Isolation of Xanthine Oxidase Inhibitors from Chrysanthemum Morifolium Dried Flowers

Tey, Zi Tong (2024) Isolation of Xanthine Oxidase Inhibitors from Chrysanthemum Morifolium Dried Flowers. Masters thesis, Tunku Abdul Rahman University of Management and Technology.

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Abstract

The xanthine oxidase (XO) enzyme serves as a cellular origin for superoxide radicals. It catalyzes the oxidation reactions, converting hypoxanthine into xanthine and further oxidizing xanthine into uric acid. Elevated uric acid production may lead to the development of hyperuricemia, which may lead to the onset of gout. Chrysanthemum morifolium has long been recognized for its anti-inflammatory and antioxidant properties due to the presence of bioactive compounds such as flavonoids and phenolic compounds. Therefore, the main objective of this study was to isolate the XO inhibitors from Chrysanthemum morifolium dried flower extracts. This was achieved through a systematic process, involving extraction, isolation, and purification. Among the methanolic crude (MC) extract, ethyl acetate (EA), petroleum ether (PE) and residual (RS) fractions, EA fraction exhibited the highest XO inhibitory potency of 42.00 ± 1.19 % (IC50 = 5.17 ± 0.32 μg/mL). A total of seven sub-fractions were obtained by sub-fractionating the bioactive EA fraction through open column chromatography, in which EA sub-fractions 2, 3, and 4 possessed > 40% XO inhibition at the final concentration of 4 μg/ml. These sub-fractions were subjected to isolation and purification using various chromatographic methods. Subsequently, spectroscopic techniques, including 1D NMR (1H, 13C, and DEPT) and 2D NMR (COSY, HMQC, and HMBC), IR, UV, and HR-ESI-MS, were employed for the elucidation of the chemical structures of the isolated compounds. Six natural compounds were isolated from the C. morifolium flower extract, namely bis(2-ethylhexyl) maleate (B1), 4',5-dihydroxy-3,3',7,8-tetramethoxy-flavone (B2), Eriodictyol-7-O-glucoside (B3), 3-methoxy-5-demethoxy-3',4'-dihydroxytangeretin (B4), Luteolin (B5) and Casticine (B6). Compound B1, B2, B3, B5, and B6 were identified as known compounds, while B4 was a new compound. All the six compounds were further subjected to XO inhibitory activity to study their XO inhibitory potency. Compound B3 (IC50 = 2.06 ± 0.01 μg/mL), B4 (IC50 = 1.65 ± 0.02 μg/mL), and B5 (IC50 = 1.15 ± 0.04 μg/mL) exhibited high XO inhibitory activity (≥70% activity at the final concentration of 3.2 μg/mL) with the binding affinity -6.4, -8.4 and -8.8 kcal/mol, respectively in the in silico analysis. Furthermore, compound B1 (IC50 = 9.84 ± 0.11 μg/mL), B2 (IC50 = 4.27 ± 0.07 μg/mL) and B6 (IC50 = 4.85 ± 0.03 μg/mL) possessed low XO inhibitory activity (< 40% inhibition) at the final concentration of 3.2 μg/mL, accompanied by the docking score of -6.2, -6.3 and -7.2 kcal/mol, respectively. Since compound B3, B4 and B5 exhibited high XO inhibitory activity, it is recommended to explore these compounds further through cell culture and animal studies for their potential clinical significance.