Comparative Study On Anti-Toxoplasma Effect Of Polysaccharide From Inonotus Obliquus With Its Compound

Toxoplasmosis is a parasitic disease which is serious harm to the health of human and animal. The methods of prevention and cure have been a hot topic in the world. Four traditional Chinese medicine were tested in this study, the Glycyrrhiza uralensis(G. uralensis), Scutellaria baicalensis(S. baicalensis), Portulaca oleracea(P. oleracea) and Inonotus obliquus. They have been shown to have the role of anti-Toxoplasma. The active ingredients of the four medicines were extracted and combined to get compound, then through the test of inhibiting Toxoplasma gondii in vitro and in vivo, pathological observation and the cytokine content of Inonotus polysaccharide compound and its group of insect resistance, provide theoretical and experimental reference for the development of anti-Toxoplasma drug use.Methods:the alcohol abstracts of G. uralensis and S. baicalensis were extracted, the polysaccharide of P. oleracea and Inonotus obliquus were extracted respectively. The content of the extracts were determined, and then the inhibition test was carried up to Toxoplasma gondii in vivo and in vitro. In vitro test:infection model was established by infecting vero cells with Toxoplasma gondii. By MTT method, the cell toxicity tests and inhibition test of single drug and different compounds were carried up. By comparing the cell proliferation rate, the effects of the four drugs extract were observed, and then the mosr effective combination was screened out. In vivo tests: infection model was estabilished by infecting normal mice with Toxoplasma gondii. Based on the test results in vitro and the Inonotus polysaccharide was compared with, by comparing the survival rates and survival time, the best combination and therapeutic doses of different extracts were selected. Through the pathologic biopsy, immunohistochemisty and transmission electron microscope (TEM), pathological changes of the main organs were observed. The levels of cytokine gamma interferon (IFN-y), leukocyte interleukin-4(IL-4) and leukocyte interleukin-8(IL-8) were detected by ELASA before and after treatment to compare the best combination and the polysaccharide of Inonotus obliquus.Results:the content determination of the extracts:the total flavonoid content of the G. uralensis ethanolic extract was13.4%, the total flavonoid content of the S. baicalensis alcohol extract was1.8%, the polysaccharide content of Portulaca oleracea was81.6%, the polysaccharide content of Inonotus obliquus was81.2%. In vitro test:the G. uralensis ethanolic extract and the S. baicalensis alcohol extract could inhibit Toxoplasma gondii in vitro, compound group of G. uralensis and S. baicalensis, compound group of four extracts both had the best effect on inhibiting Toxoplasma gondii. Cell proliferation rate in24hours and48hours were both higher than positive artemether group.In vivo test:the mice of the medium dose group of four extracts compound had the highest survival rate, and survived the longest time, the average survival days and the longest time of the mice are9.9d and13d, longer than the ones of positive control group, which wre9.1d and11d. The results showed that the best compound was the four extracts compound, and the optimal dose was the medium dose. The results of the vitro and vivo tests showed that the best group is the four extracts compound group. The observetion changes of pathological tissue section and immunohistochemistry showed that on the same day, the pathological damage of the experimental group is slightly lighter than the positive group, the quantity of Toxoplasma gondii in the organs of the experimental group is less than positive group and significantly less than the model group. TEM results showed that the experimental group and the positive Portulaca oleracea polysaccharide group showed no obvious effects on the liver cells of the infected mice. The results of detecting the cytokines levels showed that the IFN-y levels of the experimental group mice were significantly higher than the other group ones (P<0.05). The IL-4level of the experimental group mice and the positive control group mice rised significantly, and kept on a higher level than the ones before infected. Ratio of IFN-y and IL-4value showed the value of the experimental group was rising slower than the positive control group. In middle time, it appeared a rising trend and then failed back to a lower level, but the one of the positive group appeared continued decline, and keep lower level. The difference was not significant of the IL-8level of mice both the experimental group and the positive control group after infected Toxoplasma gondii (P>0.05), the peak value appeared on the fourth day, and then keep higher level, but the value of the infection group keep rising. The results of the cytokines levels showed four extracts compound group and the positive Portulaca oleracea polysaccharide group had similar action on the adjustment of cell factors level, they both had a good inhibition effect to Toxoplasma gondii and could protect the host.Conclusion:the four extracts all had a good inhibition effect to Toxoplasma gondii. The best compound combination was the four extract compound, and the optimal treatment dose of the G. uralensis ethanol extract was50mg/10g weight, the S. baicalensis ethanol extract was25mg/10g weight, the polysaccharide of Portulaca oleracea was2mg/10g weight, the polysaccharide content of Inonotus obliquus was2mg/10g weight. The compound group could protect the mice infected with Toxoplasma gondii well, increase the survival rate and survival time obviously, lighten the pathological changes and inflammation. The effect was better than the Inonotus obliquus polysaccharide. The compound group could increase the valves of IFN-y, IL-4, IL-8in the mice serum to an appropriate level, promote Thl/Th2balance through effectively adjusting cell factors levels, resist polypide, protect host, and the effect was similar to Inonotus obliquus polysaccharide.The compound had a good inhibition effect to Toxoplasma gondii in vivo and vitro, and better than the control group of polysaccharide content of Inonotus obliquus, had the advantages of enhancement effect of compound.