Evaluation of Antimicrobial, Antioxidant and Anticancer Activity of Selected Indegenous Medicinal Plants of Pakistan

Abstract

From the last few decades, herbal products have been recognized globally. Plants have a major role in human’s life as they maintain and treat numerous diseases of both animals and humans. Medicinal plants are the major source of components that can be used in drug industry. Medicinal plants have exhibited potent antibacterial, antifungal, antiviral and anticancer activities. Currently, natural products are a part of more than half of all the recent drugs in use as they are cost effective and safe to use as compared to synthetic medicines. As medicinal plants are gaining importance day by day, eight medicinal plants have been selected to estimate biological potential of crude extracts. These include Portulaca grandiflora, Cynodon dactylon, Oxalis corniculata, Boheravia diffusa, Geranium wallichianum, Melia azedarach, Lawsonia inermis, and Paeonia emodi wall. Different polar and non-polar solvents were used to prepape extracts of these plants, and evaluated for their antibacterial, antifungal, antioxidant and anticancer activities. Folin Ciocalteo and aluminium chloride methods were used to find out the total phenolic and flavonoid contents respectively. Three common methods were used to evaluate the antioxidant property of selected medicinal plants including 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing power assay and phosphomolybdenum based total antioxidant capacity. Antibacterial activity was determined by disc diffusion method against six multi drug resistant strains and two sensitive reference strains. Antifungal activity was measured by agar disc diffusion assay against five fungal pathogenic strains. These crude plant extracts were also estimated for their anticancer potential against three cancer and one normal cell line including Hep2 (hepatic cancer), DU145 (prostate cancer), MDA-MB 231 (breast cancer) and MCF-10A (normal breast cell line). Further, the most active extracts were used to estimate their role in apoptosis via flow cytometry, annexin V/FITC and DNA frangmentation assay. For prevention of metastatic cancer crude extracts of plants were investigated for their wound healing and invasive potential, and also for NF-ĸB signaling pathway through Western blotting of apoptotic genes. Selected plant extracts were active against multidrug resistant bacterial strains, as maximum zone of inhibition was observed in ethanolic extract of G. wallichianum against S. typhi (43.3±1.5 with MIC of 25 µg/ml) and second highest in methanolic extract of P. grnadiflora against P. aeruginosa (36±1 with MIC of 25 µg/ml). Highest zone of inhibition against fungus was obtained from methanolic extract of P. grandiflora and ethanolic extract of C. dactylon (18±1 and 18±1.4 with the MIC of 50 µg/ml) against C. albicans. Maximum flavonoid and phenolic contents were obtained from alcoholic (methanloic and ethanolic) extracts while from polar extracts i.e. n-hexane phytochemicals were least extracted. Methanolic extracts of P. grandiflora contained highest amount of phenolic contents (89 µg GAE/mg) while lowest in n-hexane extracts of O. corniculata and P. grandiflora (15 µg GAE/mg). Highest amount of flavonoids were found in methanolic extracts of M. azedarach and B. diffusa (72 µg QE/mg). Maximum DPPH scavenging activity was estimated in ethnolic extract of O. corniculata (IC50 of 38 µg/ml) while second highest for methanolic extract of P. grandiflora (IC50 of 40 µg/ml). Reducing power of methanolic extract of O. corniculata was 2nd highest (11.5 µg AAE/mg) while highest reducing power was obtained in methanolic extracts of P. emodi (15.29 µg AAE/mg). Polyphenols in high amount in selected plant extracts have shown high antioxidant potential thus showing a positive correlation between phenolics and free radical scavenging power of plants. P. grandiflora exhibited highest anticancer potential by its methanol (PGM) and nhexane (PGH) extracts against MDA-MB 231 breast cancer cell line with lowest IC50, thus P. grandiflora was selected for further analysis on MDA-MB 231 cells. As P. grandiflora inhibit the breast cancer cells, this inhibition was connected with the cell cycle arrest in its early phases (G0/G1), consequently apoptosis was induced by high levels of annexin V+ve cells and aggregation of cells in Sub-G1 population. Thus both the extracts PGM and PGH inhibited the proliferation and induced apoptosis through inhibition of NF-ĸB pathway and its related apoptotic genes (XIAP, Bcl-2, Bcl-xL, survivin and cyclin D1). Caspase-3 activation through PGM and PGH also indicated the persuasion of apoptosis. It was observed that PGM and PGH repudiate IĸBα phosphorylation and constitutive NF-ĸB activation in MDA-MB 231 cells. Furthermore, PGM and PGH also prevent the metastatic ability of MDA-MB 231 cells evaluated through wound healing and invasion assay. Present study concludes that selected medicinal plants of Pakistan have strong antimicrobial, antifungal, antioxidant and anticancer potential; these plants specifically P. grandiflora should be further analyzed completely through isolation and characterization of active secondary metabolites responsible for all these activities