Phytochemical characterization, anti-cancer and antimicrobial activity of isolated fractions

Materials and Methods

Microorganisms

The bacterial and fungal strains used for evaluation of antimicrobial activity were Escherichia coli (ATCC 10535), Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Pseudomonas aeruginosa (ATCC 27853), Proteus vulgaris (ATCC 13315), Candida albicans (ATCC 10231), Aspergillus niger (ATCC 16404) and A. flavons (ATCC 15517) from the stock cultures of microorganisms (Department of Microbiology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad). Sabouraud 2% (w/v)-glucose agar, Muller–Hinton agar and Muller–Hinton broth were supplied by the Merck (Germany) and RPMI 1640 broth by the Institute of Immunology.

Herbal material

The aerial parts of A. vaginalis was collected in August-September, from the Aurangabad, Maharashtra, India. The plant was identified and authenticated at the Dr. Babasaheb Ambedkar Marathwada University, Aurangabad and plant specimen was submitted to the Botany Department of the University (Accession No. 0607). The crude material was dried in the shade for five days and then powdered with the help waring blender.

Extraction and phytochemical analysis

The dried powdered plant material was successively extracted with various solvents like petroleum ether (40-60°C), chloroform, methanol and water. The four successive extracts, thus, obtained were labeled as petroleum ether extract (PE), moderately polar chloroform (SCH), polar methanol (MEE) and aqueous extract (SAE). The chemical profile of plant revealed the presence of secondary metabolites such as steroids, flavonoids, alkaloids, triterpenoids and phenolics which were identified with the help of general chemical tests (Khandelwal and Sethi, 2010).

Fractionation

The methanol extract, rich in polyphenolic compounds was selected for fractionation. Fractionation had been achieved by column chromatography using different polarities of solvents such as chloroform, n-butanol, and acetone successively to get chloroform, n-butanol and acetone fractions respectively. The residue remaining after acetone fractionation was dissolved in methanol to get methanol soluble fraction (MSF). The flowchart of fractionation is shown in Figure 1.

Figure 1: Flowchart of fractionation

In vitro cell lines study by SRB assay

In vitro anti-cancer activity has been tested by sulforhodamine B, SRB method (Skehan et al., 1990; Vanicha and Kanyawim, 2006). The cell lines were grown in RPMI 1640 medium containing 10% fetal bovine serum and 2 mM L-glutamine. For the present screening experiment, cells were inoculated into 96-well microtiter plates in 100 µL at plating densities which depended on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37°C, 5% CO₂, 95% air and 100% relative humidity for 24 hours prior to the addition of fractions.

Fraction samples were initially solubilized in dimethyl sulfoxide at 100 mg/mL and diluted to 1 mg/mL using water and stored frozen prior to use. At the time of drug addition, an aliquote of frozen concentrate (1 mg/mL) was thawed and diluted to get the required final concentrations i.e.10, 20, 40, and 80 μg/mL.

After compound addition, plates were incubated at standard conditions for 48 hours and assay was terminated by the addition of cold trichloroacetic acid (TCA). Cells were fixed in situ by the gentle addition of 50 µL of cold 30% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4°C. The supernatant was discarded; the plates were washed five times with tap water and air dried. SRB solution (50 µL) at 0.4% (w/v) in 1% acetic acid was added to each of the wells, and plates were incubated for 20 min at room temperature. After staining, unbound dye was recovered and the residual dye was removed by washing five times with 1% acetic acid. The plates were air dried. Bound stain was subsequently eluted with 10 mM trizma base, and the absorbance was read on a plate reader at a wavelength of 540 nm with 690 nm reference wavelength. The activity results of sample were compared with the reference compound adriamycin.

Percent growth was calculated on a plate by plate basis for test wells relative to control wells. Percent growth was expressed as the ratio of average absorbance of the test well to the average absorbance of the control wells.

Using the six absorbance measurements [time zero (Tz), control growth (C), and test growth in the presence of drug at the four concentration levels (Ti)], the percentage growth was calculated at each of the drug concentration levels. Percentage growth inhibition was calculated as:

[Ti/C] x 100%

Antimitotic activity

The antimitotic activity had been evaluated by using Allium cepa root (Saboo et al., 2012). A. cepa bulbs were sprouted in water for 48 hours at room temperature. The bulbs with uniform root were selected for the study. These roots were dipped in the fraction (1 mg/mL) for 3 hours and distilled water was used for dilution as well as a blank. The methothrexate was used as a reference substance for study. After 3 hours, the root tips were fixed in the fixing solution of acetic acid and alcohol (1:3). The squash preparation was made stain by acetocarmine solution. The mitotic index was calculated by following formula:

Mitotic index = Number of dividing cell X 100/ Total number of cells

Antiproliferative activity

For the antiproliferative assay, yeast Saccharocymes cerevisiae was used (Saboo et al., 2012). The yeast was inoculated with sterilized potato dextrose broth and incubated at 37°C for 24 hours and referred to as seeded broth. The seeded broth was diluted with sterilized distilled water, in order to get 25.4 x 10⁴ cells (average). For the cell viability count, solution containing 2.5 mL of potato dextrose broth and 0.5 mL of yeast inoculums were prepared in four separate test tubes. In the first four tubes, 1 mL of fractions was mixed while in the fifth tube, standard methothrexate was added. The sixth tube was kept as control. All the tubes were incubated at 37°C. After incubation, 0.1% methylene blue dye was added in all tubes containing cell suspension and observed under microscope. In the microscope, viable cells were observed as non-stained, transparent with oval shape, while dead cells get stained and appeared blue in color. The average number of cells was counted in the hemocytometer.

Antibacterial and antifungal testing

The disk diffusion assay was carried out according to Bauer et al. (1966) for the evaluation of antibacterial activity. The fresh cultures of microbial strains were used for preparation of inoculums, which aseptically cultured at 37°C on Sabouraud 2% (w/v) glucose agar medium for 18 hours and for fungi Sabouraud dextrose agar for 48 hours with saline. The agar plates were prepared approximately 4-5 mm of depth. On the solid media plates, about 100 μL suspension of bacteria was spread and sterile disks of filter paper were impregnated with 50 μL (1 mg/mL) of the fraction and then placed on inoculated plates. These plates were incubated for 24 hours at 37°C and 48 hours at 30°C for bacteria and fungi respectively. For positive control, standard tetracycline disks for bacteria and standard ketaconozol disks for fungi, respectively, were used in the same concentration. The zone of inhibition in mm was measured by the antibiotic zone reader.

Minimal inhibitory concentration

The antimicrobial activity of the fractions was determined in Muller–Hinton broth by 2-fold dilution method for the bacterial strains and RPMI 1640 for the yeast and mould (Bauer et al., 1966) and MICs were read in mg/mL after overnight incubation at 37°C. All experiments were made in triplicate.

HPLC analysis of fractions

HPLC is an important tool in qualitative analysis of the complex mixture or plant extracts.  The presence of important phytoconstituents in the sample was identified by using marker compounds (Sigma-Aldrich Chemie, Germany). Shimadzu HPLC system, a Luna C18 reverse-phase column (250 x 4.6 mm, i.d. particle size 5 µm) with LC-10AT, UV detector (Spectra System UV1000) was used. The analytical parameters such as mobile phase, wavelength, and flow rate for individual constituent have been given in Table I.

Table I: HPLC method for different phytoconstituents