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

265

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 Danwisetkanjana3, Nijsiri Ruangrungsi4 and Vithaya Meevootisom1 1 Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand 2 Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand 3 National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120, Thailand 4 Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand *Author for correspondence: Tel.: +66-2-2188380, Fax: +66-2-2545195, E-mail: Nongluksna.S@Chula.ac.th
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 [3H]hypoxanthine incorporation method. 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 prole was aected 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 identied 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 problem because 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 Development Agency through the National Center for Genetic Engineering and Biotechnology (BIOTEC) has given high priority to the search for novel bioactive compounds to treat major local diseases including malaria, tuberculosis, mycoses, HSV and cancer. Fungi have been known to be a major source of active compounds used in medicine. Healthy plants are interesting sources

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 rich source 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 (Likhitwitayawuid et al. 1998; Ito et al. 2000; Phrutivorapongkul et al. 2002). The discovery that an endophytic fungus (Taxomyces andreanae) also produced the anticancer drug paclitaxel (Taxol) derived from Pacic yew (Taxus brevifolia) was unexpected (Stierle et al. 1995). This background information led us to speculate that Thai medicinal plants might constitute another source of endophytic fungi with biological activity.

266 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 Ubonratchathani, Nakornratchasima, Chiangmai and Songkla Provinces of Thailand. The collection sites are shown in Table 1. They were identied based on their morphological characteristics. Classication was completed 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 Paralm 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 endophytic 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 a sterile potato dextrose agar (PDA) plate. After incubation at 30 C for 714 days, culture purity was determined 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 ask containing 200 ml of malt Czapek (MCz) broth or yeast extract sucrose (YES) broth (Paterson & Bridge 1994). After incubation at 25 C for 21 days under stationary condition, the fungal culture was ltered to remove mycelium. The ltered 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 freezedried 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 and weighed to constitute the crude mycelial extract. Crude extracts from the culture broth and mycelium were dissolved separately in dimethylsulphoxide (DMSO,

S. Wiyakrutta et al. 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). Briey, 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 nal concentration of 800 lg ml)1. Subsequent serial twofold dilutions were performed in a nal volume of 100 ll in a microplate. 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 incubated at 37 C for further 24 h. Visual minimum inhibitory concentration (MIC) was dened as the lowest concentration 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 presence of fungal crude extract. Briey, 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 concentration 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 to Makler & Hinrichs (1993). Samples that showed antiparasitic activity were further tested for the concentration of extract that inhibited parasite growth by 50%

Bioactive endophytic fungi

Table 1. Classication of Thai medicinal plants collected in this study. Group Club mosses Ferns Flowering vascular plants Basal families Magnoliid complex Order Selaginellales Filicales Uncertain position Piperales Magnoliales Family Selaginellaceae Schizaeaceae Chloranthaceae Aristolochiaceae Magnoliaceae Annonaceae Scientic name Selaginella involuta Spreng. Lygodium exuosum Sw. Chloranthus inconspicuus Sw. Aristolochia tagala Cham. Manglietia garrettii Craib Paramichelia baillonii Hu Polyalthia debilis Finet & Gagnep. Polyalthia parviora Ridl. Uvaria rufa Bl. Amorphophallus campanulatus Bl. ex Decne. Homalomena aromatica Smilax luzonensis Presl Stemona tuberosa Lour. Pachygone dasycarpa Kurz Stephania hernandifolia Walp. Tinospora crispa Miers ex Hook. F. & Thoms. Melientha suavis Pierre Urobotrya siamensis Hiepko Ancistrocladus tectorius Merr. Cissus repanda Vahl Tetrastigma campylocarpum Planch. Erythroxylum cambodianum Pierre Antidesma ghaesembilla Gaertn. Croton oblongifolius Roxb. Mallotus philippensis Muell. Arg. Phyllanthus emblica Linn. Sapium baccatum Roxb. Garcinia thorelii Pierre Mesua ferrea Linn. Casearia grewiaefolia Vent. Bauhinia scandens Linn. var. horseldii K. & S Larsen Erythrophleum teysmannii Craib Dalbergia nigrescens Kurz Dalbergia oliveri Gamble Derris reticulata Craib Ventilago denticulata Willd. Holoptelea integrifolia Planch. Artocarpus lakoocha Roxb. Broussonetia papyrifera Vent. Ficus hispida Linn. Streblus ilicifolius Corner Sonneratia grithii Kurz Melaleuca leucadendron Linn. var. minor Duthie Melastoma malabathricum Linn. Memecylon ovatum J. E. Smith Bombax ceiba Linn. Urena lobata Linn. Hopea odorata Roxb. Shorea obtusa Wall. Shorea roxburghii G. Don Shorea siamensis Miq. Clausena excavata Burm. f. Micromelum minutum Wight & Arn. Paramignya scandens Craib Toddalia asiatica Lamk. Eurycoma longifolia Jack Spondias pinnata Kurz Ardisia lanceolata Roxb. Diospyros lipendula Pierre ex Lecomte Diospyros mollis Gri. Sitea SK CM SK CM CM CM UB NM SK SK SK SK CM CM SK SK NM NM UB CM NM UB CM NM SK CM CM CM CM CM NM NM NM NM NM NM CM NM CM CM NM SK SK SK NM CM SK NM NM NM NM CM UB NM NM SK CM SK SK CM IDb 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

