| Puerariae Lobatae Radix is the dried root of the Pueraria lobate(wild.)Ohwi,which has been widely used in traditional Chinese medicine.P.lobate(wild.)Ohwi is contained in the “Shen Nong’s Herbal Classic” and is listed as the middle quality.It has the functions of relieving muscle and fever,promoting the production of body fluid to quench thirst,penetrating rash,promoting yang to stop diarrhea,promoting meridians and activating collaterals,detoxifying wine,etc.The modern pharmacological research shows that Puerariae Lobatae Radix mainly contains flavonoids,triterpenoids,coumarins,puerarin glycosides,alkaloids,amino acids and other chemical components,which have a variety of pharmacological effects,such as protecting cardiovascular and cerebrovascular vessels,reducing blood pressure,lowering blood sugar,lowering blood lipid,antioxidation and regulating immunity.In 2015 edition of China Pharmacopoeia,puerarin is used as the only index component to evaluate the quality of Puerariae Lobatae Radix,while the composition of traditional Chinese medicine is complex,and the pharmacological effect of multi target and complex ingredients.The quality of traditional Chinese medicine is affected by the growing areas,harvest time and etc,so it is not enough to reflect the quality of Puerariae Lobatae Radix only by one or more components.Therefore,this topic makes a spectral-effect correlation from the main chemical constituents and biological activity of Puerariae Lobatae Radix,and provides a certain basis for the subsequent quality control methods of Puerariae Lobatae Radix.And Puerariae Lobatae Radix is also a traditional Chinese medicine for food,the continuous improvement of the quality of Puerariae Lobatae Radix will also benefit the comprehensive utilization and development.ObjectiveTo explore the relationship between the chemical fingerprint and biological activity,and to provide ideas and directions for the subsequent quality control methods.Methods1.Establish the extraction method.Through the investigation of different material-liquid ratios,extraction time,extraction temperature,and extraction solvent,the single-factor experimental results are obtained,and then the optimal conditions are obtained by using the Box-Behnken response surface design to determine the optimal extraction process.2.The active ingredient content was determined.High performance liquid chromatography(HPLC)was used to simultaneously determine the content of nine active ingredients(3’-hydroxy puerarin,puerarin,puerarin apioside,3’-methoxy puerarin,puerarin 6’’-O-xyloside,daidzin,genistin,formononetinand and daidzein)in Puerariae Lobatae Radix.Establish a quantitative analysis of multi-components by single marker(QAMS)for determining contents of nine compositions in Puerariae Lobatae Radix,which is feasible and accurate.3.Based on the fingerprint atlas of Puerariae Lobatae Radix,the similarity analysis,the system cluster analysis and principal component analysis were carried out by the method of stoichiometric analysis.4.Based on the in vitro activity study,the antioxidant activity of Puerariae Lobatae Radix samples was measured in vitro to compare the differences.5.Taking the in vitro antioxidant activity and α-glucosidase activity inhibition ability as pharmacodynamic indicators,the grey-correlation analysis was used to establish the spectrum-effect relationship between the common peaks of chemical fingerprints and in vitro biological activities.Results1.The conditions of extracting the effective components of P.lobata were determined as follows: ultrasonic time 22 min,material-liquid ratio220:1,methanol concentration 72%,extraction temperature 40 ℃.2.The content of active ingredient in different origins varies greatly.The content of total isoflavones was 4.77 ~ 17.44%.But the content of puerarin in the sample(S11)was unqualified,and the highest active ingredient content was produced in Langao County,Ankang,Shaanxi Province(S16).In addition,after methodological investigation,the the quantitative analysis of multi-components by a single marker(QAMS)was was accurate,reliable and feasible.3.The HPLC fingerprint was studied.23 common peaks were obtained by similarity analysis software,and 9 of them were identified,and the similarity of the common peaks was more than 80%.The 40 batches in different regions were divided into two categories through the cluster analysis.Three principal components were extracted by principal component analysis,and the variance contribution rate was 81.835%.4.In vitro activities were studied.Puerariae Lobatae Radix from different regions had certain antioxidant activity and α-glucosidase activity inhibition,among which the antioxidant capacity and α-glucosidase activity inhibition of Pueraria from Shaanxi,Hubei and Hunan were better.5.The spectral-effect relationship was studied.Based on the antioxidant capacity and α-glucosidase activity inhibition capacity,the chromatographic peaks with a correlation of more than 0.700 were screened from the common peaks of the fingerprint,and the peaks with the three antioxidant activities were P1,P2,P5,P7(puerarin apioside),P9(puerarin 6’’-O-xyloside),P13,P15,P19,P20,P21,P22(formononetin),P23(daidzein).The peaks related to the inhibition of α-glucosidase activity were P1,P2,P3,P4(3’-hydroxy puerarin),P5,P6(puerarin),P7,P9,P11,P12(daidzein),P13,P14,P15,P16,P21,P23..Conclusion1.The content determination,fingerprint and the QAMS were established by HPLC.These methods can be used in the quality research of Puerariae Lobatae Radix,which made up for the deficiency of single index evaluation and provided a new method for the quality evaluation of Puerariae Lobatae Radix.2.The chemical composition of Puerariae Lobatae Radix samples from different areas were similar,but the content,antioxidant capacity andα-glucosidase activity inhibition ability were different.The quality of Puerariae Lobatae Radix samples from different areas may be different,and the quality of Puerariae Lobatae Radix samples from the same province may not be the same.3.Through the method of combining the in vitro antioxidant capacity andα-glucosidase activity inhibition of Puerariae Lobatae Radix with chemical fingerprinting by grey correlation analysis,the chromatographic peaks related to the activity were screened,which provided the idea and reference for the further study of the pharmacodynamic basis of Puerariae Lobatae Radix and may improve the quality evaluation method test. |
PDF Full Text Request