| ObjectiveSanweiganjiang Powder was a set prescription of Tibetan medicine composed of Rhizoma zingiberis, Amomum cardamomum and Myristica fragrans, and proved to have fairy good curative effect on hepatitis of deficiency-Cold type and hepatomegaly. The modern pharmacology research indicated that the extracts from rhizoma zingiberis, amomum cardamom and myristica fragrans possess a great anti-oxidation activity thus could help liver protection, and that Sanweiganjiang Powder’s effectiveness on liver protection may be related to its anti-oxidation capability. This dissertation discussed the methods to control and evaluate the quality and stability of Sanweiganjiang Powder through the pattern recognition and fingerprint study of its GC-MS chemical elements, and then based on the theory proposed in the discussion as well as the pharmacology experiments conducted on animals and cells, explored Sanweiganjiang Powder’s effect on liver protection and the mechanism working behind from the perspective of its anti-oxidation capability.MethodsThe volatile oil of each single medicine material in Sanweiganjiang Powder as well as the volatile oil of 12 Sanweiganjiang Powder compound medicines made up of medicine materials from 12 different places of origin were extracted by steam distillation. The chemical ingrediented in both the volatile oils were studied and analyzed by the GC-MS technique. The GC-MS fingerprint of the Sanweiganjiang Powder volatile oil was accordingly established. The quality and stability of the compound medicines’volatile oil were then studied with pattern-recognition methods such as system cluster analysis, principle component analysis, identification analysis and the chemical ingredients fingertprint to provide a scientific basis for the later quality evaluation work and the proper appliance of the results of the followed pharmacology experiments.Experiments were conducted on rats to study the effectiveness of Sanweiganjiang Powder in treating the chronic liver damage:the male SD rats were divided randomly into normal group, model group, Curcumin positive control group and Sanweiganjiang Powdermedicated group. Each group had 10 rats. Create animal models with rats suffering from CC14-induced chronic liver damage. Observed the pathology by HE staining and the fibrosis process by Masson collagen staining; dissected the rats and taken the liver tissues; calculated the liver index. Tested the level of ALT, AST, ALB and ALP in the serum, determined the serum PIIINP, CIV level as well as the MDA, SOD level and the GSH-Px vitality in the homogenate of liver tissues, and measured the protein amount of Nrf2 and Bachl in the homogenate of liver tissues by immunohistochemical method to find out the difference.The anti-oxidation and liver protection mechanism of Sanweiganjiang Powder:the male SD rats were divided randomly into normal group, model group, Curcumin positive control group, and Sanweiganjiang Powder medicated group. Each group has 12 rats. Create animal models with rats suffered from CCL4-induced chronic liver damage. The rats’ liver index, spleen index and thymus index were measured; The pathology by HE staining was observed; tested the level of ALT, AST, MDA and the GSH-Px vitality; measured the protein amount and the HO-1 amount in both the nucleus and endochylema of Nrf2 and Bach1 cells in the rats’liver by the Western-Blot method; calculated the nuclear translocation rate of Nrf2 and the ratio of Nrf2 to Bachl; measured the total level of Nrf2、 Bachl and HO-1mRNA by the RT-PCR method; and then calculated the ratio of mRNA in Nrf2 to that in Bach1Established the model of the stress-induced injury of L-02 caused by oxidation of GO: First, prepared 11 GO reagents with different concentration ranging from 0-200U per Liter and another 11 GO reagents that were to be used on the L-02 at different points of time and function for different length of time (from 1 hour to 12 hours). Used GO reagents with different concentration to do damage to the L-02 cells and used GO reagents to do the damage for different length of time, then tested the survival rate of L-02 cells by MTT method to explore the best model time and the proper concentration of GO that could properly damage the L-02 cells. Create the oxidation-induced L-02 cell damage models based on the findings in the above experiments. Tested the survival rate of cells, AST, ALT, MDA and GSH-Px level at 9 different time points during 0-22hs after modeling to explore the effect of GO-oxidation-induced L-02 damage model to provide a reliable and stable damage model for the future experiments and researches.Research on Sanweiganjiang Powder medicated serum’s anti-oxidation and liver protection effect against the GO-induced L-02 damage:After the L-02 culture, divided the cultured cells into blank serum control group, model blank serum control group, Sanweiganjiang Powder medicated serum control group, ethanol group, model ethanol group and curcumin group. Create the L-02 cell oxidation-induced damage model after the 100U/L GO reagent functions on the cells for 2 hours. Measured the cell survival rate, AST, ALT, MDA and the GSH-Px level after the medicated serum was applied to the tissues for 10 hours; checked the protein amount of Nrf2 and Bachl in the nucleus of L-02 cells by Western-Blot method; observed