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Life Science Archives (LSA)

ISSN: 2454-1354

Volume – 1; Issue - 2; Year – 2015; Page: 142 - 156

©2015 Published by JPS Scientific Publications Ltd. All rights reserved

Research Article

ENDOPHYTIC FUNGAL COMMUNITIES ASSOCIATED WITH

ETHNO MEDICINAL PLANTS FROM INDIA AND THEIR POTENTIAL

PTODUCTION OF ANTICANCER DRUG CAMPTOTHECIN

N. Lakshmi1, V. Bhuvaneswari

2, G. Kathiravan

3 and B. Shanmugapriya

2

1S.D.N.B. Vaishnav College for Women, Chromepet, Chennai, Tamil Nadu, India.

2Chikkaiah Naicker College, Veerapan chitram, Erode, Tamil Nadu, India.

3Department of Biotechnology, Vels University, Pallavaram, Chennai, Tamil Nadu, India.

Abstract

Endophytic fungi are ubiquitous, ecologically specialized group and are assumed to be widely present

in virtually all land plants. The anticancer properties of several secondary metabolites from endophytes have

been investigated recently. Following, is one of the example Camptothecin (CPT) is a monoterpenoid indole

alkaloid originally isolated from Camptotheca acuminata Decne (Nyssaceaea), a deciduous tree native to

south China, that has gained great attention for its significant antitumor activities in experimental studies. In

the present investigation, extraction of Camptothecin and its analogues from novel endophytic fungal sources

isolated from two medicinal plants namely Nerium oleander and Nyctanthus arbor-tristis were studied. Of

the taxa identified in the current study, mitosporic fungi dominated the endophyte assemblages. Therefore, in

the present investigation two hyphomyceteous fungi namely, Aspergillus flavus and Aspergillus niger were

screened for the production of Camptothecin. The production of Camptothecin was confirmed and quantified by different analytical methods. The amount of Camptothecin was found to be maximum in Aspergillus

flavus (55.5 µg/L) followed by Aspergillus niger (32.5 µg/L). UV spectroscopic analysis showed

characteristic absorption peaks at 226 nm and 269 nm, which was similar to that of the authentic

Camptothecin (Sigma aldrich). Mass spectroscopy (MS) done in the two fungal samples yielded [M + H]+

ions of CPT at m/z 349. The sodium adduct of CPT was also formed and was visible in the mass spectrum at

m/z 371 [M + Na]+. Thus, these research efforts are significant for both practical and philosophical reasons.

Article History Received : 02.04.2015

Revised : 19.04.2015

Accepted : 26.04.2015

Key words: Camptothecin, Camptotheca

acuminata, antitumor, endophytic fungi,

Aspergillus flavus and Aspergillus niger.

1. Introduction

An endophyte is an endosymbiont, often a

bacterium or fungus, which lives within a plant for

at least part of its life without causing apparent

disease. Symptomless fungal endophytes have

been discovered in the aerial plant tissues of over

300 plant species, including angiosperms,

* Corresponding author: N. Lakshmi, S.D.N.B.

Vaishnav College for Women, Chromepet, Chennai,

Tamil Nadu, India

gymnosperms, marine macro algae, mosses and

ferns (Petrini and Fisher, 1990; Clay, 1991; Lodge

et al., 1996). Studies on the distribution,

biodiversity and biochemical characteristics of

endophytes are of immense importance in plant

biology to understand and to improve plant fitness.

Plant endophytic fungi are an important and novel

resource of natural bioactive compounds with their

potential applications in agriculture, medicine and

food industry. In the past two decades, many

valuable bioactive compounds with antimicrobial,

http://en.wikipedia.org/wiki/Endosymbiont

http://en.wikipedia.org/wiki/Bacterium

http://en.wikipedia.org/wiki/Fungus

http://en.wikipedia.org/wiki/Plant

N. Lakshmi/Life Science Archives (LSA), Volume – 1, Issue – 2, Page – 142 - 156, 2015 143


insecticidal, cytotoxic and anticancer activities

have been successfully discovered from the

endophytic fungi. During the long period of co-

evolution, a friendly relationship was formed

between each endophyte and its host plant. Some

endophytes have the ability to produce the same or

similar bioactive compounds as those originated

from their host plants.

