EndophiticFungiFromThaiPlants2004

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Endophytic fungi with anti-microbial, anti-cancer and anti-malarial activities isolated

from Thai medicinal plants

Suthep Wiyakrutta1, Nongluksna Sriubolmas2,*, Wattana Panphut1, Nuntawan Thongon3, Kannawat Danwiset-

kanjana3, Nijsiri Ruangrungsi4 and Vithaya Meevootisom1

1Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand2Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330,

Thailand3National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120, Thailand4Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330,

Thailand

*Author for correspondence: Tel.: +66-2-2188380, Fax: +66-2-2545195, E-mail: [email protected]

Received 27 February 2003; accepted 12 November 2003

Keywords: Anti-malarial activity, anti-tuberculosis, anti-viral activity, biological activity, cytotoxicity, fungal

endophytes, secondary metabolites

Summary

A total of 81 Thai medicinal plant species collected from forests in four geographical regions of Thailand were

examined for the presence of endophytic fungi with biological activity. Of 582 pure isolates obtained, 360

morphologically distinct fungi were selected for cultivation on malt Czapek broth and yeast extract sucrose broth,

from which extracts were tested for biological activity. Extracts of 92 isolates could inhibit Mycobacterium

tuberculosis (MIC 0.0625200 lg ml)1) when tested by the microplate Alamar blue assay, while extracts of six

inhibited Plasmodium falciparum (IC50 of 1.29.1 lg ml

)1

) as determined by the [

3

H]hypoxanthine incorporationmethod. Strong anti-viral activity against Herpes simplex virus type 1 was observed in 40 isolates (IC50 of 0.28

50 lg ml)1). The sulphorhodamine B assay for activity against cancer cell lines revealed that 60 were active against

human oral epidermoid carcinoma cells (EC50 0.4220 lg ml)1) and 48 against breast cancer cells (EC50 0.18

20 lg ml)1). Bioactivity profile was affected by the type of culture medium. Given the high incidence of bioactive

extracts and the fact that most of the isolated fungi could not be identified due to lack of spore formation, the results

suggested that Thai medicinal plants can provide a wide variety of endophytes that might be a potential source of

novel bioactive compounds.

Introduction

There is a need to search for new antimicrobial agents

because infectious diseases are still a global problembecause of the development and spread of drug-resistant

pathogens (Pillay & Zambon 1998; Espinel et al. 2001).

Novel anti-cancer drugs are also required due to the

high worldwide mortality (Pisani et al. 1999). For

Thailand and other tropical countries, there is another

demand for new anti-malarials because of the spread of

drug-resistant malaria (Cowman & Duraisingh 2001).

The Thai National Science and Technology Develop-

ment Agency through the National Center for Genetic

Engineering and Biotechnology (BIOTEC) has given

high priority to the search for novel bioactive com-

pounds to treat major local diseases including malaria,tuberculosis, mycoses, HSV and cancer. Fungi have

been known to be a major source of active compounds

for isolation of endophytic fungi that colonize the

tissues without causing apparent harm. They can be

found in virtually all terrestrial plants (Petrini 1991;

Saikkonen et al. 1998). They are found to be a richsource of functional metabolites (Tan & Zou 2001).

Thailand is endowed with a rich biodiversity of plant

species and it has a long tradition of herbal medicine

(Panthong et al. 1986, 1991). Little is known about the

bioactive compounds in many of the medicinal plants,

although interest has increased, and more work is being

performed on their isolation and characterization (Lik-

hitwitayawuid et al. 1998; Ito et al. 2000; Phrutivor-

apongkul et al. 2002). The discovery that an endophytic

fungus (Taxomyces andreanae) also produced the anti-

cancer drug paclitaxel (Taxol) derived from Pacific

yew (Taxus brevifolia) was unexpected (Stierle et al.1995). This background information led us to speculate

that Thai medicinal plants might constitute another

World Journal of Microbiology & Biotechnology 20: 265272, 2004. 265 2004 Kluwer Academic Publishers. Printed in the Netherlands.


