Isolation of Andrographolide from Andrographis lineata Wall. ex Nees var. lawii C.B. Clarke and its Anticancer Activity against Human Ovarian Teratocarcinoma

Abstract

Introduction: Andrographolide is a well-known anticancer phytochemical often isolated from Andrographis paniculata (Burm. f.) Nees. (Acanthaceae). Though Andrographis lineata Wall. ex Nees var. lawii C.B. Clarke (ALw) which also belongs to the same family has an adequate amount of andrographolide; remained untouched for isolation of andrographolide and anticancer studies. Therefore, this study was targeted to isolate the andrographolide from the leaves of ALw and to assess its role inthe induction of apoptosis against the human ovarian teratocarcinoma (PA-1) cell line. Methods: Column chromatography, thin-layer chromatography (TLC), preparative TLC were used for the isolation and purification while melting point, ultraviolet (UV)-visible spectroscopy, Fourier transform infrared (FTIR), proton nuclear magnetic resonance ( 1 H NMR), carbon-13 (C13) nuclear magnetic resonance ( 13 C NMR) analysis were carried out for characterization of the compound. 3-(4,5-dimethylthiaxo-2yl) 2, 5-diphenyl tetrazolium bromide (MTT) assay was carried out for cytotoxicity test and further Annexin-V staining, caspase 3 activity, B-cell lymphoma-2 (Bcl-2) activity, cell cycle analysis, and DNA damage study by terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assays were carried out for apoptosis study. Results: Andrographolide was isolated from the methanolic extract of leaves of ALw which had a melting point of 230 ºC, λ max at 223 nm. FTIR results proved the presence of hydroxyl group, alkanes, carbon-carbon double bond, and a characteristic gamma lactone carbonyl. NMR data confirmed the 20 carbon structure. In the MTT assay cytotoxicity against PA-1 was at 3.7 µg/ml with other apoptotic assays supporting the induction of apoptosis by the compound at that concentration. Conclusion: ALw is proved to be an alternate source of andrographolide with potential abilities to induce apoptosis in ovarian cancer cells.