Basic Research Of Incompatibility Theory Of Traditional Chinese Medicine Based On "Eighteen Incompatible Medicaments"

This work was supported by National Basic Research Program of China (973Program): Toxicity-effect characteristics of the compatibility of "Haizao, Daji, Gansui and Yuanhua all reverse Gancao".This dissertation was divided into four chapters.Chapter1. Study on LiteratureThe compatibility of Sargassum and Glycyrrhizae radix et rhizoma is part of "Eighteen Incompatible Medicaments", which was recorded in many ancient herbal medicine books. In the2010edition of Chinese Pharmacopeia, Eighteen Incompatible Medicaments was mentioned. In the item of "attention", Sargassum was not compatible with licorice. Based on the relevant literature, the origins, material basis, biological effects and clinical application was summarized and the experimental scheme of this paper was designed.Chapter2. Toxicity-effect characteristics and mechanism of the compatibility of Sargassum and Glycyrrhizae radix et rhizoma Section1Toxicity evaluation of Sargassum and Glycyrrhizae radix et rhizomaFrom the acute toxicity test of Sargassum and Glycyrrhizae radix et rhizoma, the time-mortality curve was drawed. The results showed that:the LDso of Sargassum was30g/kg,95%confidence limit was26.1-33.9g/kg. the acute toxicity of Sargassum from different origins were studied. The results showed that after oral administration of30g/kg (19times of the upper dose of Sargassum in Chinese Pharmacopeia) Sargassum pallidum and Sargassum fusiforme, mice showed similar adverse effects. After oral administration of the maximum concentration of licorice (48times of the upper dose of Glycyrrhizae radix et rhizoma in Chinese Pharmacopeia), no significant toxicity was found in mice.In order to determine the toxic doses and target toxic organ, chronic toxicity evaluation of Sargassum and Glycyrrhizae radix et rhizoma was studied. Rats were given the water extracts of different doses of the two herbs for8weeks. The results showed that when Sargassum was used with Glycyrrhizae radix et rhizoma, the level of LDH and y-HBDH in rat serum was increased and the histopathological examination showed myocarditis lesions; the AST in rat serum was increased and spotty necrosis in liver; the CREA in rat serum was increased and renal interstitial cystitis was found. Comparing with normal group, the chloride was significantly reduced. In summary, when Sargassum was used combined with Glycyrrhizae radix et rhizoma, the body electrolyte abnormalities was found and the target toxic organs were heart, liver and kidney.Section2The effect of Sargassum in goiter ratsIn order to investigate the impact of the water extract of Sargassum on goiter and explore the dose of treatment and the possible mechanism, the effect evaluation of Sargassum in goiter rats was studied. The results of thyroid index showed that the thyroid index of the model group and the treatment group was significantly higher than the control group. The thyroid index of positive group, low dose Sargassum group and high dose Sargassum group were lower than the model group, but no significant difference was found. Sargassum and levothyroxine sodium showed swelling effect. The thyroid hormone results showed there was no significant difference between the T3, T4and TSH in model group and the treatment group.Section3the effect of Sargassum and Glycyrrhizae radix et rhizoma on the electrolytes in ratsSargassum is rich in calcium, magnesium, sodium and potassium. Glycyrrhizae radix et rhizoma is rich in inorganic elements. The glycyrrhizic acid in licorice showed glucocorticoid-like effect. Will electrolyte imbalance lead to side effect when Sargassum is used with Glycyrrhizae radix et rhizoma? With this regard, the effect of Sargassum and Glycyrrhizae radix et rhizoma on the electrolytes in rats was studied. The result of urine in rats after single and multiple doses of Sargassum and Glycyrrhizae radix et rhizoma showed that the traditional efficacy of Sargassum was not achieved by way of diuretic. The result of electrolytes in urine after single and multiple doses showed the treatment group showed that sodium, magnesium and chloride in treatment group was higher than control group. The result