Bioactive natural compounds produced

by endophytes have been promising potential

usefulness in safety and human health concerns,

although there is still a significant demand of drug

industry for synthetic products due to economic

and time-consuming reasons. Problems related to

human health such as the development of drug

resistance in human pathogenic bacteria, fungal

infections and life threatening virus claim for new

therapeutic agents for effective treatment of

diseases in human, plants, and animals that are

currently unmet (Mariana Recco et al., 2011).

Symbiotic relationship of host-microbes

producing new and interesting bioactive

compounds may find uses in pharmaceutical

industries. Some of the potentially important

compounds isolated from endophytic fungi

include cryptocin, cryptocandin, preussomerin,

phomosichalasin and torreyanic acid. Above all,

endophytes have been proved to be an effective

source of Taxol, a cytotoxic drug used in the

treatment of cancer (Wani et al., 1971). Also of

notice, endophytes have been found to be capable

of producing immunosuppressive compounds (Lee

et al., 1995). Apart from the medicinal

compounds fungal endophytes are the reservoirs

of more eco-friendly useful compounds to

mankind. Reports showed that they are producing

volatile antimicrobials from Muscodor albus

(Strobel et al., 2001), antifungal ambuic acid from

Pestalotiopsis spp. and Monochaetia sp. (Li et al.,

2001), isopectacin an antifungal and antioxidant

compound from Pestalotiopsis microspora

(Strobel et al., 2002), Napthalene an insect

repellant from Muscodor vitigenus (Daisy et al.,

2002; Azevedo et al., 2000).

There are some evidences that bioactive

compounds produced by endophytes could be

alternative approaches for discovery of novel

drugs, since many natural products from plants,

microorganisms, and marine sources were

identified as anticancer agents. The anticancer

properties of several secondary metabolites from

endophytes have been investigated recently.

Following, is one of the example Camptothecin

(CPT) is a monoterpenoid indole alkaloid

originally isolated from Camptotheca acuminata

Decne (Nyssaceaea), a deciduous tree native to

south China, that has gained great attention for its

significant antitumor activities in experimental

studies (Wall et al.,1966). Irinotecan (CPT-11)

(Masuda et al., 1992; Abigeres et al., 1995 &

1999) and topotecan (TPT) (Lilenbaum et al.,

1995; Romanelli et al., 1998 and Clements et al.,

1999), two water-soluble derivatives of CPT, have

gained approval by the Food and Drug

Administration of the United States of America

(FDA) for treating colorectal and ovarian cancer.

Other camptothecins - such as 9-

aminocamptothecin (9AC), 9- nitrocamptothecin

(9NC), and 7-(4-methyl piperazino-methylene)-

10,11- (GG211) have also showed remarkable

potential in the treatment of Carcinoma (Wall and

Wani, 1996; Giovanella, 1997; Jeha et al., 1998

and Stevenson et al., 1999).

Currently, it is believed that many of these

compounds act in defense of the harmful effects of

toxins, carcinogens, or mutagens found in the

plant or attack by external predators. The

compounds were isolated guided by bioassay on

various extracts and chromatographic fractions.

Their unique and hitherto unknown structures

were elucidated by nuclear magnetic resonance,

mass spectrometry, and X-ray analysis. Both

compounds have unique mechanisms of antitumor

activity; camptothecin uniquely inhibits an

enzyme, topoisomerase I, involved in DNA

replication. Camptothecin and analogues singly or

combined with cisplatin show efficacy against

solid tumors, breast, lung, and colorectal, which

hitherto have been unaffected by most cancer

chemotherapeutic agents (Wall and Wani, 1996).

Therefore, in the present investigation, extraction

of Camptothecin and its analogues from novel

endophytic fungal sources isolated from medicinal

plants were studied.



2. Materials and Methods

2.1. Collection and processing of plant samples

For isolating endophytes, two healthy

medicinal plants namely Nyctanthes arbor-tristis

L. and Nerium oliander L. were collected in sterile

polythene bags from their natural habitat. The

plants were collected from Chromepet, Chennai.

The samples were brought to the laboratory and

processed within 24 hours. The collected samples

were first washed thoroughly in running tap water.

