Study On The Substances And Mechanisms Against The Toxicity Of Rhubarb By Compatibility

Rhubarb is widely used in clinic, which is the important drug to cure a number of diseases in traditional Chinese medicines. Anthraquinones and tannin are its essential active components. Recently studies in vivo and vitro have demonstrated that rhubarb have the potential of hepatotoxicity and nephrotoxicity. However, so far there are not reports concerning the toxicity of rhubarb in clinic. That’s because in clinic rhubarb was used after processing or compatibility with the other drugs. But so far there are little studies about rhubarb compatibility with the other drugs to decrease the toxicity of rhubarb. To provide the basis when rhubarb is used in clinic, the research choosed proper couple drugs and compound recipes which were on the basis of preliminary works to study the compatibility of rhubarb. In this study, in vivo and in vitro experiments have been applied respectively to explore the substances and mechanisms of reducing the toxicity of rhubarb by compatibility with other drugs. The study was researched from the following several aspects:1 Study the variations of hydrolysable tannins and anthraquinones in Rhubarb and its compatibilitiesTo research the differences between Rhubarb and its compatibilities with liquorice root, prepared liauorice root, Chinese goldthread rhizome and baikal skullcap root about the contents of hydrolysable tannin and anthraquinones. The extract of rhubarb and its compatibilities were prepared by alcohol and water. An HPLC method was established to determine the contents of hydrolysable tannins, free anthraquinones and total anthraquinones in water or ethanol extract of rhubarb and its compatibilities. Gallic acid, aloe-emodin, rhein, emodin, chrysophanol and physcion as the content indexs were analyzed simultaneously by HPLC. The contents of total hydrolysable tannins decreased slightly in its compatibilities. Besides, the contents of total anthraquinones and the free anthraquinones decreased differently in its compatibilities, especially signifficently in the compatibility with Chinese goldthread rhizome. After compatibility, the contents of total aloe-emodin, rhein, emodin, chrysophanol and physcion in rhubarb were dropped. The contents of rhein reduced the largest amount. But the contents of free aloe-emodin, emodin and chrysophanol were increased slightly after compatibility with prepared liauorice root. In Xiexin Decoctions, the content of free aloe-emodin was also increased slightly. The other free anthraquinones were decreased in its compatibilities.The results showed that there were obvious differences between Rhubarb and its compatibilities about the contents of anthraquinones, which may be the main substances contributing to decrease the toxicity of rhubarb after compatibility. And rhein may be the one of the major toxic constituent of anthraquinones.2 Study the toxicity of rhubarb after compatibility in vivoTo investigate the toxicity of rhubarb on kidney and liver of rats among itself and its compatibilities with liquorice root, prepared liauorice root, Chinese goldthread rhizome and baikal skullcap root in order to guide rational medication in clinic. Total extracts of rhubarb and its compatibilities were administered to rats at oral doses of 18.0g·kg-1·d-1 (crude drug) once per day up to 30 days. According to the results of pathological section and serum biochemical parameter, the experiment was executed in vivo. Toxity of rhubarb on kidney and liver of rats occurred after administration for two weeks. Besides, there were different rise in serum biochemistry including activities of ALT, AST and Crea. After administration for 4 weeeks, activities of ALT, AST and Crea rised constantly while the concentration of BUN decreased, and the liver of rats from the rhubarb group showed slightly acidophilia and edema. Besides, the kidney of rats showed little congestion, edema and karyopyknosis. However, the toxicity of rhubarb on kidney and liver of rats was reduced in its compatibilities except for its compatibilities with prepared liauorice root.That showed the toxicity of rhubarb on kidney and liver of rats could be reduced in its compatibilities.3 Study the absorption of anthraquinones in rat after intragastric administration of total extract from Rhubarb and its compatibilitiesTo research the variation of anthraquinones in rat serum samples after ig. administration of total extract from Rhubarb and its compatibilities with liquorice root, prepared liauorice root, Chinese goldthread rhizome and baikal skullcap root. The UPLC-MS method was established to determine the concentration of free anthraquinones namely aloe-emodin, rhein, emodin, chrysophanol and physcion in rat serum