Study On Extracion, Purification, Separation And Bioactivities Of Flavonoids From Sinopodisma Houshana Huang

In this paper, Sinopodisma houshana Huang as raw materials was measured the flavonoids content and optimized the extraction process; the crude flavonoids from it was preliminary purified by using the macroreticular resin and the purification process was optimized; separated the crude flavonoids from Sinopodisma houshana Huang by chromatography and identified different flavonoids components by colour reaction and UV spectrum. At last, the antioxidant and bacteriostasis biological activity of flavonoids from Sinopodisma houshana Huang were studied, and Specific research results are as follows:(1) Adopt organic solvent to study on the effects of different extractants, extraction time, solid-liquid ratio, concentration, temperature and extraction times on extraction content of flavonoids from Sinopodisma houshana Huang. Then, optimized the extraction process by orthogonal test, the result showed that the optimum extraction parameters were extraction time2h, solid-liquid ratio1:50(m:V), methanol concentration80%(V:V), temperature85℃and the flavonoids content from Sinopodisma houshana Huang was22.70mg/g by aluminium salt spectrophotometry method under the optimum extraction process and multiple extraction until the flavonoids was approximate extracted completely.Ultrasonic technique was used to assist the extraction of flavonoids from Sinopodisma houshana Huang with alcohol. On the base of Assembled-Center of Box-Benhnken, a Response Surface Optimization Experiment was designed, and the effects of extraction temperature, ultrasonic power and alcohol concentration and their interactions on the flavonoids yield were dealt with. The equation was Y(%)=61.25014-4.14833×10-3X1—0.34199X2+0.21415X3+0.01728X1X2—3.19586×10-3X1X3+5.9452×10-3X2X3+4.45092×10-4X12—0.017597X22—9.07328×10-4X32. After ridge analysis by Design Expert8.0, the result showed that the optimum extraction parameters were:extraction temperature70℃, ultrasonic power175W and alcohol concentration53%and the flavonoids yield was85.08%under this condition. It was up to10.2%than under the same condition without ultrasonic technique. (2) AB-8and D101macroporous resin were compared in their ablility of static adsorption and dynamic adsorption and desorption with the adsorption quantity and elution rate for target. Results showed that AB-8resin had good adsorption capacity to flavonoids of Sinopodisma houshana Huang. The optimum static adsorption and dynamic and desoprtion conditions were as follows:adsorption with a ratio of resin to sample1:5, and then desorption with80%aqueous ethanol as eluent at a ratio of resin to eluent1:20and pH=4. And under such conditions, the purity of total flavonoids was19.97%, while the purity of total flavonoids of33.78%was obtained by the following optimum dynamic adsorption and desoprtion parameters of up volume3BV, up rate1mL/min and eluted volume3BV, eluted rate1.5mL/min and eluted pH=4.(3) Flavonoids separation from Sinopodisma houshana Huang by different chromatography and HPLC were studied. Only3components were isolated by paper chromatography and the trailing was obvious. It was bad that4components were isolated by silica gel thin layer chromatography. Polyamide thin layer chromatography was the better separation because7components were isolated clearly by it. Many of components were iosolated by HPLC and of6components maybe flavonoids. Sinopodisma houshana Huang maybe contain many kinds of flavonoids such as isoflavone, flavone, bi-hydrogen flavones by HPLC with ultraviolet and color reaction.(4) Oxford plate and minimum inhibition concentration method were used to study the bacteriostatic effect of the total flavonoids from Sinopodisma houshana Huang. The results showed that:the biggest antibacterial circle was19.5±1.20mm that of the total flavonoids from Sinopodisma houshana Huang against Aspergillus niger and the minimum inhibitory concentration of flavonoids against Aspergillus niger was45ug/mL, less than positive control Rutin(90ug/mL). The antibacterial circle of Escherichia coli and Staphylococcus aureus were18.08±0.75mm and15.53±0.38mm, respectively. Bacillus subtilis and Clostridium perfringens’s the antibacterial circle were small, and Candida albicans had no antibacterial circle.(5) The antioxidant activity of the total flavonoids from Sinopodisma houshana Huang was evaluated by using hydroxyl, DPPH, superoxide anion radical scavenging and the total reducing power systems. The results showed in a certain concentration range the scavenging rates of total flavonoids from Sinopodisma houshana Huang against hydroxyl free radical and DPPH were found to be89.2%and93.6%, respectively, obviously superior to those of VC and BHT. Meanwhile, it against superoxide anion was found to be68.7%was slightly superior to those of VC and BHT. The total reducing power of its absorbance was2.605, obviously superior to those of VC and BHT. So the total flavonoids from Sinopodisma houshana Huang have good antioxidant activity.