From each plant 20 segments (approx. 0.5 cm2)

from different tissues (leaf, rachis/petiole and

stem) were screened for the presence of

endophytes. The modified Standard triple ethanol

- sodium hypochlorite - ethanol surface

sterilization techniques (Fisher et al., 1993;

Dobranic et al., 1995 and Schultz et al., 1998)

were followed throughout the present study. The

segments were surface sterilized in 70% ethanol

for 5 sec, immersed in 4% Sodium hypochlorite

(NaOCl) for 90 sec, rinsed in sterile distilled water

and then dried on sterilized filter paper. The

surface sterilized segments were placed on Potato

Dextrose Agar (PDA) medium amended with

Streptopenicillin (150 mg/L). The petriplates were

then sealed with ParafilmTM

.

2.2. Incubation and isolation of endophytes

The petriplates were incubated in a light

chamber and observations were done from the

second day onwards for a period of 3 - 4 weeks for

the fungal colonies (Bills and Pollishook, 1991).

The light regime was 12 hours light followed by

12 hours darkness. The hyphae, which grew out

from the tissues, were transferred to fresh PDA

slants. They were maintained by sub-culturing.

To prevent the rapidly growing fungi from

inhibiting the slow growing species, the former

were removed as soon as they appeared on the

plates (Bills, 1996).

2.3. Identification of the fungi

The isolated endophytic fungi from selected plants

were identified down to species level with the help

of standard monographs (Guba, 1961; Ellis, 1971;

Sutton, 1980; Onions et.al., 1981 and Nag Raj,

1993). The non-sporulating sterile forms were

separated into culture groups based on their

colony morphology, hyphal mat characteristics

(texture, zonation, sectoring), presence of sclerotia

(masses of short celled, lobed and closely packed

hyphae) and pigmentation as described by Frolich

et al. (2000). Such sterile forms were included as

'species' for the analysis of the results.

2.4. Calculation of Colonization frequency (CF)

The colonization frequency of each

endophyte species was calculated by the method

of Hata and Futai (1995).

The number of colonized segments

CF % = × 100

Total number of segments observed

2.5. Relative percentage occurrence (RPO)

Relative percentage occurrence (RPO) of

each group (viz., Ascomycetes, Hyphomycetes,

Coelomycetes and sterile morphospecies) of

fungal species in each plant species was calculated

as follows (Petrini, 1991)

Density of colonization of single group

RPO= × 100

Total density of colonization

2.6. Cultivation and extraction of test fungi for

Camptothecin production

The selected endophytic fungi namely,

Aspergillus niger and Aspergillus flavus were

cultured in three different media viz., Corn Meal

Agar, Rose Bengal Agar and Sabouraud’s

Dextrose Agar medium respectively for

Camptothecin production. The methodology for

the extraction of Camptothecin was as given by Li

et al. (2012). After incubating the culture for 2 - 3

weeks, both cell homogenate and cell-free broth

were extracted four times with equal volume of

chloroform: methanol (4:1 v/v). After stripping off

the solvent, the residue was analyzed both

chromatographically as well as spectrometrically.

Blank cultures (uninoculated sterile medium) were

also maintained along with fungal cultures. The

blank cultures were autoclaved, incubated and

processed exactly in the same manner as the

inoculated fungal cultures. The blanks were also

analyzed and tested with the fungal samples.



2.7. Chromatographic analysis of test samples

TLC analysis was carried out on Merk 1 mm (20 x

20 cm) silica gel precoated plates. The plates

were developed using chloroform: methanol (9: 1

v/v) solvent system. TLC analysis exhibited spots

which were super imposable with the standard

CPT. The spots were visualized under UV.

