Protective Effects Of Bee Pollen Polyphenols On Non-alcoholic Liver Damage And DSS-induced Colitis

Bee pollen is rich in a variety of nutrients,of which polyphenols are one of the main biologically active substances.Bee pollen phenolic polyphenols have strong antioxidant activity and have preventive effects on damage caused by oxidative stress.Therefore,bee pollen has gradually attracted attention as an exogenous antioxidant.In this paper,the polyphenols in the Actinidia arguta bee pollen,Schisandra chinensis bee pollen and rape bee pollen are analyzed.In addition,this study also explored the protective effects of polyphenols on oxidative stress-induced cell damage,nonalcoholic fatty liver in high fat diet mice and DSS-induced colitis in mice,indicating that bee pollen polyphenols can effectively prevent non-alcoholic liver damage and DSS colitis.1. In this study,the A.arguta pollen spore structure was observed,the polyphenols of A.arguta pollen extract（AAPE）obtained by four extraction methods（A:ultrasonic-assisted waterextraction,B:heat reflux extractionby water,C:ultrasonic-assisted ethanol extraction,D:heat reflux extraction by ethanol solution）were analyzed,and their yield were compared.In addition,the total phenolic content,total flavonoid content,antioxidant activities（FRAP,chelating activity and DPPH×scavenging activity）were also determined.Finally,we investigated its protective effect on DNA and lymphocytes damage response to oxidative stress.The results showed that the morphology of A.arguta pollen was slightly different from other pollen of Actinidia Lindl.The A.arguta bee pollen extracted by ultrasonic-assisted ethanol had the highest total phenolic content（15.05±0.34 mg GAE/g）,strong ferrous ion chelating ability（0.37±0.023 mg Na₂EDTA/g）,DPPH×scavenging activity(IC₅₀=0.14±0.04 mg/m L)and FRAP（7.13±0.33mg Trolox/g）.It also had a strong protective effect on DNA damage caused by oxidative stress and mice lymphocytes.2. The composition of polyphenols of Schisandra chinensis bee pollen extract（SCPE）was analyzed by UHPLC-Q-Orbitrap-HRMS/HPLC-DAD-ECD.The genes related to the formation of nonalcoholic fatty liver（NAFLD）in high fat diet mice were determined by Q-PCR in order to explore the relevant mechanism of SCPE inhibition of NAFLD.16Sr RNA gene sequencing of total DNA in mice feces was performed to detect the gut microbial diversity,so as to explore the possible role of gut microbiota in the process of SCPE inhibiting NAFLD formation.The results showed that 12 polyphenols were identified in SCPE,and naringenin,rutin and chrysin were the main constituents.After high fat diet mice were administrated with SCPE at 7.86 and 15.72 g/kg BW,body weight,liver index and fasting blood glucose decreased significantly.Liver AST and ALT activities were remarkably reduced,serum TG,LDL-C and liver TC and TG contents were notably reduced.The expression of chronic metabolic inflammation genes caused by high fat diets were significantly down-regulated,and oxidative stress was reduced.What’s more,liver and epididymal fat HE staining results confirm that NAFLD caused by a high fat diet is well suppressed after oral administration of SCPE.The result of liver and epididymal adipose HE staining confirmed that NAFLD caused by a high fat diet was well suppressed after administration of SCPE.SCPE inhibits the formation of NAFLD by reducing the expression of LXR-α,SREBP-1c and FAS genes in the liver.Moreover,SCPE can improve gut health by increasing Lactobacillu in colon of high fat diet mice.3. The composition of polyphenols of rape bee pollen extract（RPE）was analyzed by HPLC-DAD-ECD.The genes related to colitis induced by dextran sodium sulfate（DSS）in C57BL/6 mice were determined by Q-PCR in order to investigate the relevant mechanism of RPE inhibition of acute colitis.In addition,16S r RNA gene sequencing of the total DNA in the intestinal contents of mice was used to detect the gut microbial diversity,and the changes of gut microbiota in colitis mice after RPE treatment were analyzed,so as to explore the role of gut microbiota in this process.The results showed that 7 polyphenols were tentatively identified in RPE,of which kaempferol had the highest content（19.87mg/g）,followed by sinapic acid and rosmarinic acid.RPE at 10.6 and 21.2 g/kg BW ameliorated colon shortening,spleen swelling and colon weight reduction,increased the activity of SOD and GSH-Px,reduced the activity of MPO,and also down-regulated the expression of colitis-related inflammation genes（i NOS,NF-κB,IL-1β,IL-6）.In particular,IL-1βexpression in the 21.2 g/kg BW RPE group was down-regulated by approximately90%compared with that of the model group.RPE improved the structure of colonic villi and glandular etc.,which was further confirmed by conventional histological assessment.In addition,RPE altered the gut microbial structure by regulating the relative abundance of Allobaculum,Bacteroide and Lactobacillus,and alleviated DSS-induced colitis.