Study On Preparation And Antioxidantive, Antibacterial Activity Of Gnaphlium Affine D.Don Flavonoids

In this study, the Gnaphlium affine D.Don was used as the material to systematically study the preparation and in-vitro antioxidantive, antibacterial activity of Gnaphlium affine D.Don flavonoids. Ultrasound-assisted was used to extraction of Gnaphlium affine D.Don flavoniods, the main factors that influence of extracting technology were discussed and the key parameters were optimized by the response surface; Metal complex method was used to separation and purification of Gnaphlium affine D.Don flavonoids, and its key technological parameters were optimized. The antioxidantive and antibacterial activity of Gnaphlium affine D.Don flavonoids and flavonoids-Zn(Ⅱ) complex were investigated by in-vitro antioxidant and antimicrobial methods and their antioxidant mechanism was preliminary discussed. The main results were showed as follows:(1) Study on the technology of ultrasound-assisted extraction of Gnaphlium affine D.Don flavonoidsThe ultrasound-assisted was used to extraction of Gnaphlium affine D.Don flavoniods, the effects of the solid-to-liquid ratio, ethanol concentration, extraction temperature, extraction time and ultrasound power on the extraction yield of Gnaphlium affine D.Don flavonoid were examined. Based on single factor experiments, by the four-factor-five-levels quadratic general revolving combination design design, the mathematical regression equation about the extraction yield of Gnaphlium affine D.Don flavonoids Yand the extraction temperature Xi, the ethanol concentation X2, the ultrasound power X3, the solid-to-liquid ratio X4 was established as follows:Y=14.95+0.63X2+0.14X3+0.47X4+0.15X1X2-0.08X1X3-0.36X2X4-0.07X12-0.36X22-0.08X3--0.25X42Through Response Surface Methodology (RSM), the key technological parameters of extraction Gnaphlium affine D.Don flavonoids were optimized as follows(after calibration):solid-to-liquid ratio of 1:35(g/mL), ethanol concentration of 75%, ultrasound power of 350 W, extraction temperature of 35℃, under the optimized conditions and extracting 50 minutes, the maximum extraction yield of Gnaphlium affine D.Don flavonoids was 15.25%.(2) Study on the key technology of separation and purification of Gnaphlium affine D.Don flavonoids by metal complex methodMetal complex method was used to separation and purification of Gnaphlium affine D.Don flavonoids. The proper metallic salt and its suitable parameter range which influenced the separation and purification of Gnaphlium affine D.Don flavonoids were determined through the single factor experiments. The orthogonal test L9(34) design was used to optimize the key technological parameters. The results indicated that the proper metallic salts was zinc acetate, the optimized technology conditions to purify Gnaphlium affine D.Don flavonoids were as follow:ethanol as solven of Gnaphlium affine D.Don flavonoids and the concentration 1.5 mg/mL, zinc acetate solution concentration 9 mg/mL, pH value 8.0, reaction time 10 min; furthermore,1.5% EDTA was as reagent to decompose complex, after being purified, the purity was increased from 20.21% to 67.47% with 2.4 times as high as the crude extracts.(3) Study on the in-vitro antioxidantive and antibacterial activity of Gnaphlium affine D.Don flavonoidsThe in-vitro antioxidantive and antibacterial activity of Gnaphlium affine D.Don flavonoids and flavonoids-Zn(Ⅱ) complex were determined and analyzed by in-vitro antioxidant and antibacterial methods with Vc and Potassium sorbate solution in the corresponding concentration as positive control. The results showed that the Gnaphlium affine D.Don flavonoids and flavonoids-Zn(Ⅱ) complex have strong in-vitro antioxidant capacity and antibacterial capacity, there were a good dose-effect relationship between the certain concentration range. The IC50 of scavenging DPPH radical (DPPH·), hydroxyl radical (·OH), superoxide anion radical (O2-·) of Gnaphlium affine D.Don flavonoids were 0.394 mg/mL、0.326 mg/mL and 0.369 mg/mL, respectively, Gnaphlium affine D.Don flavonoids-Zn(Ⅱ) complex were 0.260 mg/mL、0.374 mg/mL、0.167 mg/mL, respectively; The reducing capability of potassium ferricyanide of the flavonoids-Zn(Ⅱ) complex was highly significant higher than that of the flavonoids and Vc (p<0.01) within the concentration gradient range of the study, the reducing capability of the flavonoids was significant higher than that of the Vc (p<0.05). The Gnaphlium affine D.Don flavonoids and flavonoids-Zn(Ⅱ) complex had significant antibacterial activity against Staphylococcus aureus, Salmonella, Escherichia coli and Bacillus subtilis, and the antibacterial activity of flavonoids-Zn(Ⅱ) was better than flavonoids, the longer the action time will bring higher antibacterial rate, when the action time was 8-10 hour, their antibacterial rate all increased to the maximum. In addition, the Gnaphlium affine D.Don flavonoids and flavonoids-Zn(Ⅱ) complex have stronger inhibition effect on gram-positive bacterium than gram-negative bacterium, the MIC(minimum inhibitory concentrations) of flavonoids to four kinds of bacterias were:0.20 mg/mL、0.40 mg/mL、0.20 mg/mL、0.10 mg/mL, respectively; the MIC of flavonoids-Zn(Ⅱ) complex to four kinds of bacterias were:0.20 mg/mL、0.20 mg/mL、0.20 mg/mL、 0.10 mg/mL, respectively.