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Materials and methods

Collection of plant samples

Healthy leaves and stems were collected from 81 species

of Thai medicinal plants in the forest area of Ubon-ratchathani, Nakornratchasima, Chiangmai and Song-

kla Provinces of Thailand. The collection sites are

shown in Table 1. They were identified based on their

morphological characteristics. Classification was com-

pleted according to the phylogenetic outline provided by

Carr (2002) and the Angiosperm Phylogeny Group

(Bremer et al. 1998). The fresh-cut ends of plant samples

were wrapped with Parafilm M (3M Co. Ltd.) before

they were placed in zip-lock plastic bags and stored less

than 72 h in a refrigerator prior to isolation of endo-

phytic fungi.

Isolation of fungal endophytes

Samples were cleaned under running tap water and then

air-dried. Before surface sterilization, the cleaned stems

were cut into pieces 5-cm long. Leaves and limb

fragments were surface sterilized by immersion in 70%

ethanol for 1 min, 5% sodium hypochlorite solution for

5 min and sterile distilled water for 1 min two times. The

surface-sterilized leaves and stems were cut into small

pieces using a sterile blade and placed on sterile water

agar plates for incubation at 30oC. The hyphal tip of

endophytic fungus growing out from the plant tissue

was cut by a sterile pasture pipette and transferred to asterile potato dextrose agar (PDA) plate. After incuba-

tion at 30 C for 714 days, culture purity was deter-

mined from colony morphology. The pure endophytic

fungal cultures were deposited at the BIOTEC Culture

Collection, Pathumthani 12120, Thailand.

Fermentation and extraction

Endophytic fungal isolates were grown on PDA plates

at 30 C for 714 days depending on growth rate. Six

pieces (8 8 mm2) of the grown culture cut from the

plate were inoculated into a 1000 ml Erlenmeyer flask

containing 200 ml of malt Czapek (MCz) broth or yeastextract sucrose (YES) broth (Paterson & Bridge 1994).

After incubation at 25 C for 21 days under stationary

condition, the fungal culture was filtered to remove

mycelium. The filtered broth was then extracted with

200 ml of dichloromethane three times. The organic

phase was evaporated to dryness under reduced pressure

using a rotary evaporator and weighed to constitute the

crude broth extract. The fungal mycelia were freeze-

dried and then disrupted using a spatula and extracted

twice by soaking in a mixture of dichloromethane/

methanol (1:1, v/v) for 1 h. The two mycelial extracts

from each fungus were pooled and air-dried andweighed to constitute the crude mycelial extract. Crude

extracts from the culture broth and mycelium were

Merck) to obtain concentrations of 80.0 mg ml)1 to

1.0 g ml)1 depending on solubility. Equal amounts of

the crude extracts obtained from culture broth and

mycelium were combined.

Determination of anti-M. tuberculosis activity

Anti-M. tuberculosis activity of crude extracts was tested

by microplate Alamar blue assay using M. tuberculosis

H37Ra as a test organism according to Collins &

Franzblau (1997). Briefly, crude extract solution in

DMSO was diluted with Middlebrook 7H9 broth

(Difco) supplemented with 0.2% (v/v) glycerol (Difco),

1.0 g of Casitone (Difco) per litre, 10% (v/v) OADC

(BBL). The complete medium was referred to as

7H9GC. The crude extract was diluted to yield a final

concentration of 800 lg ml)1. Subsequent serial twofold

dilutions were performed in a final volume of 100 ll in amicroplate. M. tuberculosis H37Ra grown in 7H9GC

containing 0.05% (v/v) Tween 80 was diluted in

7H9GC to provide 5 104 cfu ml)1. A 100-ll volume

of the inoculum was added into each well. Wells

containing crude extract only were used to detect

whether the crude extract could change the Alamar

blue colour. Growth control well and sterility control

well consisted of bacteria only and medium only,

respectively. Rifampin and isoniazid were used as

positive controls. Kanamycin was used as a negative

control. On day 6 of incubation at 37 C, 20 ll of

Alamar blue solution and 12.5 ll of 20% Tween 80

were added into each well. Microplates were incubatedat 37 C for further 24 h. Visual minimum inhibitory

concentration (MIC) was defined as the lowest concen-

tration of crude extract that could prevent a colour

change.