Camptothecin standard solutions within the range

from 1 - 100 μg/ml concentrations were prepared

for HPLC analysis. The camptothecin standard

was prepared by dissolving in a solution of DMSO

and absolute HPLC grade methanol in a ratio of 5:

50 (v/v). The standard sample solutions for HPLC

were filtered using 0.2 syringe filter before

injection. The analysis of extracts was done in

High Performance Liquid Chromatographic

system (HPLC) equipped with LC8A pump, SPD-

M 10 Apphoto array detector in combination with

class LC 10 A software (Shimadzu). The presence

of camptothecin in the samples was detected by

comparing with the retention time of the standard

sample. The area of the standard was compared

with area of the sample and the amount of

camptothecin in the extracts was calculated. The

chromatographic conditions for the analysis were

as follows: mobile phase: acetonitrile : water (60:

40), column: ODS (Octadecyl silane) C18, 5

µsize, 250 X 4.6 mm (Supelco), Detector: SPD-M

10 A Vpphoto array detector, wave length: 254

nm, flow rate: 1.0 ml/min, injection volume: 25

µl, retention time: 6.4 min.

Standard concentration ×

Total area of the sample

Camptothecin

content =

Total area of the Standard

After chromatography, the two potential

Hyphomycetes fungi were analyzed

spectroscopically for further confirmation of

Camptothecin sample were taken and dissolved in

Methanol and analyzed using Beckman DU-40

with two maximum at 226 nm and 269 nm.

2.8. Mass Spectrometry analysis of test samples

In this study, the fungal test samples were

analyzed using the mass spectrometer which was

fitted with an electrospray interface. All the

interface parameters of GC–MS/MS studies were

optimized by infusing the standard solution of

CPT. The other parameters for GC–MS analysis

were set at dry gas flow of 11 l/ min, nebulizer

pressure 35 psi and drying gas temperature 320˚C.

The isolated peak width was taken as 0.8 m/z and

fragmentation amplitude value was 2.40. A mass

spectrum of CPT in methanol: chloroform [1:3]

was recorded under ESI on a Bruker Ion Trap

(Esquire 3000) mass spectrometer in the positive

ion mode, with a mass range from 50 to 800 amu.

Bruker Daltonics Esquire 5.0 software was used to

obtain the mass spectra and Chemstation Rev.

06.03 (509) software was used to acquire the GC–

MS spectra.

3. Results and Discussion

In the recent years, the quest for isolation

of new compounds from medicinal plants has

become a fascinating area of research. Plants with

ethno-pharmaceutical importance are being

exploited because of their healing properties.

However, large scale harvesting of medicinal

plants has already become a major threat to

biodiversity. As an alternative, microbes, which

live inside such plants (endophytes), may offer

tremendous potential source of therapeutic

compounds. The present study has been aimed

towards isolation and identification of endophytic

fungi associated with selected medicinal plants

and analysis of test fungi for Camptothecin

production was carried out. The plant species

chosen for this study and their medicinal uses are

presented in Table - 1. Different plant parts viz.,

leaf (including midrib), petiole/rachis and stems of

the selected symptomless host plants were surface

sterilized and incubated as per the procedure

mentioned in materials and methods. The plants

segments were studied for the presence of

endophytic fungi.



Table - 1: Medicinal plants and their uses

S. No Host plant Common name Family Tamil name Medicinal uses

1 Nerium

oleander

Rose bay,

Dogbane, Laurier

rose

Apocynaceae Arali

Cardio-tonic,

Diuretic,

Cancer

treatment

2 Nyctanthus

arbour-tristis

Night

jasmine,Coral

jasmine

Oleaceae Pavilamalligai

Cholagogue,

Laxative,

Expectorant,

Diaphoretic,

Nerium oleander is a large glabrous

evergreen shrub with milky latex; leaves three in a

whorl, shortly stalked, linear, dark green and shiny

above; flowers red, green and shiny above;

flowers red, rose-colored or white, fragrant; fruits

follicles at length separating. In the present

investigation, sixty segments of different plant

parts of Nerium oleander were surface sterilized

and screened for the presence of endophytes. A

total of 52 endophytic fungal colonies belonging

to 7 species were isolated as endophytes. The

endophytic assemblages were diverse and

comprised of 3 Hyphomycetes, 4 Coelomycetes,

and 1 non-sporulating sterile morphospecies. The

colonization frequencies and Relative percentage

occurrence (RPO %) of the endophytic fungi are

presented in Table - 2.

Nyctanthus arbor-tristis is a hardy large

shrub or small tree upto 10 m in height with grey

or greenish white rough bark and sharply

quadrangular strigose young branches; leaves

simple opposite, orate, acute or acuminate,

scabrous above with short bulbous hairs, main

nerves few, conspicuous beneath flowers, small,

white with bright orange corolla tuber, 3-7 in

head, in trichotomous cymes; fruits capsules,

compressed, separating into two, one-seeded

segments.

