| Radix puerariae is approved edible and medicinal plant by National Ministry of Health, which belongs to the drying root of Pueraria lobata (Willd)Ohwi or P.thomsonii Benth and contains abundant isoflavones and starch materials. Pueraria isoflavones proves it has the prevention and amendment of cardiovascular disease, anti-tumor, antioxidation, protecting liver, regulating endocrine system and some other pharmacological effects. The content of Pueraria isoflavones reaches as high as 7.6% in Radix puerariae, which include over thirty kinds of puerarin,daidzin and daidzein. Of them, Puerarin is the major and active ingredient, accounting for about percent 50. The content of puerarin and isoflavones are usually used as the major assessment index of Radix puerariae quality.Though Radix puerariae is abundant in our country, there exist such problems as the growth area and little processing scale, the under-developed technology of the production process and analysis detection and facility relative as well as low production added value , etc, which have restricted the healthy development of Radix puerariae industry property .Based on it, this paper just focused research on the analysis methods, the extraction and purification technology, antioxidative activity of pueraria isoflavones, in order to provide the theoretical basis for the establishment of pueraria isoflavones modernization quality evaluation system and the development and comprehensive utilization of Radix puerariae resources. The main research content is as follows:(1)The means of qualitative and quantitative analysis of pueraria isoflavones were investigated.①The HPLC detects the content of puerarin and daidzin by quantitative analysis. The conditions of determining liquid phase chromatogram are as follows: chromatogram column C18(150mm×4.6mm,5μm), the wavelength of detector ,.at 250nm and 380nm, column temperature at 30℃, sample size 5μL, rate at 0.6mL/min, adapted gradient with acetonitrile - water solution: 0～9min, acetonitrile - water(10:90); 10～19min, acetonitrile - water(15:85); 20～30min, acetonitrile - water(20:80).In this case, at least 5 components alcohol extactive can be separated well from impurities. Good linear relationships between the peak area values and concentrations have been found. The relationship of the puerarin and daidzin is good in the range of concentration. They respectively have 19.91μg/mL and 104.17μg/mL of the lowest detection limit . The recoveries for the puerarin were in the range of 99.5%～100.1% with RSD of 0.60 %. The contents of them were 45.35% and 4.89% respectively, proving that puerarin is the major component among pueraria isoflavones.②The MECC detects the content of puerarin by quantitative analysis. Theconditions of the capillary electrophoresis parameters are as follows: at 250nm,0.1MKH2PO4 -20mM borax buffer solution with pH9.0.40mmol/L SDS concentration, separation voltage 18KV. In this case, at least 5 components alcohol extactive could be separated well from impurities. A good linear relationship between the peak area values and concentrations was found in the range of puerarin concentration. The retention time and the lowest detection limit were 4.500min and 13.74μg/mL respectively. The recoveries for the puerarin were in the range of 97.2%～100.2%with RSD of 4.63%. The contents of puerarin was 42.25%, the result of them was quite similar. As daidzin content was lower, while its lowest detection on MECC higher, the measurement of content couldn't be performed.③The UV detected the content of pueraria isoflavones by quantitative analysis. At 254 nm, the rough Content of isoflavones was detected by using puerarin as chemical reference substances. There is 6.58% isoflavone content of Pueraria lobata (Willd)Ohwi. It was approved that the content of isoflavones had a close relation to material varieties, growing stage and plant tissues.④The TLC had qualitative analysis of puerarin and daidzin. On silica gel plate, puerarin and daidzin could be separated well from impurities firstly adopting ethyl acetate - acetone - methylbenzene - water (10: 8.5: 1.0: 1.7, v/v) as developing solvent and the chromogenic method of ultraviolet(254nm) - iodine vapores .The Rf value of puerarin and daidzin was 0.43 and 0.51 respectively.⑤The comparison of these analysis methods. Compared with exiting research report, HPLC has a lower the lowest detection and a wider detection range. The correctness and precision of MECC were slightly lower than HPLC. But this method had such advantages as much shorter of retention time, much better degree of separation, lower of capillary pipe and little usage quantity of organic solvents, little sample size, good accuracy, high precision and sensitivity, which could be used in quantitative analysis of pueraria isoflavones. The correctness and precision of UV were lower than HPLC and MECC. This method was simple and rapid in operation, which was used to optimize the technological parameter of distilling abstrpueraria isoflavones. TLC was simple, rapid and strongly specialized, which was used to identify qualities of Radix puerariae and the scavenging isoflavones effect in the resins during the process of macroporous resin purifying isoflavones.(2) The extraction technics of pueraria isoflavones. By ethanol refluxing and extracting process and UV method, the optimum parameters of extracting isoflavonoids had been determined. It was showed that the main factors influencing extraction efficiency were as follows: the extraction temperature > the ethanol concentration > extraction times > the ethanol amount. The extracting temperature had very significant difference. The optimum parameters was: particle size is 20, the multiple of ethanol to material is 15, the concentration of ethanol is 70%, the extraction time is 1.5h,the extraction temperature is 70℃and the extraction times is 3. Under these conditions, the extraction rate and the purity of pueraria isoflavones are 92.83% and 21.65%.(3) The purifying technology of pueraria isoflavones. The study compared adsorption properties of five macroporous resins , including S-8,NKA-9,AB-8,X-5,D4006.AB-8 resin was determined as the best sorbent. The optimum purification condition obtained was: the dried resin as reference, the flow rate on absorption is 0.5 mL/min ,100mL 80% ethanol, the flow rate on desorption is 2.0 mL/min. Under these conditions, the purity of pueraria isoflavones had much greatly improved from 21.65% to 87.55%, and The content of puerarin increases from 9.79% to 92.80%.(4) The antioxidative ability in vitro of pueraria isoflavones. When the concentration of Radix puerariae alcoholic extract and BHT were both 0.2730mg/mL, their TAC is as same as 0.98mmol/g FeSO4. The result indicates that pueraria isoflavones has strong antioxidant activity. The pueraria isoflavones has the ability to scavenging the DPPH radical .The scavenging rates are 65.25% and 82.20% when the concentrations of Radix puerariae alcoholic extract are 0.2275mg/mL and 0.2730mg/mL.The effect of scavenging will gradually increase with the rise of concentrations of Radix puerariae alcoholic extract. |
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