Intestinal Mechanism Of The Improvement Of Diabetes By Flavonoids From Oroxylum Indicum

Diabetes is one of the most serious health issues in 21St century,and caused great harm to human.So far.some problems still exist in the clinical diabetes pharmaceuticals,including high adverse-effect and unsatisfactory efficacy that limited the treatment of diabetes.In recent years,many researches revealed the correlation between diabetes and gut microenvironment,which is regarded as an important target for diabetes treatment.Natural products are widely exist in medicinal plants,vegetables,and fruits and showed relatively high safety.Flavonoids and triterpenes were found to exhibit anti-diabetic activities.However,the detailed mechanisms are still unclear,and the modulation of gut microenvironment by flavonoids still requires further elucidation.In order to solve the problems above,the researches of this thesis are focused on the following topics:Firstly,we screened 12 natural products for their inhibitory activities.Oroxylum indicum seed extracts(OISE)and triterpenes showed the best inhibition.For rat intestinal a-glucosidase,the IC50 of OISE was 43.4 ± 0.7 μg/mL.For porcine pancreatic a-amylase,the IC50 of oleanolic acid,corosolic acid,and ursolic acid were 22.6～94.1 μM.The combination of OISE with acarbose showed synergistic inhibition on rat a-glucosidase,with combination index(CI)between 0.33 and 0.54.In vivo,OISE at 50-200 mg/kg combined with acarbose at 20%of the normal dose showed synergistic postprandial blood glucose(PBG)lowering effects in both normoglycemic and diabetic mice.The combination of corosolic acid and oleanolic acid showed selectively synergistic inhibition on a-amylase.However,no significant PBG-lowering effect was found in vivo.Then,using the purified rat a-glucosidase,we identified the major active component in OISE and investigated the mechanism of synergism.Flavonoids,including baicalein and oroxin A were the major active compounds in OISE.Among the tested flavonoids,baicalein showed the best inhibition,with an IC50 value of 74.1 ± 5.6 μM.The combination of baicalein and acarbose showed the strongest synergistic inhibition on rat a-glucosidase,with CI values between 0.29 and 0.41.Structure-activity relationship,enzyme kinetics,and molecular docking studies revealed that the C-6 hydroxyl group of flavonoids is important for exerting individual and synergistic inhibition on a-glucosidase;baicalein could bind to the non-competitive binding sites of enzyme,enhance the affinity between enzyme and acarbose,and consequently lead to synergistic inhibition.In vivo studies revealed that the combination of baicalein and acarbose could synergistically reduce the PBG of mice,and reduce the effective dose of acarbose by 87.5%.These results suggested that baicalein alone or in combination with acarbose could modulate gut microenvironment of diabetic mice through the inhibition of carbohydrates digestion.After that,we investigated the intestinal pharmacokinetics of baicalein in mice.After oral administration,the intestinal content of baicalein showed peak at 1 h,and reached 87%of the administrated dose.The intestinal content of baicalein were 45%and 20%of the administrated dose at 8 h and 12 h,respectively,suggesting that the content of baicalein kept at high levels in the intestine.HPLC-MS/MS analysis revealed that the major metabolites of baicalein were glucuronidation,glucosidation,sulfation,and methylation metabolites in the small intestine;sulfation,methylation,and dehydroxylation in the large intestine.The content of metabolites showed two peaks,and the second peak at 8 h was the metabolites of enterohepatic circulation.The metabolism mechanism was investigated in vitro using tissue S9 fraction and gut microbiota.Baicalein could be transformed to its glucuronidation,sulfation,and methylation metabolites in liver and intestine.Gut microbiota could hydrolyze the glucuronide ligand and produce baicalein.These results suggested that intestine is important in baicalein metabolism in vivo.At last,we investigated the improvement of diabetes by baicalein in high-fat-high-sugar diet combined with streptozocin induced diabetic rats.After 4 weeks treatment,baicalein at 150 mg/kg/d could significantly improve the hyperglycemia.insulin resistance,and obesity in diabetic rats.Metagenomic sequencing of gut microbiota 16S rRNA V3-V4 regions revealed that baicalein treatment significantly up-regulated several potential short-chain-fatty-acid(SCFA)producing bacteria and down-regulated several opportunistic pathogens.Among the up-regulated SCFA producing bacteria,the relative abundance of Bacteroidales S24-7 and Bacteroides were increased by 74.7%and 422%,respectively,compared to the diabetic group.In addition,baicalein treatment significantly increased the content of intestinal SCFA and the thickness of inner mucus layer,enhanced the gut barrier function,reduced the circulation lipopolysaccharide(LPS),inhibited the system inflammation induced by Toll-like receptor 4(TLR4),and consequently improved the insulin resistance.Correlation analysis revealed that several gut microbiota,including Bacteroidales S24-7 and Bacteroides showed positive correlation with reduction of LPS,improvement of hyperglycemia and insulin resistance.These results suggested that the modulation of gut microbiota is an important mechanism of the insulin-sensitizing and anti-diabetic effects of baicalein.In this research,the multi-path mechanisms of the improvement of gut microenvironment modulation and diabetes by flavonoids from Oroxylum indicum was elucidated.The present research will lay a theoretical foundation of the treatment of diabetes using flavonoids.