Bupleurum - Skullcap Drug Studies On The Hepatoprotective Effect Of Effective Parts Of Its Spectrum Efficiency

It is a hot field in studying Chinese prescription to explore the material foundation. The compostion of Chinese prescription is the foundation of the effect, which can be studied by exploring the composion, chemical compounds, and basic ingredients. It is very difficult to explore the material foundation, for multifunction of one prescription, one prescription being multi-section effectiveness, and the complicated chemical ingredients. But it is possible to choose the basic herbal pair to explore chemical parts and the relationship between the consist of that pair and the effectiveness, and to get the key chemical ingredients that influence the effectiveness of that herbal pair.The classical prescription of TCM, Xiao-Chai-Hu Decoction has the effectiveness of relieving shaoyang disorder and can be used in many syndromes in clinic, which has been proved by many pharmaceuctic studies. The research that has been done before proved that the Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair had more effectiveness on antiinflammatory, antifebrile, hepatoprotection.In this research, The Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair of the Xiao-Chai-Hu Decoction will be studied on the basis of their effectiveness of hepatoprotection and their effective ingredients. On the basic of determination the effective fraction of the decoction of Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair, the serum pharmacology and serum pharmacochemistry methods were used, and the hepatoprotective effect and the fingerprint chromatogram of the serum containing drug was be researched. The relation of the effect and the chromatogram was analyzed to explore the hepatoprotective material foundation of the herbal pair.This dissertation contains the documents review and the experiment. The progress in the material foundation of compound prescription of TCM and the progress in the chemical and pharmacological research of Bupleutrum Chinese and Scutellaria baicalensis Georgi and the herbal pair were summarized in the document part. Quality control of Bupleutrum Chinese and Scutellaria baicalensis Georgi, initial investigate of Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair, tracing of the effective fraction of Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair for hepatoprotection, study on the hepatoprotective effect of the serum containing drug of Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair, study on the fingerprint chromatogram of the effective fraction of Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair for hepatoprotection and the serum containing drug and study on the correlation of the hepatoprotective effect and the fingerprint chromatogram were discussed in the experimental part.Research 1: Quality control of Bupleutrum Chinese and Scutellaria baicalensis GeorgiMethods: To determine the content of saikoside a in Bupleutrum Chinese and the content of baicalin in Scutellaria baicalensis Georgi by the method of HPLC. Results: The content of saikoside a in Bupleutrum Chinese was 0.34%, and the content of baicalin in Scutellaria baicalensis Georgi is 12.70%. Conclusion: The content of saikoside a reached the average level of Bupleutrum Chinese, the medical material was qualified. The content of baicalin reached the regulation of Scutellaria baicalensis Georgi in Chinese Pharmacopeia, the medical material was qualified. Research 2: Tracing of the effective fraction of Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair for hepatoprotectionMethods: The decoction of Bupleutrum chinese-Scutellaria baicalensis Georgi herbal pair was divided into four fractions, which are A, B, C and D, by using the alcohol precipitating method. Prepare the combining fluid of the four fractions. The rats were assigned to four groups randomly, which were the normal group, model group, the decoction of Bupleutrum chinese-Scutellaria baicalensis Georg herbal pair group (chai-qin group)and the four fractions combining group(combining group), each group had 10 rats. The last two groups administrated orally respectively the decoction of Bupleutrum chinese-Scutellaria baicalensis Georg herbal pair and the combining fluid of the four fractions in 10g?kg-1 1 time a day for 5 days and other group with equivalent distilled water. 2 hours after the last administration, 10% CCl4(7mL? kg-1) was given to rats of the model group, chai-qin group and the combining group and distilled water was given to rats of the normal group by intraperitoneal(ip) injection. The rats were killed 24 hours after the last administration with drug,the levels of ALT and AST in serum were analyzed. Results: Compared with the normal group, the content of ALT and AST in serum of the rats in the model group was significantly increased (p<0.01). Compared with the model group, the content of ALT and AST in serum of the rats in the chai-qin group and combining group were significantly decreased (p<0.05 or p<0.01). The content of ALT and AST in serum of the rats in chai-qin group and combing group had no obviously different (p>0.05). Conclusion: The chai-qin group and combining group could significantly decrease the level of ALT and AST in serum of the rats acute liver