Biosynthesis of demethylated polymethoxyflavones by a yeast isolated from aged orange peel
Creators: name: Su, Shiwei
Publisher: name: No Publisher Supplied
Year: 2022

description: Nobiletin (3′,4′,5,6,7,8-Hexamethoxyflavone) is widely proved to have significant chemopreventive effects against various diseases. In the past few years, it has been recognized that the biological benefits of orally administrated nobiletin mainly came from its demethylated metabolites which were produced in organs or by gut microbiota. The most widely reported demethylated metabolites were 3'-demethylnobiletin (3'DMN), 4'-demethylnobiletin (4'DMN), and 3',4'-didemethylnobiletin (3'4'DDMN). Other mono-demethylated-nobiletins (DMNs) were also detected in pharmacokinetic and bio-efficacy studies. However, because of the lack of standards, the demethylation positions were not known. It was reported that 6-demethylnobiletin (6DMN), 7-demethylnobiletin (7DMN) were produced after incubating nobiletin with human liver microsomes. Therefore, we assume that 6DMN, 7DMN are two important metabolites of orally administered nobiletin. However, there is no reported method to produce these compounds.The overall goal of this dissertation is to find out a practical and efficient synthetic method for 6DMN and 7DMN. The research will help deepen our understanding of the bio-beneficial effects of the two compounds and the mode of action of nobiletin as to its chemopreventive effects.Firstly, microbes that was able to metabolize nobiletin were isolated, identified and characterized. In this research, a strain of microbe, which was able to metabolize nobiletin into two new compounds, was obtained. ITS results showed that the isolated microbe belonged to yeast family, Filobosidum mangnum specie (NRRL Y-68198). The morphology and growth properties were disclosed under different culture conditions. To maximize the demethylation efficiency, operational factors were optimized.Secondly, the nobiletin metabolites (NMs) were extracted, purified and analyzed. LC-MS results showed that the two NMs had molecular weight of 388 Da, which meant that the metabolites of nobiletin occurred after losing one methyl group. However, comparing with the HPLC profiles of available demethylated nobiletin standards, 5-demethyl-nobileitn (5DMN), 3'DMN, 4'DMN, 3'4'DDMN, NMs demonstrated different retention times. For the identification of NMs, batch fermentation, solvent extraction, silica gel chromatography, preparative HPLC were applied to obtain pure NMs. Notably, the extraction and purification process were optimized, with the purpose of enhancing the efficiency and minimizing the loss of NMs. 1H and 13C NMR was used for the identification of chemical structures of the two NMs. Results demonstrated that they were 7DMN and 6DMN, respectively.Thirdly, the anti-adipogenesis ability of 7DMN and 6DMN were studied on the 3T3-L1 cell model. In this study, all the three compounds showed significant suppressive effect on the lipid accumulation of differentiating 3T3-L1 cells. However, in comparison with nobiletin, 7DMN did not show significant difference, while 6DMN demonstrated much stronger efficacy. 6DMN exerted its effect on both the early differentiation and the terminal differentiation phases of 3T3-L1 cells. Flow Cytometry results showed that 6DMN blocked mitotic clonal expansion (MCE) in the S phase. Western blotting method reveals that the expression of early transcription factors as well as lipogenesis specific genes were greatly attenuated. The expression and phosphorylation of AMP-activated protein kinase (AMPK) in the early and late differentiation processes were significantly enhanced by 6DMN. Taken together, 6DMN was much more potent than nobiletin and 7DMN in suppressing lipogenesis.The fourth part of the thesis studied the demethylation mechanisms of the isolated microbe. Biochemically, cleavage of a methyl aryl ether bond is catalyzed by CYP 450s in eukaryotes. To disclose the role of CYP450s in the demethylation process, four kinds of CYP450 inhibitors were added into the fermentation media. Results demonstrated that the production of 7DMN and 6DMN were suppressed after the usage of CYP450 inhibitors. A time-course monitoring of substrate and metabolites was carried out to uncover the metabolic pathway. Multiple PMFs and demethyl-PMFs were employed to test substrate specificity of the enzyme.Lastly, a nobiletin nano-dispersion system was fabricated by using media-milling technique. With the purpose of increasing the accessibility of nobiletin for transformation, nobiletin nanodispersion system was fabricated in hydrophobically modified starch (HMS) by media milling technique. Results showed that after 3 hours of milling process, the particle size stabilized at 800 nm. Compared with HMS/nobiletin sludge, the nanodispersion contributed to much higher nobiletin water solubility and demethylation efficiency. Moreover, the production of DMNs by using the nano-dispersion system was 3 times higher than that by using DMSO/nobiletin solution. In addition, the nano-dispersion system had two more advantages: 1) gave higher bulk extraction efficiency; 2), can be used to encapsulate crude orange peel extract for bio-demethylation.