The Separation And Pharmacodynamic Evaluation Of The Effective Antibacterial Components From Terminalia Chebula Retz. And Rhizoma Polygoni Cuspidati

On the basis of existing study of this laboratory,two Chinese herbs,Terminalia Chebula Retz.and Rhizoma Polygoni Cuspidati which have effective antibacterial activity to the aquatic pathogenic bacteria,were selected for further study.Main constituents were separated from the selected two Chinese herbs by supercritical fluid extraction and high-speed counter-current chromatography?HSCCC?.Microcalorimetry was used to evaluate the inhibition effect of the separated components on Aeromonas hydrophila?B11?and Aermonas veronii?B59?,then the best bacteriostasis concentration of effective antibacterial components were measured.According to the results of the inhibition,one of monomer constituent with high antimicrobial active was selected for the further experiment in vivo.Zerbrafish was used as experimental fish to discuss its efficacy against Aeromonas hydrophila.The efficacy was evaluated by the non-specific immune parameters and the mortality of fish.The mian results are as follows:1.Ultrasonic assisted extraction was used to extract tannis from Chinese herb Terminalia Chebula Retz.To maximize the extraction of tannis,the L₉?3⁴?orthogonal array was designed for investigating the effects of ethanol concentration,solid-liquid ratio,ultrasonic temperature and extraction time.Optimum conditions as follows:the use of 60%ethanol for extracting tannis with 1:20 solid-liquid ratio?m/V?at the temperature 50?and extraction time 80 min.Four factors on the influence of gallic acid extraction rate from Terminalia Chebula Retz.were in size of order:A?ethanol concentration?>D?extraction time?>B?solid-liquid ratio?>C?ultrasonic temperature?.Under this condition,the gallic acid was extracted in amount of 10.80 mg/g.2.The main antibacterial components of Terminalia Chebula Retz.were extracted and isolated using ultrasonic assisted extraction and HSCCC.The solvent system used to separate its crude extract was optimized by analytical HSCCC.And the separation system was n-hexane-ethyl acetate-methanol-water?1:5:1:5,v/v?.Four kinds of components?No.T1?T4?were isolated by preparative HSCCC.Microcalorimetry was used to test the inhibition effect of separated components on strain B11 and strain B59.The results showed that four separated components had antibacterial activities to experimental strains,bacteriostatic strength in the order were T3>T2>T4>T1.The optimum antibacterial concentrations of component T3 to strain B11 and strain B59 were 0.56 mg/mL and 0.74 mg/mL.By HPLC analysis,the purity of T3 was 96.4%,which was identified as gallic acid by mass spectrometry.3.The main antibacterial components of Rhizoma Polygoni Cuspidati were extracted and isolated using ultrasonic assisted extraction and HSCCC.By the analytical HSCCC,the solvent system was determined as chloroform-methanol-water?4:3.8:2,v/v?,and 5 kinds of components?No.R1?R5?were isolated by preparative HSCCC.The inhibition results showed that component R1 and component R5 had good inhibition to the two pathogenic bacteria.The antibacterial activity of R1 was better than R5.The optimum concentration of component R1 was 1.58 mg/mL to strain B11 and 1.14 mg/mL to strain B59.By HPLC analysis,component R1 contain two constitutes,wherein one is rheum emodin.4.Zebrafish was used as experimental fish,a 14-day feeding experiment was conducted to evaluate the efficacy of gallic acid.Four practical diets were formulated containing gallic acid with the mass of 0.3%,0.6%,1.2%,2.4%,respectively.Four kinds of non-specific parameters?SOD,CAT,ACP,AKP?were measured during the days of 0th,4th,10th and 14th.The results showed that gallic acid can enhance non-specific immune function of fish.The activities of four kinds of non-specific parameters in group 0.6%and group 1.2%were all increased significantly than control group at 10th day?P<0.05?.After 14 days of feeding with gallic acid-diets or control diet,groups of zebrafish were infected with Aeromonas hydrophila?B11?artificially.Based on the artificial infection test,the accumulative mortalities of fish feeded with gallic acid were lower than control group.Zebrafish of group1.2%and group2.4%had significantly lower mortality compared with control group?P<0.05?,which reduced by 25%and 20%respectively.In summary,ultrasonic extraction method is suitable for the extraction of gallic acid from Terminalia Chebula Retz.,and under the optimum conditions,the gallic acid was extracted in amount of 10.80 mg/g.According to the results of HSCCC and microcalorimetry,4 kinds of separated components?No.T1?T4?of Terminalia Chebula Retz.all had antibacterial activities to experimental strains,and component T3 was the main antibacterial ingredient,which identified as gallic acid;Component R1 and R5 of Rhizoma Polygoni Cuspidati had inhibition effect against two strains.Gallic acid can improve non-specific immunity of fish and then strengthen their resistance to disease,which have good preventive effect on bacterial disease.