injury model induced by CCl4, and the effect had no different. It showed that in the dividing process of the fraction A, B, C and D, the active constitutent of hepatoprotection effect in Bupleutrum chinese-Scutellaria baicalensis Georgi herbal pair had not lost.Research 3: Tracing of the effective fraction of Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair for hepatoprotectionExperiment 1: Relation between the hepatoprotection effect and Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair and its different combination.Methods: 1)The orthogonal design was used to combine the fraction A, B, C and D, and the different combination was the combination 1-8. The rats were assigned to eight groups randomly, which were the orthogonal combination group 1-8, each group had 5 rats. Each groups administrated orally respectively the different combination of fraction A, B, C and D in 10g?kg-1 1 time a day for 5 days. 2 hours after the last administration, 10% CCl4(7mL? kg-1) was given to rats of all the groups. The rats were killed 24 hourss after the last administration with drug,the levels of ALT an AST in serum were analyzed. 2)The rats were assigned to four groups randomly, which were the normal group, model group, chai-qin group and the optimal combining group(the optimal group of hepatoprotection effect comes from orthogonal analysis, optimal group), each group had 10 rats. The last two groups administrated orally respectively the decoction of Bupleutrum chinese-Scutellaria baicalensis Georg herbal pair and the optimal combining fluid in 10g?kg-1 1 time a day for 5 days and other group with equivalent distilled water. 2 hours after the last administration, 10% CCl4(7mL? kg-1) was given to rats of the model group, chai-qin group and the optimal group and distilled water was given to rats of the normal group by intraperitoneal(ip) injection. The rats were killed 24 hourss after the last administration with drug,the levels of ALT an AST in serum were analyzed. Results: 1)According to analysis of orthogonal design, fraction C and D could differently decrease the level of ALT and AST in serum of model group rats (p<0.05 or near 0.05). The optimal arrangement was the combination of fraction C and fraction D. 2)Compared with the normal group, the content of ALT and AST in serum of the rats in the model group was significantly increased (p<0.01). Compared with the model group, the content of ALT and AST in serum of the rats in the chai-qin group and optimal group were significantly decreased (p<0.05). The content of ALT and AST in serum of the rats in chai-qin group and optimal group had no obviously different (p>0.05). Conclusion: The effective fractions of Bupleutrum chinese-Scutellaria baicalensis Georgi herbal pair are C fraction and D fraction. The combination of C and D could significantly decrease the level of ALT and AST in serum of the rats acute liver injury model induced by CCl4, and the effect was same to the effect of Bupleutrum chinese-Scutellaria baicalensis Georgi herbal pair.Experiment 2: Subdivision of the effective fraction of Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair and the hepatoprotection effect of the different combinationMethods: The effective fraction of Bupleutrum chinese-Scutellaria baicalensis Georgi herbal pair (fraction C and D) was subdivided into four fractions, which areⅠ,Ⅱ,ⅢandⅣ, by using the acetoacetate, n-butanol extracting method and alcohol precipitating method. The orthogonal design was used to combine the fractionⅠ,Ⅱ,ⅢandⅣ, and the different combination was the combination 1-8. The rats were assigned to eight groups randomly, which were the orthogonal combination group 1-8, each group had 6 rats. Each groups administrated orally respectively the different combination of fractionⅠ,Ⅱ,Ⅲ, andⅣin 10g?kg-1 1 time a day for 5 days. 2 hours after the last administration, 10%CCl4(7mL? kg-1) was given to rats of all the groups. The rats were killed 24 hours after the last administration with drug,the levels of ALT an AST in serum were analyzed. 2)The rats were assigned to four groups randomly, which were the normal group, model group, chai-qin group and the optimal combining group(the optimal group of hepatoprotection effect comes from orthogonal analysis, optimal group), each group had 10 rats. The last two groups administrated orally respectively the decoction of Bupleutrum chinese-Scutellaria baicalensis Georg herbal pair and the optimal combining fluid in 10g?kg-1 1 time a day for 5 days and other group with equivalent distilled water. 2 hours after the last administration, 10% CCl4(7mL? kg-1) was given to rats of the model group, chai-qin group and the optimal group and distilled water was given to rats of the normal group by intraperitoneal(ip) injection. The rats were killed 24 hours after the last administration with drug,the levels of ALT an AST in serum were analyzed. Results: 1)According to analysis of orthogonal design, fractionⅠandⅢcould differently decrease the level of ALT and AST in serum of model group rats (p<0.05 or near 0.05). The optimal arrangement was the combination of fractionⅠand fractionⅢ. 