Study On Quality Control And Extraction,purification And Antioxidant Activity Of Total Flavonoids From Sauropi Folium

Objectives:To establish HPLC fingerprints of Sauropi Folium in different harvest periods,evaluate the quality difference combined with chemical pattern recognition,and identify the chemical components by UPLC-Q-TOF-MS;The content determination method of total flavonoids in Sauropi Folium was established,the total flavonoids were extracted and purified,and the chemical composition identification and antioxidant activity of the purified products were studied.The experimental results will provide a scientific basis for the quality control of Sauropi Folium and the development and application of its flavonoids.Methods:（1）The HPLC fingerprints of Sauropi Folium in different harvest stages were established.Data mining of HCA,PCA and OPLS-DA were performed in combination with chemical pattern recognition to screen out the quality markers among the Sauropi Folium in different harvest stages.The UPLC-Q-TOF-MS method was used to analyze and identify the chemical constituents in Sauropi Folium.（2）A method was established for the determination of total flavonoids in Sauropi Folium with rutin as reference substance and the extraction yield of total flavonoids as the index.The extraction conditions of total flavonoids from Sauropi Folium were determined by single factor experiments.The static and dynamic adsorption-desorption experiments of macroporous resin were conducted to compare the purification effects of different resins（D101,AB-8,HPD100,HPD600,DM301 and NKA9）on the total flavonoids in Sauropi Folium and to screen the resin type.The purification parameters such as concentration,p H,and volume of the loading solution,as well as the volume fraction and elution dosage of the ethanol eluent were screened by single factor experiments.The adsorption mechanism of macroporous resin was analyzed by combining adsorption kinetic model and adsorption isothermal model fitting.The chemical components of the purified products were analyzed and identified by HPLC and UPLC-Q-TOF-MS.（3）Ascorbic acid（VC）was used as the reference substance to evaluate the in vitro antioxidant activity of total flavonoids from Sauropi Folium based on its total antioxidant activity and the capability of scavenging DPPH,OH and ABTS radicals.Results:（1）Twenty-one common peaks were identified from the HPLC fingerprint of Sauropi Folium in different harvest periods,and the similarity was more than0.9.However,the RSD values of the relative peak areas of the common peaks were mostly higher than 20%,indicating that the overall quality of the eleven batches of medicinal materials was relatively stable,with good similarity but significant content difference.The content of chemical constituents in Sauropi Folium was higher in summer and autumn,when the best harvest time was obtained.The eleven batches of Sauropi Folium were divided into three categories by HCA,and four principal components,PC1,PC2,PC3 and PC4,were extracted from PCA,with the cumulative contribution rate reaching94.446%.Nine quality markers showing differences among the medicinal materials were screened out by OPLS-DA.A total of 47 chemical components were identified by UPLC-Q-TOF-MS.（2）The extraction process of total flavonoids from Sauropi Folium was as follows:50 times of water was added and the extraction was carried out in a water bath under reflux for two times,90 min for the first time and 60 min for the second time.After filtration,the filtrate was collected and concentrated to a crude drug concentration of 1g/m L for subsequent use.The purification effect of total flavonoids from Sauropi Folium was superior in AB-8 resin.The adsorption kinetic behavior of total flavonoids conformed to the quasi-two-stage kinetic model（R²=0.9988）,and the isothermal adsorption data conformed to the Freundlich model（R²=0.9839）.The optimal purification process parameters were as follows:1.5 BV of loading solution with concentration of0.167 mg/m L and p H=5 was loaded onto the column at the flow rate of 1m L/min,fully adsorbed,and then washed with 4BV pure water to remove impurities;finally,4 BV 50%ethanol solution was used for elution at the flow rate of 2m L/min.The retention rate and purity of total flavonoids were 98.42%and22.17%,respectively,3.06 times higher than that before purification.After UPLC-Q-TOF-MS analysis,it was identified that the purified products might contain flavonoids such as quercetin-3-O-gentiobioside,kaempferol-3-O-α-L-rhamnose-β-D-glucose-β-D-glucoside and kaempferol-3-O-gentiobioside.It may also contain methionine,（-）-lyoniresinol-3a-O-β-D-glucopyranoside,ferulic acid,dibutyl-2,2-dimethylmalonate,and 4H-1-Benzopyran-4-one.（3）The total antioxidant activity and the abilities of scavenging DPPH,OH and ABTS radicals of total flavonoids were positively correlated with the concentration.Under the same reaction time,the total oxidative activity of total flavonoids in Sauropi Folium was significantly stronger than that of VC.The IC₅₀ values of DPPH radical scavenging activities of total flavonoids and VC were 74μg/m L and 85μg/m L,respectively.The IC₅₀ values of OH radical scavenging activities were 376.38μg/m L and 262μg/m L,respectively.The IC₅₀values of ABTS radical scavenging activities were 10.937μg/m L and 11.705μg/m L,respectively.Conclusions:The HPLC fingerprint of Sauropi Folium in different harvest periods was established.Based on the fingerprint,combined with chemical pattern recognition to evaluate the quality differences of Sauropi Folium in different harvest periods,a more perfect,effective and reasonable evaluation method of Sauropi Folium quality was constructed.The established extraction and purification process of total flavonoids from Sauropi Folium was stable and reliable,which could effectively improve the purity of total flavonoids and laid the foundation for component analysis and activity research.The total flavonoids from Sauropi Folium had strong antioxidant activity in vitro and there was a dose-effect relationship with the concentration,which had clinical development significance.