samples after ig. administration of total extract from Rhubarb and its compatibilities.Compared with the serum of rhubarb, the concentration of each compound of anthraquinone in rat serum was different after oral the extract of rhubarb compatibilities. Five compounds of anthraquinone were improved or lessened absorption by different compatibility. The concentration of rhein in rat serum was higher than the other compounds of anthraquinone in serum. Besides, the concentration of rhein in rat serum was dropped dramatically after compatibility with Chinese goldthread rhizome. The rhein showed the higher absorption in rats when oral the extract of rhubarb alone.And the concentration of rhein in rat serum after ig. administration of total extract from Rhubarb compatibility with Chinese goldthread rhizome was reduced sharply. Besides the other compounds of anthraquinone were absorbed differently in rat serum after ig. administration of total extract from rhubarb compatibility with liquorice root, prepared liauorice root and baikal skullcap root, which contribute to decrease the toxicity of rhubarb. The results also showed that the absorption of anthraquinones was differences in male and female rats. The concentration of anthraquinones in femal rat serum was higher than in male rat serum. The concentration of anthraquinones in rat serum was related to gender in rat.The results of experiment in vivo showed that the toxicity of rhubarb on kidney and liver of rats was reduced notable when it compatibility with Chinese goldthread rhizome. And compared with the other drugs, the content of rhein was lowest after compatibility with Chinese goldthread rhizome whether in the extract or in rat serum. So rhein may be the one of the major constituent of toxic substance in rhubarb.4 Study the influence of serum containing the extract of rhubarb and its compatibilities on proliferation of LO-2 liver cells and HK-2 kidney cells to investigate the attenuation mechanism of rhubarb compatibility with other drugs.To study the toxicity of serum containing the extract of rhubarb and its compatibilities on liver cell L-O2 and kidney cell HK-2 for investigate preliminarily the mechanism against the toxicity of rhubarb by compatibility with liquorice root, prepared liauorice root, Chinese goldthread rhizome and baikal skullcap root.MTT method was used to observed the effects of serum containing the extract of rhubarb and its compatibilities on cell growth. The morphological changes of cells were observed by inverted microscope. The LDH leakage and the ATP level were also measured.The inhibition of serum containing rhubarb was larger on the growth of L-O2 cells than on the growth of HK-2 cells. And the serum from female rats containing rhubarb was superior to the serum from male rats containing rhubarb in inhubiting the growth of L-O2 cells and HK-2 cells. After culture cells with the serum containing rhubarb, the LDH leakage of cells increased. And the ATP level of L-O2 cells descended when culture cells with the serum containing rhubarb. But when culture cells with the serum containing rhubarb compatibility with Chinese goldthread rhizome and the serum containing Xiexin Decoction, compared with the serum containing rhubarb, the LDH leakage of cells decreased and the ATP level elevated.The serum containing rhubarb could injure L-O2 cells by the way of destroying the plasma membrane integrity and reducing the mitochondrial function. The toxicity of the serum containing rhubarb could be reduced in the serum containing its compatibilities by affecting the LDH leakage the ATP level of cells to some extent.Combined with the results of experiment for determination of anthraquinones of rhubarb and its compatibilities in ratserum, the order of the effects of serum containing the extract of rhubarb and its compatibilities from female rats on cells was consistent with the concentration order of emodin in serum from each group of female rats. Meanwhile, The order of the effects of serum containing the extract of rhubarb and its compatibilities from male rats on cells was consistent with the concentration order of rhein in serum from each group of male rats. Among the serum containing the extract of rhubarb and its compatibilities from female rats, the lowest concentration of emodin was the serum containing Xiexin Decoction from female rats.And the lowest concentration of rhein was the serum containing the rhubarb compatibility with Chinese goldthread from male rats compared with the serum from male rats in the other groups. Infer from the result, emodin have a greater toxic effect for the female rats, while the rhein for the male’s. After compatibility of rhubarb, Chinese goldthread and Xiexin Decoction have a better effect on the reduce of toxicity.