Fresh plant materials were collected from the Indira Gandhi Agricultural University, Sarkanda, Bilaspur, Chhattisgarh, India, in August 2015. The plants were identified and authenticated by the Department of Botany, V.Y.T.P.G. Autonomous College, Durg, Chhattisgarh, India. The plant materials, stems and leaves, were separated and air dried. The dried stems and leaves were powdered and extracted with methanol by using a Soxhlet apparatus for 72 hours (h) at 55 - 60°C. The extract was filtered and concentrated under vacuum in a rotary evaporator at a temperature of less than 50°C. Methanol was then added to increase the volume of the concentrated extract to 100 mL. For the cytotoxic evaluation, the whole plant was extracted with methanol by using the procedure described above, after which the concentrated extracts were lyophilized and kept at 4°C until use.
The bacterial strains were obtained from the Microbial-type Culture Collection, Chandigarh, India. The bacterial strains studied for antibacterial activity were Escherichia coli (E-coli) (MTCC 739), Staphylococcus aureus (S. aureus, MTCC 2940), Micrococcus luteus (M. luteus) (MTCC 2470), Bacillus subtilis (MTCC 121), Staphylococcus aureus sub sp aureus (S.aureus Sub.aureus, MTCC 96), and Pseudomonas aeruginosa (MTCC 2453). The fungal strains were obtained from the GD Rungta College of Pharmaceutical Science & Research, Bhilai, Chhattisgarh, India. The fungal strains studied for antifungal activity were Botrytis cinerea (KACC 43524), Fusarium oxysporum (F. oxysporum) (KACC 42109), Penicillium expansum (P. expansum) (KACC 40815), Penicillium chrysogenum (KACC 40399), Rhizoctonia solani (KACC40136), Fusarium moniliforme (KACC 41031), Geotrichum candidum (OKI 605/8402), Candida albicans (C. albicans) and Yarrowia lipolytica (Y. lipolytica) (KACC 41237).
Agar well diffusion was employed for the antibacterial assay of the plant extracts [4, 5]. A loop full of the bacterial strain was inoculated in 5 mL of each nutrient broth in a test tube and incubated on a rotary shaker at 140 rpm for 24 h. Mueller Hinton Agar no. 2 was prepared for the study. The test bacterial strain (1 mL) was inoculated into the media (inoculums size: 108 cells/mL) when the temperature had reached 40 - 42°C, and care was taken to ensure proper homogenization. The experiment was performed under strict aseptic conditions. After the medium had solidified, a well was made in the plates with the help of a cup-borer (0.85 cm). The test compound was introduced into the well, and the plates were incubated for 24 h at 37°C. Microbial growth was determined by measuring the diameter of the zone of inhibition. Methanol was used as the control. The control activity was deducted from the experimental activity, and the result obtained was plotted. Experiments were performed in triplicate.
For the antifungal activity, the agar well bioassay using PDA medium (Hi- Media, 39 g) was employed [6, 7]. Fungi were inoculated, and the procedures were similar to those mentioned above. The treated fingi and the controls were kept in an incubator at 37°C for 24 to 72 h, and the zones of inhibition were measured. Three to four replicates were maintained for each treatment.
The cytotoxicity assays were performed using the MTT method . Human colon cancer cells (HCT-116) and leukemia cells (K-562) were used for the MTT assay. The cells were harvested (1 × 105 cells/well) and inoculated into 96- well microtiter plates. The cells were washed with phosphate buffered saline (PBS), and the cultured cells were then incubated for 72 h with the test sample at a final concentration of 25 - 800 μg/mL. One hundred μL of 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg Ml-1 in PBS, pH 7.2) were added to each well, and plates were incubated for 4 h at 37°C. After incubation, 200 μL of dimethyl sulfoxide (DMSO) were added to each well, the absorbance of each well was measured at 570 nm by using a microplate reader, and the surviving cell fraction was calculated. The inhibition of cell viability was calculated using % cytotoxicity = (1-[absorbance of treated cells/absorbance of untreated cells]) × 100.