Source of endophytic fungi
Plant materials were obtained from the National Park, Pahang, Malaysia in June, 2007. Two different locations, Kuala Keniam (KK) and Kuala Trenggan (KT), where medicinal plants could be found in abundance were selected for sampling. Chosen parts from individual plants were collected and stored at 4°C until used. All plant samples were identified by Kamaruddin Saleh of the Forest Research Institute of Malaysia (FRIM) and were deposited in the herbarium at the Faculty of Pharmacy, Universiti Teknologi MARA, Shah Alam, Malaysia.
Isolation of endophytic fungi
Isolation of endophytes from the 43 plant samples was carried out as described by Strobel et al.,  but with minor modifications. Plant samples, which included leaves, stems, roots, rhizomes, flowers, fruits and bark, were washed under running tap water for 10 min followed by immersion in 70% EtOH for 1 min and in NaOCl (2.5% - 5.25%) for 3 min, drained and immersed in 70% EtOH again for 30 sec. Finally, the samples were rinsed with sterile d.H2O. Each plant sample was cut aseptically into 1 cm long segments. The cut surfaces of the segments were placed on petri dishes containing potato dextrose agar (PDA) (Oxoid) supplemented with chlortetracycline HCL (50 μg/ml, Sigma) and streptomycin sulphate (250 μg/ml, Sigma) at 28°C. Pure cultures were then transferred to PDA plates free of antibiotics and maintained in the culture collection of the Collaborative Drug Discovery Research (CDDR) Group, UiTM, Malaysia. For investigations of biological activity, the endophytes were cultivated for 14 days on PDA plates at 28°C.
Semipolar extraction of fungal cultures
Crude endophytic extracts were prepared as described by Lang et al.,  but with slight modifications. Endophytic cultures (five plates per fungus) were homogenized and transferred to a 500 ml conical flask filled with 250 ml EtOAc (Merck) and left to stir overnight at room temperature. The mixture was filtered through Whatman No.1 filter paper, after which Na2SO4 (40 μg/ml, Merck) was added to further remove the aqueous layer within the mixture. The mixture was then transferred to a round bottom flask and dried using a rotary evaporator. The resultant extract was dissolved in 1 ml of dimethyl-sulfoxide (DMSO) (Sigma) and kept at 4°C as stock solution.
Human chronic myeloid leukemic, K562 (ATCC CCL - 243), murine leukemic, P388 (ATCC TIB 63), human colorectal carcinoma, HCT116 (ATCC CCL - 247) and human breast adenocarcinoma, MCF7 (ATCC HTB - 22) cell lines were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA. All cell lines were cultured in RPMI 1640 (Sigma) supplemented with 10% heat inactivated fetal bovine serum (FBS) (PAA Laboratories) and 1% penicillin/streptomycin (PAA Laboratories). Cultures were maintained in a humidified incubator at 37°C in an atmosphere of 5% CO2.
Cytotoxicity of extracts at various concentrations (0.01 - 100 μg/ml) was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) (Sigma) assay, as described by Mosmann, 1983  but with minor modification, following 72 h of incubation. Assay plates were read using a spectrophotometer at 520 nm. Data generated were used to plot a dose-response curve of which the concentration of extract required to kill 50% of cell population (IC50) was determined. Cisplatin (Mayne Pharma) and tamoxifen (Dynapharm), which are both established chemotherapeutics, were used for comparison. Cytotoxic activity was expressed as the mean IC50 (± standard deviation) of three independent experiments.
The crude extracts of the 300 endophytic fungi were tested against Bacillus subtilis (ATCC 6633), Micrococcus luteus (ATCC 10240), Staphylococcus aureus (ATCC 25923), Escherichia coli ( ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). Antibacterial activity was determined using the disc diffusion method according to the National Committee for Clinical Laboratory Standards (NCCLS) . Pre-warmed Mueller-Hinton agar (MHA) (Oxoid) plates were seeded with 107 - 108 cfu suspension of test bacteria. Endophytic extracts (10 μl) dissolved in DMSO (1 mg/ml) were pipetted (10 μl) onto sterile paper discs (6 mm diameter, Oxoid) and placed onto the surface of inoculated agar plates. Gentamicin sulphate (10 μg, Oxoid) was used as the positive control. Plates were incubated at 37°C for 48 h. Antibacterial activity was expressed as the diameter of the inhibition zone (mm) produced by the extracts.