Extraction of plant materials for antibacterial activity. P. integerrima leaves, barks, gall were collected from local market and first shade dried and then grinded in Mortar and pestle. Plant material was collected, stored and processed following standard protocols by WHO and institutional, national, and international guidelines and legislations, therefore no risks were associated in any steps of handling to disposal. For extraction purpose cold maceration technique was used16. Powdered plant material 10gm was individually macerated in 100mL of distilled H2O, methanol and ethanol and kept for 24 hours on a rotary shaker. These extracts then filtered 4-5 times through Whatman filter paper till all soluble compounds were completely extracted. Then the evaporation of extract was carried out by using rotary evaporator (Buchi Rotavapor R-300, Japan) and dried residues were used to check the antibacterial efficacy.
Bacterial culture collection. Different bacterial pathogens were obtained from Ayub Medical Complex lab Abbottabad including Staphylococcus aureus, E. coli, Proteus vulgaris, Bacillus subtilis, Pseudomonas aeruginosa. Liquid inoculums were prepared by adding pure bacterial strains in 10mL normal saline and used further.
Agar well diffusion method used to evaluate the antibacterial efficacy of the different parts of plant. Mueller Hinton agar (Oxoid) plate surface was inoculated by spreading 50 µL inoculums, McFarland 0.5 BaSO4 standard used for balancing the turbidity of used inoculum as 108 CFU mL-117. Then 8mm borer used to make 5 holes aseptically on agar plate. In 4 wells 50 µL of leaf, bark and gall extracts (in concentration of 200, 150, 100, and 50µL) poured. For negative control pure dimethyl sulfoxide (DMSO) poured in one agar well and disc of Ciprofloxacin taken as positive control in the center of plate and all agar plates were incubated for 24 hours at temperature 37oC18. The inhibitory effects measured by the zones of inhibition in mm around the wells.
Extraction and fractionation of plant materials for anticancer activity. Shade dried leaves, galls and barks of P. integerrima soaked with methanol for one week at room temperature. By using rotary evaporator, methanolic extract of each portion of plant was evaporated at 40oC temperature. Then for the process of liquid-liquid partitioned in separatory funnel, the crude methanolic extract of each portion of plant was suspended in water and fractionated with n-hexane, chloroform, ethyl acetate in sequenced manner, to get their respective fractions13. All standard procedures were adopted and experiment performed in fume hood16.
Amount of 10mg of each crude and fractionated extracts of P. integerrima bark, gall and leaves in hexane, chloroform and ethyl acetate carefully weighed in 1.5ml Eppendorf tubes and dissolved in DMSO. After vortex mixing, 1 mg of each of the crude extract and fraction was used in cancer cell culture.
Anticancer activity through cell viability assay. Plant extracts along with their subsequent fractions evaluated for their anti-proliferative activity against HeLa and BHK-21 by utilizing the dimethyl–2–thiazolyl–2,5–diphenyl–2H–tetrazolium bromide (MTT) based cell metabolic assay19, 20. After sub-culturing, grown cell colonies were harvested using proteolytic trypsin enzyme. The cellular content along with sufficient culture media was transferred to a 15 ml conical tube and centrifuged for 5 minutes at 150 speed and 37°C. After careful removal of supernatant, cell pellet dissolved in 1ml culture media. Cells counted by pipetting out a volume of 10 µL from cell suspension and dropped onto Neubauer counting chamber to count cells under microscope. Cell suspension diluted accordingly to make 2.5x104 cells mL-1, from this suspension with the help of a multi-channel micro-pipette 90 μL added in each well of 96-wells microliter plate and left for 24 hours incubation in a cell culture incubator set at 5% CO2 and 37˚C. Each plant extracted sample poured into these wells at 100 µg/mL of final concentration and 10 μL of media was used in control well. Carboplatin at a final concentration of 100 µM was used as a standard for cell lines. For IC50 determination, three-fold dilutions used and 10 μL of MTT (5mg/mL PBS) after 24 hours placed in all wells. After incubation of 4 hours in a cell culture incubator 100 µL of stopping reagent (isopropanol and 10% sodium dodecyl sulfate) poured to solubilize formazan crystals and kept at room temperature with subsequent agitation for half an hour. Absorbance recorded using a 96–well microtiter plate reader (Bio–TekELx 800TM) at 570 nm, background signal (690 nm) was subtracted and results were evaluated as inhibition values percentage.
Analysis of active ingredients by GC-MS. Identification and separation of various extracts of plant were carried out on Perkin Elmer Clarus 600 gas chromatograph (GC) connected with mass spectrometer (MS). Plants extracts dissolved in respective organic solvents with concentration of 5mg.mL-1 and 1µL was injected to GC-MS and protocol was adopted as described by Shah et al., (2016)21. Different separated compounds identified by comparing their mass spectra to the Finnigan NIST-05 (National Institute of Standard and Technology, USA) Mass spectrum library and available literature. Relative concentration of each identified compound found in injected sample was determined by comparing peak area of the compound to the sum of all peaks’ area in a total ion current (TIC) chromatogram.
Statistical Analysis. Two factor ANOVA with replication applied by using Data analysis Tools of MS Excel 2010. It was used for the analysis of significance difference (at p > 0.05) among zone of inhibition by using different doses and different types of solvent extracts. PRISM 5 software used for the determination of IC50 value.