Determination of activity against Plasmodium falciparum

Plasmodium falciparum K1, maintained in continuous

culture in human erythrocytes as described by Trager &

Jensen (1976), was used as a test organism. In vitro

activity against P. falciparum K1 was screened by

growth assessment of erythrocytic stage in the presenceof fungal crude extract. Briefly, crude extract solution in

DMSO (20 mg ml)1) was diluted 1:100 in RPMI-1640

(Roswell Park Memorial Institute-1640; Gibco). A 25-ll

volume of the diluted sample was pipetted into each well

of a 96-well microplate and 200-ll of 1.5% erythrocyte

suspension with 12% parasitaemia at early ring stage

was added. A well containing the corresponding con-

centration of DMSO was used as growth control. This

was done in duplicate. The microplates were incubated

in a 3% CO2 incubator at 37 C. After 24 h of

incubation, parasite lactate dehydrogenase activity was

determined to assess the parasitaemia according toMakler & Hinrichs (1993). Samples that showed anti-

parasitic activity were further tested for the concentra-

266 S. Wiyakrutta et al.


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Table 1. Classification of Thai medicinal plants collected in this study.

Group Order Family Scientific name Sitea IDb

Club mosses Selaginellales Selaginellaceae Selaginella involuta Spreng. SK 1

Ferns Filicales Schizaeaceae Lygodium flexuosum Sw. CM 2

Flowering vascular plantsBasal families Uncertain position Chloranthaceae Chloranthus inconspicuus Sw. SK 3

Magnoliid complex Piperales Aristolochiaceae Aristolochia tagala Cham. CM 4

Magnoliales Magnoliaceae Manglietia garrettii Craib CM 5

Paramichelia baillonii Hu CM 6

Annonaceae Polyalthia debilis Finet & Gagnep. UB 7

Polyalthia parviflora Ridl. NM 8

Uvaria rufa Bl. SK 9

Monocots Alismatales Araceae Amorphophallus campanulatus

Bl. ex Decne.

SK 10

Homalomena aromatica SK 11

Liliales Smilacaceae Smilax luzonensis Presl SK 12

Pandanales Stemonaceae Stemona tuberosa Lour. CM 13

Eudicots (Tricolpates) Ranunculales Menispermaceae Pachygone dasycarpa Kurz CM 14

Stephania hernandifolia Walp. SK 15

Tinospora crispa Miers ex Hook. F.& Thoms. SK 16

Core eudicots Santalales Opiliaceae Melientha suavis Pierre NM 17

Urobotrya siamensis Hiepko NM 18

Caryophyllid clade Caryophyllales Ancistrocladaceae Ancistrocladus tectorius Merr. UB 19

Rosid clade Vitales Vitaceae Cissus repanda Vahl CM 20

Tetrastigma campylocarpum Planch. NM 21

Eurosids I Malpighiales Erythroxylaceae Erythroxylum cambodianum Pierre UB 22

Euphorbiaceae Antidesma ghaesembilla Gaertn. CM 23

Croton oblongifolius Roxb. NM 24

Mallotus philippensis Muell. Arg. SK 25

Phyllanthus emblica Linn. CM 26

Sapium baccatum Roxb. CM 27

Clusiaceae Garcinia thorelii Pierre CM 28

Mesua ferrea Linn. CM 29

Flacourtiaceae Casearia grewiaefolia Vent. CM 30Fabales Fabaceae Bauhinia scandens Linn. var.