of electrolytes in serum after multiple doses showed when Sargassum was used combined with Glycyrrhizae radix et rhizoma, the sodium and magnesium contents were higher than Sargassum group, and there was significant difference comparing with the control group. The results indicated that when the two herbs used together, electrolytes imbalance would be found, which might be one of the possible mechanism of the incompatibility of the two herbs.Chapter3. Research on chemical components of toxicity-effect of Sargassum and Glycyrrhizae radix et rhizomaSection1Chemical constituents from SargassumSo far, the study of the chemical composition of Sargassum was focused on the hierarchical structure of purification and analysis of Fucoidan. Little study was conducted on the other components. For further study of the active ingredient of Sargassum, seven components were isolated from the alcohol extract of Sargassum and were identified as palmitic acid-α-monoglycerides (1), stearic (2), palmitic acid (3), saringosterol (4), liquiritin (5), mannitol (6) and uracil (7). Compounds1,5and7were isolated from Sargassum pallidum for the first time.Section2Analysis and evaluation of chemical composition in Sargassum from different origins Part1Analysis and evaluation of inorganic elements in Sargassum from different originsBased on the microwave digestion-ICP-AES method and iodine ion selective electrode method,30inorganic elements were analyzed. The results showed that19to26inorganic elements were contained in Sargassum. The average content of inorganic element from high to low were in sequence of K>Ca>Na>Mg. the mean value of Cu, Hg, Pb, As and Al were7.83,1.54,3.07,3.02,109.45,1434.26mg/kg, respectively. No significant difference was showed in most elements between Sargassum pallidum and S. fusiforme.Part2The speciation of arsenic in Sargassum from different orginsBased on ICP-AES, the total arsenic was determined in18Sargassum samples from different origins. The speciation of arsenic in water extract of Sargassum was determined using HPLC-HG-AFS. The results showed that the speciation in decoction of Sargassum was As(Ⅲ), DMA and As(V). The total arsenic content was20.12-170.7mg/kg (dry weight). The main speciation of arsenic in water extract was inorganic arsenic and the content was9.836-97.73mg/kg,24.99%-89.54%of the total arsenic. The total arsenic in Sargassum was significantly higher than the limit standard total arsenic<2.0mg/kg of Green standards of medicinal plants and preparations for foreign trade and economy. Inorganic arsenic was significantly higher than the limit standard inorganic arsenic<1.5mg/kg of Hygienic standard for marine algae and algae products.Part3Analysis and evaluation of nucleosides, nucleobases and free amino acids in brown seaweeds from different regionsIn this study, the contents of nucleosides, nucleobases and free amino acids in common brown seaweeds from major production areas in China were determined by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-TQ-MS). The total contents of nucleosides and nucleobases varied from48.46to4105u.g/g. The free amino acids contents ranged from0.14to22.76mg/g. Furthermore, principle component analysis (PCA) was performed to classify the samples. The results showed that chemical profiles of nucleosides, nucleobases and free amino acids in Sargassum pallidum and Sargassum fusiforme were different from Sargassum muticum, Sargassum thunbergii and Saccharina japonica.Section3Study on the mechanism based on the interaction of multiple components for Sargassum and Glycyrrhizae radix et rhizomaPart1The influence of decoction in chemical components was studied based on UPLC/Q-TOFMS and MassLynx4.1statistical software for data analysis. The contents of saponins glycyrrhizin A3,22-Acetoxyl-glycyrrhizin and glycyrrhizin G2was significantly increased in co-decoction of Sargassum and Glycyrrhizae radix et rhizoma comparing to the combined decoction of the two herbs, while the contents of flavonoids liquiritin, liquiritigenin-4’-Apiosyl-glucoside and licuraside were significantly reduced.Part2Influence of the chemical constituent of the different proportions of Sargassum and Glycyrrhizae radix et rhizomaThe changes of the chemical constituents when Sargassum