The Study On Mechanism Of Decreased Toxicity By Glycyrrhiza Uralensis Fisch Combined With Diosorea Dulbifera Rhizome Based On The Theory Of "Gan Can Relieve Detoxication"

Objective:Investigate the hepatotoxicity caused by Dioscorea bulbifera L. and whether Glycyrrhiza uralensis Fisch. can abatement this effect or not as well as the relevant mechanism with a method of in vivo and vitro together. Then provide a experimental basis for the safety and rational use of Dioscorea bulbifera L. and other difficult cases of toxic traditional Chinese medicine in clinical, in addition, to enrich and develop the property theory of gan can relieve spasm and slow detoxification " and Compatibility theory of’mutual detoxication、 mutual restraint".Methods1. Observe the survival rate of LO2cells under different dose of Dioscorea bulbifera L. and different culture time with Dioscorea bulbifera L.by MTT metablism. Analyze the effect of Dioscorea bulbifera L.-containing serum and combined with Glycyrrhiza uralensis Fisch.-containing serum on the rate of LO2cells survival; in order to determine the hepatotoxicity caused by using Dioscorea bulbifera L. only and its compatibility of Glycyrrhiza uralensis Fisch. we tested the activity of GPT, GOT and LDH in the culture media after cell culture with colorimetric method.2. Use real-time dynamic detection method to monitor the fluorescence intensity of the hepatocyte staining by Fluoesceim diacetate with the laser scanning confocal microscope, then definite the effect of Dioscorea bulbifera L. and its compatibility of Glycyrrhiza uralensis Fisch. on LO2cells membrane permeability by compared with the reduced rate of fluorescence intensity.3. To investigate the effect of Dioscorea bulbifera L. and its compatibility of Glycyrrhiza uralensis Fisch. on the function of mitochondria, SD rats were grouped by Dioscorea bulbifera L. extracting solution, Dioscorea bulbifera L. and Glycyrrhiza uralensis Fisch. extracting solution and distilled water, take the rat liver tissue one hour later, then cryogrinding and use gradient centrifugation to extract mitochondria. Colorimetric assay for the activity of main complex phosphoesterasum in mitochondria respiratory chain.Results1. At higher doses of Dioscorea bulbifera L. or longer culture duration, lower percentage of cell survival is noticed. Percentage of LO2cells survival is significantly lower in the Dioscorea bulbifera L. group than the control (P<0.01) while Glycyrrhiza uralensis Fisch. can partially antagonize the toxicity of Dioscorea bulbifera L. Also Dioscorea bulbifera L. raises the activity of GOT、GPT、LDH while Glycyrrhiza uralensis Fisch. counteracts the toxicity of Dioscorea bulbifera L.2. When scanning stopped at ten minutes, there was found a significant difference between the blank group and Dioscorea bulbifera L groups in reducing the fluorescence intensity of cells (P<0.05), while the Glycyrrhiza uralensis Fisch groups had no obvious influence on the fluorescence intensity of cells. The changes of fluorescence intensity of cells were recorded by LSCM.3. There was found a significant difference between the blank group and Dioscorea bulbifera L groups in inhibit the activity of Complex Phosphoesterasum Ⅰ, Ⅳ in the mitochondria of LO2cells (P<0.05), while Glycyrrhiza uralensis Fisch had no obvious influence on these Phosphoesterasums. High dose of Dioscorea bulbifera L can decrease the activity of amber acid dehydrogenase slightly (P<0.05), while other groups had no obvious difference with the blank group and compatibility group.Conclusions1. Toxicity of Dioscorea bulbifera L. on LO2cells is related to the does and the time of cells culture. Glycyrrhiza uralensis Fisch. can partially shield LO2cells from the the toxic effect of Dioscorea bulbifera L.2. Dioscorea bulbifera L.-containing serum has an effect on the hepatocyte membrane permeability in a short time after administration, while its compatibility group of Glycyrrhiza uralensis Fisch. and the serum group have no excellent effects on it. The hepatic injuries caused by Dioscorea bulbifera L. maybe via a coup injury mechanism, but this injury can be weaken by compatibility of Glycyrrhiza uralensis Fisch.3. Glycyrrhiza uralensis Fisch offers partial protection by blocking the activity of Dioscorea bulbifera L, which exert its effect on the electronic respiratory chain of the mitochondria.