267

Monocots

Alismatales

Araceae

Eudicots (Tricolpates)

Liliales Pandanales Ranunculales

Smilacaceae Stemonaceae Menispermaceae

Core eudicots Caryophyllid clade Rosid clade Eurosids I

Santalales Caryophyllales Vitales Malpighiales

Opiliaceae Ancistrocladaceae Vitaceae Erythroxylaceae Euphorbiaceae

Clusiaceae Flacourtiaceae Fabaceae

Fabales

Rosales

Rhamnaceae Ulmaceae Moraceae

Eurosids II

Myrtales

Lythraceae Myrtaceae Melastomataceae Memecylaceae Malvaceae Dipterocarpaceae

Malvales

Sapindales

Rutaceae

Asterid clade

Ericales

Simaroubaceae Anacardiaceae Myrsinaceae Ebenaceae

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Table 1. (Continued) Group Euasterids I Order Gentianales Family Rubiaceae Scientic name Anthocephalus chinensis Rich. ex Walp. Gardenia sootepensis Hutch. Gardenia sp. Hymenodictyon excelsum Wall. Ixora javanica DC. Morinda elliptica Ridl. Randia wittii Craib Aganosma marginata G. Don Alstonia macrophylla Wall. Atherolepis pierrei Cost. var. glabra Kerr Strychnos kerrii A. W. Hill Strychnos nux-blanda A. W. Hill Jasminum nervosum Lour. Linociera microstigma Gagnep. Myxopyrum smilacifolium Bl. Thunbergia laurifolia Linn. Clerodendrum paniculatum Linn. Trevesia palmata Vis. Crassocephalum crepidioides S. Moore Elephantopus scaber Linn. Pluchea indica Less.

S. Wiyakrutta et al.

Sitea SK UB UB CM SK NM NM CM SK CM NM CM NM NM UB CM SK SK SK SK SK

IDb 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81

Apocynaceae

Loganiaceae Lamiales Oleaceae

Euasterids II

Apiales Asterales

Acanthaceae Verbenaceae Araliaceae Asteraceae

a b

CM Chiangmai; NM Nakornratchasima; SK Songkla; UB Ubonratchathani. Plant identication number.

(IC50) using the [3H]hypoxanthine incorporation method according to Desjardins et al. (1979). The experiment was done in duplicate. Determination of anti-Herpes simplex virus activity and cytotoxicity Crude extracts were tested at a nal 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 medium (MEM; HyClone) were added to a nal concentration of 2 104 cells ml)1 in a volume of 200 ll. Acyclovir (in nal 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 in 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 by performing the experiment without addition of virus. Ellipticine (in nal concentrations of 0.258 lg ml)1) was used as a positive control.

