Instrument For column chromatographic technique, column grade G60 (60-120 mesh with 0.04-0.063 mm particle size, Ar. 7734, ASTM, Merck-Germany) silica gel was used as stationary phase. For 1 H and 13C NMR spectra recording, a Bruker 400 MHz spectrometer were used taking deuterated chloroform (CDCl3) as the internal standard. Preparation of media was done under a Laminar flow (Biological Safety Cabinet; Thermo Forma. Class 11 A1). HIRAYAMA autoclave (Hirayama Mfg. Corp.) was used for media sterilization.
Collection of plant and sterilization process Young medicinal plant Gynura procumbens was collected from local nursery of Dhaka, Bangladesh. Different plant parts were cut into small fragments. The surface of each plant barks was sterilized using 70% EtOH, 3% NaOCl (sodium hypochlorite), and sterile H2O (Schulz et al., 1993). The clean plant parts were successively kept into respective solution for 3 min. The surface sterilization efficiency for every segment of tissue was confirmed by imprint method (Schulz et al., 1993). Potato-carrot agar medium was used for culture and growth of endophytic fungi. The medium was prepared using grated potato and carrot (200 g/L each) boiled for 30 min. The boiling mixture was cooled and smashed. The extract was adjusted to a volume of 1L by the addition of water. 17.5 g/L agar was used for the solidification of media.
Isolation of endophytic fungi The plant parts obtained from surface sterilization process were inoculated in potato-carrot agar media autoclaved on sterilized petri dish. After 20 days of inoculation period, three fungal strains from the barks were isolated. In the replication process of common fungi, one strain (Sh-1) was found to show optimum growth for further cultivation. This fungal strain isolated from bark showed maximum growth in minimum time and solvent extract showed spots which were well separated in thin layer chromatography (TLC). Sh-1 was chosen for chemical and biological screening.
Culture extraction Sh-1 was fermented in large scale (3 inch ×3 inch, 5×100 petri dishes, 20 days, room temperature) at potato-carrot agar semi-solid medium. The fungal broth from 500 Petri dishes was homogenized using an Ultra-Turrax. The resultant mixture was extracted with EtOAc (ethyl acetate). The EtOAc was collected by filtration and the filtrate was evaporated to dryness.
Antibacterial Activity assay Sh-1 fungal strain extract was studied for antibacterial activity against six bacteria- Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Bacillus coccus, Bacillus subtilis and Clostridium tetani in agar disc diffusion assay. Standard tetracycline was used as positive control. The zones of inhibition produced by compounds and tetracycline were recorded in mm and compared. 7-9 mm, 10-12 mm, 13- 15 mm, and above 15 mm zone of inhibition were considered as insignificant, mild, moderate and significant activity, respectively (Rob et al., 2005).
Antifungal activity assay Crude fungal extracts of the plant G. procumbens were tested for antifungal activity using disc diffusion method. The crude fungal extract of ethyl acetate was tested for antifungal activity against fungi Pseudomonas aeruginosa. All the extract was tested at 400 µg/disc concentration.
Chemical studies of the extract The crude extract (600 mg) was dissolved in n-hexane and silica gel was added to it. A column (60 cm) was packed with normal phase G-60 silica gel using n-hexane as the equilibrating solvent. The dried extract mixed with silica gel was carefully applied to the top of the column and initial elution was bring out with n-hexane. After the application of sample, solvent having increasing polarities from 100% n-hexane to 100% ethyl acetate (EtOAc) was added. After running pure EtOAc, mixture of EtOAc and methanol was run. The eluted fractions were collected in test tubes, monitored by TLC and on the basis of Rf values, eleven different fractions (F-1 – F11) were obtained. Among the fractions, the F-3 portion which was eluted with 20% EtOAc (n-hexane/ethyl acetate 8:2) gave single spot in TLC with a Rf value ~0.3 (n-hexane/ethyl acetate 8:2) and obtained as pure compound 1 (S-1) (10 mg).
Properties of isolated compounds Compound 1 (S-1): white needles and crystalline (10 mg), IR (KBr): ? 3428, 2955, 1652, 1458, 1370, 1055, 960, 840 cm-1. 1 H NMR (400 MHz, CDCl3): δ ppm 0.62 (3H, s, H-18), 0.80 (3H, d, H-26), 0.82 (3H, d, H-27), 0.91 (3H, d, H-28), 0.93 (3H, s, H-19), 1.03 (3H, d, H21), 3.63 (1H, bm, H-3α), 5.16 (1H, dd, H-22), 5.19 (1H, dd, H-23), 5.37 (1H, m, H-7) and 5.56 (1H, dd, H-6). 13C NMR (100 MHz, CDCl3): δ ppm 38.39 (C-1), 31.98 (C2), 70.48 (C-3), 40.79 (C-4), 139.79 (C-5), 119.61 (C-6), 116.30 (C-7), 141.37 (C-8), 46.26 (C-9), 37.04 (C-10), 21.12 (C-11), 39.10 (C-12), 42.84 (C-13), 54.57 (C-14), 23.01 (C-15), 28.31 (C-16), 55.74 (C-17), 12.07 (C-18), 16.29 (C-19), 40.45 (C-20), 21.12 (C-21), 135.58 (C-22), 131.99 (C-23), 42.84 (C-24), 33.10 (C-25), 19.97 (C-26), 19.67 (C-27) and 17.63 (C-28).