Study On Extraction And Purification Process Of Total Isoflavonoids From Stem Of Pueraria Lobata (Willd.) Ohw And Its Antioxidant Activity

Radix puerariae is traditional Chinese medicine in common use and have many pharmacological effects,Which is proved medical and edible dual purpose by National Ministry of Health.There are more than thirty kinds of isoflavonoids in Radix puerariae,such as puerarin,daidzin and daidzein,of them,puerarin is the major and active ingredient.Along with expanding application,market demand beomes larger,so it is in short supply.The major ingredient in stem of Pueraria lobata(Willd.) Ohw is isoflavonoids.After a series of the feasibility research on stem of Pueraria lobata(Willd.) Ohw,people think out that stem of Pueraria lobata(Willd.) Ohw have many kinds of isoflavonoids which Radix puerariae have.Of which,puerarin and daidzein have high content.However,stem of Pueraria lobata(Willd.) Ohw was all threw away after Radix puerariae was dug,which lead to resources greatly waste,and destroyed the environment.Therefore, deeply research on application of stem of Pueraria lobata(Willd.) Ohw have great meanings on expanding Radix puerariae officinal resource,keeping zoology balance in nature,protecting environment and resource recycle,and also have far-reaching meanings on pharmacy and food industry.Using the stem ofPueraria lobata(Willd.) Ohw as the main material,and using alkali water as the impregnant to research on its total flavonoids extraction technology,making extraction liquid purifying technology better,identified flavonoids ingredients within the extraction liquid by HPLC, and the antioxidative ability in vitro of total flavonoids was also discussed.The main content and results of research were as follows:(1) The contents of total flavonoids in different parts of Pueraria lobata(Willd.) Ohw were contrasted and analyzed,the results indicted Radix puerariae's total flavonoids content was the highest,obtained 4.81%,however,the stem of Pueraria lobata(Willd.) Ohw gathered in the late autumn were also rich in total flavonoids,the content was 2.73%;The season when the stem of Pueraria lobata(Willd.) Ohw were gathered had a great impact on total flavonoids,in autumn and winter,the contents of total flavonoids were the highest.(2) Using coloration reactions and TLC to qualitative analyze the flavonoid ingredients.The TLC test showed that the stem ofPueraria lobata(Willd.) Ohw had many kinds of isoflavonoids as the same as Radix puerariae,puerarin and daidzein,the two kinds of isoflavonoids were discovered, the Rf value of them was 0.45 and 0.83 respectively.(3) The alkali water extraction technics of total flavonoids from stem of Pueraria lobata(Willd.) Ohw.Taking the extraction ratio of total flavonoids as the index,single factor test and orthogonal test were made.The results showed that the primary and secondary order of factors influencing extraction efficiency were as follows:the extraction times＞the extraction temperature＞the ratio of material to liquid.The extracting times had very significant difference.The optimum extraction technics parameters was particle size is 40,the ratio of material to liquid is 1:40,the extraction temperature is 65℃,the extraction times is 2.0h.(4) The microwave-alkali water treatment extraction technics of total flavonoids from stem of Pueraria lobata(Willd.) Ohw.Taking the extraction ratio of total flavonoids as the index,the area of test was ascertained by single factor test.The optimum microwave- alkali water extraction technics were studied by using response surface analysis,the optimum extraction technics parameters was the ratio of material to liquid is 1:22,the microwave power is 728W,the extraction times is 47S.The results showed that the primary and secondary order of factors influencing extraction efficiency were as follows:the extraction times＞the microwave power＞the ratio of material to liquid.(5) Taking total flavonoids from stem of Pueraria lobata(Willd.) Ohw as viewed object, through the comparison of static absorption and desorption properties of four kinds of macroporous resins,including S-8,AB-8,D-101,NKA-9.AB-8 resin was determined as the best sorbent to purify total flavonoids from stem of Pueraria lobata(Willd.) Ohw.Respectively taking AB-8 resin and ethanol as sorbent and eluant,through the study of purification technics of total flavonoids from stem of Pueraria lobata(Willd.) Ohw,the best purification technics obtained was taking the wet resin 40g as reference,total flavonoids concentration is 1.21 mg/mL,velocity of solution is 2mL/min, the feed volume is 150mL,180mL 70%ethanol,the flow rate on desorption is 3.0mL/min.By vacuum desiccation,The content of total flavonoids obtained 80.25%.(6) Identified total flavonoids from stem of Pueraria lobata(Willd.) Ohw by HPLC,The conditions of determining liquid phase chromatogram are as follows:chromatogram column Hypersil BDS C18,column temperature at 30℃,the wavelength of detector at 250nm,sample sizes 20μl,adapted gradient desorption:0～15min,methanol:H₂O,(25:75);15～30min,methanol:H₂O (25:75);rate at 1.0mL/min.In this case,major components of extractive from stem of Pueraria lobata(Willd.) Ohw can be separated well.(7) It is analysised by HPLC that the two major efficent ingredient puerarin and daidzein are in purified flavonoids.Contrasted HPLC graph fore-and-aft purification,it is thought out that with extractive from stem of Pueraria lobata(Willd.) Ohw purified,puerarin and other flavonoids were enriched,the ratio of apex area was increased 15.36%,daidzein was at a 8.25%percent loss.(8) Total flavonoids from stem ofPueraria lobata(Willd.) Ohw have strong·OH scavenging ability and deoxidize ability,moreover,after purified by AB-8 resin,the ability obviously increased. Whereas,a little weaker than Radix puerariae extractive.