horsfieldii K. & S Larsen

NM 31

Erythrophleum teysmannii Craib NM 32

Dalbergia nigrescens Kurz NM 33

Dalbergia oliveriGamble NM 34

Derris reticulata Craib NM 35

Rosales Rhamnaceae Ventilago denticulata Willd. NM 36

Ulmaceae Holoptelea integrifolia Planch. CM 37

Moraceae Artocarpus lakoocha Roxb. NM 38

Broussonetia papyrifera Vent. CM 39

Ficus hispida Linn. CM 40

Streblus ilicifolius Corner NM 41

Eurosids II Myrtales Lythraceae Sonneratia griffithii Kurz SK 42

Myrtaceae Melaleuca leucadendron Linn. var.

minor Duthie

SK 43

Melastomataceae Melastoma malabathricum Linn. SK 44

Memecylaceae Memecylon ovatum J. E. Smith NM 45

Malvales Malvaceae Bombax ceiba Linn. CM 46

Urena lobata Linn. SK 47

Dipterocarpaceae Hopea odorata Roxb. NM 48

Shorea obtusa Wall. NM 49

Shorea roxburghii G. Don NM 50

Shorea siamensis Miq. NM 51

Sapindales Rutaceae Clausena excavata Burm. f. CM 52

Micromelum minutum Wight & Arn. UB 53

Paramignya scandens Craib NM 54

Toddalia asiatica Lamk. NM 55

Simaroubaceae Eurycoma longifolia Jack SK 56

Anacardiaceae Spondias pinnata Kurz CM 57

Asterid clade Ericales Myrsinaceae Ardisia lanceolata Roxb. SK 58Ebenaceae Diospyros filipendula Pierre

ex Lecomte

SK 59

Di lli G iff CM 60

Bioactive endophytic fungi 267


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(IC50) using the [3H]hypoxanthine incorporation meth-

od according to Desjardins et al. (1979). The experiment

was done in duplicate.

Determination of anti-Herpes simplex virus activity andcytotoxicity

Crude extracts were tested at a final concentration of

50 lg ml)1. A 10-ll volume of crude extract in 10%

DMSO was dispensed into each well of a 96-well

microplate followed by the addition of 30 pfu of Herpes

simplex virus type 1 ATCC VR-260. Then, Vero cells

ATCC CCL-81 cultivated in minimum essential med-

ium (MEM; HyClone) were added to a final concentra-

tion of 2 104 cells ml)1 in a volume of 200 ll.

Acyclovir (in final concentrations of 0.62510 lg ml)1)

and 10 ll of 10% DMSO were used as positive and

negative controls, respectively. After incubation at

37 C i n a 5% CO2 incubator for 72 h, viability of

Vero cells was determined by sulphorhodamine assay as

described by Skehan et al. (1990). Activity against

Herpes simplex virus type 1 (anti-HSV-1 activity) was

determined at the concentration of the crude extract that

showed no toxicity to the Vero cells. Crude extract

inhibiting less than 25% of viral growth was regarded as

inactive. Crude extract that could inhibit more than

50% of viral growth was further tested to determine the

concentration that inhibited 50% of viral growth (IC50).

This was performed in triplicate. Cytotoxicity of the

crude extracts against Vero cells was determined byperforming the experiment without addition of virus.

Ellipticine (in final concentrations of 0.258 lg ml)1)

Determination of activity against cancer cells

Crude extracts were tested against a human oral

epidermoid carcinoma (KB) cell line ATCC CCL-17

and a breast cancer cell line (BC-1) at a final concen-

tration of 20 lg ml)1

. A 10-ll volume of crude extract in10% DMSO was dispensed into each well of a 96-well

microplate. Ellipticine (in final concentrations of 0.258

lg ml)1) and doxorubicin (in final concentrations of

0.041.25 lg ml)1) were used as the positive control and

10 ll of 10% DMSO was used as the negative control.

