5-Demethylnobiletin (5-DMN), a hydroxylated polymethoxyflavone (OH-PMF) identified in aged citrus peels, has demonstrated health benefiting effects in previous studies. 5-DMN undergoes biotransformation in vivo, yielding 5,3'-didemethylnobiletin (5,3'-DDMN), 5,4'-didemethylnobiletin (5,4'-DDMN) and 5,3',4'-tridemethylnobiletin (5,3',4'-TDMN). However, the anti-cancer effects of 5-DMN and its in vivo metabolites against HepG2 cells remain unclear. In this study, an efficient chemical synthetic method was developed to obtain 5-DMN and its 3 metabolites, and their molecular structures were confirmed by 1H NMR and LC-MS. Cytotoxicity, cell cycle arrestment, apoptosis and caspase-3 expression were investigated to evaluate the anti-liver cancer effects of these OH-PMFs on HepG2 cells. The results showed that all 4 compounds inhibited the proliferation of HepG2 cells in a concentration-dependent manner. Their anti-proliferative activity was exerted through inducing G2/M phase arrestment, cell apoptosis and promoting expression of a key apoptotic protein called cleaved caspase-3. Our results indicated that 5,3'-DDMN and 5,3',4'-TDMN showed a stronger inhibitory activity on cell proliferation than 5-DMN, followed by 5,4'-DDMN. The expression of cleaved caspase-3 was the highest in cells treated with 5,4'-DDMN, implying that the apoptosis induced by other OH-PMFs might be mediated by other apoptotic execution proteins. Our research reveals the application potential and scientific evidence for the production and functionality of OH-PMFs.

Yanping Xin;Ting Zheng;Man Zhang;Ruiqiang Zhang;Siyue Zhu;Dongli Li;Denggao Zhao;Yanyan Ma;Chi-Tang Ho;Qingrong Huang

School of Biotechnology and Health Sciences,Wuyi University,Jiangmen 529020,China;International Healthcare Innovation Institute(Jiangmen),Jiangmen 529020,China;Department of Food Science,Rutgers University,New Jersey 08901,USA

食品科学与人类健康（英文）

[1]Yanping Xin;Ting Zheng;Man Zhang;Ruiqiang Zhang;Siyue Zhu;Dongli Li;Denggao Zhao;Yanyan Ma;Chi-Tang Ho;Qingrong Huang-.5-Demethylnobiletin and its major metabolites: efficient preparation and mechanism of their anti-proliferation activity in HepG2 cells)[J].食品科学与人类健康（英文）,2022(05):1191-1200

Demethylnobiletin,polymethoxyflavone,didemethylnobiletin,DDMN,tridemethylnobiletin,TDMN,arrestment

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