Study On Hydrolysates Of Acteoside In Vitro And Its Inhibition Of Xanthine Oxidase

Acteoside is one typical phenylethanoid glycoside and widely exist in dicotyledonous plants.Studies have shown that it has a lot of biological activities,such as antioxidant,hepatoprotect,anti-inflammatory,neuroprotective and immune regulation.Acteoside was chosen as the main object in this thesis.The in vitro simulative gastrointestinal digestion products of acteoside,the in vitro co-culture products of acteoside and intestinal flora of rats,and inhibitory effect of acteoside and its main digestion products on xanthine oxidase activity were studied.The results of the study are summarized as follows.1.According to the ultraviolet absorption properties of acteoside,caffeic acid and hydroxytyrosol,and combined with HPLC detection conditions of three substances,simultaneous determination of three substances by HPLC were established.The HPLC chromatographic were separated on a Waters Symmetry C18 column(5?m,250mmx4.6mm)and detected by 2998 photo-diode-array detector(PAD).The mobile phase was methanol-0.1%aqueous formic acid(40:60).Analyses were performed at 30? with a flow rate of 0.7mL/min and detection at 280nm.The sample volume was 10?L.The standard curve equation of acteoside is y=4.474x+5.224,R2=0.99996;the average recovery is 102.71%,the RSD is 1.65%.The standard curve equation of caffeic acid is y=20.687x-3.392,R2=0.99953;the average recovery is 101.24%,the RSD is 1.15%.The standard curve equation of hydroxytyrosol is y=4.716x-17.901,R2=0.99987;the average recovery was 98.81%,the RSD was 1.79.The results indicated that this methodology is simple,accurate,and reproducible.2.Acteoside was hydrolyzed for 4h by hydrochloric acid solution(pH2),artificial saliva,artificial gastric juice,artificial small intestinal juice,respectively and was digested for 4h according to the order of the organism digestion,and main hydrolysates were detected by HPLC and LC-MS.The results showed that only a few ester bond and glycosidic bond of acteoside could be broken to generate caffeic acid and hydroxytyrosol.3.The acteoside and intestinal flora of SD rats were co-cultured for different time,and main hydrolysates were detected by HPLC and LC-MS.The results showed that acteoside was metabolized to caffeic acid,hydroxyltyrosol and 3-methoxyphenylacetic acid,etc.As incubation time progressed,phenolic hydroxyl hydrogen ions of caffeic acid and hydroxyltyrosol were further replaced by-CH3 and-SO3H,and hydroxyltyrosol derivatives were further oxidized into acid.But it still need deeply test to determine whether the main product of the final reaction is 3-methoxy benzene acetic acid or not.4.In this study,inhibiting xanthine oxidase activity of acteoside hydrolysate,acteoside,caffeic acid and hydroxytyrosol were investigated.Results showed that the inhibiting effect on xanthine oxidase of hydrolyzed acteoside was stronger than before hydrolysis,the inhibitory effect of coculture of acteoside and intestinal bacteria on xanthine oxidase increased most significant.Acteoside,caffeic acid and hydroxytyrosol all have the inhibitory effect on xanthine oxidase,their IC50 values were 81.15?g/mL,32.06?g/mL,27.04?g/mL,respectively.It meant that the inhibitory effects on xanthine oxidase in vitro of caffeic acid and hydroxytyrosol are stronger than acteoside.Therefore,it was speculated that the in vivo inhibiting xanthine oxidase action of acteoside may due to its metabolic products.