrylimide

3.075 mL

50x TAE 200 uL

APS 70 uL

TEMED 10 uL

Milli-Q Water 8.3 ml

The gels were run in 1UTAE (40 mM Tris, 20 mM acetate, 1 mM Na2EDTA (pH 7.8)

and a linear gradient of the denaturants urea and formamide increasing from 35 to

65%. One hundred percent denaturant is defined as 7 M urea and 40% formamide

(v/v; deionised). PCR products obtained from DNA extracts of fungal isolates and an

admixture of each PCR product from the isolates was used as positive control in

DGGE. The fingerprints of the inoculated samples were carried out with final PCR

products. Gels were run for 6 h at 150 V in 1UTAE at a temperature of 60³C. DGGE

bands were visualised by ethidium bromide staining and UV illumination. Digital

images acquired by CCD camera were inverted using the Easy Image Plus software.

2.8 Phylogenetic Analysis

The BLAST search program (http://blast.ncbi.nlm.nih.gov) was used to compare the

nucleotide sequence similarity of the ITS regions of related fungi. The closely related

sequences obtained were aligned through CLUSTAL W using MEGA version 6.0


33

software (Tamura et al., 2013) and a neighbor-joining tree was constructed using the

same software. One thousand bootstrap replications were used as a statistical support

for the nodes in the phylogenetic tree. The aligned sequences were submitted to

GeneBank of NCBI for obtaining the accession numbers.

2.9 Diversity Analysis

The endophytic fungal diversity was estimated by using the Shannon diversity index

(H) and Simpson‟s diversity index (1-D) for both domesticated and wild types of

samples. The colonization density, colonization rates and isolation rates of fungal

diversity were calculated as the percentage of segments infected by one or more

isolates from the total number of segments of each plated tissue. All samples were

analyzed in triplicate. The data are presented as the mean ± standard error of the mean

(SEM) and differences were evaluated by using one-way analysis of variance

(ANOVA).

2.10 ACC Deaminase Activity of Endophytic Fungi

ACC deaminase activity was assayed according to a modification of the method of

Honma and Shimomura (1978) which measures the amount of α-ketobutyrate produced

upon the hydrolysis of ACC. The number of μmol of α-ketobutyrate produced by this

reaction was determined by comparing the absorbance at 540 nm of a sample to a

standard curve of α-ketobutyrate ranging between 10 and 200μmol. A stock solution of

100 mmol L-1 α-ketobutyrate was prepared in 0.1 mol L-1 Tris-HCl (pH 8.5) and

stored at 4°C. Just prior to use, the stock solution was diluted with the same buffer to

make 10 mmol L-1 solution from which a standard concentrations curve was generated.

In a series of known α-ketobutyrate concentrations, 2 mL of the 2, 4-dinitrophenyl-

hydrazine reagent (0.2% 2, 4-dinitrophenyl-hydrazine in 2 mol L-1 HCl) was added, the


34

contents were vortexed and incubated at 30ºC for 30 min, during which time the α-

ketobutyrate was derivitized as a phenylhydrazine. The color of phenylhydrazine was

developed by the addition of 2 mL, a 2 mol L-1 NaOH, the absorbance of the mixture

was measured after mixing by using spectrophotometer at 540 nm.

For determining ACC deaminase activity, Endophytic fungal strains were grown in

rich medium (TSB) for 4 days at 28°C. The cells were then harvested by

centrifugation, washed with 0.1 M Tris-HCl (pH 7.5), and incubated for another 4

days in Dowkin and Foster minimal medium containing 5 mM ACC as the sole source

of nitrogen. The fungal cells were collected by centrifugation (Holguin and Glick

2001) and suspended in 5 mL of 0.1 mol L-1 Tris-HCl, pH 7.6, and transferred to

microcentrifuge tube. The contents of the tubes were centrifuged at 16000 rpm for 5

min and supernatant was removed. The pellets were suspended in 2 mL 0.1 mol L-1

TrisHCl, pH 8.5. Thirty μL of toluene were added to the cell suspension and vortexed

for 30 seconds. Two hunderedμL of the toluenized cells were placed in a fresh

microcentrifuge tube, 20 μL of 0.5 mol L-1 ACC were added to the suspension,

vortexed, and then incubated at 30°C for 15 min, following the addition of 1 mL of

0.56 mol L-1 HCl, the mixture was vortexed and centrifuged for 5 min at 16000 rpm

at room temperature. Two mL of the supernatant was vortexed together with 1 mL of

0.56 mol L-1 HCl. There upon, 2 mL of the 2, 4- dinitrophenylhydrazine reagents

(0.2% 2, 4-dinitrophenylhydrazine in 2 mol L-1 HCl) was added to the glass tube, and

the contents were vortexed and then incubated at 30°C for 30 min. Following the

addition and mixing of 2 mL of 2 mol L-1NaOH, the absorbance of the mixture was

measured by using spectrophotometer at 540 nm (Shaharoona et al., 2006).


35

2.11 Quantification of Extracellular Enzymes

To quantify extracellular enzymes, the method of Marx et al., 2001 was adopted with

some modifications. Briefly, all the substrates were obtained from Sigma-Aldrich Co.

Ltd in crystalline form. Ten milliliters of a 10 mM stock solution of each 4-

methylumbelliferone (MUB) substrate was prepared, while the assay procedures were

the same for each substrate. Depending on the substrate, a 7-MUB standard was used.

A 10 mM stock solution of pure MUB was prepared in methanol (0.1762 g of 4-

methylumbelliferone in 100 mL). This stock solution was diluted in MES buffer to 1

μMand stored at 4°C.

The endophytic fungi grown in Czapek broth were harvested using centrifugation (4°C,

12,000 rpm for 10 min). The pure and fresh culture filtrates (CF) were syringe filtered

(0.22 μm) to remove traces of turbidity. For each type of enzyme analysis, a minimum

of three replicates for each substrate (CF + buffer + substrate), a quenched standard

(sample + buffer+ 4-MUB), and a substrate control (media + buffer + substrate) were

maintained. The total volume of liquid in the cuvette was 2 mL CF or buffer or media

and 100 μL substrate or 4-MUB with different types of CF obtained from endophytic

fungi. The pre-optimized fluorescence spectrophotometer (Shimadzo, Tokyo, Japan)

was used to read the absorbance with 360 nm excitation and 460 nm emission at time

zero and 30-minute intervals for 2 hours.

The Bradford reagents are more commonly used for protein assay in which under

acidic conditions the red form of the dye is converted into its bluer form to bind to the

protein being assayed. It is the simple and reliable method used for protein

determination (Bradford, 1976). The protein-dye complex formed has absorption

maxima at 595 nm, this complex is detected in the assay using spectrophotometer or


36

micro plate reader (Reisner et al., 1975). Practical advantage of this method is that

reagent is simple to prepare and the colour is developed rapidly and is relatively

stable.Fluorescence-based MUB standards were used to analyze the presence of three

enzymes (β-1,4-glucosidase, 1,4-β-cellobiosidase, and phosphatase).

2.12 Reagents

2.12.1 Substrates

All substrate analogues were obtained from sigma-Aldrich Co. Ltd, in a crystalline

form. A list of substrates tested is given below (4-MUB = 4-methylumbel-liferone and

7-AMC = 7-amino-4-methyl coumarin). They allow the targeting of a wide range of

enzymes involved in the hydrolysis of C, N and P compounds. Ten ml of a 10 mM

stock solution of each MUB/AMC-substrate was prepared and assay procedures were

the same for each substrate.

4-MUB-β-D-glucoside

4-MUB-β-D-galactoside

4-MUB-7-β-D-xyloside

4-MUB-β-D-glucuronide

4-MUB-β-D-cellobioside

4-MUB-N-acetyl-β-glucosaminide

4-MUB-phosphate

L-leucine-7-AMC

L-tyrosine-7-AMC

L-arginine-7-AMC

The substrates were pre-dissolved in 1ml of ethylene glycol monomethylether

(methylcellosolve), except 4-MUB-β-D-glucuronide, 4-MUB-phosphate, L-leucine-7-


37

AMC and L-arginine-7-AMC, which were dissolved in sterile deionised water. All

substrates were made up to 10ml with sterile deionised water and the resulting stock

solutions of 10 mM were kept at 4°C. New working solutions of the enzyme substrate

were prepared for each assay by diluting the stock solution to 1000, 100 and 10 µM in

sterile 25ml universal bottles. All dilutions were made in sterile (autoclaved) buffer.

2.12.2 Buffers

In order to standardize the method, all enzymes assays were carried out in buffered

conditions; this also stabilizes the fluorescence intensity of MUB, which is highly

dependent on pH (Chrost and Krambeck, 1986). The choice of buffer depended on the

particular enzyme. Glycosidases and acid phosphatase were assayed in 0.1M MES

buffer (2- [N-Morpholino] ethanesulfonic acid, pH 6.1) (sigma-Aldrich Co. Ltd), and

peptidase in 0.05M Trizma buffer, pH 7.8.

2.12.3 Standards

Depending on the substrate tested either a 4-MUBor a 7-MUB standard was used. A 10

mM stock solution of pure MUB was prepared in methanol (0.1762 g of 4-

methylumbelliferone in 100 ml). This stock solution was diluted in MES buffer to 1 µM.

For the AMC standard, 0.1752g of 7-amino-4-methyl coumarin was dissolved in 100 ml

methanol (to prepare a 10 mM stock solution), then diluted in Trizma buffer to 1 µM.

2.12.4 Cultrul Filtrate

For each sample, 2ml of fresh CF was taken into cuvette. A 100µl of substrate or 4-

MUB with different types of CF obtained from endophytic fungi.

