Optimization Of Polysaccharide Extraction Process And In Vitro Antioxidant And Bacteriostatic Study Of Actaea Heracleifolia J.

Actaea heracleifolia J.is a medicinal and edible homologous of Actaea L.in the Ranunculaceae.It is a high-quality and authentic medicinal material in Northeast China,and its main medicinal parts are rhizomes.A.heracleifolia J.is rich in saponins,polysaccharides,alkaloids and other active substances.Among them,plant polysaccharides have antibacterial,antioxidant,and anti-inflammatory effects,and are widely used in food,medicine,health products and other fields.In this study,Heilongjiang wild A.heracleifolia J.was selected as the test material,and an efficient extraction scheme was established by response surface experiment.The extraction efficiency of polysaccharide was used as the evaluation index,and the extraction efficiency of two methods was compared and analyzed to explore the best extraction process of polysaccharide from A.heracleifolia J.The optimal conditions for the removal of impurities were screened and purified by DEAE-52 cellulose column chromatography.The antioxidant and antibacterial activities of purified A.heracleifolia J.polysaccharide were determined in vitro,in order to improve the added value of A.heracleifolia J.,and provide theoretical reference for the future development of A.heracleifolia J.from Heilongjiang Province.Findings were as follows:1.The traditional water extraction method and atmospheric pressure internal boiling method were used to extract A.heracleifolia J.polysaccharide.On the basis of single factor,the response surface method was selected to determine the best extraction process.The optimal extraction conditions of the traditional water extraction method were as follows: liquid-solid ratio of 36:1 mL/g,extraction time of 3 h,extraction temperature of 80℃,and the average extraction yield of 2.84%.The optimal extraction conditions of atmospheric pressure internal boiling method were as follows: liquid to solid ratio of 36:1 mL/g,extraction time of 51 min(including infiltration time),extraction temperature of 100℃,ethanol volume fraction of 60%,and the average extraction yield of 3.60%.The atmospheric pressure internal boiling method improves the extraction efficiency while ensuring the leaching of effective components.2.The purification and separation of polysaccharide from A.heracleifolia J.were studied.The results showed that the removal effect of TCA method was better than that of Sevage method,and the best effect was achieved when the concentration of TCA was 15%,with a protein removal rate of 71.20% and a polysaccharide retention rate of 65.98%.The pigment removal ability of five resins(S-8,AB-8,X-5,HPD-100,D101)was compared and analyzed by resin adsorption method.The results showed that under the static adsorption condition,the polar resin S-8 had the best pigment removal effect on A.heracleifolia J.The loading concentration,loading volume and elution volume of S-8 resin were investigated.The optimal adsorption conditions for dynamic pigment removal were as follows: loading concentration 0.5 mg/mL,rotation speed 17.5 r/min,loading volume 600 mL,and elution volume 400 mL.The purified polysaccharide was separated by DEAE-52 cellulose column chromatography,and four kinds of polysaccharide fractions were obtained,namely: the neutral polysaccharide fraction CHP1 in aqueous phase,and the acidic polysaccharide fractions CHP2,CHP3 and CHP4 in salt phase.3.The antioxidant activity of four polysaccharide fractions of A.heracleifolia J.after purification was studied in vitro by scavenging DPPH free radical,ABTS free radical,hydroxyl free radical and total reducing power.The results were as follows: the DPPH radical IC50 of the four fractions were 0.376 mg/mL,0.669 mg/mL,0.654 mg/mL and 0.129 mg/mL,respectively,and the scavenging ability was in the order of CHP4>CHP1>CHP3>CHP2.The IC50 values of ABTS free radicals were 0.62 mg/mL,0.36 mg/mL,0.28 mg/mL and 0.15 mg/mL,respectively,and the scavenging ability was CHP4>CHP3>CHP2>CHP1.The IC50 values of hydroxyl radical were 0.097 mg/mL,0.048 mg/mL,0.036 mg/mL and 0.039 mg/mL,respectively,and the scavenging ability was CHP3>CHP4>CHP2>CHP1.The reducing power was CHP4>CHP3>CHP2>CHP1.In the range of 0.05 mg/mL～1.6 mg/mL,the antioxidant capacity gradually increased with the increase of polysaccharide concentration.4.The inhibition zone and minimum inhibitory concentration were used as evaluation indexes to determine the antibacterial activity of the purified polysaccharide fractions of A.heracleifolia J.in vitro.The results showed that the inhibitory effects of the four components on Bacillus subtilis were CHP3>CHP4>CHP1>CHP2.The antimicrobial activity against Escherichia coli was in the order of CHP4>CHP3>CHP2>CHP1.The inhibitory effect on Staphyloccocus aureus was in the order of CHP4>CHP3>CHP1>CHP2.The inhibitory effects on the activity of the test strains were as follows: S.aureus > B.subtilis > E.coli.