the expression of Nrf2 and Bachl by immunofluorescence.Results1. Findings on the stability of Sanweiganjiang Powder’s chemical ingredients.102 chemical compounds were found in the volatile substances of the Rhizoma zingiberis contained in Sanweiganjiang Powder, among which 82 are identified. The mass of the 82 compounds identified accounted for 99.21% of the total mass. For Amomum cardamomum 49 compounds were found, among which 37 were identified and the mass of which accounted for 99.8 of the total. From Myristica fragrans,58 were found,40 identified and the mass accounted for 98.04%.A total of 55 compounds were found in the volatile substances of the compound medicine, among which 48 were identified. Eucalyptol was found to be the No.1 major component of the compounds that accounted for more than 50% of all the volatile substances in amount. The other 5 major components, ranked according to their functions, were β-Phellandrene, β-pinene,4-terpineol, a-pinene and a-terpineol. The mass of the 6 major components mentioned above accounted for more than 80% of the total of volatile oil, therefore were believed to characteristic ingredients of Sanweiganjiang Powder volatile oil.The components in volatile oil were not merely a combination of all the volatile oils from the single medicine materials. Great changes were detected in the components of the volatile oil of the compound medicine:Many material peaks identified in the single medicine materials could not be detected in the compound medicine, such as the myristicin. The average ratio of myristicin in the 12 compound samples was around 2.5%, which was much lower than the number 16.6% measured in the single medicine samples of Myristica fragrans. This, to some extent, justified the compatibilities of Sanweiganjiang Powder’s component materials.The volatile substances in 12Sanweiganjiang Powder made up from materials of different place of origin showed the slightest difference in the types of their major chemical compounds. The samples showed a high overall similarity and a stable and reliable quality. The characteristic fingerprint of common pattern could be used to estimating the Similarity degree of different medicinal herbs, and could serve as the evaluating indicators for future experiments on the quality and stability of Sanweiganjiang Powder. For the choosing of the medicine materials, the Sanweiganjiang Powder made up of nutmegs from Canton, cardamon fruits from Hainan and rhizoma zingiberis from Sichuan demonstrates the best quality as well as the highest stability thus was recommended the medicine of choice.2. Findings of experiments on Sanweiganjiang Powder’s curative effect against rats’ chronic liver damage:The HE and Masson staining experiments indicated that compared with that of the model group, the liver cell damage in Sanweiganjiang Powder group was attenuated, the hepatic lobule was essentially intact and the cells on the hepatic cell cords were in regular line-up. Only small amount of dead cells and fibrosis tissues were found but the density of collagen fiber drops dramatically. The liver index in Sanweiganjiang Powder group also fell substantially compared with that of the model group.Compared with that of the model group, the serum tests showed that the Sanweiganjiang Powdergroup’s serum ALT, AST and ALP level all dropped substantially. So did the PⅢNP、CIV level in their serum collagen according to the collagen test. Besides, the MDA level in the rats’liver tissues fell while their GSH-Px vitality and SOD level rose dramatically according to the antioxidation test on their liver homogenate vitality.Immunohistochemical experiments showed that compared with that of the normal group, the amount of Nrf2 protein in the liver cell cytoplasm of rats from the model group was much higher versus their Bachl protein was much lower. However, compared with the model group, the Sanweiganjiang Powder group showed a lower Nrf2 amount and a higher Bach1 level in their liver cell cytoplasm.3. Findings on the mechanism behind Sanweiganjiang Powder’s anti-oxidation and liver protection effect.Compared with that of the model group, both the liver and spleen index of rats from Sanweiganjiang Powder group dropped substantially. But no significant difference was found on their thymus index. HE staining experiment showed that the hepatic lobule of rats from Sanweiganjiang Powder group was essentially intact with only small amount of fatty degeneration, dead cells and fibrogenesis found, and the fibrosis level degraded distinctly. The serum ALT, AST levels and the liver tissue homogenate MDA level of rats from Sanweiganjiang Powder group also saw a clear dropped while their GSH-PX vitality rose significantly.The protein test by Western-Blot method showed, compared with that of the normal group, the total amounts of protein Nrf2, Bach1 and HO-1 in both the nucleus and cytoplasm of the liver cells of rats from the model group rose distinctly and the nuclear translocation rate of Nrf2 and Bachl also rose, while the ratio of nucleus Nrf2 to Bachl dropped. It was also found that compared with that of the model group, the Nrf2 expression level of rats from Sanweiganjiang Powder group experiences an obvious rose in their liver nucleus and a dropped in their cytoplasm. The nuclear translocation rate of Nrf2 