2.12.5 Microplate set-up

The plate set-up varies according to the aim of the investigation (e.g. estimation of kinetic

parameters or simple measurement of enzyme activity rates under optimum substrate, pH

and temperature conditions, in different soils). Each plate therefore includes a minimum

of three replicates for each substrate at each substrate concentration (sample + buffer +


38

substrate), a quenched standard (sample + buffer + 4-MUB/7-AMC), a substrate control

(sterile water + buffer + substrate) and an optional abiotic control (autoclaved soil +

buffer + substrate) (Sinsabaugh, personal communication). The total volume of liquid in

each well of the microplate was 200 µl. A typical plate set-up for kinetic studies of

glucosidase in two different soils is described below and represented schematically in

Figure 2.1.

Figure 2.1:- Schematic representation of a microplate set-up for the study of kinetic

parameters of β-glucosidase in the pure and fresh culture filtrates (CF) of endophytic

fungi grown in Czapek broth. Adopted from (MC Marx, M Wood- 2001).

1. For the enzyme assay and the standards 20 µl were withdrawn from the CF under

continuous stirring with a 4-channel pipette and dispensed into the microplate. At

the same time, the negative controls were set up, one in sterile water, to check for

contamination of the substrate or buffer, and another control in autoclaved soil

(optional), to investigate abiotic cleavage of the analogue.

2. Depending on the final substrate concentration in the wells, the appropriate amount

of sterile buffer was added into each well with a digital multi-channel pipette.


39

3. The standards were added to give final amount of 0, 10, 20, 30, 40, 50, 60 and 70

pmol 4-MUB/7-AMC well-1

.

4. Various aliquots of the 10, 100 and 1000 µM working substrate solutions were

added into the wells in order to establish the substrate saturation curves. The final

substrate concentrations in the wells were 2, 8, 20, 50, 80, 200, 300, 500, 700 and

900 µM.

Inhibition of the reaction and maximization of the fluorescence intensity through

alkalinisation of the mediumas suggestedby Freeman et al., (1995), was found to be

unnecessary because of the high sensitivity of the analytical equipment used to

measure fluorescence intensity of MUB is very low (Chrost and Krambeck, 1986), a

change in fluorescence was detected. The elimination of the alkalinisation and

purification step makes the microplate method very quick and offers potential for

routine soil analysis.

2.12.6 Fluorescence Readings

The fluorescence intensity was measured by a computerized microplate fluorimeter

(BioLuminTM

960, Kinetic fluorescence/absorbance, Molecular Dynamics, Inc).

This analytical equipment allows incubation of the microplate at a constant

temperature and automatically repeats measurements after a given time interval

without having to interrupt the reaction. For the measurements described here the

fluorimeter was programmed to shake the microplate for 5 S in order to homogenise

the reaction medium, then to pre-incubate the microplate for 10 min at 30°C (bring

microplate and its content up to temperature), before starting the first reading cycle.

From then on measurements were taken every minute for 35 min at 30°C. After

finishing the readings, the computer software automatically fitted a regression line

to the data and calculated the rate of fluorescence increase in each well. Since the


40

rate of fluorescence was measured rather than the absolute amount of fluorescence

at the of the incubation period, there was no need to incubate a „time zero blank‟

into the microplate for background fluorescence. Initial experiments demonstrated

that background fluorescence did not change during the incubation time.

2.13 Indole Acetic Acid Quantification of Endophytic Fungi

Estimation of indole-3-acetic acid (IAA) in the culture broth was done using

colorimetric assay. The endophytes cultured in 20 mL LB broth without (0 g/L) and

with 0.1 g/L of L-tryptophan and incubated at 30±2°C in shaking incubator at 200 rpm

for seven days. The fungal cultures were then centrifuged at 10,000xg for 10 min at 4°C

and the cell free cultures were filtered through 0.45µm cellulose acetate filter

(DISMIC®, Denmark). The filtrates were acidified to pH 2.8 with 1 N HCland extracted

3 times with 20 mL ethyl acetate. The ethyl acetate fractions were combined and

evaporated under vacuum at 45°C in a rotary evaporator. The residue was re-suspended

in 3 mL 50% methanol. One milliliter of supernatant was mixed with 2 mL Salkowski

reagent (12 g FeCl3/L of 7.9 M H2SO4) and kept in dark for 30 min. The resultant

reddish color was read after 30 min at 535 nm in ELISA Spectrophotometer (BioRad,

USA). The amount of IAA was calculated with standard of pure IAA (Sigma-Aldrich.,

Korea, Ltd) prepared separately.Indol-Acetic-acid may be produced by two pathways;

(1) Tryptophan dependent and (2) tryptophanindependent pathways. One set of the

fungal strains were grown in the Czapek broth without supplementing any precursor

(Tryptophan) for IAA production to check IAA production via tryptophan-independent

pathway. For the investigation of tryptophan dependent production of IAA endophytic

fungi were grown in Czapek medium containing different concentration of

L-tryptophan. This precursor was added to the media in 500, 1000, 1500 and 2000µg

50mL-1

.Strains were harvested after seven days of growth through filtration.


41

2.14 Extraction and Purification of Compounds

The cultural filtrates were extracted by implying the equal volume of ethyl acetate

(EtOAc) three times. Both the extracts were compiled and concentrated in vacuo to get

the crude extract. The extracts were compiled and dried over sodium sulfate

(anhydrous) and concentrated in vacuo to afford the crude extract. The EtOAc extract

was then subjected to silica gel column chromatography using gradients of ethyl

acetate/n-hexane system to afford five fractions (Fr.A to Fr.E). Fr.E was further

subjected to recycling preparative HPLC (JAI) analysis. Compound (mg) was purified

at a retention time of 23 min by using ethyl acetate/n-hexane (6:4) in a silica gel

column with the flow rate 3.5 mL/min after five recycles.

2.15 NMR Spectroscopy

Nuclear magnetic resonance spectroscopy is a powerful tool for structure elucidation

of different natural products. The NMR spectra (1H and 13C) were recorded on

Bruker spectrometer operating at 600-MHz (150-MHz for 13C). The δ-values on the

chemical shift scale are reported in ppm, while the J values of the coupling constants

were noted in Hz.

2.15.1 HSQC

Hetero-nuclear Multiple Quantum Correlation. It is 2D experiment which correlates the

chemical shift of proton with the chemical shift of the directly bonded carbon. On the

bottom axis is a proton spectrum and on the other is a carbon. The cross peaks give the

shift of the corresponding proton and carbon with each other. This experiment utilizes

the one-bond coupling between carbon and proton (J=120-215 Hz).

2.15.2 HMBC

Hetero-nuclear Multiple Bond Correlation. This experiment utilizes multiple bond

couplings over two or three bonds (J=215Hz. The Cross peaks are between protons and


42

carbons that are two or three bonds away (and sometimes up to four or five bonds away).

Direct one-bond cross-peaks are suppressed. This experiment is analogous to the proton-

proton COSY experiment in that it provides connectivity information over several bonds.

2.15.3 COSY

This is the correlation spectroscopy which is good for determining basic connectivity

via J- couplings, through-bond.

2.15.4 NOESY

Nuclear Overhauser Effect Spectroscopy: This allows one to see through-space

effects, useful for investigating conformation and for determining proximity of

adjacent spin systems.

2.15.5 Sample Preparation for NMR

NMR instruments require 5 mm tubes (5mm outer diameter of the tube) whereas

length of NMR tube is usually 7 or 8 inches. The samples were dissolved in 0.6 mL of

CDCl3 genetely mixed and transferred into NMR tube. The solution height in the

NMR tube must be within 3.5-4.5 cm. Marker pen were used for a short code written

at the top of the NMR tube.For 1H spectra of organic compounds with a molecular

mass less than 500, the quantity of material required is usually 3-10 mg. More

samples were needed for 13

C, which is ~6000 times less sensitive than 1H. Bruker 600

mhz instrument with a cryo-probe optimised for 13

C measurements.

2.16 Anticancer Activities

Breast cancer cell line (MCF-7) was used for thescreening of cytotoxicity of the crude

extracts and bioactive pure compounds obtained from endophytic fungi. The cell lines

were culturedin Advanced DMEM with 10% NBCS (inactivated) and5mM l-

glutamine, and then grown at 37C in a humidatmosphere with 5% CO2 in air. The 3-


43

(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide(MTT) colorimetric assay

developed by Mosmann, 1983.

2.17 Statistical Analysis

All samples were analyzed in triplicate. The data are presented as the mean ± standard

error of the mean (SEM). Differences were evaluated using one-way analysis of

variance (ANOVA). Differences were considered significant at P < 0.05 and were

calculated by GraphPad Prism Version 5.0 for Windows (GraphPad Software, San

Diego, CA, USA). The mean values were compared using Duncan‟s multiple range

tests at P < 0.05 (SAS 9.1, Cary, NC, USA). The Unscrambler version 9.0 by Camo

was used for principal component analysis to understand the correlation of enzyme

production abilities by the endophytic fungi.

Chapter 3 Results

44

RESULTS

3.1 Morphological Characteristics of Endophytic FungiColonies

3.1.1 Morphology of Endophytic Fungi Isolated from C. acutangula

The fungal endophytes shows “circular” form of colony on agar plates, and all

colonies were flat with entire margins displaying off white color with the passage of

time the color changed into yellow and brownish-black. F1 (Paecilomyces variotii)

colony was raised showing filamentous fungi strongly resembles to Penicillium but

can be differentiated by its loose branches and cylindrical-conidiogenous cells with

tapering-tips on PDA plates. The colour of P. variotii colony initially white, and

becomes yellow, yellow-brown. The FEF6B (Alternaria sp) flat colony and was

covered by grayish, short aerial hyphae. Initially, the surface was grayish white and

converted into dark-brown due to the production of pigments. FEF7 (Aspergillus

nidulans), were showing septate hyphae with a woolly-colony texture and white

mycelia. The green colour of colonies is due to pigmentation of the spores. BSR

(Epicoccum nigram), colonies were bright shades of yellow, orange and often with

brown or black and fast growth were noted on PDA.

