The Chemical Constituents And Their Hypoglycemic Activity In Vitro Of Ribes Meyeri Leaves And Fruits

Diabetes mellitus is one of the major metabolic diseases threatening human health.It has the characteristics of high mortality rate,high disability rate and high medical cost.At present,most of the hypoglycemic drugs are chemosynthetic,which have significant effects but also produce many toxic and side effects.In recent years,edible plant ingredients as a means of prevention and treatment of diseases have gradually become the focus of research.Ribes meyeri Maxim.is a unique edible Ribes L.plant in Xinjiang.It has biological activities such as antioxidant and hypoglycemic.However,few studies have been done on its chemical constituents and biological activities.In this paper,HPLC-QTOF-MS/MS was established for the qualitative analysis of the chemical components of the leaves and fruits of R.meyeri.Quantitative analysis of the chemical components of the leaves and fruits of R.meyeri by UPLC-MS/MS;On this basis,the DPPH and ABTS free radical scavenging experiments were used to study the antioxidant activity of the methanol extracts and different extractions of R.meyeri leaves and fruit.Theα-glucosidase,α-amylase,and PTP1B inhibitory activity experiments and the glucose consumption of 3T3-L1 preadipocytes was used to study their hypoglycemic activity in vitro,aiming to discover the active fractions and active constituents in the leaves and fruits of R.meyeri.The main research results are as follows:In the qualitative analysis,77 phenolic compounds,including 44 phenolic acids and 33 flavonoids,were identified in the methanol extract of R.meyeri leaves under the negative ion mode.A potential new compound,a quercetin glucoside with tetra-saccharide groups was found.And an UPLC-MS/MS method was established for the simultaneous determination of 19 polyphenols（gallic acid,epigallocatechin,protocatechuic acid,chlorogenic acid,p-hydroxybenzoic acid,epicatechin,caffeic acid,p-coumaric acid,rutin,ferulic acid,isoquercitrin,salicylic acid,trans cinnamic acid,kaempferol-3-O-rutinoside,astragalin,luteolin,quercetin,apigenin and hydroxygenkwanin）in the R.meyeri leaves.The results showed that there were significant differences in the contents of 19 polyphenols in the methanol and four extracts of R.meyeri leaves.Among them,rutin,epigallocatechin,kaempferol-3-O-rutinoside,isoquercitrin,protocatechuic acid,epicatechin and chlorogenic acid had the highest content in the ethyl acetate extract,and the content of rutin reached 86.07±4.06 mg/g.The chromatographic conditions are as follows:acetonitrile-0.1%formic acid water as mobile phase and gradient elution.19 polyphenols were separated well within 8 min in this condition.The 19 polyphenols showed good linear relationship（R²≥0.9967）,stability,repeatability and precision（RSD≤6.98%）,and the recoveries ranged from 82%to 105%,which indicated that the method was good.In the qualitative analysis of R.meyeri fruits,26 anthocyanins were identified under positive ion mode.The main anthocyanins in the R.meyeri were cyanidin derivatives,delphinidin and pelargonin derivatives.In negative mode,17 phenolics,including phenolic acids and flavonoids,were identified.In addition,in positive ion mode,using multiple response monitoring（MRM）,a UPLC-MS/MS method was established for the simultaneous determination of four anthocyanins in the R.meyeri fruits,including delphinidin-3-O-glucoside,delphinidin-3-O-rutinoside,cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside.The results showed that the contents of cyanidin-3-O-rutinoside in methanol extract and anthocyanin extract of R.meyeri fruits were the highest,which reached 22.01±0.37 mg/g and 79.36±1.01 mg/g,respectively.In order to optimize the separation efficiency of the four anthocyanins,three different mobile phase systems were investigated.When acetonitrile-5%formic acid water was used as mobile phase,the separation efficiency of the four anthocyanins compounds was the best and the peak shape was good,and baseline separation was achieved within 8 min.Methodological studies showed that the linear relationships of the four anthocyanins were good in the concentration range（R²≥0.9941）.The average recoveries were 83%～94%with RSD≤5.77%.The results showed that the method was accurate,simple,rapid and reproducible.The antioxidant activities of R.meyeri leaves and fruits were evaluated by DPPH and ABTS methods.The antioxidant activity of each extract was lower than that of ascorbic acid（Vc）for DPPH radical,but the ethyl acetate extract of R.meyeri leaves had the highest DPPH scavenging rate compared with other extracts.The antioxidant activities of the extracts were lower than those of the positive control（Vc）,except that the ethyl acetate extract of R.meyeri leaves was higher than that of Vc.The results showed that the ethyl acetate extract and anthocyanin extract of R.meyeri had better inhibitory activity againstα-glucosidase andα-amylase,and the IC₅₀of both extracts were higher than that of acarbose.There was no significant inhibitory activity on PTP1B in all extracts.The extracts of R.meyeri leaves and fruits all promoted glucose uptake by 3T3-L1 preadipocytes.It was suggested that R.meyeri leaves and fruits had potential hypoglycemic active components,and the active components and mechanism of action were worthy of further study.