Studies On The Anti-diabetic Activity And Regulating Gut Microbiota Of Extracts From Enteromorpha Prolifera In Mice

Diabetes mellitus is a chronic metabolic disease,which is caused by relatively insufficient insulin secretion in body and leads to high blood glucose level.There are a large number of people with diabetes and the morbidity is gradually ascending.It is the third most serious chronic disease that threatens human health.Type 2 diabetes account for 90-95%of all diabetes and its cause is insulin resistance.The modes of action of common hypoglycemic drugs are relatively simple and most of them have side effects when taken for a long time.Therefore,it is very worthwhile to study and develop functional ingredient from natural products that help decrease blood glucose level.Seaweeds are rich in bioactive substances and easy to obtain.Enteromorpha prolifera belongs to the family Ulvaceae.It is widely distributed in China’s coastal areas and usually grows at intertidal area.E.prolifera is rich in functional nutrients.It has a long history as food and traditional Chinese herbal medicine.In this study,hypoglycemic effects and mechanisms of water and ethanol extracts of E.prolifera were evaluated and investigated.The results are as follows:Water-ethanol extract from E.prolifera（EPW）and its flavonoid-rich fraction less than 3000 Da（EPW3）were studied.The IC50 value of EPW3 forα-glucosidase inhibitory activity was 3.36 mg/mL slightly better than of EPW as 3.88 mg/mL.The major active compounds of EPW3 identified by ultra performance liquid chromatography-mass spectrometry（UPLC-MS）were eriodictyol-glucuronide,kaempferol,O-glucuronide derivatives,and sweroside.The hypoglycemic effects of EPW and EPW3 treatments in vivo and regulation on gut microflora of mice were evaluated in type 2 diabetic mice induced by high-fat and high-sugar diet and streptozotocin（STZ）injection.The results showed that both EPW and EPW3 treatments significantly decreased the fasting blood glucose（FBG）level（p<0.05）and EPW3 treatment significantly increased the glucose tolerance of mice（p<0.05）after 28 days of oral administration.Histopathological results showed that EPW and EPW3treatments protected the liver and kidney from damage caused by diabetes.Meanwhile,the mRNA transcriptional levels of IRS1,PI3K,AKT and JNK1/2 genes which are associated with glucose metabolism were detected by quantitative real-time PCR.Compared to the model group,both EPW and EPW3 treatments significantly up-regulated the mRNA transcription of PI3K and AKT in liver tissues（p<0.01）and down-regulated of JNK1/2 transcription.Western blot analysis showed that both EPW and EPW3 treatments could up-regulate the gene expression of IRS1,PI3K,and AKT.Effect of EPW and EPW3treatments on gut microbiota of mice showed that the abundance of the Bacteroidetes and Actinobacteria in the model group were significantly lower than those in the normal group,while Fermicotes and Verrucomicrobia were significantly higher than those in normal group.After the treatment of EPW and EPW3,Bacteroidetes increased and Firmicutes decreased significantly in the gut of diabetic mice.Moreover,EPW3 treatment increased Actinobacteria.EPW and EPW3 treatments alleviated and administrated type 2 diabetic mice by activating IRS1/PI3K/AKT and balancing gut microbiota.Ethanol extract（EPE）was obtained by extracting the E.prolifera with 55%ethanol and then being filtered by ultrafiltration.The IC50 value of EPE for theα-glucosidase inhibitory activity was 1.81 mg/mL.The major active compounds identified by UPLC-MS were estr-5（10）-ene-3,17-diol,regiolone,luteolin-6-C-glucoside,and neoeriocitrin.EPE treatment significantly decreased FBG level（p<0.05）in diabetic mice,improved oral glucose tolerance（p<0.05）and decreased damage of liver and kidney caused by diabetes.The transcription and expression of the target genes PI3K and JNK1 were detected by RT-qPCR and Western blot,respectively.It was found that EPE treatment significantly up-regulated the expression of PI3K and down-regulated the expression of JNK1（p<0.05）.The analysis of gut microbiota in mice showed that EPE treatment increased the abundance of Bacteroidetes and decreased Verrrucomicrobia.By comparison between water and ethanol extracts of E.prolifera,the finding was that ethanol extracts of E.prolifera had better hypoglycemic effect in mice.In addition,E.prolifera oligosaccharides（EPO）,GA₁R₂X₁S₂ and GA₁Man₁R₁X₁S₂,were obtained from E.prolifera polysaccharide by mild acid hydrolysis and had the inhibitory activity onα-glucosidase with IC50value of 16.86 mg/mL.