The Bioassay Guided Isolation of Xanthine Oxidase Inhibitors from Chrysanthemum Morifolium Ramat

Chan, Veng Vooh (2018) The Bioassay Guided Isolation of Xanthine Oxidase Inhibitors from Chrysanthemum Morifolium Ramat. Final Year Project (Bachelor), Tunku Abdul Rahman University College.

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Abstract

Gout disease is known as arthritis. It is caused by excess of uric acid in bloodstream, result in hyperuricemia. Human body consists of xanthine oxidase which play an important role to degrade purine into uric acid. The aim of this project is to investigate the xanthine oxidase inhibitory capability of Chrysanthemum morifolium Ramat. Flower extract and to isolate the xanthine oxidase inhibitors by using bioassay-guided fractionation. In this study, the C. morifolium was extracted by using 80% methanol (crude extract); the extract was then partitioned in ethyl acetate (EA), petroleum ether (PE) and residual (RS) fractions. Allopurinol, a commercial XO inhibitor, was used as a positive control in this study. The formation of uric acid was measured at 295nm using a microplate reader. In the XOI assay, the EA fraction showed highest XO inhibition of 46.43 ± 29.59%. Therefore, the most active EA fraction was subjected to repeated fractionation by gravity column chromatography. Elution of compounds was done by using a gradient of increasing polarity of solvents as mobile phase, with a stepwise gradient of hexane: ethyl acetate from 7:3 (hexane: ethyl acetate) to 0:10 and then eluted sequentially with methanol and chloroform. These sub-fractions were further analysed for its XO inhibitory potential. The sub-fractions (SF2, SF3, SF4 and SF6) demonstrated high XO inhibition of around 65-75%. The SF2 was chosen for further purification and was screened for its XO inhibitory activity. The results showed a significant XO inhibition of 96.58 ± 4.823% in SF2.2 and qualitative phytochemical determination of the compounds in SF2.2 showed the presence of phenolic and flavonoid compounds. However, further identification of the bioactive compound is required. The findings in this study suggested that C. morifolium can be a potent natural anti-hyperuricemic agent.