went up, while the Bachl levels dropped substantially in both their liver cell nucleus and the cytoplasm. No significant changes were found in the nuclear translocation rate of Bachl. The nucleus Nrf2/Bachl ratio as well as the total amount of HO-1 protein both rose dramatically, suggested Sanweiganjiang Powder’s regulating effects on Nrf2 and Bach1 expression level in liver tissues as well as on helping increase the expression level of the anti-oxidation enzyme HO-1.The 2-△△CT statistics showed, compared with that of the normal group, one could be seen a substantial rise in the mRNA expression levels of Nrf2 and Bachl in liver tissue and a drop in the HO-1 mRNA expression level. The comparison between the model group and Sanweiganjiang Powder group obviously showed that the use of Sanweiganjiang Powder can dramatically increased both the Nrf2 and HO-1 mRNA expression levels in the liver tissue and decreased its Bachl mRNA expression level. Similar condition was found on the two proteins’ total amounts and their expression levels. The correlation analysis indicates the HO-1 mRNA expression level was in proportion to the Nrf2/Bach1 ratio (r:0.987, P< 0.05)4. Results of creating the GO-oxidation-induced L-02 cell damage model:Created the L-02 cell oxidation-induced damage model after the 100U/L GO reagent functioned on the cells for 2 hours. it was found that after the 100U/L GO reagent functioned on the cells for 2 hours, the survival rate of L-02 cells as well as the GSH-Px vitality plunge while the ALT,AST and MDA level rose substantially. The damage differs at different points of time: the oxidation-induced damage aggravated during the first 10 hours, and gradually attenuated from the 11th hour to the 22th hour. Then the body started to recover from the L-02 damage.5. The findings on Sanweiganjiang Powder medicated serum’s antioxidant and protective effect against L-02 damage caused by GO:With the appliance of the medicated serum of Sanweiganjiang Powder, the survival rate of L-02 cells increased substantially, the levels of ALT、AST、MDA dropped in cell culture supernatant and the vitality of GSH-Px increased. Results found through immunofluorescence techniques and Western blot advocated that the medicated serum of Sanweiganjiang Powder could increase the expression of Nrf2 in the nucleus of cells and at the same time decrease that of Bach1.ConclusionThe main chemical compounds in the volatile substances of Sanweiganjiang Powder made up of different herbal medicines from 12 different regions demonstrated slight difference in categories and types. All the samples showed a high overall similarity and a stable and reliable quality, among which the one made up of myristica fragrans from Canton, amomum cardamon fruits from Hainan and rhizoma zingiberis from Sichuan demonstrated the best quality, the least quality difference among samples as well as the highest stability thus could be the medicine of choice. The previous research on the recognition patterns of GC-MS could be applied to the quality stability control of the main chemical compounds in the volatile components of Sanweiganjiang Powder.Sanweiganjiang Powder could lower the liver function index indicators such as ALT、 AST、ALP as well as collagen indicators-PⅢNP、CIV. It could also reduce the amount of MDA and SOD, the antioxidant indicators. Besides, it could increase the vitality of GSH-Px, lower the amount of Nrf2 protein and increase the Bachl protein level in the cytoplasm of liver cells. Therefore, its effected against the chronic liver damage may be attributed to its anti-oxidation capability.Sanweiganjiang Powder could help balance body’s oxidation and antioxidation mechanism:it increased the Nrf2 level in the liver cells, lower the Bachl level, increased the nuclear translocation rate of Nrf2, revitalizing the GSH-Px and reduced the ALT、AST、 MDA level. On the gene’s level, Sanweiganjiang Powder’s influence on the expression of mRNA in Nrf2 and Bachl appeared to be coherent with its influence on that of all the protein:it increased mRNA expression in Nrf2, reduced mRNA expression in Bach1, causing a distinct rise in the expression of gene HO-1mRNA in Nrf2.The Nrf2 and Bachl maintained a certain level of balance and may lose the balance when interfered by external elements. The ratio of Nrf2/Bach1 in the nucleus of cells showed body’s oxidation and antioxidation condition. During the time when body was damaged by oxidation, the ratio of Nrf2/Bachl in the nucleus of cells appeared lower than that of the normal ones while in the process of antioxidant therapy, an distinct rise was found in the ratio. On the gene’s level, the ratio of the mRNA amount in Nrf2 to that in Bachl also accords with the ratio of the protein amount in Nrf2 to that in Bachl. As for the gene level, the expression of Ho-1 mRNA and the protein level of HO-1 from the medicated group were also in proportion to the relevant ratio of Nrf2 to Bachl.The medicated serum of Sanweiganjiang Powder could increase the expression of Nrf2, reduce the expression of Bachl in the nuclei of L-O2 cells, promote the ratio of nucleus Nrf2/Bach1, vitalize the GSH-Px in L-O2 cells, lower the amount of ALT、AST、MDA, thus has the antioxidant and protective effect against L-O2 damage caused by GO. |
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