FEF5A (Penicillium purpurogenum) were showing white to light green mycelia while

the back side of the plate were light pink. FEF1 (Fusarium oxysporum), initially, the

mycelia of the isolates on PDA plates were delicate, white to creamy and pink, the

manual of Booth 1971 was used for the identification of these fungi and were having

dispersed form of colony with black sporos organization.

3.1.2 Morphology of Endophytic Fungi Isolated from B. sacra

The endophytic fungi isolated from B. sacra were characterized on the basis of their

morphological characteristics. FEF1 (Fusarium oxysporum) were also isolated from

Chapter 3 Results

45

B.sacra, the mycelia of the isolates on PDA plates were delicate, white to creamy and

silightly pink in colour. The Strain FEF2 (Penicillium spinulosum) was white in

colour and forming a circular and fragile body, While it‟s a difficult task to

differentiate most of penicillium species phenotypically. The FEF3 (Aspergillus

caespitosus) colonies of Aspergillus caespitosus varying on different media, in

Czapek solution agar were slow growing. The mycelium was largely submerged and

very tough to tear. Aspergillus caespitosus was characterized particularly by clusters

of irregular ovoid structure, initially, colorless becoming reddish purple with the

passage of time. FEF4 were recorded as uncultured species. FEF5 (Alternaria sp) was

common in both B. sacra and C. acutangula. These were also having flat colony.

Initially, the surface was grayish white and converted into dark-brown due to the

production of different pigments. FEF6 (P. citrinum), the colonies of P. citrinum were

floccose, showing slow growth. The mycelium was white to greyish-orange in colour.

3.2 Diversity of Endophytic Fungi with C. acutangula and B. sacra

For sampling, a total of 80 plants parts from C. acutangula and 67 different parts from

B. sacra were selected for the isolation of endophytic fungi; the different pieces were

sterilized for isolation of endophytic fungi. A total of 25 endophytic fungal strains

were isolated and only 12 were selected for further processing (Table 3.1 and 3.2).

The selected endophytes were grouped on the basis of different parameters: colour of

aerial hyphae, surface texture, colony, shape, height from the medium, base colour,

growth rate and pattern (Arnold et al., 2007). The overall morphological analysis

suggested that all the selected stains possessed different characteristics.

Chapter 3 Results

46

Table 3.1:- Endophytic fungi isolated from C. acutangula.

Strain

Code

Part used for

Isolation

Fungal endophytes Plant species

F1 Stem Paecilomyces variotii C. acutangula

FEF6B Root Alternaria sp. C. acutangula

FEF7 Stem Aspergillus nidulans C. acutangula

BSR Root Epicoccum nigram C. acutangula

FEF5A Stem Penicillium

purpurogenum

C. acutangula

FEF1 Leaf Fusarium oxysporum C. acutangula

Table 3.2:- Endophytic fungi isolated from B. sacra

The colonization rate in C. acutangula shows the significant results and is much

higher in stem as compare to root. The colonization rate for the stem part was 2.28

whilst for root it was 0.681. Similarly, the isolation rate was significantly higher in

stem (1.46) as compared to roots (0.435). The diversity indices for fungal endophytes

as analysed by Shannon-Weiner indices (𝐻1= 0.9077) and Simpson indices (1/𝑙=

1.778) indicated differences in parts variation and species richness. The rate of

Strain Code Part used for

Isolation


FEF1 Leaf Fusarium oxysporum B. sacra

FEF2 Leaf Penicillium spinulosum B. sacra

FEF3 Stem Aspergilluscaespitosus B. sacra

FEF4 Leaf Uncultured B. sacra

FEF5 Leaf Alternaria sp. B. sacra

FEF6 Leaf Penicillium citrinum B. sacra

Chapter 3 Results

47

colonization also much higher in the leaves (3.12%) of B. sacra compared to the

stems (1.7%), and isolation rate was higher in leaves (4.11%) compared to stems

(1.22%).The diversity indices for endophytic fungi from B.sacrawere also analysed

by Shannon-Weiner indices (𝐻1= 0.8877) and Simpson indices (1/𝑙= 1.06) indicated

differences in parts variation and species richness.

The fungal species did not differ significantly between plant species, whereas they are

different from each other in their habitation. These endophytic fungi were further

identified by extracting the gDNA, amplification of PCR, sequencing of 18S rRNA

region and bioinformatics tools (BLASTn search).

3.3 Sequencing and Identification of Endophytes

The ITS sequences of the rRNA gene region from the 10 endophytic fungi revealed

that the fragment lengths ranged from 600–900 bp. The sequences were aligned in

MEGA 6.0 and BLASTn searched to correlate them with the highly homologous

fungal strains. Most of the fungal sequences showed 95–100% homology with related

fungi (Table 3.3). The phylogenetic analysis of these strains showed 94–99%

homology with ITS sequences of rRNA genes of related species. The evolutionary

history was inferred using the Maximum Parsimony method.

Chapter 3 Results

48

Table 3.3:- Sequence Similarities of Endophytic Fungi Isolated from C. acutangula

and B. sacra

ID

Name

Length

Identities

Match Total (%)

F1 Paecilomyces variotii 997 722 756 100%

FEF2 Penicillium spinulosum 568 567 571 98%

FEF6B Alternaria sp. 701 620 691 99%

FEF7 Aspergillus nidulans 820 768 801 98%

BSR Epicucum nigram 924 561 564 98%

FEF5A Penicillium purpurogenum 920 776 810 100%

FEF6 Penicillium citrinum 867 781 809 100%

FEF1 Fusarium oxysporum 823 756 780 98%

FEF4 Uncultured. 742 690 710 94%

FEF3 Aspergilluscaespitosus 802 735 792 95%

3.4 Phylogenetic Analysis

The 18S rDNA sequences from these endophytic fungi revealed that the fragment

lengths range from 520 – 997 bp. The sequences were aligned in MEGA 6.0 and then

were BLASTn searched to correlate them with the highly homologous fungal strains.

Most of the fungal sequences showed 94-99% homology with related fungi (Table

3.3). The isolated fungi belonged to Paecilomyces, Fusarium, Penicillium, Aspergillus

and Alternaria species. Based on 94 – 99% sequence similarity, we identified the

fungal strains as Aspergillus nidulans, Aspergillus caespitosus, Paecilomyces variotii,

Fusarium oxysporum, Alternaria sp. Penicillium purpurogenum, Penicillium

Chapter 3 Results

49

spinulosum, Phoma sp. Epicucum nigram and Penicillium citrinum (Table 3.1 and

Table 3.2). The phylogenetic analysis of these strains showed 94 – 99% homologies

with ITS sequences of related species. The sequences were deposited in the GenBank,

NCBI for accession numbers.

Table 3.4:- Gene Bank Numbers of Endophytic Fungi Isolated from C. acutangula

and B. sacra

Strain

Code

Gene Bank

Number


F1 KY921608 Paecilomyces variotii C. acutangula

FEF6B KY921609 Alternaria sp. C. acutangula

FEF7 KY921610 Aspergillus nidulans C. acutangula

BSR KY921611 Epicoccum nigram C. acutangula

FEF5A KY921612 Penicillium

purpurogenum

C. acutangula

FEF1 KY921613 Fusarium oxysporum C. acutangula

FEF1 KY474347 Fusarium oxysporum B. sacra

FEF2 KY474348 Penicillium spinulosum B. sacra

FEF3 KY474349 Aspergilluscaespitosus B. sacra

FEF4 KY474350 Uncultured endophyte B. sacra

FEF5 KY474351 Alternaria sp. B. sacra

FEF6 KY474352 Penicillium citrinum B. sacra

Chapter 3 Results

50

Evolutionary Relationship of Endophytic Fungal Strains from B. sacra

Figure 3.1:- The evolutionary history was inferred using the Neighbor-Joining method

(Saitou and Nei 1987). The optimal tree with the sum of branch length = 7.97184890 is

shown. The percentage of replicate trees in which the associated taxa clustered together

in the bootstrap test (2000 replicates) are shown next to the branches (Felsenstein J,

1985). The tree is drawn to scale, with branch lengths in the same units as those of the

evolutionary distances used to infer the phylogenetic tree. The evolutionary distances

were computed using the Maximum Composite Likelihood method (Tamura et al.,

2004) and are in the units of the number of base substitutions per site. The analysis

involved 59 nucleotide sequences. All positions containing gaps and missing data were

eliminated. There were a total of 94 positions in the final dataset. Evolutionary analyses

were conducted in MEGA6 (Tamura et al., 2013).

Chapter 3 Results

51

Evolutionary Relationship of Endophytic Fungal Strains from C. acutangula

Figure 3.2:- Evolutionary relationships of taxa of endophytic fungal strains isolated from

Caralluma-acutangula. A neighbour joining tree was constructed of homologous ITS

sequences using MEGA-6.0 with 1K-bootstrap replication. Botrybasidium-subcoronatum

was used as an out group.

Aspergillus sp. BAB-3919

Aspergillus nidulans strain BPPTCC 6038

Aspergillus nidulans strain NRRL 2395

Aspergillus sp. BAB-4426

LF1

SF2

Paecilomyces variotii strain LXM5

Paecilomyces variotii strain GF59

Paecilomyces variotii strain SCSAAF0011

Paecilomyces variotii isolate D SC2

LF3

Fusarium proliferatum isolate Fp-1

Fusarium proliferatum strain PA3

Fusarium proliferatum strain MA84

Epicoccum nigrum strain A168




Penicillium purpurogenum strain KCTC6820

Penicillium purpurogenum isolate Tian1

Penicillium purpurogenum strain KCTC16073

Penicillium purpurogenum strain HS-A82

SF5

6B

Alternaria sp. B13

Alternaria sp. CPCC 480375

Alternaria sp. GE

Alternaria sp. SPS-04

SF3

Botryobasidium subcoronatum voucher KHL s.n.

