Cancer is a malady of uncontrolled proliferation of defected cells. Mass of irregularly divided cells might stay at one place or advances to other tissues. Many therapies for cancer are in practice including surgery, radiotherapy and chemotherapy. Chemotherapy is most extensively used method but it has severe side effects. Natural products being originated from biological resources are biological friendly and have fewer side effects so they can provide valuable template for drug development. Bistotra amplexicaulis is a famous herb used in natural therapeutics for curing many ailments especially hepatic disorders but the underlying mechanism for this activity was scientifically unexplored. The main objective was to isolate cancer chemo-preventive and/or chemotherapeutic compounds from this plant species. For initial screening of antioxidant and cytotoxic potential against HepG2 cell line, crude extract of B. amplexicaulis rhizome was prepared by using maceration technique and the percentage yield obtained was (18.72 %). Different fractions were prepared by solvent-solvent extraction of crude extract including nhexane (0.04 %), chloroform fraction (0.06 %), ethyl acetate fraction (4 %), nbutanol fraction (8 %). The capacity of plant extracts as anticancer and antioxidant was measured by employing cytotoxic assay (MTT assay) and range of antioxidant assays (DPPH radical scavenging, ABTS radical scavenging, superoxide scavenging, hydroxyl scavenging, and hydrogen peroxide scavenging assay). nhexane fraction was proved as highly active against HepG2 cancer cell line in MTT assay with the higher percentage inhibition (89 %) at 20 μg/ml. The results of the antioxidant assays showed that ethyl acetate fraction was most active in DPPH radical scavenging assay, ABTS radical scavenging assay, total antioxidant assay, superoxide radical scavenging assay, hydroxyl radical scavenging assay with the IC50 values of (5.76 ± 0.03 μg/ml), (0.74 ± 0.1 μg/ml), (72.55 ± 0.09 ascorbic acid equivalents), (6.86 ± 0.19 μg/ml), (0.96 ± 0.16 μg/ml) respectively. On the basis of the above findings both n-hexane and ethyl acetate fraction were considered for isolation of active compounds. For the isolation and purification of compound from the active fractions of Bistorta amplexicaulis, fractions were subjected to normal phase open column chromatography, reverse phase open column chromatography, reverse phase low pressure liquid column chromatography, gel filtration chromatography and high pressure liquid column chromatography. One compound from n-hexane fraction and three compounds from ethyl acetate fraction were isolated. The chemical structure of these compounds were established by nuclear magnetic resonance spectroscopy including proton nuclear magnetic resonance spectroscopy (1H NMR) and carbon nuclear magnetic resonance spectroscopy (13C NMR) and electron spray ionization mass spectrometry (ESI-MS). Compound 1 with ESI-MS (m/z 284 calc. for C16H12O5); 1H NMR 400 MHz and 13C NMR100 MHz (CD3OD) was identified as 1,5-dihydroxy-3-methoxy-7-methylanthracene-9,10-dione, compound 2 with ESIMS (m/z 574 calc. for C35H58O6); NMR 400 MHz and 13C NMR 100 MHz (C5H5N) was identified as daucosterol, compound 3 with ESIMS (m/z 290 calc. for C15H14O6); NMR 400 MHz and 13C NMR 100 MHz (CD3OD) was identified as -(-) catechin, Compound 4 with ESIMS (m/z 452 calc. for C24H20O9), white powder; NMR 400 MHz and 13C NMR 100 MHz (CD3OD) known as cinchonain Ib. Finally isolated compounds were screened for cytotoxic potential against Human non-small cell lung cancer cell line NCI-H1299. Human hepatocellular carcinoma cell line SK-Hep-1 and for antioxidant potential in ABTS assay. Results indicate that compound 1 was the most potent inhibitor on SK-Hep-1 cell proliferation with 30.26 μM of IC50 value, compound 2 showed the IC50 value of 78 μM and compound 4 showed the IC50 value of 89 μM. Compound 3 was found inactive in anti cytotoxic assay. All of the compounds were also tested for their antioxidant potential in ABTS radical scavenging assay. Results showed that compound 3 had higher antioxidant potential with the IC50 value of 40 μM. This is the first report on detailed antioxidant and cytotoxic evaluation of crude extract and polarity based fractions of Bistorta amplexicaulis. This is also the first report on the establishment of protocol for the purification of two of the high market valued compounds from Bistorta amplexicaulis. Further isolation is required to find more compounds for antioxidant and cytotoxic potential. A comprehensive study on mechanism of action of compounds in in-vivo model is required. The current retail price of two of these compounds is very high so a company based on commercialization of this technology would be an industry which has the potential to create jobs in Pakistan.
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