New insights into the antioxidant and apoptotic potential of Glycyrrhiza glabra L. during hydrogen peroxide mediated oxidative stress: An in vitro and in silico evaluation

Abstract

Plant-derived substances (phytochemicals) are well recognized as sources of pharmacologically potent drugs in the treatment of several oxidative stress related disorders. Our study aims to evaluate the antioxidant and apoptotic effects of Glycyrrhiza glabra L. in both cell free and cell culture system. Plant fractions have been prepared with hexane, chloroform, ethyl acetate, methanol and water and their antioxidant properties are reviewed. Potent antioxidant activity has been well established in both in vitro and in silico studies which is believed to be responsible for the anticancerous nature of the plant. Results obtained indicate that methanol fraction of G. glabra L. exhibited maximum scavenging activity against DPPH and nitric oxide free radicals comparable to standard antioxidant L-AA. Administration of methanol fraction also considerably reduced the malondialdehyde produced due to lipid peroxidation in mammalian liver tissues. Moreover, the levels of antioxidant enzymes SOD, CAT, GST, GPx and GR in the oxidative stress induced tissues were refurbished significantly after treatment with plant's methanol fraction. Moreover, methanol fraction was found to be nontoxic to normal human cell line whereas it inhibited cancer cells HeLa and HepG2 considerably. Apoptosis was established by DAPI fluorescent staining and western blot analysis of pro apoptotic protein caspase-8, caspase-3 and anti-apoptotic protein Bcl-2.There is an up regulation in the levels of pro apoptotic caspase-8 and caspase-3 and down regulation of anti-apoptotic Bcl-2. Furthermore, GC-MS analysis of the methanol fraction revealed the presence of many compounds. In silico experiments using Autodock 4.2 tools showed strong affinity of plant compounds towards antioxidant enzymes (proteins) thus validating with the conclusions of antioxidant enzyme assays and establishing a role in cancer pathogenesis.

Keywords: (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); 2,4-Dinitrophenylhydrazine (DNPH); Antibiotic-antimycotic solution; Anticancer; Antioxidant; Catalase (CAT); Dimethyl sulphoxide (DMSO); Ferric chloride; Fetal bovine serum; Glutathione peroxidase (GPx); Glutathione reductase (GR); Glutathione s transferase (GST); Glycyrrhiza glabra L.; Griess reagent; HeLa; HepG2; Hydrogen peroxide (H(2)O(2)); Hypochlorous acid; Molecular docking; Oxidized glutathione; Reduced glutathione; Sodium nitroprusside; Superoxide dismutase (SOD); Trichloroacetic acid; l-Ascorbic acid (PubChem CID: 54670067). 2,2-diphenyl-1-picrylhydrazyl (DPPH); l-Nitro-arginine methyl ester (L-NAME); tetramethoxypropane.