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 nal concentration of 20 lg ml)1. A 10-ll volume of crude extract in 10% DMSO was dispensed into each well of a 96-well microplate. Ellipticine (in nal concentrations of 0.258 lg ml)1) and doxorubicin (in nal 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 modied 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 assay according to Skehan et al. (1990). The crude extracts exhibiting cytotoxicity against cancer cell lines were further tested for the eective concentration that inhibited 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 could be classied into 40 families and 23 orders (Table 1). All were owering plants except for one club moss and one fern. There were one species of the Basal families, six

Bioactive endophytic fungi 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 spores when 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 dierentiate amongst the isolates. Molecular methods are required for classication of these isolates. Therefore, identication of these endophytic mycelia sterilia was not performed in this study. Out of a total of 582 preliminary isolates, 360 morphologically distinct isolates (112 isolates per plant, as shown in Figure 1) were selected for biological activity screening without attempting identication.

269

Number of active crude extracts (samples)

50 45 40 35 30 25 20 15 10 5 0 0.0625 3.125 12.5 25

-1

MCz culture YES culture

37

26

15 10 1 1 1 0 0 3 3 5 2 10

50

100

200

MIC (g ml )
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.

15 12 9 6 3 0

Anti-M. tuberculosis
(A)

Number of fungal endophyte isolates

15 12 9 6 3 0

Anti-P. falciparum
(B)

Number of fungal endophyte isolates

Anti-HSV-1
Number of fungal endophyte isolates

(C)
9 6 3 0

Cytotoxicity against KB cells

Number of fungal endophyte isolates

(D)
9 6 3 0

Cytotoxicity against BC cells

Number of fungal endophyte isolates

(E)
9 6 3 0

Number of fungal endophyte isolates

15 12 9 6 3 0

Cytotoxicity against Vero cells

(F)

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81

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

270
Number of active crude extracts (samples)

S. Wiyakrutta et al.
25
A

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 streptomycin (8 lg ml)1) (Inderlied & Salnger 1999). Activity against the malarial parasite P. falciparum was rare, being found in only six isolates (2%, Figure 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 activiTable 2. IC50 (lg ml)1) of active crude extracts with anti-P. falciparum activity. Endophytic fungus isolate IC50 (lg ml)1) MCz culture Cgre02 Fhis02 Gspe07 Gspe11 Le03 Stub03 NA no activity. 7.0 8.0 3.96 6.2 1.7 1.6 YES culture NA 9.1 1.2 NA 4.4 8.4

20 15 10 5 0

19

20

15 12 13

15

0.42-5.0
Number of active crude extracts (samples)

5.1-10.0

10.1-20.0

25
B

MCz culture YES culture

16 14 13 14

20 15 10
6

5 0

0.18-5.0

5.1-10.0

10.1-20.0

EC50 (g ml-1 )
Figure 4. Number and EC50 of active crude extracts with cytotoxicity against KB cells (A) and BC cells (B). For abbreviations, see Figure 2.

14 12 10 8 6 4
2 3 7 6 5 3 1 1 0 3 2 11

MCz culture YES culture

2 0 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

Figure 3. Number and IC50 of active crude extracts with strong antiHSV-1 activity. For abbreviations, see Figure 2.

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 antiviral 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 classied into three groups as strong activity (EC50 0.185.0 lg ml)1), medium activity (EC50 5.110.0 lg ml)1) and weak activity (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 EC50 value for BC-1 cells (0.18 lg ml)1) was lower than that for KB cells (0.42 lg ml)1). Comparing with EC50 values 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 results for KB and BC-1 cancer cells revealed that 12 of

Number of active crude extracts (samples)

Bioactive endophytic fungi these isolates did not inhibit cancer cells. On the other hand, ve isolates that were non-toxic to Vero cells did inhibit KB or BC-1 cancer cells. These could be good candidates for further study. Eects of culture medium on in vitro bioactivity Culture media were found to aect bioactivities expressed by endophytic fungi both in terms of occurrence and intensity. With respect to target organism or cell, the eect of medium was highly variable and unpredictable. It was found that 22 isolates of endophytic 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 YES broth, 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 antiM. tuberculosis activity in both MCz and YES broths, 11 gave equal activity with one very active isolate at an MIC 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 aect 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 conrm 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 dierential between the two media suggested that at least two culture media containing dierent carbon and nitrogen sources should be used to screen endophytic fungi for bioactive compounds.

271 those of respective therapeutic drugs. Thus, Thai medicinal 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 activity, cytotoxicity test and anti-malarial activity. The work was nancially supported by the Biodiversity Research and Training Program (BRT) and the National Center for Genetic Engineering and Biotechnology (BIOTEC).

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