was used with Glycyrrhizae radix et rhizoma with different proportions was studied based on the UPLC-TOFMS method. The results showed that liquiritigenin-4’-Apiosyl-glucoside, liquiritin and glycyrrhizic acid was significantly increased.Part3The effect of the content of total arsenic and speciation of arsenic with different proportion of Sargassum and Glycyrrhizae radix et rhizomaThe changes of the content of the chemical constituents in the different proportion of Sargassum and Glycyrrhizae radix et rhizoma were studied based on the technology of ICP-OES and HPLC-HG-AFS. The results showed that total arsenic, As(Ⅲ) and DMA were increased. The effect of dissolution on As(Ⅴ) was not significant. The results indicated the decoction of the two herbs would increase the content of arsenic, leading to the enhanced toxicity. The increased toxic components might be one of the mechanisms of incompatibility.Section4Pharmacokinetic properties of arsenic species after oral administration of Sargassum pallidum extract in ratsA high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) was developed for simultaneous determination of the common arsenic species including arsenite, dimethylarsinate, methylarsonate and arsenate. This method was applied to study the pharmacokinetic profile of these arsenic species in rats after oral administration of S. pallidum extract at different doses. As(V) and DMA in the S. pallidum could be readily absorbed and eliminated in rats. A trend of dose-dependence was shown for DMA and As(V) in the drug concentration-time profiles.Chapter4. Study on the relationship of dose-toxicity-effect of Sargassum and Glycyrrhizae radix et rhizoma based on the goiter model in ratsThe research was based on the goiter model to study the dose-toxicity-effect of Sargassum and Glycyrrhizae radix et rhizoma.26groups were set based on the low dose, high dose,2times of high dose in Chinese Pharmacopoeia of Sargassum pallidum, Sargassum fusiforme and Glycyrrhizae radix et rhizoma. The thyroid hormones and tissue slice was investigated to evaluate the pharmacodynamics of Sargassum combined with Glycyrrhizae radix et rhizoma. The biochemical and histopathological indicators was investigated to evaluate the toxicity of the compatibility of the two herbs. A comprehensive analysis was conducted to evaluate the relationship of dose-toxicity-effect.The result of dose-effect showed Sargassum and Sargassum-Glycyrrhizae radix et rhizome could increase the T3, T4, FT3and FT4levels in rats and restored the imbalance caused by propylthiouracil. The effect did not affected on the low dose of Sargassum pallidum and low dose of Glycyrrhizae radix et rhizoma in Chinese Pharmacopoeia. FT4was significantly higher than normal group when the low dose of Sargassum pallidum used with high dose and2times of high dose of Glycyrrhizae radix et rhizome in Chinese Pharmacopoeia. The effect did not affected on the low dose of Sargassum Jusiforme and low dose and high dose of Glycyrrhizae radix et rhizome in Chinese Pharmacopoeia. T4was lower when the low dose of Sargassum fusiforme used with2times of high dose of Glycyrrhizae radix et rhizome in Chinese Pharmacopoeia. The effect did not affected on the high dose of Sargassum Jusiforme and low dose and high dose of Glycyrrhizae radix et rhizome in Chinese Pharmacopoeia. FT4was significantly higher than normal group when the high dose of Sargassum fusiforme used with2times of high dose of Glycyrrhizae radix et rhizome in Chinese Pharmacopoeia.The results of dose-toxicity showed in goiter rats, Cr of the high dose of Sargassum pallidum in Chinese Pharmacopoeia was significantly higher than normal and model group, suggesting toxicity of kidney. TP of the low dose of Sargassum fusiforme in Chinese Pharmacopoeia was significantly higher than normal and model group, suggesting toxicity of liver. BUN of2times of high dose of Sargassum fusiforme in Chinese Pharmacopoeia was significantly higher than normal and model group, suggesting toxicity of kidney. Toxicity of liver and kidney might be induced when Sargassum pallidum used with Glycyrrhizae radix et rhizome. Sargassum fusiforme and Glycyrrhizae radix et rhizome combination would affect the liver function and triglycerides in serum.