Study On Quality Evaluation Of Several Varieties Of Puerariae Radix From Sichuan Province

Puerariae Radix including P.lobata and P.thomsonii has been recorded in the current version of China Pharmacopoeia,the former is the dried roots of P.lobata（Willd.）Ohwi,and the latter is the dried roots of P.thomsonii Benth,Due to the rich resources of Puerariae Radix in Sichuan,besides the Pharmacopoeia varieties,appeared wild or cultivated of P.omeiensis used as P.thomsonii or Plobata.The resources of P.omeiensis just after P.lobata and P.thomsonii in Sichuan.Based on literature,medicinal origin and market research,this research used the classification and identification of classical methods,combined with modern instrument analysis technology,studied the mainstream varieties and quality status through the system evaluation of the quality research on Puerariae Radix from Sichuan.The results are as follows:1 The experiment made systematic comparative studies on the morphologicalhistologythroughcombinedconventionalcrudedrug identification methods with microscopic-chemical identification method,It found out main difference characteristics of the original plants,medicinal properties and microscopic of the three different varieties,and the polysaccharide,flavonoid composition distribution in the organization.The results provided reference bases for plants identification.1.1 The differences among the leaves,trichomes,inflorescence length and the corolla and calyx color,etc were the mainly identification characteristic of original plant morphology.1.2 Medicinal properties were identified mainly from the surface color,the strength of medicinal mealiness,the annular texture on section and smell etc.1.3 The identification on microscopic characteristics mainly through the number of crystal sheath fiber bundles,fibre bundles and stone celles,the size and distribution of the catheters in the root transverse section and from the number of stone cells,secretory tissue and crystal fibers,starch grains of single grains and compound grains situation in powder characteristics,etc.1.4 Chemical microscopic showed that polysaccharides and flavonoids components in different varieties of medicinal materials were mostly had different degrees of coloring in the cortex,phloem and xylem.Due to the different of varieties,the wild or cultivated,its`medicinal materials contained different polysaccharides and flavonoids.2 The TLC identification method of puerariae radix crude drugs was improved by increasing reference substances daidzein and daidzien relative to the TLC identification method of puerariae radix in the current edition of China pharmacopoeia.The similarities and differences among the puerariae radix herbs were found out by TLC analysis.These results provided a reference basis for variety identification and chemical composition analysis of puerariae radix herbs.There were corresponding spots`locations to three reference substance appeared in varieties,but fluorescence intensity were mostly different,which indicated that there were different content of three components in varieties.Samples from different places which were same variety,its chromatographic spoted spots size and the fluorescence intensity were discrepancy,indicated that there are differences between the samples of different origin on the components content.3 The research established NIR analysis and UPLC fingerprint analysis methods on three puerariae radix and made comparative analysises on the different regions and varieties of puerariae radix drugs.The results provided references for identification of three varieties,and laid the groundwork for puerarin quality of medicinal materials.3.1 P.thomsonii and P.omeiensis had absorption peak in 850⁹50 nm wavelength in NIR original map,first derivative map,but P.lobata without,and P.thomsonii consistent with P.omeiensis on peaks.P.lobata had absorption peak shapely different from P.thomsonii and P.omeiensis in 1000¹162 nm wavelength of first derivative map.The second order number processing map of P.lobata in 1180¹210 nm,1695¹775 nm,and 2095²195 nm wavelength were different with P.thomsonii and P.omeiensis.There were no significant difference P.thomsonii with P.omeiensis in second order number processing map.3.2 The study determined P.lobata UPLC fingerprint 29 common peaks,P.thomsonii UPLC fingerprint 18 common peaks,P.omeiensis UPLC fingerprint 16common peaks,and identified P.lobata seven peaks,P.thomsonii and P.omeiensis six peaks.Three varieties were highly similar,P.lobata could be identified by 13,14,16,23,25,26,29 peaks with other,and P.thomsonii could be identified by 9 and 19 peaks with P.omeiensis.4 Based on the requirement of the inspection item in the current version of the Chinese pharmacopoeia,this experiment made samples detections on moisture,total ash,acid insoluble ash,sulfur dioxide residue,and the heavy metals and harmful elements and pesticide residue.4.1 The moisture and total ash of each sample conformed to the requirement of2015 China pharmacopoeia.There were differences in acid insoluble ash content of samples with different varieties.4.2 SO₂ residue determination results showed that whether pick-your-own medicinal materials or goods medicinal materials,which were non SO₂ smoked much within 50 mg·kg^-1.But sulfur smoked samples of SO₂ residues difference was remarkable,11 samples of sulfur smoked SO₂ residue in minimum of 110.3 mg·kg^-1,up to 645.35 mg·kg^-1,there were 4 batch than pharmacopoeia regulation,accounted for 36%of the sulfur smoked samples.4.3 The residue detection results of 12 different batch samples on the lead,cadmium,mercury,arsenic and copper showed that some samples were not up to the standard.4.4 Pesticide residues determination results showed that the all test samples conformed to the standard,and had not been detected BHC,DDT and PCNB residues.5 According to the current edition of China pharmacopoeia,this research made comparisons and analysis of different varieties alcohol extract content between different samples.The study laided a foundation for further