97

97

100

83

56

100

96

100

100

100

83

100

93

100

100

100

0.1

Chapter 3 Results

52

3.5 ACC Deaminase Activity of the Endophytic Fungi

ACC deaminase was observed in endophytic fungal strains by their growth in

Dworking and Foster (DF) minimal medium containing ACC (Figure 3.3), P. citrinum

(FEF6) strain had the highest value of deamination of ACC (358nmol α- ketobutyrate

mg-1

h-1

) followed by P. variotii (F1). In this way, the endophytic fungal strain

Epicucum nigram (BSR) had the lowest value (62nmol α- ketobutyrate mg-1

h-1

) of

ACC deaminase activity.

Figure 3.3:-ACC deaminase activity by the isolated endophytic fungi in (DF)

minimal medium containing ACC. Different letters in the column shows that values

are significantly different (p<0.05) from each other as evaluated from DMRT

(Duncan's Multiple Range Test) test.

3.6 Indole Acetic Acid Quantification of Endophytic Fungi

Our results showed that P. citrinum (FEF6) and P. variotii (F1) have the highest

results (1.64 and 1.54 nM/mL) for IAA production, while Epicucum nigram (BSR)

and Phoma Sp. (BSL), produced a little amount (0.21 and 0.30 nM/mL) (Figure 3.4).

0

50

100

150

200

250

300

350

400

F1 FEF2 FEF6B FEF7 BSR FEF5A FEF6 FEF1 FEF4 FEF3

Chapter 3 Results

53

Similarly, the endophytic fungi P. spinulosum, A. caespitosus and Alternaria

speciesalso revealed some prominent results for IAA production. The production of

IAA is dependent on the type of pathway utilized by endophyte. In our studies, for

the estimation of IAA in culture broth sophisticated methodologies were used as

colorimetric assay in addition with L-tryptophan which favoured the high amount of

IAA. Then endophytic fungal IAA was also quantified by using Waters Ultra

Performance Liquid Chromatography (UPLC) system.

Figure 3.4:- Indole acetic acid production by endophytic fungi. Different letters in the

column shows that values are significantly different (p<0.05) from each other as

valuated from DMRT (Duncan's Multiple Range Test) test.

3.7 Quantification of Extracellular Enzymes from Endophytic Fungi

To recognise the extracellular enzymes producing capability of isolated endophytic

fungi during axenic conditions. All the endophytic strains were grown in broth

medium for 14 days and were centrifuged to obtain pure culture. Fluorescence-based

MUB standards were used to analysed the presence of three enzymes (β-1,4-

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

F1 FEF2 FEF6B FEF7 BSR FEF5A FEF6 FEF1 FEF4 FEF3

Chapter 3 Results

54

glucosidase , phosphatase and 1,4- β –cellobiosidase . Standard curve readings were

taken for the florigenic substrate excitation and emission in the presence of buffer and

in combination with MUB standard. A curve fitting values for Cellulases (R2=0.98; y

= 6.35), phosphatases (R2=0.96; y = 113.04) and glucosidases (R

2=0.98; y = 36.4)

was recorded on fluorescence spectrophotometer.

The different fungal strains showed varying concentrations of cellulases,

phosphatases and glucosidases in their pure culture filtrates. The endophytic fungal

strain P. citrinum and P. variotii displayed very prominent results in the culture media

while P. spinulosum, A. caespitosus strain and A. nidulans strain also encoded for a

higher amount of extracellular enzymes as compare to F. oxysporum, P. spinulosum

and Alternaria sp.

Table 3.4:- Extracellular enzymes produced by endophytic fungi

ID NAME OF

ENDOPHYTIC FUNGI

Cellulases (μM

-1min

1mL)

Phosphatases (μM

-1min

-1mL)

Glucosidases (μM

-1min

-1mL)

F1 Paecilomyces variotii 5.65±0.00 0.41±0.00 35.22±0.01

FEF2 Penicillium spinulosum 0.41±0.00 2.12±0.00 46.82±0.00

FEF6B Alternaria sp. 0.64±0.00 0.02±0.00 16.66±0.00

FEF7 Aspergillus nidulans 21.12±0.00 0.68±0.00 18.73±0.00

BSR Epicucum nigram 0.67±0.00 1.22±0.00 31.23±0.00

FEF5A Penicillium purpurogenum 35.66±0.00 0.53±0.00 38.33±0.00

FEF6 Penicillium citrinum 0.38±0.00 3.31±0.00 42.24±0.16

FEF1 Fusarium oxysporum 15.24±0.00 0.70±0.00 03.23±0.00

FEF4 Uncultured endophyte 05.64±0.00 0.35±0.00 52.44±0.00

FEF3 Aspergilluscaespitosus 15.33±0.00 0.72±0.00 32.13±0.00

Chapter 3 Results

55

3.7.1 Glucosidases, Cellulases and Phosphatases

In case of glucosidases A. nidulans, P. variotii, P. purpurogenum and Alternaria has

significantly higher concentration where P. spinulosum, A. caespitosus and P. citrinum

were encoding for very little amount of glucosidases. The phosphatase production in P.

citrinum, P. variotii and P. spinulosum was significantly higher. The P. purpurogenum,

A. caespitosus and Alternaria sp. also produce significantly higher amount of enzymes.

While A. nidulans, Fusarium oxysporum and Alternaria sp. encodes for a very little

amount of phosphates enzymes (Figure 3.5). The cellulases activity showed significant

results for most of the isolated endophytic fungi. The production of cellulases enzymes

was much higher in P. variotii followed by F. oxysporum, A. caespitosus and P.

spinulosum (Figure 3.6). While the endophytic fungal strains A. nidulans and P.

purpurogenum encodes for a very little amount of these enzymes.

The capability to produce extracellular enzymes, indol-acetic acid and proficiency of

ACC deaminase by endophytic fungal species from Caralluma acutangula and

Boswellia sacra was the central part of the present research work. The isolated

endophytes from the different parts (root, leaf and stem) of Caralluma acutangula and

Boswellia sacra showed very prominent results in different biological activities.

Extracellular enzymes are encoded by endophytic fungi for penetrating the host cell

wall, as well as contribute in biocontrol.

The enzymes like glucosidases, cellulases and phosphatases were estimated by using

florigenic substrates and standard curve readings were taken in combination with

MUB standard. The endophytic fungi A. nidulans, P. variotii, P. purpurogenum and

Alternaria has significantly higher concentration where P. spinulosum, A. caespitosus

and P. citrinum were encoding for very little amount of glucosidases. The previous

data also described the purification, crystallization and different properties of α-

Chapter 3 Results

56

glucosidase from the mycelia of Mucor (Wei et al., 2013) α-glucosidases from

Aspergillus niger and Asp. Nidulans have so far been isolated from culture filtrate.

While α-glucosidase is also reported from the different species of Penicillium (Hsu et

al., 2013; Liu et al., 2014).

Even though, the diversity of fungal species estimated about 1.5 million and a very

little number is identified (Hawksworth, 1991) only few genera like Aspergillus,

Penicillium, Rhizopus and Trichoderma etc. produce more than 50% fungal enzymes

used in various industrial processes (Ostergaard and Olsen, 2010). The wide variety

uses of these enzymes enhance the interests of many researchers for the exploration of

a low coast and sustainable resource. In connection to this, the endophytic microbial

floras have the potential to encode for these enzymes. In our research, the isolated

endophytic fungi showed very noticeable results for phosphatase production by

endophytic fungi. The productions of phosphatase were significantly higher in P.

citrinum, P. variotii and P. spinulosum. The strain P. purpurogenum, A. caespitosus

and Alternaria sp. also produce significantly higher amount of these enzymes. While

A. nidulans, F. oxysporum and Alternaria sp. comparatively encodes for a very little

amount of phosphates enzymes.

There are different physical, chemical and enzymatic processes known for the

hydrolysis of cellulose. In addition to, the cellulases enzymes produced by endophytic

fungi Pycnoporus sanguineus isolated from Baccharisdra cunculifolia also showed

very proficient results in the hydrolysis of cellulose (Onofreet et al., 2015). While in

our results the isolated endophytic strains revealed significant results for most of the

strains. The production of cellulases enzymes was much higher in P. variotii followed

by F. oxysporum, A. caespitosus and P. spinulosum. While the endophytic fungal

strains A. nidulans and P. purpurogenum encodes for a very little amount of these

enzymes. It was also observed that by keeping the pH at 5.5 subsidise in the greater

amount of cellulase production in liquid media.

Chapter 3 Results

57

3.8 Extraction and Purification of Compounds

The cultural filtrates were extracted by implying the equal volume of ethyl acetate

(EtOAc) three times. Both the extracts were compiled and concentrated in vacuo to

get the crude extract. The extracts were compiled and dried over sodium sulfate

(anhydrous) and concentrated in vacuo to afford the crude extract. The EtOAc extract

was then subjected to silica gel column chromatography using gradients of ethyl

acetate/n-hexane system to afford five fractions (Fr.A to Fr.E). Fr.E was further

subjected to recycling preparative HPLC (JAI) analysis. Compound (mg) was purified

at a retention time of 23 min by using ethyl acetate/n-hexane (6:4) in a silica gel

column with the flow rate 3.5 mL/min after five recycles.

