Extraction, Purification, Physicochemical Properties And Biological Activity Of Polysaccharide Of Baphicacanthis Cusiae Rhizoma Et Radix

Objective:Baphicacanthis Cusiae Rhizoma et Radix, the dried rhizome and root of Baphicacanthus cusia (Nees) Bremek. from isatis Acanthaceae. Its nature is cold, bitter taste. It can enhance the fuction of intestines and stomach, having good effect of clearing detoxication and the antibacterial effect. For the South China, It commonly used as traditional Chinese medicine, but there are less domestic and foreign report of Baphicacanthis Cusiae Rhizoma et Radix polysaccharide (BCP) study. The experiment of BCP ultrasonic extraction process makes a systematic research, and separation of BCP, purification, and further purified components were preliminary pharmacological experiments and to improve BCP extraction rate and the development of a corresponding BCP food and drug, it also can provide BCP use theoretical basis.Method:In this study, using response surface methodology (response surface methodology, RSM) to optimize ultrasound assisted extraction of BCP. First of all, on the basis of the single factor experiments, extraction time, extraction temperature, the ratio of water to raw material of experimental factors were investigated. On the basis of the results of single factor experiments, a 3-factor,3-level of response surface was designed, then response surface condition for testing operations can get from the calculated quadratic linear fitting equation, then the optimum extraction conditions were obtained, finally three parallel test operations are processed under the optimum extraction conditions. Comparing with the theoretical value and actual value differences, to verify the theoretical model is match with the actual experimental results. Then under the optimum extraction conditions of extraction BCP, DEAE-cellulose-52 ion exchange column chromatography according to the polarity gradient elution polysaccharide will be isolated from different time elution liquid classified collection, concentration and drying, and further purified with Sephadex G-100 column chromatography. Using phenol sulfuric acid method to measure total sugar content, protein content was measured by the method of Bradford method, using sulfuric acid carbazole method to measure the content of uronic acid, sulfate radical content was measured by chloride-gelatin method, which can evaluate chemical properties of BCP. Purification of BCP in vitro antioxidant experiment, clearing of superoxide anion free radical, hydrogen peroxide and DPPH activity were investigated, to evaluate its antioxidant activity. And then xylene induced mouse ear edema and acetic acid induced mouse peritoneal permeability increase experiment were studied, using the effect of BCP of mice ear swelling degree and the peritoneal permeability to evaluate BCP anti-inflammatory activity. By using the MTT method to detect the effect of BCP on growth of HepG2 cells to evaluate the BCP in vitro antitumor activity.Result:1. Optimum extraction technology of BCP extraction temperature was 60℃, extraction time was 35min, the ratio of water to raw material was 24.5:1 (v:m, mL·g-1). Under the optimum conditions, extraction rate theory value for 8.43% and its actual yield was 8.37±0.36%(n=3), which was consistent with the theoretical value. Therefore, the RSM method for optimization of extraction parameters is reliable. The theoretical value is in good Consistent with the theoretical value.2. Crude BCP preliminary isolation by DEAE cellulose 52 column chromatography, then got two fraction, respectively B1 (put off by deionized water) and B2 (put off by 0.3 mol/ml NaCl). Then using Sephadex chromatographic column to separate two fractions (B1 and B2), then obtained BCP-1 and BCP-2.3. Basic physical and chemical properties of crude BCP and two different purified components (BCP-1, BCP-2):the polysaccharide content in crude BCP is 79.07%. After purification, polysaccharide content increased significantly, the polysaccharide content in BCP-1 is 89.71%, BCP-2 contents 92.56%. The content of protein in the crude polysaccharide is 0.34%; protein content in BCP-1 is 0.23%, the protein content of BCP-2 is 0.26%; the protein content in two purified polysaccharide are all decreased. Thirdly, uronic acid content in crude BCP is 1.60%; uronic acid content of BCP-1 is 2.24% and BCP-2 with the content of 2.57%. Sulfate radical content in crude BCP is 1.72%; BCP-1 and BCP-2 content 0.76% and 4.74%, respectively. Sulfate radical content of BCP-2 significantly higher than other components.4. Antioxidant ativities of crude BCP and pure purified components (BCP-1, BCP-2) were studied, the results are as follows:(1) BCP, BCP-1, BCP-2 have good superoxide anion free radical scavenging activity, of which BCP-2 clearing superoxide anion free radical ability was the strongest. At the concentration of 2000μg/mL, The clearance rate of crude BCP, BCP-1, BCP-2 and Vc were 66.3%,84.8%,87.4%,80.3%; the crude BCP and its purified components reducing power order:Vc> BCP-2> BCP-1> crude BCP. The clearance rate is similar between BCP-2 and VC at a concentration of less than 120 μ g/ml. But the clearance rate of Vc gradually higher than BCP-2 when the concentration is higher than 120 μg/ml. Crude BCP and purification of synthetic Fe2+ ability were process growth trend between the concentration of 40 μ g/ml to 2000 μ g/ml, BCP-2 synthetic ability of ferrous ion was strongest in these concentration Crude BCP had the weakest ability for synthetic Fe2+.5. BCP has obvious effect of treat xylene induced mouse ear swelling and acetic acid induced mouse peritoneal permeability increased.6. BCP can effectively inhibit the activity and cell proliferation of human hepatocarcinoma cells in vitro. Conclusion:The results showed that the response surface method to optimize the ultrasonic assisted extraction process parameters of BCP is stable and reliable; BCP and its purified components have obvious anti-oxidative effect; BCP has a certain anti-inflammatory and anti-tumor activity. BCP has a good biological activity, with in-depth study significance, in order to further develop the corresponding drugs and functional health products for the new platform.