study of types and varieties of puerarin herbs quality.Determination results showed that the alcohol extract content in different varieties and regions were varied.P.lobata samples alcohol extract were consistent with current edition pharmacopoeia,which mainly came from the Sichuan Beichuan,Pingwu,Qingchuan region of were higher.P.thomsonii alcohol extract content consistent with current edition pharmacopoeia,except the sample 5,6,P.omeiensis sample alcohol extract content was 7.43%⁷.43%,49,52 samples content less than10.0%.6 The study established ultraviolet spectrophotometry determination methods of the flavones and polysaccharide content in puerariae radix drugs,and determined samples,analyzed the different species,different origins puerarin class of the flavonoids and polysaccharide composition.The reasults provided referential basises for further study on different varieties.Results indicated that the different regions,different sources of puerarin,puerariae radix of flavonoids and polysaccharide content were different.Wild P.omeiensis flavonoids content higher than cultivated,cultivated P.omeiensis polysaccharide was higher than the wild P.omeiensis,and total flavonoids and polysaccharides closed to P.thomsonii.Samples of P.lobata flavonoids content were2.62%².62%,the polysaccharide content were 11.03%¹1.03%,flavonoids content were different.But no obvious difference was found between polysaccharide content.P.lobata flavonoids content were high than P.thomsonii and P.omeiensis,nearly 40 times,but P.thomsonii and P.omeiensis polysaccharide content were higher than puerarin,nearly six times.7 The study established UPLC-DAD wavelength switched multicomponent determination method,and made determination and analyzed the differences between the varieties,origins puerarin herbs and the correlation between each component.It provided referential basis for further quality study on different varieties of puerarin medicinal materials.7.1 The results showed that each component content in the sample were different.According to the current edition of the China pharmacopoeia standards to measure the content of puerarin,P.thomsonii samples 1,2,4,8,9,10,13 samples of content were higher than other and conform to the requirement of pharmacopoeia,respectively from Cangxi,Wangcang,Quxian,Jiangyou,Jianyang,Mianyang,Shehong,but the rest since the mining samples did not conform to the standard.45,47,55,58,50,51,61commodity P.thomsonii conformed to the standards,of which 50,55,57,58 sample content were higher,the rest of commodities P.thomsonii did not conform to the regulations,smoked and non smoked was no significant difference.Wild P.omeiensis sample 21 to 27 content were higher,over 0.3%,cultivated samples 19,20,29,30,31and goods 49 were close to P.thomsonii,52 sample higher than 0.3%,the rest of the sample content lower than it.Samples of P.lobata,in addition to 38,39,70 sample content lower than 2.4%,the rest of samples conformed to the requirements of the pharmacopoeia,32³7 samples content were higher,which produced in Pingwu,Qingchuan,Beichuan areas.Commodities of P.lobata samples of 65,67,68,69content were higher.7.2 The DPS processing analysis software analysis on the correlation among3’-hydroxy puerarin,puerarin,daidzein,genistein glycosides,daidzien and genistein showed that contents influenced each other on the component.8 The research were first time to use TLC-since the imaging technology and DPPH antioxidant activity clearance IC₅₀0 as index,made test and analyzed the correlation between sample IC₅₀0 and each component content.Provided referential basis for further quality research and evaluation of puerarin medicinal materials.8.1 TLC-since the imaging results revealed that 3’-hydroxy puerarin,puerarin,daidzien,daidzein,genistein glycosides,genistein six spots corresponding Rf value position of the spots had DPPH scavenging activity,and other spots position had weak DPPH scavenging activity.8.2 According to the results,50%ethanol extract of P.lobata had the strongest antioxidant activity,P.thomsonii extract with 70%ethanol had strongest antioxidant activity.The composition of the antioxidant activity of pecking order:3’-hydroxy puerarin>genistein glycosides>daidzein>genistein>daidzein>puerarin.8.3 Determination of antioxidant antioxidant activity of 71 samples showed that,with the 3’-hydroxy puerarin,puerarin,daidzien,genistein glycosides,daidzein,genistein were higher,the smaller the IC₅₀0 became,samples`antioxidant ability were stronger,each component content influenced the degree of the sample aontioxidant capacity,puerarin and 3’-hydroxy puerarin antioxidant capacity of the sample`s effect were more obvious.8.4 Applicated DPS software to analyize the correlation between component content and IC₅₀.The results showed that six component content and IC₅₀0 presented a significant negative correlation.The correlation between puerarin and IC₅₀0 of the most significant,followed by 3’-hydroxy puerarin.3’-hydroxy puerarin,puerarin,daidzien,genistein glycosides,daidzein,genistein had a significant impact on P.lobata and P.thomsonii antioxidant capacity,the effects of puerarin was the most significant.The higher content was,the stronger antioxidant capacity became.9 Based on flavonoids,polysaccharides,3’-hydroxy puerarin,puerarin,daidzien,daidzein,genistein glycosides,genistein content and IC₅₀0 9 indexes in samples,it`s quality had been analyzed by useing comprehensive weighted scoring method and gray correlation analysis evaluation method,and combined with clustering analysis method.The reasults provided a reference methods for further research of puerarin drugs and quality evaluation.Thethree methods comprehensive analysis results were basically identical.P.thomsonii samples 8,9,50,51 sample quality were better,8,9 samples were from Jianyang,Jiangyou.The quality of P.omeiensis samples from 21 to 27 were good,P.lobata 32³7,40 produced in Pingwu,Qingchuan,Beichuan and Cangxi area),65,67,68,67 sample quality were better,followed by 41 and 42 sample produced in Wangcang,Qingbaijiang respectively.