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

Objective:Ribes meyeri stem as research object,the qualitative and quantitative method for 14 phenolic components were established,anti-diabetes and anti-oxidation activities were screened of different fractions,the correlation was analyzed between activities and the content of total polyphenols（TPC）and total flavonoids（TFC）,the extraction process of flavonoid aglycones from the stem of Ribes meyeri stem was optimized.Method:The different factions of Ribes meyeri.stem（dichloromethane faction ethyl acetate faction n-butanol faction water faction）were prepared by methanol extration and polar fractional solvent extraction method.A UPLC-PDA-MS/MS method was established to analyze qualitatively the phenolic compounds.Chromatographic conditions are as follows:column BEH C₁₈（5cm×5mm,1.7μm）,column temperature 35℃,injection volume 1.0μL,PDA scanning wavelength range 200 nm～400 nm,methanol-water（containing 0.1%formic acid）was used as the mobile phase,the gradient elution was performed at a flow rate of 0.3 m L/min.The retention time,mass spectrometry information,and maximum ultraviolet absorption wavelength of the compounds of the samples by comparing with those of the standard compounds.The chemical composition was speculated.The contents of polyphenol（TPC）and total flavonoids（TFC）in four fractions were determined by spectrophotometry.A UPLC-ESI-MS/MS method was established to quantify 14 kinds of phenolic compounds,Waters ACQUITY BEH Shield RP18 column（100 mm×2.1 mm,1.7μm）was used,with acetonitrile-water（0.1%formic acid）as mobile phase,the gradient elution was performed at a flow rate of 0.3 m L/min.using mass spectrometry of multiple reaction monitoring（MRM）mode for quantitative analysis.At the same time,Optimization of acid hydrolysis extraction process of flavonoid aglycones（kaempferol,quercetin,isorhamnetin,luteolin,apigenin）of Camellia oleifera stem by single factor experiment（hydrochloric acid concentration,extraction temperature,extraction time）and orthogonal experiment.The DPPH method and the ABTS method were used to investigate the anti-oxidant activity of different fractions of Ribes meyeri stem,and theirα-glucosidase,α-amylase,and PTP1B inhibitory activities were measured.Results:In the qualitative analysis of the polyphenols,it was found that there were 14 kinds of compounds in the Ribes meyeri stem,which were epigallocatechin,protocatechuic acid,epicatechin,caffeic acid,rutin,isoquercetin,kaempferol-3-O-rutin,vicularin,astragalin,quercetin,quercitrin,luteolin,kaempferol,and genkwanin,the compounds had a good linear relationship（r²≥0.9917）,stability,repeatability,and precision（RSD≤4.84%）in the test range,the recovery range was 91.50%to 102.02%（RSD≤4.39%）,indicating that the method was good.Among the four fractions,the content of polyphenols and flavonoids in the ethyl acetate fractions was the highest（965.51mg GAE/g,162.04 mg CE/g）,among which the contents of protocatechin acid,caffeic acid,epigallocatechin and epicatechin were the highest.The optimal extraction process of flavonoid aglycones from Ribes meyeri stem was as follows:5%hydrochloric acid-methonol,extraction temperature 70℃,extraction time 100 min,In this optimal extraction condition,the content of 5 kinds of flavonid aglycone（kaempferol,quercetin,isorhamnetin,luteolin and apigenin）increased significantly.among which the contents of kaempferol and luteolin reached 1.23±0.02 mg/g and 1.25±0.02 mg/g,the contents of quercetin and isorrhamtin reached 0.70±0.02 mg/g and 0.66±0.03 mg/g.Among fractions,ethyl acetate fraction showed the best antioxidant and hypoglycemic activities,the IC₅₀ values of DPPH and ABTS free radical scavenging activities were 0.34 and 0.18 mg/m L,respectively,and the IC₅₀ values of the inhibitory activities ofα-glucoidase,α-amylase and PTP1B were 134.81 mg/L,457.91 mg/L and 0.43 mg/L,respectively.Conclusion:The methods established of UPLC-PDA-MS/MS and UPLC-ESI-MS/MS in this study can be used for the qualitative and quantitative analysis of the polyphenols in the Ribes meyeri stem.The four factions from the possesed good antioxidant and hypoglycemic activities,among which the ethyl acetate fraction had the best activity.Protocatechuic acid,caffeic acid,epigallocatechin,and epicatechin may be the active constituents among the polyphenols in the Ribes meyeri stem.The optimized acid hydrolysis extraction process can effectively improve the extraction efficiency of the flavonoid aglycone from Ribes meyeri stem,which can provide a reference for the in-depth study of the constituents and biological activities of plant of Ribes L.,and provide a theoretical basis for the development and utilization of the plant.