| "Sangao",namely,high blood lipid,high blood pressure and high blood sugar,is known as the " invisible killer" of human health.The high blood lipid not only can lead to atherosclerosis,fatty liver,coronary heart disease and other cardiovascular diseases,also is closely related with hypertension and hyperglycemia-related diseases,which both threaten human health.Hyperlipidemia is mainly caused by the continuously high levels of triglycerides(TG),total cholesterols(TC)and lipids(LP),especially the triglycerides is mainly factor.TG is hydrolyzed by lipolytic enzymes(lipase)in intestinal mucosal cells to glycerol(diglycerides or monoesters)and fatty acids.And then TG is produced by a large number of fatty acids again in the liver under the action of fatty synthetase,causing blood lipid content rise.At the same time,a lot of free radicals can be produced by cell metabolism,and excess free radicals in the blood are more likely to increase lipid content by oxidizing or modifying lipoprotein cholesterol.The researches have shown that antioxidant compounds,as flavonoids and polyphenols,with strong free radical scavenging ability can downregulate TG content in serum to achieve the purpose of hypolipidemic by inhibiting the key enzymes(LP)catalytic activity during the TG synthesis.A large number of studies confirmed that the extracts of various parts of mulberry had hypolipidemic effect,and there were many reports about it in mulberry leaves,roots and fruits.But,it was rarely reported that active substances were isolated from twigs with inhibition activity of lipase in tracking.The studies showed that adiabatic environment(overgrowth,drought stress,etc.)or induced treatment(physical induction,chemical induction,bioinduction,etc.)could affect the contents and species of secondary metabolites in plants,so this research was about the hypolipidemic in twigs after induced.And found the natural lipase inhibitor with the tracking of inhibiting lipase activity.It could provide theoretical basis for the development of mulberry resources,and to provide the material source for the further enrichment of hypolipidemic components.1 Screening hypolipidemic activity from mulberry twigsIn order to located in the hypolipidemic activity extraction fraction scientifically and rapidly,the contents of total polyphenols flavonoids and the related hypolipidemic activities were tested,including inhibiting the lipase,lower cholesterol,scavenging DPPH free radical(DPPH?),protecting DNA activities in this study.The results showed that there was a significant difference in all hypolipidemic activities of different extracted components.The total content of polyphenols and flavonoids(247.79 mg/g)extracted from ethyl acetate extract(TEE)was significantly higher than that of petroleum ether extract(TPE)(57.19 mg/g),n-butanol extraction(TBE)(138.20 mg/g)and the remainder of the aqueous phase(TWE)(43.15 mg/g)(P < 0.05).The lipase inhibitory activity of TEE(IC50 1.06 ± 0.08 mg/m L)was significantly stronger than TPE(IC50 > 12.80 mg/m L),TBE(IC50 6.39 ± 0.98 mg/mL)and TWE(IC50 11.21 ± 1.98 mg/m L).And the scavenging DPPH? ability of TEE(IC50 0.24 ± 0.01 mg/m L)was also much stronger than that of TPE(IC50 0.77 ± 0.10 mg/mL),TBE(IC50 10.35 ± 1.12 mg/mL)and TWE(IC50 12.18 ± 1.80 mg/m L).Hence,the activity of TEE was the best.2 Separation and identification of active ingredients with inhibition activity in lipaseIn order to directly locate in the hypolipidemic activity components,the lipase inhibition activity substances were isolated and identified from TEE with the tracking of inhibiting lipase activity.Firstly,the TEE had been separated by different techniques such as column chromatography of different fillers(eg: silica gel,alumina,sephadex LH-20 gel),recrystallization,and so on.Then a high-throughput screening method and TLC-autoradiography were used to isolated with the tracking of inhibiting lipase activity.So six compounds with significant inhibition activity of lipase were isolated from 85.60 g of TEE by this approach.In addition,the physical and chemical properties of the compounds were analyzed by TLC-autoradiography,HPLC,NMR,and MS.The results showed that MT4-7 were respectively rutin,quercetin,morin and oxyresveratrol;The structure of MT1 and MT’2 should be further determined.3 Detection of lipase inhibiton activity and mechanism by monomer compoundsFirstly,the results of inhibition lipase activity of six compounds from TEE showed that MT’2(IC50 0.12 mg/mL)had the highest inhibition activity,followed by quercetin(IC50 2.04 mg/m L),MT1(IC50 0.99 mg/mL),rutin(IC50 1.89 mg/mL),morin(IC50 2.19 mg/m L),and oxyresveratrol(IC50 13.80 mg/mL).Secondly,the results found that quercetin and lipase combined through hydrogen bonding with molecular docking in AutoDock program.1)The H atom of 5-OH from quercetin combined with H atom of ASP-248 residues from lipase,and the hydrogen bond distance was 2.8 ?.2)There were formed two hydrogen bonds between the H atom of 7-OH from quercetin and O atom of ASP-332 and ARG-266 residues from lipase,and the hydrogen bond distance were 2.2 ? and 2.3 ? respectively.3)The H atom of 7-OH from quercetin combined with H atom of LYS-240 residues from lipase,and the hydrogen bond distance was 2.4 ?.The atoms of quercetin occupied the active site of lipase,and inactivated the lipase.The lipase activity residues was bound to quercetin by hydrogen bonding,so that it could not hydrolyze the fat,thereby reducing the origin of TG in the blood and lowering the level of TG.In this study,a number of hypoglycemic-related activity indicators were studied in the induction of mulberry twigs,and explored the mechanism of lipase and quercetin,isolated from mulberry twigs,in the molecular level.It not only can provide a new way for the natural sources of lipase inhibitors,but also provide a new ideas for the development of treating hyperlipidemia drug. |
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