Cells at exponential growth phase were harvested and

diluted to 105 cells ml)1 with Dulbeccos modified

Eagles medium (HyClone) for KB cells and with

MEM for BC-1 cells. The cell suspension was mixed

gently before aliquots of 190 ll were plated. After

incubation at 37 C in a 5% CO2 incubator for 72 h, cell

growth was determined by sulphorhodamine assayaccording to Skehan et al. (1990). The crude extracts

exhibiting cytotoxicity against cancer cell lines were

further tested for the effective concentration that inhib-

ited 50% of cancer cell growth (EC50). EC50 was the

average of triplicate experiment.

Results and discussion

Collected plants and isolation of endophytic fungi

All 81 species collected were vascular plants and couldbe classified into 40 families and 23 orders (Table 1). All

were flowering plants except for one club moss and one

Table 1. (Continued)

Group Order Family Scientific name Sitea IDb

Euasterids I Gentianales Rubiaceae Anthocephalus chinensis Rich. ex Walp. SK 61

Gardenia sootepensis Hutch. UB 62

Gardenia sp. UB 63

Hymenodictyon excelsum Wall. CM 64Ixora javanica DC. SK 65

Morinda elliptica Ridl. NM 66

Randia wittii Craib NM 67

Apocynaceae Aganosma marginata G. Don CM 68

Alstonia macrophylla Wall. SK 69

Atherolepis pierreiCost. var. glabra Kerr CM 70

Loganiaceae Strychnos kerriiA. W. Hill NM 71

Strychnos nux-blanda A. W. Hill CM 72

Lamiales Oleaceae Jasminum nervosum Lour. NM 73

Linociera microstigma Gagnep. NM 74

Myxopyrum smilacifolium Bl. UB 75

Acanthaceae Thunbergia laurifolia Linn. CM 76

Verbenaceae Clerodendrum paniculatum Linn. SK 77

Euasterids II Apiales Araliaceae Trevesia palmata Vis. SK 78

Asterales Asteraceae Crassocephalum crepidioides S. Moore SK 79Elephantopus scaber Linn. SK 80

Pluchea indica Less. SK 81

a CM Chiangmai; NM Nakornratchasima; SK Songkla; UB Ubonratchathani.b Plant identification number.



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species of the Magnoliid complex, four Monocots and

68 Eudicots.

All collected specimens were found to harbour various

endophytic fungi. This is consistent with previous

reports (Petrini 1991; Saikkonen et al. 1998). Most of

the endophytic fungi did not produce conidia or sporeswhen cultured on common mycological media tested

(i.e., cornmeal agar, malt extract agar, potato dextrose

agar, Sabourauds dextrose agar and yeast extract

sucrose agar). However, they did exhibit characteristic

colony and microscopic morphology that could be used

to differentiate amongst the isolates. Molecular methods

are required for classification of these isolates. There-

fore, identification of these endophytic mycelia sterilia

was not performed in this study. Out of a total of 582

preliminary isolates, 360 morphologically distinct iso-

lates (112 isolates per plant, as shown in Figure 1) were

selected for biological activity screening without

attempting identification.

0

3

6

9

Anti-M. tuberculosis

0

3

6

9

Anti-P. falciparum

0

3

6

9

Anti-HSV-1

0

3

6

9

Cytotoxicity against KB cells

0

3

6

9

12

15

12

15

Cytotoxicity against BC cells

0

3

6

9

12

15

1 3 5 7 9 1113 15 1719 21 2325 27 2931 33 3537 39 4143 4547 4951 53 5557 59 6163 65 6769 71 7375 77 7981

Cytotoxicity against Vero cells

Plant ID

Numberoffungal

endophyteisolates

Numberoffungal

endophyteisolates

Num

beroffungal

endophyteisolates

Numberoffungal

endophyteisolates

Numberoffungal

endophyteisolates

Numberoffungal

endophyteisolates (A)

(B)

(C)

(D)

(E)

(F)

Figure 1. Number of endophytic fungi isolated from each Thai medicinal plant and screened for bioactivities (j, active isolate; (, inactive

1 1 03 2

15

37

1 03 5

10 10

26

0

5

10

15

20

25

30

35

40

45

50

0.0625 3.125 12.5 25 50 100 200

MIC (g ml-1

)Numberofactivecrudeex

tracts(samples)

MCz culture

cultureYES

Figure 2. Number and MICs of active crude extracts with activity

against M. tuberculosis. MCz is malt Czapek broth, YES is yeast

extract sucrose broth.