In the present investigation, 20 segments

of different tissues of Nyctanthus arbor-tristis

were surface sterilized and screened for the

presence of endophytes. Out of 60 segments

inoculated, 69 fungal colonies were obtained.

These colonies were classified into 11

species of fungi (1 Zygomycetes, 6

Hyphomycetes, 2 Coelomycetes, and 2 non-

sporulating sterile morphospecies). Colonization

frequency (CF %) and relative percentage

occurrence (RPO %) of the endophytic fungi are

illustrated in Table - 3.

The presence of sterile forms as

endophytes continues to frustrate the mycologists

because of their uncertain taxonomy. Moreover,

they demand the use of molecular techniques for

classification (Bills, 1996). The non-sporulating

sterile forms recovered during the current study

were separated into culture groups based on their

colony morphology, hyphal mat characteristics

(texture, zonation, sectoring), presence of sclerotia

(masses of short celled, lobed and closely packed

hyphae) and pigmentation as described by

Frohlich et al. (2000).

Analysis of the fungal endophytes obtained

during the course of the study shows that many

potential pathogenic species or genera have also

been encountered as endophytes. Genera that are

found to be in common include Alternaria,

Nigrospora, Cladosporium, Colletotrichum,

Fusarium and Phoma. These results suggest that

an endophytic stage at times may be an important

part in the life cycle of pathogens. Species known

to be pathogenic that has been isolated as

endophytes are not necessarily always pathogenic

strains. It has been demonstrated that if the

infection is not latent, then the alternative

hypothesis is that a mutation of a virulent

http://en.wikipedia.org/wiki/Apocynaceae



pathogen has occurred and the fungus has become

a non-pathogenic strain of the pathogen (Freeman

and Rodrigues, 1993).

Saprotrophic taxa that were isolated as

endophytes in the present study include

Nigrospora sphaerica, Aspergillus niger and

Phoma sp. The above-mentioned fungi are known

to biodegrade cellulose and lignin and their

ecological role is primary in decomposing dying

plant material.

Endophytic fungi have been recognized as

a repository of novel compounds of immense

value in agriculture, industry and medicine (Naik

et al., 2009). To date, many valuable bioactive

compounds and anticancer activities have been

obtained from the endophytic fungi (Verma et al.,

2009; Aly et al., 2010; Yu et al., 2010;

Kharwar et al., 2011). On the whole, the percent

contribution of Hyphomycetes (57 %) as

endophytes was very high followed by

Coelomycetes (31 %) non-sporulating sterile

morphospecies (8%), and Zygomycetes (3 %)

(Fig-1). Among the different plant parts viz., stem,

leaf lamina and petiole studied for the presence of

endophytes, more numbers of isolates were

recovered from leaf lamina (67), followed by stem

(36) and petiole (17) (Fig 2).

Apart from trying to understand the

biology of endophytes, another motivation to

study these fungi is its ability to produce a novel

bioactive compound Camptothecin. Wani et al.

(1971) from North Carolina discovered that an

extract of the Yew tree bark has antitumor activity

and the compound was named "Paclitaxel" or

"Taxol". Interest in developing the drug increased

after the mechanism of action of tubulin

polymerization was studied by Horowitz in 1980.

It was discovered that paclitaxel promotes tubulin

polimerization and stabilizes microtubules against

depolymerization (Schiff et al., 1979; Schiff and

Horowitz, 1980). However, Taxol supply from

the original source cannot meet the rising demand

of clinical uses because of the scarcity and slow

growth of Taxus yew trees and the low Taxol

(0.02% dry weight) content in the bark (Cragg et

al., 1993). Moreover, the extraction procedure is

complex and expensive. Hence, alternative

sources were explored for anticancer drug supply

including chemical synthesis, cultural techniques

and extraction from different microbial sources.

Camptothecin, a quinoline indole alkaloid

and its analog, 10 –hydroxy Camptothecine are

potent inhibitors of eukaryotic topoisomerase I.

