In Vitro Study Of Interactions Between Citrus Flavonoids And Gut Microbiota

Citrus is an important medicine food homologous material,which is considered as a natural plant source of various functional components.Flavonoids are one of the most important functional components in citrus,especially in the peels,which exhibit multiple health-promoting effects.Citrus flavonoids mainly exist in the form of glycosides in nature.Among them,hesperidin and naringin are the main ones with highest content in citrus,which have drawn special attention due to various significant bioactivities,such as antioxidant,anti-inflammatory,anti-tumor and anti-cardiovascular diseases.After oral absorption,citrus flavonoids,especially those in glycosides,are difficult to be absorbed by the small intestine.Most of them directly enter the large intestine,which inevitably interact with gut microbiota.On the one hand,the rich enzyme systems of gut microbiota may catalyze the metabolic biotransformation of flavonoid glycosides to produce metabolites with distinct structures and biological activities.On the other hand,gut microbiota is easily affected by dietary components,and flavonoid glycosides and their metabolites may also regulate the structures and functions of the gut microbiota,which is closely related to human health.Therefore,the interaction between citrus flavonoids and gut microbiota is of special significance to human health.However,there is no systematic study on the interaction between citrus flavonoids,especially aglycones and intestinal flora.Therefore,in this study,hesperidin and naringin,the main citrus flavonoids,were selected as the research objects,and the metabolism of citrus flavonoids(especially the metabolites in flavonoid form)and its regulation on gut microbiota were systematically studied by the simplest anaerobic fermentation in vitro.The results of this study would provide new insight for the reveal of health-promoting effect mechanisms for citrus flavonoids,as well as scientific guidance for the design and application of functional foods based on citrus flavonoids.The main results were summarized as follows:(1)In vitro metabolism of citrus flavonoids by gut microbiota: Citrus flavonoids hesperidin and naringin were fermented in the feces of healthy adult men.The metabolites of flavonoids derivatives were systematically investigated by HPLC and LC-MS.The results showed that naringin and hesperidin showed similar fermentation rules: in the first 8 hours of fermentation,hesperidin and naringin could be rapidly degraded,while the contents of their corresponding aglycones,hesperidin and naringenin,increased rapidly.After that,the content of aglycones decreased rapidly,and almost no parent compounds and aglycones could be detected after 12 h.At the same time,the level of aglycone(hesperetin)detected in hesperidin groups were higher than that of naringin(naringenin)at the same dosage,and the degradation rate of hesperetin was significantly lower than naringenin.These results indicated that the aglycone of hesperidin may play a more important role in the health-promoting effects,especially in the regulation of gut microbiota.In addition,five other flavonoid derivative metabolites were detected in hesperidin fermentation samples,among which three 7-O-?-D-glucoside of hesperetin derivatives were identified as hesperidin metabolites for the first time.One more acetylation metabolite was also detected in naringin.The metabolic transformation pathways of hesperidin and naringin under gut microbiota was speculated.The results further improved the in vivo metabolic pathway of citrus flavonoids,and laid the foundation for the elucidation of the health-promoting effect mechanisms.(2)Effects of citrus flavonoids on gut microbiota: The effects of citrus flavonoids hesperidin and naringin at high(250 ?M)and low(50 ?M)doses on human gut microbiota were systematically investigated through in vitro liquid fermentation.The results showed that the incubation of hesperidin and naringin both at high dosage and low dosage lead to significant increase of the four main short chain fatty acids(SCFAs)(acetate,propionate,isobutyrate and butyrate).And almost all the SCFAs except propionate significantly increased along with the extension of fermentation time.The level of SCFAs in hesperidin treatment group was significantly higher than that of naringin group with the same dosage,and high dose generated more SCFAs than low dose.These results indicated that hesperidin and its metabolites may be more conducive to the production of SCFAs.16 S rRNA sequencing results showed that citrus flavonoids treatment could significantly regulate the abundance of gut microbiota,including a variety of bacteria closely related to human health.And the regulation tended to be beneficial for human health.Further gene function prediction analysis showed that hesperidin and naringin at high dosage could significantly increase the relative abundance of genes related to energy metabolism and short chain fatty acid biosynthesis.And they were in higher relative abundance in hesperidin treatment than that of naringin treatment,which was consistent with the content of SCFAs.The results facilitated the reveal of in vivo health-promoting effect mechanisms for citrus flavonoids,and provided scientific guidance for functional food design based on citrus flavonoids.