3.9 Chromatography and Spectroscopic Techniques for Identification

The mycelial mats and the culture filtrate were extracted completely with ethyl acetate

(EtOAc). Both the extracts were compiled and dried over sodium sulfate (anhydrous)

and concentrated in vacuo to afford the crude extract (7.5 g). The ethyl acetate extract

(7.5 g) of the endophytic fungus Penicillium citrinum was then subjected to repeated

column chromatography (silica gel, n-hexane, DCM/n-hexane, and MeOH/DCM) to

get various sub-fractions (PC1-PC14). The sub-fraction PC12, which was obtained at

5% MeOH/ DCM were subjected to recycling preparative High Performance Liquid

Chromatography (HPLC) by JAI for the final purification. Compound 1 (3.8 mg) was

obtained at a retention time of 27 min through a 1H/2H column by using chloroform

at a flow rate of 3.5 mL/min. Along with some semi-pure compounds.These semi-

pure compounds were loaded on preparative TLC plates and the known compounds-

4(12.5 mg) and 5 (14.1 mg) were purified at DCM/n-hexane (90:10) and DCM/n-

hexane (95:5), respectively. The sub-fraction PC5 afforded compound 2 (3.3 mg) and

3 (2.7 mg) by repeated silica gel column chromatography at DCM/n-hexane (15:85)

and DCM/n-hexane (20:80), respectively.

Chapter 3 Results

58

Compound 1: Amorphous powder, 1H-NMR (600 MHz, CDCl3), δ 7.34-7.31 (5H,

overlap signals, H-33 to H-37), 5.54 (1H, br s, H-12), 5.14 (1H, d, J = 12.7 Hz, H-

31a), 5.04 (1H, d, J = 12.7 Hz, H-31b), 2.31 (1H, s, H-9), 1.51 (1H, br s, H-18), 1.37

(3H, s, CH3-23), 1.24 (3H, s, CH3-27), 1.15 (6H, s, CH3-25 and CH3-26), 0.92 (3H,

overlap signal, CH3-30), 0.80 (3H, s, CH3-28), 0.76 (3H, d, J = 6.3 Hz, CH3-29). 13

C-

NMR (150 MHz, CDCl3), δ 208.4 (C-3), 198.9 (C-11), 173.1 (C-24), 165.5 (C-13),

130.3 (C-12), 128.6-128.5 (C-32 to C-37), 66.9 (C-31), 59.9 (C-9), 59.0 (C-18), 58.5

(C-5), 57.7 (C-4), 53.4 (C-4), 44.8 (C-14), 43.8 (C-8), 41.0 (C-22), 40.9 (C-1), 39.3

(C-19 and C-20), 37.1 (C-10), 36.7 (C-2), 33.9 (C-17), 32.7 (C-7), 28.8 (C-28), 21.1

(C-23 and C-30), 20.4 (C-27), 18.3 (C-26), 17.4 (C-29), 13.4 (C-25). ESI-MS m/z:

581.06 [M + Na]+for C37H50O4Na (Appendix- I).

Compound 2: Colorless oil, 1H-NMR (600 MHz, CDCl3), δ 1.40-1.23 (14H, overlap

signal, H-2 to H-8), 0.86 (6H, t, J = 7.1 Hz, CH3-1 and CH3-9). 13

C-NMR (150 MHz,

CDCl3), δ 31.9-29.4 (C-2 to C-8), 14.1 (C-1 and C-9). ESI-MS mlz: 150.79 [M +

Na]+for C9H20Na (Appendix- II).

Compound 3: Colorless lequid, 1H-NMR (600 MHz, CDCl3), δ 5.32-5.19 (2H,

overlap signal, H-3 and H-4), 3.63 (2H, m, H-1), 1.53 (2H, m, H-2), 0.83 (3H, t, J =

4.8 Hz, H-10). 13

C-NMR (150 MHz, CDCl3), δ 130.9 (C-4), 128.9 (C-3), 66.5 (C-1),

32.8 (C-2), 29.3-29.0 (C-5 to C-9), 14.1 (C-10). ESI-MS mlz: 178.94 [M + Na]+for

C10H20ONa(Appendix- III).

Compound 4: Gummy solid, 1H-NMR (600 MHz, CDCl3), δ7.18 (1H, t, J = 8.6 Hz,

H-4), 7.12 (1H, d, J = 7.3 Hz, H-6), 6.90 (1H, d, J = 8.8 Hz, H-3), 6.89 (1H, t, J = 7.4

Hz, H-5). 13

C-NMR (150 MHz, CDCl3), δ 173.1 (C-8), 154.5 (C-2), 131.2 (C-6),

Chapter 3 Results

59

129.3 (C-4), 123.1 (C-1), 121.2 (C-5), 117.3 (C-3), 36.8 (C-7). ESI-MS mlz: 150.79

[M + Na]+for C8H8O3Na (Appendix- IV).

Compound 5: Colorless powder, 1H-NMR (600 MHz, CDCl3), δ7.48 (2H, dd, J =

8.1, 11.2 Hz, H-4 and H-5), 7.23 (2H, d, J = 7.4 Hz, H-3 and H-6), 5.64 (1H, d, J =

7.3 Hz, H-1 of glu), 4.41-3.42 (6H, overlap signal, H-2 to H-6 of glu). 13

C-NMR (150

MHz, CDCl3), δ 174.5 (C-8), 150.1 (C-2), 127.8 (C-3 and C-6), 126.4 (C-4 and C-5),

124.1 (C-1), 101.5 (C-1 of glu), 77.2-70.5 (C-2 to C-5 of glu), 63.4 (C-6 of glu), 39.4

(C-7). ESI-MS mlz: 336.84 [M + Na]+for C14H18O8Na (Appendix- V).

3.10 Characterization of Compounds

The ethyl acetate extract (7.5 g) of the endophytic fungus Penicillium was subjected

to repeated column chromatography followed by the final purification through

recycling HPLC to afford five known compounds: 11-oxoursonic acid benzyl ester

(1), n-nonane (2), 3-decene-1-ol (3), 2-Hydroxyphenyl acetic acid (4), and

Glochidacuminosides A (5).

Chapter 3 Results

60

O

O

O

CH3

CH3 CH3

H3C

CH3

CH3

H3C

H

H

O

OH

OH

O

OGlu

OH

O

OH

1

2

3

1211

10

9

8

76

54

19

18

17

16

13

1514

2021

22

23 24

25 26

27

28

29

30

1

4

3132

33

34

35

36

37

5

2

3

12

38

7

654

12

38

7

654

Figure 3.5:- Structures of Compounds 1-5: 11-oxoursonic acid benzyl ester (1), n-nonane

(2), 3-decene-1-ol (3), 2-Hydroxyphenyl acetic acid (4), Glochidacuminosides A (5)

Compound 1 was isolated in the form of amorphous powder which gave characteristic

pink color on the TLC plates after spraying with ceric sulphate reagent (Figure 3.5a).

This indicates the terpenoid skeleton in the molecule. The 1H NMR spectrum of 1

displayed seven methyl signals at 1.37, 1.24, 1.15, 1.15, 0.92, 0.80, and 0.76, which

were indicative of ursane skeleton. The olefinic proton H-12 appeared as broad singlet

Chapter 3 Results

61

at δ5.54, indicating the presence of trisubstituted double bond. The downfield 1H

NMR absorptions at δ5.14 and 5.05 (1H each, d, J = 12.7 Hz, H-31) and HMBC

correlation of H-31 to C-28 (δ173.1), and the aromatic carbons (C-32 to C-37),

showed the presence of benzyl ester at C-28. The presence of conjugated ketone

moiety at C-11 was evident by the 13

C NMR signals and the HMBC interactions of H-

12 with C-11 (δ198.9) and C-13 (δ165.5). Another downfield signal at δ208.4 was

assigned to the ketone functionality at C -3. The study of 13

C NMR (BB and DEPT)

data showed signals for seven methyls, nine methylenes, eleven methines and ten

quaternary carbons for a triterpene skeleton having O-benzyl substitution. Further

comparison of the spectral data with the literature valuesestablished the structure of

compound 1 as benzyl ester of 3,11-dioxo-ursolic acid. This compound has earlier

been reported in a Chinese patent on ursolic acid derivatives.

Figure 3.5a: Demonstrate Compound 1; 11-Oxoursonic acid benzyl ester

Compound 2 was isolated as a colorless oil and showed pseudo molecular ion peak at

m/z 150.79 corresponding to the molecular formula C9H20Na for the sodium adduct.

The 1H NMR spectrum of 2 showed a triplet for six protons due to two terminal

methyl groups (CH3-1 and CH3-9) at δ 0.86 (6H, t, J = 7.1 Hz). The methylenes

Chapter 3 Results

62

moieties associated with the long chain of 2 appeared as overlap signal at δ 1.40-1.23.

The 13

C NMR spectrum of compound 2 showed signals due to two terminal methyl

groups in the aliphatic hydrocarbon chains at δ 14.1, whereas the remaining

methylenes of the chain appeared at a range from δ 31.9 to 29.4. The final structure of

2 as n-nonane was confirmed through comparison with the literature data.

Figure 3.5b: Demonstrate Compound 2; n-nonane

Compound 3 was isolated as a colorless liquid. The molecular formula C10H20O was

established on the basis of MS, 1D and 2D NMR spectra. The ESI-MS showed the

pseudo molecular ion peak at m/z 178.94, suggesting the molecular formula

C10H20ONa in the form of sodium adduct. The 1H NMR spectrum displayed a triplet

at δ 0.83 (3H) and a broad singlet at δ 1.23 corresponding to the straight chain

hydrocarbon. The presence of hydroxyl group at C-1 was also inferred from 1H NMR

spectrum which displayed signals at δ 3.63 (αH, H-1) which showed the presence of

hydroxyl at C-1 position. The olefinic double bond at C-3 was indicated by the

presence of overlap signal between δ 5.32-5.19, which was further supported through

13C NMR spectrum. The

13C NMR spectrum (BB and DEPT) showed characteristics

signals due to oxygenated methylene at δ 66.5, whereas the down field signals at δ

130.9 and 128.8 were assigned to the double bond at C-3 position. The signals

observed between δ 29.3 and 29.0 indicated the presence of a long chain hydrocarbon.

