Study On The Inhibitory Effect Of Walnut Green Husk Extract On α-Glucosidase

Walnut green husk is a by-product of walnut processing,which can reach millions of tons every year and contains rich bioactive substances,including flavonoids,polysaccharides,polyphenols,quinones,pigments and so on,so it has antioxidant,antibacterial,anti-inflammatory and other functions.Therefore,further development and utilization of walnut green husk is of great significance to improve the economic benefits of walnut industry,reduce the waste of resources and environmental pollution.At present,the inhibition of α-glucosidase activity in walnut green husk has not been reported.The research on the screening of α-glucosidase inhibitors from walnut green husk has not been carried out yet.In this study,distilled water,70% ethanol/water(v/v)and 70%acetone/water(v/v)were used to extract active substances from walnut green husk,and the inhibitory effects of different extracts on α-glucosidase were evaluated.In addition,ultrafiltration assisted LC-MS/MS was used to screen and identify the 70% ethanol/water(v/v)extract with the strongest inhibitory activity,to obtain the strong inhibitory effect and high content of Sclareol Glycol.At the same time,the molecular mechanism of the inhibition of α-glucosidase activity was investigated by enzymatic inhibition kinetics,fluorescence spectroscopy,atomic force microscopy,circular dichromatography,molecular simulation and sucrose loading test in mice.The main research results of this paper are as follows:(1)Extraction,screening and identification of active substances from walnut green husk.The extraction rates of distilled water,70% ethanol/water(v/v)and 70% acetone/water(v/v)were 22.58%,25.80% and 20.24%,respectively.The half inhibitory concentrations of α-glucosidase were 5.07 mg/mL,3.32 mg/mL and 4.06 mg/mL,respectively.The extraction of 70% ethanol/water(v/v)was screened and identified by ultrafiltration assisted LC-MS/MS.The main active components of the extract were Betulinic acid、Hederagenin、Fumaric acid、Asiatic acid、Chlorogenic acid、Oleanolic acid、Ellagic acid、Corosolic acid、Schisandrin B 、 Ursolic acid 、 Sclareol Glycol and other compounds.Examines corresponding material standard product inhibition effect of alpha glycosidase enzymes,results showed that the activity of Ellagic acid is strongest,Sclareol Glycol followed,but in70% ethanol/water(v/v)of walnut green husk extract ultrafiltration Sclareol Glycol content in the sample is far higher than that of Ellagic acid,thus the subsequent choose Sclareol Glycol further research.(2)Inhibitory mechanism of Sclareol Glycol on α-glucosidase.The results of enzymatic kinetics study showed that the α-glucosidase activity was reversibly inhibited by Sclareol Glycol,and the inhibition type was competitive,and there was a single inhibition site.Fluorescence spectrum analysis showed that the endogenous fluorescence of α-glucosidase could be quenched statically by Sclareol Glycol.Atomic force microscopy(AFM)observation showed that the interaction of Sclareol Glycol withα-glucosidase changed the microenvironment around the enzyme molecules,leading to the aggregation of α-glucosidase molecules.Circular dichromatographic analysis showed that Sclareol Glycol could induce the conformational change of α-glucosidase,which resulted in the decrease of α-helix content,and the increase of β-Sheet,β-Turn and Random coil content.Molecular docking simulation showed that Sclareol Glycol could form hydrogen bonds with the amino acid residues ASP69 and GLU277 at the active center ofα-glucosidase and interact with each other to reduce the binding with the substrate.The results of sucrose loading in mice showed that Sclareol Glycol significantly reduced the blood glucose level in mice,and the effect was similar to that of acarbose.Compared with the negative control,the blood glucose level in mice was decreased by 20.89% after 120 min of intragastric administration of Sclareol Glycol.In summary,the main components inhibiting α-glucosidase activity in walnut green husk were Ellagic acid and Sclareol Glycol,among which Sclareol Glycol played a major inhibitory effect on the enzyme and had hypoglycemic activity.This study clarified the material basis and mechanism of the hypoglycemic effect of walnut green husk,and provided a theoretical basis for the further research and development of related functional health food.