The roots of Millettia speciosa Champ., a medicinal-edible plant, contain bioactive polysaccharides (MSCPs) that demonstrate potential prebiotic effects. Studies in diet-induced obese (DIO) mice have shown that MSCPs can modulate gut microbiota dysbiosis, suppress appetite, and ultimately alleviate obesity. However, the mechanism by which MSCPs regulate appetite and ameliorate obesity-associated metabolic dysfunction through modulating the gut microbiota still requires further investigation. In this study, we observed that MSCPs improved diet-induced obesity to the same extent as the pair-fed group, suggesting that MSCPs can ameliorate obesity in DIO mice by reducing appetite and energy intake. Through RT-qPCR, Western blot, and immunohistochemical experiments, it was found that MSCPs regulate the expression of appetite-related peptides and activate the JAK2/STAT3 signaling pathway to improve obesity in DIO mice; however, MSCPs did not ameliorate obesity in leptin receptor-deficient (db/db) mice, indicating that the leptin pathway is a critical target for treating DIO mice. Furthermore, MSCPs beneficially modulate gut microbiota; fecal microbiota transplantation experiment (FMT) restored the gut microbiota structure in mice, and the FMT group still alleviated obesity through the same signaling pathway. Additionally, 16S rRNA gene sequencing revealed that both MSCPs and FMT upregulated beneficial microbiota such as Lachnospiraceae_UCG_006 and Faecalibaculum, and promoted the production of short-chain fatty acids (SCFAs). Notably, in db/db mice, although MSCPs significantly elevated SCFAs levels, they failed to ameliorate obesity or metabolic disorders, further confirming that the leptin pathway serves as a critical requirement for MSCPs-mediated obesity improvement. In summary, MSCPs may alleviate obesity in DIO mice by enriching Lachnospiraceae_UCG_006 and Faecalibaculum to promote SCFAs production, thereby modulating the leptin pathway.

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.