Because of this activity, several semi synthetic

derivatives of CPT topotecan and camptostar are

used clinically. The production of Camptothecin

from a microbial source has many advantages over

other sources. Industrial production of bioactive

compounds like Camptothecin requires

reproducible, dependable productivity. If a fungus

is the source organism, it can be grown in tank

fermenters to produce an inexhaustible supply of

Camptothecin. The added advantage is that the

fungi usually respond favorably to routine cultural

techniques. Normally tissue cultures of

Camtotheca accuminata for Camptothecin

extraction will take a longer time when compared

with the fungi. Also, tissue culture needs

specialized techniques and conditions have to be

maintained whereas in fungi one can easily alter

the cultural conditions for the production of

different bioactive compounds.

Touseef et al. (2006) isolated

ectomycorrhizal fungi from Camtotheca

accuminata which demonstrated the ability to

produce Camptothecin. The presence of

Camptothecin in this fungus was confirmed by

mass spectrometry, chromatography, and

biochemical techniques. He also suggested that

improved culture techniques, the addition of

activators and the application of genetic

engineering methods may permit the

commercialization of Camptothecin production.

Techniques previously applied in the confirmation

of Camptothecin from Camptotheca species and

fungi include Thin layer chromatography (TLC),

High performance thin layer chromatography

(HPLC), Ultraviolet (UV), Infrared (IR), and Mass

spectrometry (MS) Many other investigators have

supported these confirmatory techniques (Li et al.,

1996; Strobel et al., 1996 ; Li et al., 1998;

Baloglu, 1999; Wang et al., 2000). Hence, in the

present study TLC, HPLC, UV and MS methods

were employed for confirming the presence of

Camptothecin in the fungal sample.



Table - 2: CF % and RPO % of endophytic fungi recovered from Nerium oleander

Therefore, in the present investigation two

endophytic hyphomycetous fungi namely,

Aspergillus flavus and Aspergillus niger obtained

from the selected medicinal plants were screened

for the production of Camptothecin. The

methodology for the extraction of Camptothecin

was as given by Li et al. (2002). The selected test

fungi were grown in the medium as mentioned

earlier. After the incubation period of 16 days, the

fungal samples were extracted with methanol and

were used for further analysis. Analytical thin

layer chromatography reference standard and test

sample fractions was conducted on silica gel

GF254 analytical plate developed with

Chloroform/methanol (90:10, v/v). Blue

fluorescent spot detected by long UV were

observed at Rf values of 6.9, which indicates the

presence of Camptothecin in the fungal test

samples. Among the three different media tested

the fungi grown in SDA showed the best result.

Hence these fungi were further analyzed by using

High Performance Liquid Chromatography

(HPLC), Ultra Violet (UV) and Mass

Spectrometry (MS) analysis.

S.

No. List of fungi isolated

Colonization frequency of endophytic fungi

RPO Stem Leaf lamina Petiole

NIC CF % NIC CF % NIC CF %

Hyphomycetes - - - - - -

50 1 Alternaria alternate 6 42.8 - - - -

2 Aspergillus niger - - 8 29.6 2 18.1

3 Cladosporium cladosporioids 3 21.4 7 25.9 - -

Coelomycetes - - - -

40.3

4 Colletotrichum sp. 2 14.8 - - - -

5 Phoma herbarum - - 4 14.8 - -

6 Phoma pomorum 3 21.4 - - 4 36.3

7 Phyllosticta sp. - - 8 29.6 - -

Sterile morphospecies - - - - 9.6

8 Sterile form 1 - - - - 5 45.4

Total No. of isolates observed 14 - 27 - 11 - -

No. of species recorded 4 - 4 - 3 - -



Table - 3: CF % and RPO % of endophytic fungi recovered from Nyctanthus arbor-tristis

S.