2)Compared with the normal group, the content of ALT and AST in serum of the rats in the model group was significantly increased (p<0.01). Compared with the model group, the content of ALT and AST in serum of the rats in the chai-qin group and optimal group were significantly decreased (p<0.05). The content of ALT and AST in serum of the rats in chai-qin group and optimal group had no obviously different (p>0.05). Conclusion: The effective fractions of Bupleutrum chinese-Scutellaria baicalensis Georgi herbal pair areⅠfraction andⅢfraction. The combination ofⅠandⅢcould significantly decrease the level of ALT and AST in serum of the rats acute liver injury model induced by CCl4, and the effect was same to the effect of Bupleutrum chinese-Scutellaria baicalensis Georgi herbal pair.Research 4: study on the hepatoprotective effect in vitro of the serum containing drug of Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pairMethods: 1)The rats were administrated orally the Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair,prepare the serum containing drug 1.5 hours after the administration. The serums were added orderly to rat hepatocytes in 5%, 10% and 20%, which were injured by CCl4 in 15mmol/L. The levels of ALT and AST in the clear supernatant liquid were analyzed to find out the optimal concentration of the serum containing drug. 2)Prepare the normal serum and the serum containing drug 5min,15min,25min,50min,1.5h,2.5h,5h,8h (serum containing drug No.1,2,3,4,5,6,7,8)after the administration. The rat hepatocytes were assigned to ten groups randomly, which were the normal group, model group and the serum containing drug groups (No.1-8). The serums were added to rat hepatocytes correspondingly in 10%, which were injured by CCl4 in 15mmol/L (except the normal group). The levels of ALT and AST in the clear supernatant liquid were analyzed to evaluate the hepatoprotective effect of the serum containing drug in different times. Results: Compared with the normal group, the content of ALT and AST in the clear supernatant liquid of the model group was significantly increased (p<0.01). Compared with the model group, the content of ALT in the clear supernatant liquid of the group of the serum containing drug No.5, 6, 7, 8 was significantly decreased (p<0.01), and the content of AST in the clear supernatant liquid of the group of the serum containing drug No.5, 6, 7, was significantly decreased (p<0.01 or p<0.05 ). Conclusion: The serum containing drug 1.5, 2.5, 5 hours after the administration of the Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair had the good hepatoprotection effect to hepatic injury model of hepatocyte injured by CCl4.Research 5: study on the fingerprint chromatogram of the effective fraction of Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair for hepatoprotection and the serum containing drugMethods: To establish a fingerprint of Bupleutrum Chinese, Scutellaria baicalensis Georgi, the effective fraction of Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair and its serum containing drug by the method of HPLC, and to compare the fingerprint with that of the standard substance of saikoside a and baicalin and that of the normal serum. The HPLC fingerprints were obtained from SHIMADZU 20A instrument. The HPLC separation was performed on a C18 analytical column gradient eluted with acetonitrile-0.15% H3PO4at the flow rate of 1.0 mL/min. The temperature of column was 30℃. The UV detection wavelength was 203 nm. Results: The fingerprints of Bupleutrum Chinese, Scutellaria baicalensis Georgi, the effective fraction of Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair and its serum containing drug were set up. Conclusion: The majority of the chromatographic peaks of the effective fraction of Bupleutrum Chinese-Scutellaria baicalensis Georgi herbal pair in the fingerprint were come from Scutellaria baicalensis Georgi. The peak of saikoside a and baicalin in the fingerprint of effective fraction were higher than that of the medical material. There were 16 new peaks in the fingerprint of serum containing drug, in which there were 8 peaks represented the prototype constituent of the effect fraction.Research 6: study on the correlation of the hepatoprotective effect and the fingerprint chromatogramMethods: To establish the fingerprint of the serum containing drug 5min,15min,25min,50min,1.5h,2.5h,5h,8h (serum containing drug No.1-8)after the administration by the method of HPLC. The chromatographic condition was same to the former. Correlating analysis was made to discern the interrelationship between each peak and the pharmacologic data of the hepatoprotective effect of the serum containing drug in different times. Results: In bivariate correlating analysis, the content of ALT was found having good interrelationship with the peak 13 and 16(p<0.05), the content of AST was found having good interrelationship with the peak 8, 9, 11 and 16(p<0.05). In enter regression analysis, the peak 1, 3, 5, 6, 11, 13, 16 were entered (p<0.1). In stepwise regression analysis, the peak 16 was found having good regression with ALT and AST (p<0.05), and the standardized coefficients were -0.83 and -0.76. Conclusion: In the fingerprint of the effective fraction of Bupleutrum chinese-Scutellaria baicalensis Georgi herbal pair, the chemical compositions represented by peak 13 and 16 were the active substance of the hepatoprotective effect of the herbal pair, and the composition represented by peak 16 was more important.