Study Of Extraction, Purification, Identification And Biological Activities Of Anthocyanidin From Bacca Sedge

Based on the raw material of Bacca sedge, the ultrasound-assisted technology was selected to extract anthocyanidin. On the basis of single factor experiments, the experiment of response surface methodology was carried on to optimize the extraction conditions of anthocyanidin from Bacca sedge. Macroporous resins were selected to purify anthocyanidin from Bacca sedge. Six kinds of anthocyanidin standard substances in plants were used as comparisons to determine the category and contents of anthocyanidin from Bacca sedge by HPLC（High-performance liquid chromatography）. The linear relationship, precision and recoveries of the method of HPLC were detected. The chemical luminescence method was used to measure the superoxide anion free radicals, hydrogen peroxide, hydroxyl radical scavenging and DNA damage protection. The experiments on scavenging effect of DPPH and antibacterial activity were carried on. The main results were as follows:（1） The single factor experiments showed that, when the ratio of material to liquid was 1:30 g/mL, the concentration of hydrochloric acid was 3 mol/L, ethanol volume ratio was 3:1, the extraction time was 15 min, and the extraction power was 200 W, the extraction yield of anthocyanidin from Bacca sedge was the best.Based on the single factor experiments, the experiment of response surface methodology showed that, the optimum extraction conditions of anthocyanidin from Bacca sedge: ratio of material to liquid was 1:32 g/m L, the concentration of hydrochloric acid was 2.6 mol/L, ethanol volume ratio was 4.6:1, and the extraction time was 16 min. Three validation experiments were performed under the optimum extraction conditions. The extraction yield of anthocyanidin from Bacca sedge was 7.18%, which was closed to the theory predicted value（7.26%）. The results showed that the fitting degree of model matched well, and the analysis and forecasting of the extraction condition of anthocyanidin from Bacca sedge were reliable by the fitted regression equation.（2） Through the static absorption and desorption experiments, the adsorption time of S-8 mcroporous resin was 0.5 h¹.0 h, and the advisable desorption of time was about 3.0 h⁴.0 h. When the absorption and desorption reached to the balance, the absorption rate was 92.17%, and the desorption rate was 87.23%. Compared with other 6 mcroporous resin, the absorption rate and desorption rate of S-8 mcroporous resin was higher. Besides, the absorption and desorption time were both shorter. As a result, the S-8 mcroporous resin was suitable to purify anthocyanidin from Bacca sedge.The static desorption experiment of mcroporous resin showed that, when pH was 1.0, and the concentration of ethanol was 70%, the results of static desorption were more effective. Dynamic experiment showed that, when the sampling rate was 1.0 mL/min, and the sample concentration was 2.00 mg/m L, the results of dynamic absorption were more effective. When the elution flow rate was 1.0 mL/min, the result of dynamic desorption was more effective.（3） The category and contents of anthocyanidin from Bacca sedge were detected by HPLC.The peak time of six kinds of anthocyanidin standard substances including delphindin, cyanidin,petunidin, pelargonidin, peonidin and malvidin,were 8.585, 9.986, 10.252, 11.832, 12.464 and 12.697 min in order. The peak time was about in 7 minutes, and the baseline was separated basically. The main anthocyanidins from Bacca sedge were cyanidin and pelargonidin. The cantents were 689 mg/k and 80 mg/kg in order. When detected anthocyanidins from Bacca sedge by HPLC, the linear relationship was well, the rate of precision and recovery were higher. As a result, the HPLC was an quantitative analysis method.（4） The experiment of antioxidant and DNA damage protection showed that, with the increasing of the concentration of the sample, the scavenging rate increased, and the dose-effect relationship was presented. In the chemical luminescence system of pyrogallicl- luminol, when the concentration was 12.5 mg/mL, the scavenging rate of superoxide anion free radical was 46.45%, and the IC₅₀ was 15.37 mg/m L. In the chemical luminescence system of luminol-H₂O₂, when the concentration was 10 mg/m L, the scavenging rate of hydrogen peroxide was 50.05%, and the IC₅₀ was 8.14 mg/m L. In the chemical luminescence system of FeSO4- luminol-H₂O₂,when the concentration was 1000 mg/m L, the scavenging rate of hydroxyl free radical was 96.78%, and the IC₅₀ was 88.89 mg/mL. In the chemical luminescence system of CuSO₄-Phen-VC- H₂O₂-DNA, the half inhibition concentration IC₅₀ of DNA damage protection was 0.85 mg/mL.The scavenging effect experiment of DPPH showed that when the concentration of anthocyanidin from Bacca sedge was 106.67 μg/mL, the scavenging rate reached the maximum value 62.81%.The results of antibacterial experiment showed that, the anthocyanidin from Bacca sedge had bacteriostatic effects on staphylococcus aureus, enterobacter sakazakii and salmonella aureus. With the increasing of the concentration, the antimicrobial effect increased. The antibacterial effect of salmonella aureus was better than the staphylococcus aureus and enterobacter sakazakii.