The position of the double bond and the hydroxyl group was further confirmed

through mass fragmentation as well as from the HMBC correlations. Thus, on the

basis of above discussion coupled with the literature value, compound 3 was

confirmed as 3-decene-1-ol.

Chapter 3 Results

63

Figure 3.5c: Demonstrate Compound 3;3-decene-1-ol

Compound 4 was obtained as colorless powder. The molecular formula C8H8O3 was

deduced from MS and 13

C NMR spectral data. The ESI-MS showed the pseudo

molecular ion peak at m/z 175 [M + Na]+, suggesting the molecular formula C8H8O3 for

compound 4. The analysis of 1H NMR spectrum revealed the presence of a singlet for a

methylene moiety at δ 3.69 and four signals for one proton each in the downfield

aromatic region (δ 7.18 to 6.89). These aromatic signals showed splitting in the form of

two doublets (δ 7.12, J = 7.3 Hz and 6.90, J = 8.8 Hz) and two triplets (δ 7.18, J = 8.6

Hz and 6.89, J = 7.4), which indicated the presence of ortho-disubstituted benzene ring

in compound 4. The 13

C NMR spectrum (BB and DEPT) indicated the presence of eight

signals which were resolved into one methylene, four methane and three quaternary

carbons. The downfield signal at δ 173.1 was assigned to the carboxylic functionality

whereas the methylene carbon appeared at δ 36.8. The sp2 methine carbons of the

aromatic ring appeared at δ 131.2, 129.3, 121.2, and 117.3, whereas the signals at

δ154.5 and 123.1 were assigned to C-2 and C-1 respectively. The position of individual

groups in the molecule was further confirmed through HMBC interactions. Thus, the

structure was finally confirmed as 2-Hydroxyphentlacetic acid which was in complete

agreement with the reported values.

Chapter 3 Results

64

Figure 3.5d: Demonstrate Compound 4; 2-Hydroxyphenyl acetic acid

Compound 5 was isolated as a gummy solid and the structure was assigned on the

basis of comparison of the MS and NMR data in the reported literature. The ESI-MS

showed the pseudo molecular ion peak at m/z 336.84 [M + Na]+, which suggested the

molecular formula C14H18O8 for compound 5. The spectral data of compound 5 was

similar to that of compound 4 with the additional signals for a sugar moiety in the

molecule. The aromatic signals showed splitting in the form of a doublet at δ 7.23

(2H, d, J = 7.4 Hz, H-3 and H-6) and a doublet of doublets at δ7.48 (2H, dd, J = 8.1,

11.2 Hz, H-4 and H-5), which indicated the presence of ortho-disubstituted benzene

ring in compound 5. The anomeric proton was observed at δ 5.64 as doublet (J = 7.3

Hz), indicating the beta linkage of the sugar. The remaining protons of the sugar

moiety were observed at δ 4.41 to 3.42. The assignments of the sugar moiety were

further confirmed through 13

C NMR spectra which indicated the presence of anomeric

carbon at δ 101.5, a methylene carbon at δ 63.4 and four methine signals at δ 70.5 to

77.2. The identity of the sugar moiety was thus indicated as β-D-glucopyranoside

which was further confirmed through co-TLC with the authentic sample. The position

of the sugar moiety and the side chain was further confirmed through HMBC

interactions. The structure was finally established as 2-Hydroxyphentlacetic acid-2-O-

β-D-glucopyranoside, previously isolated from the leaves of Glochidionacuminatum

and commonly known as glochidacuminosides A.

Chapter 3 Results

65

Figure 3.5e: Demonstrate Compound 5; Glochidacuminosides A

3.11 Enzyme Inhibitory Activities of Secondary Metabolites

Our aforementioned screening for α-glucosidase inhibition assay showed that the

highest α-glucosidase inhibitory activity was revealed from ethyl acetate extract of

endophytic fungi P. citrinum (FEF6).The α-glucosidase inhibitory activities of the

isolated bioactive compounds from endophytic fungus P. citrinum FEF6 demonstrates

that the characterized compounds 2-Hydroxyphenyl acetic acid (4), 11-oxoursonic acid

benzyl ester (1) and Glochidacuminosides A (5) endure high level of potential for the

inhibition of α-glucosidase (Figure 3.6). Among these compounds, compound 1 showed

anIC50 273.87±1.59 μg/mL. The urease inhibition assay showed that compound 2, 3 and

4 have a very weak role in urease enzyme inhibition while compound1 (11-oxoursonic

acid benzyl ester) and 5 (Glochidacuminosides A) shows high level of urease inhibitory

activities (Figure 4).

Chapter 3 Results

66

Figure 3.6:- Ureaseand α-Glucosidase enzyme inhibition activities of the secondary

metabolites isolated and characterized from the endophytic fungi. The bars represent

the mean values of three replications with standard error.

3.12 MTT Assay on Breast Cancer Cell Line

Breast cancer cell line (MCF-7) was used for thescreening of cytotoxicity of the

cultural filtrate and bioactive pure compounds obtained from endophytic fungi. The

Chapter 3 Results

67

cell lines were cultured in advanced DMEM with 10% NBCS (inactivated) and5mM

l-glutamine, and then grown at 37Ċ in a humidatmosphere with 5% CO2 in air. The

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide(MTT) colorimetric

assay developed by Mosmann, 1983. Anticancer activities of endophytic fungal

cultural filtrate and pure compounds were assessed by using breast cancer cell

(MCF-7) lines. Various low concentrations of CF were prepared and tested against

the grown cell cultures. The screening results showed that P. citrinum, P.

purpurogenum and P. variotii extract hasgreatly reduced the cancer cell viability as

compared to other extracts (Figure 3.7).

Figure 3.7:- Effect of cultural filtrate of endophytic fungi on the viability of MCF-7

breast cancer cells in culture

Breast cancer cell line, MCF-7, was incubated with indicated concentrations of the

samples from endophytic fungi for 24 hours. The effect on cell proliferation was

detected by performing MTT assay as described in „Methods‟ section. All results are

expressed as percentage of control ± SE of triplicate determinations.

0

10

20

30

40

50

60

70

80

90

F1 FEF1 FEF5A FEF6 FEF6B FEF7 FEF2

150µg/ml

300µg/ml

Ce

ll V

iab

ility

(%

)

Chapter 3 Results

68

Our aforementioned screening for MTT assay showed that the highest activity was

shown in the ethyl acetate extract of endophytic fungi P. citrinum (FEF6), P.

purpurogenum (FEF5A), and P. variotii (F1).

The activities of the isolated bioactive compounds from endophytic fungus P.

citrinum (FEF6) demonstrate that the characterized compounds 2-Hydroxyphenyl

acetic acid (4), 11-oxoursonic acid benzyl ester (1) and Glochidacuminosides A (5)

endure a significantlyhigher potential to inhibit cell proliferation.The compound1 (11-

oxoursonic acid benzyl ester) and 5 (Glochidacuminosides A), show significantly

higher level of cells growth inhibition (Figure 3.8).

Figure 3.8:- Effect of pure compounds on the viability of MCF-7 breast cancer cells

in culture.

0

20

40

60

80

100

120

Control C1-PBLT C4-PCF-5D C5-PCF11

100. 50 μg/ml

100 μg/ml

Chapter 4 Discussion

69

DISCUSSION

Section 1: Endophyte Diversity Assessment

In present research work, a range of endophytic fungi were evaluated by using

denaturing gradient gel electrophoresis (DGGE) for the identification of various

endophytic fungi. The medicinally valuable plants „Caralluma acutangula and

Boswellia sacra’ offered an array of endophytic fungi. The surface-sterilized samples

were assessed by means of isolation techniques and phylogenetic analysis. With these

approaches we were able to detect differences in the relative abundance of endophytic

fungi.The rate of colonization was higher in the stem part of C. acutangula while

leaves of B. sacra showed higher colonization rate.

Mycorrhizal fungi colonize in near vicinity of plant roots or penetrate into plant root

tissues in case of endomycorrhiza, whereas endophytic fungi reside within all tissues

of the plants and are symptomless (Khan et al.,2013; Stone et al., 2004). Historically,

endophytic fungi were divided into two major groups „Clavicipitaceous and

Nonclavicipitaceous endophytes‟ on the basis of their hosts, taxonomy, ecological

function and phylogenetic analysis. However, nonclavicipitaceous endophytes are

highly diverse in nature and now further grouped into three different classes, based on

their life history and ecological consequences (Arnold, 2007). Endophytic fungi

greatly contribute in stress conditions and favours host growth regardless of the

habitat of origin. Diverse kinds of fungal endophytes establishes important niche with

plants through secretion of bioactive constituents, regulating the growth of host even

underharsh environmental conditions. Endophytic microbes associated with

medicinally important plants have been shown to enhance host plant growth and

development. This could possibly be owing to the production of various constituents


70

during the symbiotic processes with the host plants (Khan et al., 2013; Kusari et al.,

2013). Endophytes are also able to colonize more than one host species of the same

plant family within the same habitat. Endophytic fungi often shows single host-

specificity at plant species level which could be also influenced by environmental

conditions (Cohen 2006).

To further understand the interactions in in-vitro environments of culturable

endophytes from medicinal plants, we isolated and identified endophytic fungal

species from C. acutangula. To our knowledge, there have beenno previous studies on

this plant and its associated endophytic microorganisms. In the current study, we

grouped endophytic fungi on the basis of colony shape, thickness, colour of aerial

hyphae, growth rate and pattern, margin characteristics, surface texture, and growth

depth into medium (Arnold et al., 2007). The endophytes were identified by genomic

DNA extraction, PCR techniques, nucleotide sequencing, and phylogenetic analysis

as described by Khan et al., (2011).