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In vitro bioactivities of endophytic fungi

Extracts from cultures of the 360 selected endophytic

fungi gave a wide variety of biological activities in six

screening assays (Figure 1). Approximately one-fourth

(26%, 92 isolates) had anti-M. tuberculosis activity(Figure 1A). As shown in Figure 2, 13 of these isolates

(one isolate gave activity in both media and 12 isolates

showed activity in one medium only, either MCz broth

or YES broth) are potential candidates for further

development because despite testing as crude extracts

they are active at MICs in the range of 0.0625

25 lg ml)1 that are comparable to MIC values of

known antituberculosis drugs, e.g. isoniazid (MIC

0.050.2 lg ml)1), rifampin (0.5 lg ml)1), pyrazinamide

(20 lg ml)1), ethambutol (15 lg ml)1) and streptomy-

cin (8 lg ml)1) (Inderlied & Salfinger 1999).

Activity against the malarial parasite P. falciparum

was rare, being found in only six isolates (2%, Fig-ure 1B), with IC50 in the range of 1.29.1 lg ml

)1

(Table 2). These isolates should be studied further in the

anti-malarial compound project. A good number of

isolates (110 or 31%) were active against HSV-1

(Figure 1C) in the Vero cell assay. Among these, 40

isolates showing strong activity could inhibit viral

growth higher than 50% with IC50 in the range of

0.2850 lg ml)1 (Figure 3; 36 isolates exhibited activi-

ties in one medium only, either MCz broth or YES

broth, four exhibited activities in both media). The best

anti-HSV-1 activities found were IC50 of 0.28

1.8 lg ml)1 which are comparable to that of the anti-

viral drug acyclovir (Swierkosz & Hodinka 1999). These

crude extracts with very strong activity will be the

priority to search for anti-viral compound(s).

Activities against KB and BC-1 cancer cell lines were

found in 60 isolates (17%, Figure 1D) and 48 isolates

(13%, Figure 1E), respectively. They were classified into

three groups as strong activity (EC50 0.185.0 lg ml)1),

medium activity (EC50 5.110.0 lg ml)1) and weakactivity (EC50 10.120.0 lg ml

)1). Based on the number

of strongly active crude extract (Figure 4), KB cells were

found to be generally more susceptible to the crude

fungal extracts than BC-1 cells. But the lowest EC50value for BC-1 cells (0.18 lg ml)1) was lower than that

for KB cells (0.42 lg ml)1). Comparing with EC50values of tamoxifen against human breast cancer cells

(i.e., 0.52 lg ml)1 for MCF-7 cell line and 0.93 lg ml)1

for T47D cell line) (Hawariah & Stanslas 1998), the

active isolates with cytotoxicity to BC-1 cells especially

those with strongly active crude extracts will be selected

to study further.Cytotoxicity against Vero cells was found in extracts

of 74 isolates (21%, Figure 1F). A cross-check with the

2

11

0

5

1

3

7

6

3 3

1

2

0

2

4

6

8

10

12

14

0.28-2.0 2.1-5 5.1-10.0 10.1-20.0 20.1-30.0 30.1-50.0

IC50(g ml-1)Numberofactivecrudeextracts(sample

s)

MCz

YES culture

culture

Figure 3. Number and IC50 of active crude extracts with strong anti-

19

15

13

20

12

15

0

5

10

15

20

25

0.42-5.0 5.1-10.0 10.1-20.0Numberofactivecrude

extracts(samples)

A

6

14 13

3

1614

0

5

10

15

20

25

0.18-5.0 5.1-10.0 10.1-20.0

EC50 (g ml-1)

Numberofactivecrudeextracts(samples)

MCz culture

YES culture

B

Figure 4. Number and EC50 of active crude extracts with cytotoxicity

against KB cells (A) and BC cells (B). For abbreviations, see Figure 2.

