Characterization And Purification Of Flavonols From Morella Rubra Sieb.et Zucc. And The Structure-activity Mechanism Of Flavonols For Inhibition On α-glucosidase

In the present study,flavonols profile and their anti-diabetic activity were investigated in different tissue parts of Chinese bayberry（Morella rubra Sieb.et Zucc.）.In vivo anti-hyperglycemic effect of flavonol-rich fruit extract from white bayberry was evaluated in diabetic KK-A^y mice.Flavonol components were identified and quantified in leaves,stem and young fruits of 12 bayberry cultivars.Antioxidant activity andα-glucosidase inhibitory activity of their extracts were also determined to evaluate in vitro anti-diabetic potential of different bayberry tissues.Rapid and efficient purification system were built to obtain flavonols with high purity from bayberry leaves.Enzyme inhibitory effects and structure-activity mechanism of natural flavonol monomers were explored withα-glucosidase as the target enzyme.The main results were as follows:’Shui Jing’fruit extracts（SJE）rich in flavonols including myricetrin and quercitrin,and showed excellent in vivo anti-hyperglycemic effect in diabetic KK-A^y mice.SJE（200 mg/kg bodyweight）significantly reduced fasting blood glucose,elevated glucose tolerance,and insulin sensitivity in diabetic mice.It markedly decreased insulin,leptin,glucagon,triglyceride（TG）,total cholesterol（TC）,low-density liptein cholesterol（LDL-c）and alanine aminotransferase（ALT）levels in mice.Liver weight and hepatic lipid accumulation were also significantly reduced by SJE.Gene and protein expression analysis showed SJE may exert anti-diabetic effect by improving glucose and lipid metabolism in liver through an AMPK-dependent pathway.Total phenolics and antioxidant activity in three tissue parts of different bayberry cultivars were in the order of leaf>stem>young fruit.By HPLC and LC-MS techniques,ten flavonols were identified in different tissues of Chinese bayberry,including six myricetin glycosides,i.e.,myricetin-3-O-rhamnoside（myricitrin）,myricetin hexoside,myricetin-O-galloyl-hexoside,myricetin-O-acetyl-deoxyhexoside,myricetin deoxyhexoside gallate and its isomer,as well as four quercetin glycosides,i.e.,quercetin-3-O-rhamnoside（quercetrin）,quercetin-3-O-galactoside（hyperoside）,quercetin-3-O-glucoside（isoquercetrin）and quercetin-O-galloyl-hexoside.Myricitrin were the predominant flavonols in the three tissues of bayberry,and its content in leaf or stem was much higher than that in young fruit.Interestingly,results showed thatα-glucosidase inhibitory activities of extracts from leaf or stem were also stronger than that of young fruit.There were markedly positive correlation between theα-glucosidase inhibitory activities and flavonol contents.By solid phase extraction（SPE）combined with high speed counter current chromatography（HSCCC）,a rapid and efficient separation and purification system were established to obtain high purity of myricitrin from bayberry leaves.After the first-step purification by C18 Sep-Pak^?SPE column,the purity of myricitrin was increased from 1.22%to 39.79%.Further HSCCC purification with the solvent system of petroleum ether-ethyl acetate-methanol-water（2:6:2:5,v/v/v/v）resulted in a final purity of more than 98%for myricitrin,wich was analyzed and confirmed with the standard substance by HPLC and LC-MS.Inhibition activities onα-glucosidase were investigated among 24 flavonols,including six aglycones and eighteen glycosides.Among them,myricetin（3’,4’,5’-OH）possessed the strongestα-glucosidase inhibitory activity with an IC₅₀ value of 33.20±0.43μmol/L,followed by quercetin（3’,4’-OH）and kaempferol（4’-OH）,with IC₅₀values of 46.51±0.54μmol/L,and 65.36±0.27μmol/L,respectively,indicating their inhibitory activities were enhanced by increasing number of hydroxyl groups on B-ring.Notably,their inhibitory activities onα-glucosidase were stronger than that of the positive drug acarbose(IC₅₀=419.60±2.41μmol/L).In addition,quercetin-3-α-L-arabinofuranoside showed the most potentα-glucosidase inhibitory activity among the tested flavonol glycosides,while there was no glycoside derivatives showed better inhibition effects onα-glucosidase than that of acarbose.Myricetin,quercetin and kaempferol were selected as the representative chemicals to study interaction mechanism between flavonols andα-glucosidase by kinetic analysis,fluorescence spectrum analysis,molecular docking and molecular dynamics simulations.Results showed that flavonols could interact withα-glucosidase,quench its intrinsic fluorescence statically,and occupy the entrance to active center of the enzyme.Furthermore,the B-ring of flavonols deeply penetrated to the active pocket ofα-glucosidase,and interacted with catalytic key residues including ASP352 and GLU277 by forming hydrophobic force and hydrogen bonds.Their binding induced conformational changes of enzyme,blocked the recognition and combination procedure,and thus resulted in the inhibition activity onα-glucosidase.Based on the inhibitory activity data of 24 flavonols onα-glucosidase,two 3D-QSAR models were established by the methods of Co MFA and Co MSIA,respectively.Related pharameters of the models were both up to standanrds,and the predicted p IC₅₀values of flavonols in training set and test set were very close to their experimental p IC₅₀values with deviations less than 1,indicating the built 3D-QSAR models were reliable and had good prediction ability for the inhibitory activity of flavonols onα-glucosidase.Futher analysis on counter maps revealed effects of steric field,electrostatic field,hydrophobic field,hydrogen bond receptor field and hydrogen bond donor field onα-glucosidase inhibitory effects of flavonols,and elucidated the structure-activity relationships among flavonols interacted withα-glucosidase.In addition,inhibitory effects of 379 kinds of natural flavonol derivatives onα-glucosidase were predicted by the built 3D-QSAR models.Some flavonols showed lower predicted p IC₅₀ values compared with the experimental p IC₅₀value of acarbose.Such results show screening references,and provide a batch of candidate compounds with great potential on development of natural flavonolα-glucosidase inhibitors with stronger activity,higher bioavailability and lower toxic and side effects.Our findings showed that different tissues of Chinese bayberry rich in flavonols showed great anti-diabetic potential in vivo and in vitro.Myricetin showed the strongestα-glucosidase inhibitory activity among hundreds of flavonols tested by experiment or predicted by 3D-QSAR models in this study,indicating myricetin as well as plant resources rich in myricetin such as Chinese bayberry leaves and stem may have great application potential on the development of functional food or drugs for the treatment and management of diabetes.