Study On Screening α-Glucosidase Inhibitors And Anti-Diabetic Mechanism In Sargassum Pallidum

Sargassum pallidum（S.pallidum）is mainly distributed in coastal areas of China like Shangdong and Liaoning province,and used for both food and medicine.S.pallidum was rich in polyphenols,polysaccharides,fatty acids,terpenes and so on,and showed good antioxidant,antibacterial,hypoglycemic,antitumor and immune enhancement activities.Currently,the study of the active constituents in S.pallidum mainly focuses on polysaccharide,while there is limited reports about the small molecule compound.The kinds ofα-glucosidase inhibitors of S.pallidum and its antidiabetic mechanism are also unavailable.Theα-glucosidase inhibitors from four brown seaweeds（S.pallidum,Ecklonia kurome,Sargassum fusiform and Undaria pinnatifida Suringar）were screened in the study.The phenolics of S.pallidum were studied systemically,including extraction,phytochemical composition,antioxidant and hypoglycemic effects.The inhibition mechanism of quercetin-3-O-glucuronide（Q3GA）6-gingerol（6G）and poricoic acid A（PA）onα-glucosidase and non-enzymatic glycation were investigated.The effect of S.pallidum phenolics extracts on the hypoglycemic activity and gut microbes in type II diabetic mouse was also evaluated.The main results were as follows:（1）The phytochemical composition,antioxidant andα-glucosidase inhibition activity of four brown seaweeds were compared in this study.The crude extracts of our brown seaweeds showed low content of phenolics,weak antioxidant capacity andα-glucosidase inhibitory.After purification,the total phenolics content of enriched extracts of four brown seaweeds were 2-4times higher than that of crude extracts.The enriched extracts of Sargassum fusiform had the highest total phenolics content（TPC）with 60.15 mg GAE/g E,and showed the strongest antioxidant activity.The enriched extracts of S.pallidum indicated the bestα-glucosidase inhibitory effect with IC₅₀ value of 270.7μg/m L,which was 6.5 times lower than acarbose.Forty-five compounds were identified in four brown seaweeds extracts.A total of twenty-one potentialα-glucosidase inhibitors were separated from four brown seaweed extracts by ultrafiltration,and the phenolics and terpenoids like Q3GA,xylariacin B and PA were the major constituents.（2）Box-Behnken design（BBD）were used to optimize the ultrasound-assisted extraction of phenolics in S.pallidum.The optimum condition was ethanol concentration 8.5%,extraction time 55 min,extraction temperature 42℃,ultrasonic power 494 W,and the total phenolic content of S.pallidum extract was 1.81 mg GAE/g DM,which was close to predicted value.The soluble phenolics of S.pallidum mainly existed in bound form,esterified and glycoside-bound phenolics fractions showed the highest total phenolics（68.30 mg GAE/g E）and flavonoids content（83.49 mg Qu E/g E）,respectively.A total of twenty-two compounds were identified in S.pallidum phenolics extract,Q3GA,PA and 6G showed high content.The antioxidant activity of bound phenolics fractions was stronger than free phenolics fraction,and glycoside-bound phenolics fractions had the highest CAA value（18.18μmol QE/g E）.Esterified-bound phenolics indicated the strongestα-glucosidase inhibitory with IC₅₀ value of27.09μg/m L,and the three phenolics fractions were mixed typeα-glucosidase inhibitors.（3）Theα-glucosidase inhibitory of PA,6G and Q3GA was investigated,and Q3GA showed the strongestα-glucosidase inhibition ability with IC₅₀ value of 0.05 mg/m L,which was 27.2 times lower than acarbose.PA,6G and Q3GA could bind with the active sites ofα-glucosidase by hydrogen bond to prevent the substrates to enter the active center of enzyme,reduced reaction rate of enzyme,and reached the effect of inhibitingα-glucosidase activity.Q3GA showed mixed-mode inhibition ofα-glucosidase activity,quenched the fluorescence ofα-glucosidase with a static process,and the main driving forces were hydrogen bonding and van der Waals forces.There is only one class of binding site for Q3GA onα-glucosidase.The addition of Q3GA enhanced the polarity of the microenvironment of Trp residues,reduced the percentages ofα-helix andβ-turn,increased the percentages ofβ-sheet and random coil,made the structure ofα-glucosidase more compact,and promoted the inhibitors entering the active center of the enzyme.（4）The non-enzymatic glycation inhibition activity of S.pallidum,PA,6G and Q3GA was evaluated,and the results indicated they could suppress the generation of fructosamine,5-hydroxymethylfurfural（5-HMF）and advanced glycation end products（AGEs）,which was stronger than aminoguanidine（AG）.Q3GA,6G and PA quenched the fluorescence of ovalbumin（OVA）with a static mechanism,changed the hydrophilic microenvironment of the tyrosine（Tyr）and Trp residues,and the number of binding sites were 2,1 and 1,respectively.The binding of 6G and PA with OVA were driven by hydrogen bonds and van der Waals interactions,while that for Q3GA was hydrogen bonds and hydrophobic interaction.Q3GA,6G and PA formed hydrogen bonds with Ser103,Leu101 and Thr 91 of OVA,influenced the reaction between OVA and fructose,and then inhibited the production of early,middle and advanced glycation products.（5）Oral administration of S.pallidum phenolics extract had significant improvement effect on a high-fat-diet/streptozocin induced model of type II diabetes mice with the following improvements:（a）significantly reduced fasting blood glucose and insulin levels,improved the glucose tolerance,and relieved the hyperlipidemia symptoms;（b）increased the antioxidant enzyme activities and alleviated oxidative stress in mice,decreased the PEPCK activity and increased the HK activity in liver,inhibited gluconeogenesis and promoted glycogen synthesis;（c）regulated hepatic glucose metabolism through PI3K/Akt/FOXO1/G6pase/GLUT2 signaling pathway,promoted the transport of glucose,and kept glucose homeostasis in liver;down-regulate the expression levels of FAS and ACC-1 for lipid metabolism in liver;（d）decreased the ratios Firmicutes/Bacteroidetes（F/B）and Proteobacteria levels in gut,enhanced the growth of beneficial bacteria（Lactobacillaceae and Peptostreptococcaceae）,and inhibited the production of harmful bacteria（Helicobacteraceae and Desulfovibrionaceae）,which may benefit to intestinal health in diabetic mice.The study could provide guidance for the development and utilization of S.pallidum in the field of diabetes mellitus,and also may give reference for the development of high value-added food of S.pallidum.