Abstract
Background: Plants contain secondary metabolite used as drugs/medicines for the treatment of various diseases. Aristolochia tagala is used for the treatment of several diseases. Our study reported the chemopreventive potential of crude aqueous-methanol extract against diethylnitrosamine-induced hepatocellular carcinoma in BALB/c mice. A few articles have reported the presence of pharmacologically active compounds. Objective: Identification of biologically active compounds can give an insight into the mechanism of action of A. tagala and its potential development into modern drugs for the treatment of various diseases including cancer. Materials and Methods: Aqueous methanol extract (ATC) was prepared from roots of A. tagala and fractionated by column chromatography. The compounds present in ATC were identified and characterized by liquid chromatography (LC)–high-resolution mass spectrometry. ATC as well as the fractions (FI–FIV) were tested for their cytotoxic effect in HeLa cells by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and apoptotic events were analyzed by flow cytometry. The fraction that showed the most efficient cytotoxic effect against HeLa cells was purified by high-performance thin-layer chromatography. Purified compounds were again assayed for their apoptotic and cytotoxic effect. The most active compound was identified and characterized by electrospray ionization high-resolution mass spectrometry and LC–tandem-mass spectrometry. Statistical analysis was carried out using one-way anova followed by Tukey's multiple comparisons test. Results: A total of 21 compounds were identified; aristolochic acid I, aristolactam IIIa, β-sitosterol, kaempferol, and stigmasterol were previously reported in A. tagala and other compounds in other species of Aristolochia, and some compounds were reported to have anticancer, anti-inflammatory activities. From our study, compound S7 showed the highest cytotoxic and apoptotic activity and was identified as aristolochic acid I. Aristolochic acid earlier has been reported to have antitumor and anticancer effects, but lately, it has also been reported to have nephrotoxic effect. Conclusions: A. tagala was found to contain a number of compounds with reported biological activity. This plant and its related species can, therefore, be exploited for the extraction and isolation of these compounds with no toxicity. Abbreviations used: ATC: Aqueous-methanol extract, AA-I: Aristolochic acid I, CQA: Caffeoylquinic acid, CHCl3: Chloroform, MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, ESI: Electrospray ionization, FITC: Fluorescein isothiocyanate, HCC: Hepatocellular carcinoma, HPLC: High-performance liquid chromatography, HRMS: High-resolution mass spectrometry, LC-MS/MS: LC-tandem-mass spectrometry, MeOH: Methanol, PBS: Phosphate buffered saline, PVDF: Polyvinylidene difluoride, RF: Radio frequency, R F: Retardation factor, XEVO-TQD: Xevo triple quadrupole
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