BMC Complementary and Alternative Medicine BioMed Central

Background: Endophytes, microorganisms which reside in plant tissues, have potential in producing novel metabolites for exploitation in medicine. Cytotoxic and antibacterial activities of a total of 300 endophytic fungi were investigated.

thesized by Pestalotiopsis microspora, a fungus that colonizes the Himalayan yew tree, interest in studying such endophytes for their medicinal potential has grown tremendously [3]. To date, endophytes have been most extensively studied for their ability to produce antibacterial, antiviral, anticancer, antioxidants, antidiabetic and immunosuppressive compounds [1]. Their study is expected to become an important component in the production of new natural bioactive products.
Only a few studies on endophytic fungi from Malaysian plant species have been conducted so far. The current study was undertaken to investigate this biodiversity and to isolate and screen endophytic fungi with cytotoxic and antibacterial activities from medicinal plants collected from two locations in the National Park, Pahang, Malaysia.

Isolation of endophytic fungi
Isolation of endophytes from the 43 plant samples was carried out as described by Strobel et al., [4] 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.H 2 O. 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., [5] 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 Na 2 SO 4 (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.
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 [6] 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 (IC 50 ) was determined. Cisplatin (Mayne Pharma) and tamoxifen (Dynapharm), which are both established chemotherapeutics, were used for comparison. Cytotoxic activity was expressed as the mean IC 50 (± standard deviation) of three independent experiments.

Antibacterial activity
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) [7]. Pre-warmed Mueller-Hinton agar (MHA) (Oxoid) plates were seeded with 10 7 -10 8 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.

Results and discussion
A total of 300 endophytes were isolated from 43 plants found at two different locations (Kuala Keniam and Kuala Trenggan) within the National Park, Pahang, Malaysia (Table 1). Of the total endophytes obtained, 70.0% were isolated from plants at Kuala Keniam, and the remaining from Kuala Trenggan. Relatively greater distribution of endophytes was found within leaf (48.7%), stem (25.7%) and root (16.3%) samples compared to other segments (9.3%, including flower, fruit, rhizome and bark) of the plants. Ardisia colorata (laloh, local name) was found to Cytotoxicity of the extracts against P388 and K562 cell lines is shown in Table 2. Generally, the extracts were found to be more effective against P388 than the K562 cell line. Nearly half (47.6%) of the extracts showed activity (IC 50 of < 10 μg/ml) against P388 compared with 25% active against K562. These values were within the cutoff point of the National Cancer Institute criteria for cytotoxicity (IC 50 < 20 μg/ml) in the screening of crude plant extracts [8].

Conclusion
In conclusion, this preliminary screening of rainforest fungal endophytes revealed their potential to yield potent bioactive compounds for drug discovery programmes. Extract KK29FL1, a Sporothrix sp., showed very potent cytotoxic effect indicating its possible potential for development as an anti-cancer drug and warrants further investigation.