| Radix Puerariae, which distributes widely in china, is a kind of traditional Chinese herbal medicine and also ate as food. Nowadays, most of people adopt macroporous absorption resin separate and purify flavonoids from Radix Puerariae, In this article, adopt natural clarifying agent purify flavonoids and compared with macroporous absorption resin, the major contents and results of theresearch are as follows:1. Using coloration reaction to analyze the flavonoid ingredient of Radix Puerariae, confirmed that the main ingredient was isoflavonoids. The total flavonoids ingredient was analyzed qualitatively according to TLC. Chloroform-methanol-water(9:4:0.5,v/v), ethylacetate-acetone-methylbenzene-water(10:8.5:1.0:1.7,v/v),ethylacetate-acetone-meth ylbenzene-water (11:9:1.0:1.7,v/v) was adopted as developing solvent respectively. The result showed, chloroform-methanol-water (9:4:0.5,v/v) had good efficient, the sample could be separated well. The puerarin emerged shallow blue fluorescence spot from the chromogenic method of ultraviolet (254nm) and emerged light blue fluorescence spot from the chromogenic method of ultraviolet (365nm). The flavonoids from Radix Puerariae had the same Rf value with puerarin, the Rf value was 0.51. It showed that the flavonoids from Radix puerariae contain puerarin. Puerarin could be well detected on the silica gel plate.2. The extraction technology of flavonoids was discussed, effect of solvent concentration, time, temperature and the ratio of solid to solvent on yield of flavonoids from Radix Puerariae was determined individually. On the basis of single factor experiment, response surface central composite rotatable experiment was designed to optimize the extract process. The optimal conditions of extraction was as follows:solvent concentration 61.24%, extraction time 1.97 h, extraction temperature 80.86℃, and the ratio of solid to extraction solvent 1:27.70. Under this condition, the result was 6.60%.3. Using ZTC1+1 natural clarifying agent and AB-8 macroporous resin to purify flavonoids respectively. The result showed that, under the treatment of natural ZTC1+1 clarifying agent, the purity of flavonoids was 23.93% (without clarification of purity was 14.03%), it showed that clarifying agent could remove something and it was possible to use in purity technology. The condition affect the purification technology by AB-8 resin was all examined, the optimum absorption and desorption condition was as follows(resin column length of 50cm, the diameter 2cm):flavonoid concentration was 3.0mg/mL, the volume of sample was 55mL, absorption rate was 0.5mL/min, the desorption solvent was 70% ethanol of 200mL,the desorption velocity was 1.OBV/h, in this condition, the purity of flavonoids could up to 71.21%.4. The structure of flavonoids in Radix Pueraria was proved by TLCã€UVã€IR and HPLC. The result showed that flavonoids had characteristic ultraviolet and infrared absorption peak. By HPLC, we could detect that the content of puerarin was 20.63% before purified, the content of puerarin arrived 52.03% after purified.5. The vitro scavenging action of flavonoids in Radix Puerariae on superoxide anion, hydroxide radical and H2O2 was determined by Pyrogallol-Luminol system, CuSO4-Phen-Vc-H2O2 system and Luminol-H2O2 system respectively. The IC50 is 0.1136mg/mL,0.0201 mg/mL,0.0570mg/mL, scavenging action on the DPPH free radical and the reduction was also determined, the IC50 of scavenging action on the DPPH free radical is 0.0162. Flavonoids of Radix Puerariae had good ability of scavenging reactive oxygen, and the scavenging activity would increase with the rise of concentration. |