Table 2. IC50 (lg ml)1) of active crude extracts with anti-P. falciparum

activity.

Endophytic fungus isolate IC50 (lg ml)1)

MCz culture YES culture

Cgre02 7.0 NA

Fhis02 8.0 9.1

Gspe07 3.96 1.2

Gspe11 6.2 NA

Lfle03 1.7 4.4

Stub03 1.6 8.4

NA no activity.



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these isolates did not inhibit cancer cells. On the other

hand, five isolates that were non-toxic to Vero cells did

inhibit KB or BC-1 cancer cells. These could be good

candidates for further study.

Effects of culture medium on in vitro bioactivity

Culture media were found to affect bioactivities

expressed by endophytic fungi both in terms of occur-

rence and intensity. With respect to target organism or

cell, the effect of medium was highly variable and

unpredictable. It was found that 22 isolates of endo-

phytic fungi with anti-M.tuberculosis activity gave

activity in both MCz broth and YES broth, while 37

and 33 isolates were active only in MCz broth and only

in YES broth, respectively. Activity against HSV-1 was

found in the same manner. Both medium cultures of 25

isolates showed anti-HSV-1 activity, whereas 37 and 48

isolates were active only in MCz broth and only in YESbroth, respectively. The numbers of fungi that showed

anti-KB cell activity and anti-BC-1 cell activity in one

medium only, either MCz broth or YES broth, were

equal. They were 13 and 15 isolates, respectively. The

higher numbers were found to be active in both media,

i.e. 34 isolates for anti-KB cell activity and 18 isolates

for anti-BC-1 cell activity.

Level of activity with the two media was also highly

variable and unpredictable with target organisms. For

example, only 22 of the 92 fungal isolates that gave anti-

M. tuberculosis activity in both MCz and YES broths,

11 gave equal activity with one very active isolate at anMIC of 0.0625 lg ml)1. Considering fungal isolates

giving extracts with MIC of 25 lg ml)1, YES broth

gave higher number of fungi with strong activity

(Figure 2). Similar high variations were seen with the

IC50 for P. falciparum (Table 2) and HSV-1 (Figure 3),

and with the EC50 for cancer cells (Figure 4).

It is well known that culture medium can affect the

presence or absence of secondary metabolites and/or

their level of production by fungi (Paterson & Bridge

1994). MCz and YES broths have previously been

recommended as media for production of secondary

metabolites by fungi (Paterson & Bridge 1994) and our

results confirm that they are suitable for screening large

numbers of isolates. On the other hand, more selective

media or optimized custom media might give better

expression for some phenotypes. In addition, the high

differential between the two media suggested that at

least two culture media containing different carbon and

nitrogen sources should be used to screen endophytic

fungi for bioactive compounds.

Conclusion

A high number of endophytic fungal cultures give crudeextracts that exhibit bioactivities. Some of them are

strongly active in vitro against M. tuberculosis, HSV-1

those of respective therapeutic drugs. Thus, Thai medic-

inal plants should be another potential source of

bioactive endophytic fungi. The strong bioactive isolates

will be studied further to isolate and elucidate the active

metabolite(s) and to identify the isolates to various

taxonomic levels.

Acknowledgements

We are grateful to Professor Timothy W. Flegel for his

kind assistance in critically reading the manuscript. We

thank the Bioassay Research Facility of the BIOTEC for

screening in anti-M. tuberculosis activity, anti-viral activ-

ity, cytotoxicity test and anti-malarial activity. The work

was financially supported by the Biodiversity Research

and Training Program (BRT) and the National Center

for Genetic Engineering and Biotechnology (BIOTEC).

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