Isolation And Purification On Isoflavonoids From Radix Puerariae And Preliminary Research On The Stems And Leaves Of Puerariae Lobata (wild) Ohwi

Radix Puerariae is famous Chinese traditional medicine herb,which contains abundant isoflavones,such as puerarin,daidzin and daidzein are the major ingredient. It has been experimentally demonstrated that puerarin has effects on ceardiovascular disease,angina and arrhythmia,and daidzin and daidzein have the prevention and amendment of women's illness.Puerarin was analyzed and utilized from Radix Puerariae with the low value.Moreover,the study was concentrated on Radix Pueraria and Pueraria omeiensis.The stems and leaves of Pueraria lobata(wild) Ohwi also has different active components,but only isflavones were utilized while other active components and their biological effect were seldom researched.The isolation and purification on isoflavonoids from Radix Puerariae and preliminary research on the stems and leaves of Puerariae Lobata(wild) Ohwi,which supply found for establishing the modernization quality standard of Pueraria lobata (wild) Ohwi and comprehensive utilization of Pueraria lobata(wild) Ohwi in Maoshan were studied in the paper.The simulataneous determination of puerarin,daidzin and daidzein in Pueraria lobata(wild) Ohwi of Maoshan had never been reported.The former treatment in Chinese Pharmacopoeia monography which was only used for determination of purarin is studied with suitability for simulataneous determination of puerarin,daidzin and daidzein.Therefore,the former treantment and analytical method by Reverse Phase High Performance Liquid Chromatography(RP-HPLC) was established for simultaseeous determination of puerarin,daidzin and daidzein in radix,stems and leaves of Pueraria lobata(wild) Ohwi.The total isoflavone was extracted with 70% EtOH solution by ultrasonic method for 20min at 50℃,comparing with extraction method according to the Chinese Pharmacopoeia monography.The linear range of puerarin,daidzin and daldzein in Radix Puerariae were 0.2140～1.2840μg(r=0.9998), 0.0178～0.1066μg(r=0.9997) and 0.0044～0.0026μg(r=0.9994),respectively.All of the three compounds showed good linearities.The recoveries of average recovery were 99.46%,104.29%and 111.54%,respectively.And the RSD of average recovery were 2.20%,4.60%and 6.60%(n=5),respectively.The precision,stability and reproducibility of the method were good.The contents of puerarin,daidzin,and daidzein by ultrasonic method were 3.98%,0.36%and 0.039%respectively.The contents of puerarin,daidzin,and daidzein by ultrasonic method were superior to that by extraction method according to the Chinese Pharmacopoeia monography.The contents of puerarin,daidzin,and daidzein in stems of Pueraria lobata(wild) Ohwi were 40mg/kg,32mg/kg and 6.6mg/kg,respectively.And the contents of puerarin, daidzin,and daidzein in leaves of Pueraria lobata(wild) Ohwi were 16mg/kg, 50mg/kg and 12mg/kg,respectively.The method was accurate,fast,simple,good separation and used for study the isoflavone puerarin,daidzin,and daidzein in radix,stems and leaves of Pueraria lobata(wild) Ohwi.The contents of puerarin,daidzin, and daidzein in stems and leaves of Pueraria lobata(wild) Ohwi were obviously lower than in radix of Pueraria lobata(wild) Ohwi.The purification of isoflavonoids from Radix Pueraria had been reported,which was mainly purified for puerarin.So the purification condition of puerarin,daidzin, and daidzein from Radix Pueraria with macroporous resion was studied.The isflavones of Radix Pueraria was purified with four kinds of macro porous adsorbing resins by the adsorption rate and desorption rate.The AB-8 resin was selected and its optimal purification conditions were explored.The results were as follows:AB-8 resin had the best adsorption capacity for total flavonoids with 116.33mg/g.The results of adsorption thermodynamic of AB-8 resin for total flavonoids of Pueraria lobata(wild) Ohwi showed that the adsorption isotherm could be described by Langmuir type.On the basis of single factor