We isolated and identified various strains of Paecilomyces, Epicucum, Fusarium,

Penicillium, Aspergillus, and Alternaria species using molecular techniques. Based

on ITS sequence homology, we identified these fungal strains as A. nidulans, P.

variotii, F. oxysporum, E. nigram, P. purpurogenum and Alternaria sp. These

strains were assessed for their potential to produce ACC deaminase, extra-cellular

enzymesand IAA during their growth. The advantageous effects of endophytic fungi

and bacteria have been regarded for their potential to produce biologically active

secondary metabolites and other substances. The production of such substances can

maintain their own metabolism as well as supports the growth of their hosts

(Saxena, 2014). These substances can range from secondary metabolites to various


71

enzymes. In case of metabolites, endophytic microbes have recently been also

known to produce different plant hormones (Hardoim et al., 2008). The distribution

of identified endophytic fungi in plant organs differs in diversity and abundance. As

our results showed that higher colonization rate was observed in the stem (2.41) of

C. acutangula, whereas for leaves and roots was 1.26 and 1.11, respectively. The

isolation rate was much higher (1.82) for the stem of C. acutangula. The diversity

indices analyzed by Shannon-Weiner (𝐻1= 0.8424) and Simpson (1/𝑙= 1.662)

indices indicated differences in plant parts and species richness (Wei et al., 2013).

While the leaves of B. sacra showed higher colonization rate. The study of Maria et

al., (2005) also reported fungal endophytes from medicinal plants and isolated 18

endophytic fungi from the stem and leaf segments of host plants. The dominant

species were Curvularia clavata, C. lunata, C. pallescens and F. oxysporum. The

highest species richness as well as frequency of colonization of endophytic fungi

was found in the leaf segments rather than the stem and bark segments of the host

plant species. The greatest numbers (11 species) of endophytic fungi were found

within Callicarpato mentosa, whereas Lobeliani cotinifolia harbored the lowest

number of fungal endophytes (5 species). The study also provides evidence that

fungal endophytes are host and tissue specific (Mariaet al., 2005).

However, the biodiversity of fungal species is estimated at about 1.5 million, a limited

number of species have been identified, includingonly fewgenera, such as Aspergillus,

Penicillium, Rhizopus, and Trichoderma, which produce >50% of the fungal enzymes

used in various industrial processes (Rytioja etal., 2015; Gawas-Sakhalkar etal.,

2012).The wide variety of uses of these enzymes has enhanced the interest of many

researchers to explorethese as a low cost, sustainable resource.


72

Section 2: Potential Role of Endophytes

The endophytic fungi from Caralluma and Boswellia species are rarely employed for

endophytic fungal diversity, extracellular enzymes production and their bioactive

compounds. The genus Penicillium is most prevalent followed by Aspergillus, Alternaria

and Paecilomyces in C. acutangula, while Phoma, Fusarium and Aspergillus were

screened from B. sacra. Recently, Penicillim species has been also reported from

different medicinal plants (Nagerabi et al., 2014). Penicillium is an important genus of

ascomycetous fungi which greatly contribute in the production variety of drugs and a

large number of its species produce mycotoxins (Diblasi et al., 2015).

The most famous and economically important being penicillin is produced by Penicillium

chrysogenum. Mycophenolic acid produced by Penicillium brevicompactum and

Compactins produced by Penicillium solitum, while several other secondary metabolites

from Penicillium species showed potential anticancer activities including alternariol,

fumitremorgin C, hadacidin, paclitaxel, penicillic acid, PR-toxin, and viridicatumtoxin.

Fumitremorgin C and paclitaxel appear to show most promise in cancer treatment

(Samson and Frisvad 2004; Houbraken et al., 2011). In 2011 five new di- and tri-citrinols

were added to the known citrinin family from P. citrinum all of them showed cytotoxic

activity against human leukemia HL-60, colon cancer HCT-116, and cervical cancer KB

cell lines. The endophytic fungus Penicillium chrysogenum was reported by Zhou et al.,

in 2011 obtained from Lycopodium serratum have the ability to produce huperzine A, the

drug which is used for Alzheimer's disease, memory and age-related memory impairment

(Zhou et al., 2011). The P. citrinum as an endophyte with bioactive compounds in the

bark of Taxus cuspidate has been also reported by Yong et al.,in 2003.

The genus Paecilomyces comprises a number of species that are able to produce a wide

collection of bioactive secondary metabolites of different chemical classes and with

different biological activities, such as antimicrobial and cytotoxic activities (Kyong et


73

al., 2001; Wang et al., 2002). Initially, the production of bioactive secondary

metabolite polygalactosamine from Paecilomyces sp. with anti-tumor activity was

reported by Ishitani et al., in 1988. The species of Paecilomyces marquandii was

reported by Radics et al. (1987) for the production of leucinostatins A, D and K as

peptide antibiotics. Similarly, the antitumor antibiotics; saintopin and UCE1022 with

topoisomerase- dependent DNA cleavage activity were described in last ten to fifteen

years (Long and Balasubramanian 2000; Fan et al., 2001). In some of the reported

studies Paecilomyces sp. have been used for the production of leu-cinostatins A, D, H

and K as peptide antibiotics, polygalactosamine which showed antitumor activities;

saintopin as antitumor antibiotics with topoisomerase dependent DNA cleavage

activity. Similarly, Aspergillus has usually been found as a saprophytic fungus, and

producing a variety of mycotoxins such as patulin and cytochalasin E (Liu et al., 2009;

Lopez-Diaz, T.M. and Flannigan 1997). The apoptotic antitumor activity antioxidant

and immune-stimulating activities of Paecilomyces japonica were reported by Fujji et

al., (1994), while the Paecilomyces variotii has been exploited by sachan et al., (2006)

for the biotransformation of p-coumaric acid (Nam, 2001). Recently, a new compound

3H-oxepine-containing alkaloid, varioxepine A, was isolated from endophytic

fungus Paecilomyces variotii. This compound has the ability to inhibit the growth of

some pathogenic fungus (Zhang et al., 2014; Zhang et al., 2015).

The relationships exist between endophytes and their host plants, ranging from

mutualism or symbiosis to antagonism or slight pathogenesis (Schulz and Boyle,

2005; Khan et al., 2013).Fungal endophytes have also a varied relationship between

pathogen and host plant and alsoinfer how they limit pathogen damage to the host

(tropical trees). Endophytes represent a ubiquitous yetcryptic component of terrestrial

plant communities. The fundamental aspects of endophytic interactions with hosts are


74

unknown. In contrast to vertically transmitted endophytes, horizontally transmitted

endophytes of woody angiosperm are thought to contribute little to host defence

mechanism. The fungal endophytes helpto decrease both leaf necrosis and leaf

mortality when T. cacao seedlings are challenged with pathogen (Phytophthora sp.).

Further endophyte mediated protection was greater in mature leaves, which bear less

intrinsic defence against fungal pathogens than do young leaves (Arnold et al., 2003).

Endophytes may have developed friendly relationships with their hosts during

evolution and may be host or tissue specific. Host or tissue specificity of endophytic

fungi must depends on certain factors i.e. endophyte colonization or active ingredients

within host tissues (Arnold, 2007). Fungal endophytes produce bioactive metabolites

that mediate in the plant–endophyte interaction (Strobel, 2003). In addition, fungal

endophytic metabolites are useful resources for natural products which effectively

have wide range of application in medicine, agriculture, and industry (strobel and

Daisy 2003; Selim et al., 2012). Fungal endophytes have the ability to produce

numerous extracellular enzymes; such as pectinases, cellulases, lipases, amylases,

laccases, and proteinases. These fungal enzymes play the key role in biodegradation

and hydrolysis processes which are significantly important mechanisms against

pathogenic infection and to obtain their nutritional need from the host plants (Sunitha

et al., 2013).

Many bioactive metabolites are originated from microbial organisms, fungi are the

core important groups of eukaryotic organisms that have wide capacity to produce

numerous metabolites with antimicrobial activities and possess potential application

as drugs. Phytohormones such as auxins are produced in the different parts of the

plant and these are mainly responsible for the regulation of plant developmental


75

processes. The array of IAA within various parts of the plant is a key factor for

plant growth and at the same time its additional supply can support the host in

stress condition.

In our study, P. variotii and A. nidulans showed considerable IAA production. The

production of IAA is dependent on the type of pathway utilized by endophytes;

therefore, IAA in the culture broth was estimated by the presence and absence of L-

tryptophan. Similar results for IAA were found by Mei and Flinn, where IAA

producing endophytic fungi improved plant growth under stress conditions. Thus,

previous findings about IAA-producing endophytic fungi strongly support our current

results. The results conclude that endophytic fungi associated with medicinally

important plants possess a unique potential to produce bioactive metabolites, which

might improve the growth of the host in severe environmental conditions. Such

bioactive fungal strains may have applications for crop improvement and industrial

production of important constituents (Mei and Flinn 2010; Fu et al., 2015).

The functional role of IAA in plant growth in addition to the capacity of fungal

endophytes to produce IAA has gained great attention due to their impact on the

concentration and distribution of IAA in plant tissues. Little is known about the

biology and ecology of fungal endophytes; subsequently, isolation and

characterization of fungal endophytes that colonize different plant species of various

habitats and ecosystem is potentially useful.