No. List of fungi isolated

Colonization frequency of endophytic fungi

RPO Stem Leaf lamina Petiole

NIC CF % NIC CF % NIC CF %

Zygomycetes

1 Rhizopus stolonifer - - 4 10 - - 5.79

Hyphomycetes

2 Alternaria alternate 4 18.1 - - 2 28.5 -

3 Aspergillus flavus - - 8 20 - - -

4 Aspergillus ochraceous 5 22.7 - - - -

5 Cladosporium cladosporioids - - 9 22.5 3 42.8 -

6 Fusarium sp. 4 18.1 - - - - -

7 Nigrospora sphaerica - - 3 7.5 - - -

8 Penicillium citrinum 3 13.6 - - 2 28.5 62.31

Coelomycetes

9 Colletotrichum sp. - - 8 20 - - -

10 Phoma sp. 4 18.1 5 12.5 - - 24.63

Sterile morphospecies

11 Sterile form 2 - - 3 7.5 - - -

12 Sterile form 3 2 9 - - - - 7.24

Total no. of isolates observed 22 - 40 - 7 - -

No. of species recorded 6 - 7 - 3 - -



Figure – 1: RPO% of test fungi isolated from

two medicinal plants

Figure – 2: Influence of different plant parts on

distribution of Endophytes

Further the presence of Camptothecin was

confirmed and quantified using HPLC analysis.

The fungal test sample was subjected to HPLC

analysis for further quantification. The retention

time of the sample was found to be 6.1 min when

compared to that of the authentic Camptothecin

(5.1 min) (Fig - 3a and 3b). As per the formula

mentioned in Materials and Methods, the amount

of Camptothecin content in the sample was found

to be 32.5 µg/L for Aspergillus niger and 55.5

µg/L for Aspergillus flavus.

The fungal compound having

chromatographic properties comparable to

Camptothecin when subjected to Ultraviolet (UV)

spectroscopic analysis gave characteristic

absorption peaks at 226 and 269 nm. This

confirmed the presence of Camptothecin (Fig - 4a

and 4b). Mass spectrum corresponded to the [M +

H]+ ions of CPT at m/z 349 (Fig - 5a and 5b). The

sodium adduct of CPT was also formed and was

visible in the mass spectrum at m/z 371 [M +

Na]+. Therefore the chromatographic and

spectroscopic analysis of fungal samples showed

the presence of Camptothecin by comparing this

with authentic.

The fungal filtrate may contain not only

Camptothecin but also other bioactive compounds.

Analysis of all will involve tremendous time and

expenditure. In the present study particular

attention was paid to Camptothecin, an anticancer

drug. The significance of finding a fungus

capable of producing Camptothecin should not be

understated, since such a discovery will

revolutionize the search for effective

pharmaceutical agents.

4. Conclusion

Thus these research efforts are significant

for both practical and philosophical reasons. First,

they could have a profound effect on the supply

issues concerning the important anticancer

compound Camptothecin. A fungus or bacterium

capable of producing Camptothecin at a rate of

50mg/liter would represent an inexhaustible

source of the drug. From both an ecological and

an economic viewpoint, a microbial source would

supplant reliance on the yew. We would no longer

be confronted with the choice of saving lives or

saving yews. If any of the microbial sources

isolated can provide reasonable, reliable quantities

of Camptothecin, more drug would be available

for both studies and treatment regimen, at a lower

cost to patient and at no cost to the environment.

0%

10%

20%

30%

40%

50%

60%

ZygomycetesHypomycetesCoelomycetesSterile Morphospecies

3%

57%

31%

8%

Rela

tive p

erc

en

tag

e o

ccu

ren

ce

RPO% of test fungi isolated from two medicinal plants

0%

10%

20%

30%

40%

50%

60%

70%

Leaf lamina Petiole Stem

67%

17%

36%

No

. o

f co

lon

ies o

bserv

ed

Plant parts

Influence of different plant parts on the distribution of endophytes



Authentic Camptothecin

Aspergillus niger

Figure - 3a: HPLC analysis of Camptothecin

extracted from Aspergillus niger


Aspergillus flavus

Figure – 3b: HPLC analysis of Camptothecin

extracted from Aspergillus niger


Figure - 4: UV-Visible spectrometer analysis of

Camptothecin extracted from Aspergillus niger

and Aspergillus flavus

Fig 4a: Fungal Camptothecin: Aspergillus niger



Fig - 4b: Fungal Camptothecin: Aspergillus flavus


Figure - 5a: Gas chromatography-mass spectrometry analysis of Camptothecin extracted from

Aspergillus niger




Figure 5b: Gas chromatography-mass spectrometry analysis of Camptothecin extracted from

Aspergillus flavus



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