tests,the optimal conditions of isolation and purification for the total flavonoids of Pueraria lobata(wild) Ohwi by AB-8 resin were obtained.The results showed that the concentration of total flavonoids in the crude extracts was 9.63 g/L,the flow rate was 1.5mL/min.And after the in-purities were eluted down by distilled water,the adsorbed total flavonoids were gradiently eluted down by 10%ethanol,30%ethanol,50%ethanol and 70%ethanol.The elution was made through fraction of it,and then was utilized by vacuum concentration and lyprilization.The isoflavones in the elution(10%ethanol) were constituted mainly by puerarin as its main active component,and the content of puerarin was 87.17%.The isoflavones in the elution(30%ethanol) were constituted mainly by puerarin and daidzin as its main active components,and the content of puerarin and daidzin were 53.72%and 18.41%.The isoflavones in the elution(50%ethanol and 70%ethanol) were constituted mainly by daidzein as its main active components,and the content of daidzein were 72.41%and 74.31%,respectively.The refined isoflavones of the different ingredients were extracted from the crude extracts.The contents of puerain,daidzin,and daidzein in stems and leaves of Pueraria lobata(wild) Ohwi of Maoshan were low.To further study on other active components in the stems and leaves of the Puerariae lobata(wild) Ohwi,the effective components of different extracting parts were studied.The three extracts from stems and leaves of Pueraria lobata(wild) Ohwi were extracted by water vapour distillation-ether extraction,ethanol extraction and water-extraction and alcohol-precipitation method.The chemical constituents of the three extracts were analyzed.The volatile oil from water vapour distillation of the stems and leaves of Pueraria lobata(wild) Ohwi by GC-MS,using CH₃OH(10%H₂SO₄)-C₆H₆(1:1) as the methyl esterification solvent firstly.153 compounds were separated,70 of them were identified.The contents of alcohols,ketones,alkene hydrocarbon,alkyl hydrocarbon,aldehydes,carboxylic acids,aromatic hydrocarbon,ester,phenolic compounds and ohers were 7.44%,4.41%,0.65%,0.76%,0.43%,71.68%,0.65%, 3.55%,0.25%and 1.35%,respectively.The contents of the total flavonoids in the stems and leaves of Puerariae lobata (wild) Ohwi were determined by two kinds of UV.The contents of the total flavonoids which were determined by UV regarding rutin and puerain as reference substance were 5.66%and 1.95%,respectively.Coarse polysaccharide from the stems and leaves of Pueraria lobata(wild) Ohwi which was extracted by water and precipitated by ethanol were determined.The average gain ratio of coarse polysaccharide was 8.39%.The content of the polysaccharide in coarse polysaccharides was 30.31%.It is now clear that the stems and leaves of Pueraria lobata(wild) Ohwi in Maoshan are rich in polysaccharide.To study the scavenging free-radical activity of volatile oil,ethanol extraction and coarse polysaccharide from the stems and leaves of Pueraria lobata(wild) Ohwi in vitro by using DPPH,phenanthroine and pyrogallic acid methods.The results were as follows:volatile oil,ethanol extraction,and coarse polysaccharide from the stems and leaves of Pueraria lobata(wild) Ohwi all have good ability to scavenge radicals and the ability of coarse polysaccharide was best.By the scavenge activity of DPPH radicals,the IC₅₀ were 3.3011,1.0767 and 0.1229 mg/mL of volatile oil,ethanol extraction and coarse polysaccharide(the IC₅₀ was 0.0091mg/mL of Vc);By the scavenge activity of hydroxyl radicals,the IC₅₀ were 3.0755,1.5615 and 0.7511 mg/mL of volatile oil,ethanol extraction and coarse polysaccharide(the IC₅₀ was 0.3477mg/mL of Vc);By the scavenge activity of superoxide radicals,the IC₅₀ were 2.3424,1.1460 and 0.9229 mg/mL of volatile oil,ethanol extraction and coarse polysaccharide(the IC₅₀ was 0.2299 mg/mL of Vc);From it,the antioxidation of ethanol extraction and coarse polysaccharide can be studied ulteriorly.So the stems and leaves of Pueraria lobata(wild) Ohwi has worth to research.