The Separation And Antibacterial Research Of The Effective Constituents From Rhus Chinensis Mill., Scutellaria Baicalensis Georgi., And Rheum Palmatum Linn

In this study, ten Chinese herbs were firstly screened for controling three familiar aquatic pathogenic bacteria, of which, three effective herbs, Rhus chinensis Mill., Scutellaria baicalensis Georgi and Rheum palmatum Linn. were selected for further study. Main constituents were isolated from the selected three Chinese herbs using supercritical fluid extraction and high-speed counter-current chromatography(HSCCC), and their inhibition effect on Vibrio alginolyticus(G1-1), Edwardsiella tarda(G1-2), Aeromonas hydrophila(B18) was determined using microcalorimetry. The antibacterial activities of different constituents were compared to determine the effective constituents of the three Chinese herbs, and the best bacteriostasis concentration were measured, so as to provide scientific, effective and theoretical basis for reference in aquatic animal bacterial disease control and prevention. The results are as follows:1. Ten Chinese herbs were comminuted with ultra-pulverization, and then tested antibacterial effect by alone and double compounds against three major pathogenic bacteria of cultivated eel. The results show that all these ten kinds of chinese herbs have certain degrees of inhibiting and killing the three pathogenic bacteria. Five Chinese herbs have a good antibacterial effect by using only one herb, they were Rhus chinensis Mill., Punica granatum Linn.,Rheum palmatum Linn., Rhizoma polygoni Cuspidati, Scutellaria baicalensis Georgi. Of which ten combinations of two Chinese herbs designed by Cross-test method were used to examine the bacteriostatic effects against above three pathogenic bacteria. The results showed that the combinations of Rhus chinensis Mill.and Punica granatum Linn., Punica granatum Linn. and Scutellaria baicalensis Georgi, were showed synergistic action against the three pathogenic bacteria, and the synergy rate was 100%. The combinations of Rhus chinensis Mill. and Rheum palmatum Linn., Rhus chinensis Mill. and Scutellaria baicalensis Georgi, Punica granatum Linn. and Rhizoma polygoni Cuspidati, were showed synergistic action with the synergy rate of 67%. The combinations of Rhus chinensis Mill. and Rhizoma polygoni Cuspidati, Punica granatum Linn. and Rheum palmatum Linn., Rhizoma polygoni Cuspidati and Scutellaria baicalensis Georgi,were showed synergistic action with the synergy rate of 33%. However the combinations of Rheum palmatum Linn. and Rhizoma polygoni Cuspidati, Rheum palmatum Linn. and Scutellaria baicalensis Georgi, were not showed synergistic action.2. The main constituents of the three Chinese herbs, Rhus chinensis Mill., Scutellaria baicalensis Georgi and Rheum palmatum Linn. were extracted and isolated using supercritical fluid extraction and HSCCC. The solvent system which were used to isolate and purify three Chinese herbs were screened and determined by analytical HSCCC. The system of ethyl acetate-ethanol- water(5:1:5, v/v) were used to separate the main components of Rhus chinensis Mill., the system of ethyl acetate- methanol- water-acetic acid(5:0.5:4.5:0.05, v/v) were used to separate the main components of Scutellaria baicalensis Georgi, and the system of n-hexane-ethyl acetate- methanol- water(1:1.5:1.5:0.8, v/v) were used to separate the main components of Rheum palmatum Linn.. Each component was collected by absorption peak, and the main components of each Chinese herbs were prepared by the preparative HSCCC, and then the purity of each component was analyzed. The results showed that the purity of seven kinds of main components from Rhus chinensis Mill.(No. W1 to W7) were 97.7%,97.6%,86.9%,83.1%,48.7%,98.5%,94.2%. The purity of five kinds of main components from Scutellaria baicalensis Georgi(No. H1 to H5) were 85%,94.3%,95.6%,89.5%,47.6%. The purity of 5 kinds of main components from Rheum palmatum Linn.(No. D1 to D5) were 90.3%,86.9%,94.1%,80.3%,43.0%.3. The inhibition effect of the main Constituents separated from Rhus chinensis Mill. on Vibrio alginolyticus(G1-1), Edwardsiella tarda(G1-2), Aeromonas hydrophila(B18) were determined by microcalorimetry. The optimum concentrations to these three pathogenic bacteria were confirmed respectively by the relationship between growth rate constants of bacteria and concentrations of herb. The results showed that seven constituents of Rhus chinensis Mill. had the inhibiton to the three major pathogenic bacteria, and W4, W5, W7 have good inhibiton to the three major pathogenic bacteria, the optimum concentration was between 0.10mg/m L and 0.62mg/m L. The same constituent had different inhibitory action to different pathogenic bacteria.4. The inhibition effect of the main constituents separated from Scutellaria baicalensis Georgi on Vibrio alginolyticus(G1-1), Edwardsiella tarda(G1-2), Aeromonas hydrophila(B18) were determined by microcalorimetry. The optimum concentrations to these three pathogenic bacteria were confirmed respectively by the relationship between growth rate constants of bacteria and concentrations of herb. The results showed that H5 had inhibitory action to the three pathogenic bacteria, the optimum concentration was 0.25mg/m L to 1.92mg/m L; but H1, H2, and H3 promoted the growth of G1-2 and B18; and H4 promoted the growth of B18. The same constituent had different inhibitory action to different pathogenic bacteria.5. The inhibition effect of the main constituents separated from Rheum palmatum Linn.on Vibrio alginolyticus(G1-1), Edwardsiella tarda(G1-2), Aeromonas hydrophila(B18) were determined by microcalorimetry. The optimum concentrations to these three pathogenic bacteria were confirmed respectively by the relationship between growth rate constants of bacteria and concentrations of herbs. The results showed that W1 had good inhibitory effect to the three major pathogenic bacteria, the optimum concentration was 1.05mg/m Lto 2.0mg/m L; and D4 had certain inhibitory effect to them, the optimum concentration was 2.89mg/m L to 9.82mg/m L; but D3 promoted the growth of G1-1 and G1-2, D2 and D5 promoted the growth of B18. The same constituent had different inhibitory action to different pathogenic bacteria.6. In summary, Rhus chinensis Mill., Scutellaria baicalensis Georgi and Rheum palmatum Linn.have good antibacterial activities on the pathogenic bacteria of G-1, G1-2, and B18. For the extracted and isolated chemical constituents from the three Chinese herbs by HSCCC, and the inhibition effect of the main constituents on the pathogenic bacteria by microcalorimetry, we found that the bacteriostatic constituents of W4,W5, and W7 of Rhus chinensis Mill. had the inhibition effect against three pathogenic bacteria; the bacteriostatic constituents of H5 of Scutellaria baicalensis had the inhibition effect against three pathogenic bacteria; the bacteriostatic constituents of D1 of Georgi and Rheum palmatum Linn.had the inhibition effect against three pathogenic bacteria.