Section 3: Bioactive Metabolites from Endophytes

Endophytes may yield a plethora of bioactive secondary metabolites that may be

involved in the host-endophyte relationship and also have the capability to encode for

the same type of metabolites as produced by their host plant (Strobel 2004). Natural

http://en.wikipedia.org/wiki/Pattern_formation#Biology


76

products from endophytic microbial flora have a broad spectrum of biological

activities i.e. antifungal, antibacterial, antidiabetic and anticancer etc. Therefore,

bioactive secondary metabolites from endophytic fungi can be grouped into several

categories like steroids, alkaloids, terpenoids, phenol and lignin (Qawasmeh et al.,

2012; Xiao et al., 2014). There are different Endophytic fungi which are reported

from genus Boswellia. The genus Alternaria is the most prevalent followed by

Aspergillus and Rhizopusstolonifer. Recently, Penicillim species has been also

reported from Boswellia (Nagerabi et al., 2014).

In our study we quantified the endophytic fungal extracts and tested for α-glucosidase

inhibition which revealed significant inhibition for α-glucosidase enzymes.The ethyl

acetate extract of P. citrinum (FEF6) and P. spinulosum (FEF2) showed prominent

results. While in urease inhibitory activity of endophytic fungi extracts the isolated

endophytic fungi showed more than 60% rate of inhibition. The endophytic fungus P.

citrinum (FEF6) revealed increased rate of inhibition.Previously, Singh et al., (2015)

evaluated the maximum inhibitory activity of an endophyte Cladosporium sp. isolated

from T. cordifolia (TN-9S) they observed thatpurified inhibitor is a phenolic

compound with amine groups and it can also inhibited the activity of α-glycosidase in

vivo condition. There is a variety of organisms which encodes for the production of

urease enzymes including plants, fungi, bacteria and invertebrates, and it also occurs

in soils as a soil enzyme (Krajewska 2009).Khan et al., (2015) isolated the endophytic

fungus Bipolaris sorokiniana LK12 from the leaves of Rhazyastricta. They

characterized bipolarisenol from B. sorokiniana evaluated for its potential role in

urease inhibition. Their results suggest that Bipolarisenolinhibit urease in a dose-

dependent manner with high IC50 (81.62 ± 4.61 µg·mL−1).


77

In addition, cellulase enzymes produced bythe endophytic fungi, Pycnoporus

sanguine isolated from Baccharis dracunculifolia effectively hydrolyzed cellulose

(Onofreet al., 2015). The production of cellulase enzymes was considerable higher in

F. oxysporum, whereas it was the lowest inAlternaria sp. P. variotii and P.

purpurogenum produced high amounts of cellulases, followed by A. nidulans and

Alternaria sp. It was also observed that by keeping the pH at 5.5, a greater amount of

cellulase was produced in liquid media.

It has been reported that endophytic microbial flora can significantly contribute to the

production of phytohormones (Marwah et al., 2007; Kuldau G and Bacon C, 2008). In

our study, P. variotii and A. nidulans showed considerable IAA production. The

production of IAA is dependent on the type of pathway utilized by endophytes;

therefore, IAA in the culture broth was estimated by the presence and absence of L-

tryptophan.Similar results for IAAwere foundby Mei and Flinn (Mei and Flinn, 2010),

Where IAA producing endophytic fungi improved plant growth under stressconditions.

Thus, previous findings about IAA-producing endophytic fungi strongly support our

current results (Fu et al., 2015).The results conclude that endophytic fungi associated

with medicinally important plants possess a unique potential to produce bioactive

metabolites, which might improve the growth of the host in severe environmental

conditions. Such bioactive fungal strains may have applications for crop improvement

and industrial production of important constituents.

The production of enzymes such as ACC (1-aminocyclopropane-1-carboxylate)

deaminase is common in bacteria while some of fungal strains also encoding genes for

ACC deaminase. The fungal stain P. citrinum and a very few other fungal strains are

documented for the production of ACC (Jiaet al., 1999, Yim et al., 2013). The


78

endophytic microbial flora also contain the enzyme 1-aminocyclopropane-1-

carboxylate (ACC) deaminase and this enzyme can cleave the ethylene precursor ACC

to α-ketobutyrate and ammonia and thereby lower the level of ethylene in developing or

stressed plants (Hontzeas et al., 2005). ACC deaminase-producing endophytes promote

plant growth under stress conditions and also playing a very important role in the

process of nodulation, specific to plant species.

Our results suggested a significantly higher amount of ACC deaminase production by

P. purpurogenum and P. variotii strains. Of the endophytic fungal strains grown in DF

minimal medium containing ACC, P. purpurogenum showed the highest productionof

ACC at 355nmol α- ketobutyrate mg-1

h-1

among the isolated strains. Previously, P.

citrinum and some other fungal strains were found to produce ACC (Yim et al., 2013;

Jia et al., 1999). The endophytic microbial flora also contained the enzyme 1-

aminocyclopropane-1-carboxylate (ACC) deaminase which was confirmed by the

total protein content inthe pure growth cultures of P. purpurogenum and P. variotii.

However, extracellular enzyme production by endophytes isessential to help the

microorganism penetrate the host cell wall, and can contribute to the defence

responses and mineral uptake in host plants (Weiet al., 2013). Enzymes, such as β-

glucosidases, cellulases, and phosphatases, were determined using florigenic

substrates and standard curve readings were taken in combination with MUB

standards. In our study, the extracellular enzyme, β-glucosidase, encoded by

endophytic fungi isolated from C. acutangula revealed significant results; P. variotii

and P. purpurogenum produced higher amounts of β-glucosidases than other strains.

The previous data also described the purification, crystallization, and different

properties of α-glucosidase from the mycelia of Mucor (Kato et al., 202), andα-


79

glucosidases from Aspergillus niger and A. nidulans (Hsu et al., 2013) have been

isolated from culture filtrates;Since α-glucosidase has also been reported from the

different species of Penicillium (Liu etal., 2010; Qureshietal., 2013).

In our study the ethyl acetate extract of endophytic fungus P. citrinum (FEF6) was

subjected to repeated column chromatography followed by recycling HPLC to givethe

compounds: 11-oxoursonic acid benzyl ester (1), n-nonane (2), 3-decene-1-ol (3), 2-

Hydroxyphenyl acetic acid (4), and Glochidacuminosides A (5). Compound 1 was

isolated in the form of amorphous powder which gave characteristic pink colour on

the TLC plates after spraying with ceric sulphate reagent. Compound 2 and 3were

isolated as a colourless liquids while Compound 4 and 5 were obtained as colourless

powder and as a gummy solid respectively. The α-glucosidase inhibitory activities of

the isolated bioactive compounds from endophytic fungus P. citrinum FEF6

demonstrates that the characterized compounds 2-Hydroxyphenyl acetic acid (4), 11-

oxoursonic acid benzyl ester (1) and Glochidacuminosides A (5) exhibited high level

of potential for the inhibition of α-glucosidase. While assay for urease inhibition

showed that compound 2, 3 and 4 have a very weak role in urease enzyme inhibition

as compare to compound 1 (11-oxoursonic acid benzyl ester) and 5

(Glochidacuminosides A) which shows a substantial level of urease inhibitory

activities. The anticancer activities for bioactive secondary metabolites showed that

the three compounds, 11-oxoursonic acid benzyl ester (1), 2-Hydroxyphenyl acetic

acid (4), and Glochidacuminosides A (5) from endophytic fungi Penicillium citrinum

bears a considerable level of inhibition of cancer cell proliferation and apoptosis using

human breast cancer cell line (MDA-MB-231). In our results the cell viability was

greatly arrested by 11-oxoursonic acid benzyl ester (1) followed by 2-Hydroxyphenyl

acetic acid (4). Similarly Hu et al., (2008) isolated xylarenones A, Band xylarenic


80

acid from endophytic fungus Xylaria sp. NCY2, obtained from Torreya jackii CHUN

and were evaluated for antitumor and antimicrobial assays in in-vitrocondition which

demonstrated that these compounds exhibit moderate antitumor activities against

HeLa cells line (Hu et al., 2008).


81

Section 4: Conclusion

In conclusion, diverse fungal endophytes have established important niches with plants

through the secretion of bioactive constituents, regulating host growth during harsh

environmental conditions. Endophytic fungi were isolated from medicinally important

plants i.e.Caralluma acutangula and Boswellia sacra. The isolated endophytic fungi

were identified on the basis of their morphological traits and by using genomic DNA

extraction, PCR amplification and sequencing the internal transcribed spacer regions,

whereas a detailed phylogenetic analysis of the same gene fragment was made with

homologous sequences. The endophytic fungi were identified as Penicillium citrinum;

Paecilomyces variotii, Aspergillus nidulans, Fusarium oxysporum, Epicucum nigram,

Penicillium purpurogenum, Penicillium spinulosum, Aspergillus caespitosus, Phoma

and Alternaria sp. Furthermore, the endophytic fungi P. citrinum, P. variotii,

Aspergillus nidulans, Fusarium oxysporum, Penicillium purpurogenum and Alternaria

sp. were assessed for their potential to produce anti-cancerous metabolites by

performing MTT assay and extracellular enzymes such as cellulases, phosphatases and

glucosidases in growth media. The P. variotii, P. citrinum and F. oxysporum showed

significantly higher amount of phosphatases and glucosidases as compared to other

strains. The NMR spectra were recorded on Bruker spectrometer operating at 600-MHz

(150-MHz for 13C) which give singals for five different bioactive secondary

metabolites from endophytic fungi. Additionally, endophytic P. citrinum, P. variotii

and F. oxysporum showed significantly higher potential of indole acetic acid

production.The above mentioned activities were for the first time elucidated for our

isolated endophytic fungi. We also concluded that the isolated endophytic fungi

produce bioactive constituents that could provide a unique niche of ecological

adaptation by symbiosis and greatly contribute to healthy life of their host plantand can

be utilized for future drug discovery.

References

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