| Background and purpose:Liver fibrosis leads to a variety of serious harm to human health. It makes heavy burden for the family and public. Research and development of the drug for restrict liver fibrosis; there is an important social and economic significance especially for traditional Chinese medicine. Combine the theory of traditional Chinese medicine and modern pharmacology research, and combination of ancient and modern clinical practice, screening of Chinese medicine treatment of liver fibrosis; extract the active in gradient combination of prescription. And then experimental the method of studies and clinical trials, it’s an effective means of research and development of Traditional Chinese Drug. Therefore, under this guidance of this idea, we examine a large number of Chinese ancient pharmacology literature, and modern pharmacology literature at domestic and foreign of modern pharmacology research. Finally we screen out of Radix et Rhizoma Glycyrrhizae and Radix Sophorae Flavescent. Glycyrrhizae have the effects of antiinflammatory, antivirus, liver detoxification and enhance immune function and role. Its active ingredient is glycyrrhizin. Radix Sophorae Flavescent is having the effects of remove heat, damp to relieve fire, and insecticidal diuretic. Its use is for the hot dysentery, hematochezia, jaundice and urodialysis. Its active ingredient is matrine. Modern pharmacological studies have shown that glycyrrhizin and matrine combined have the treatment of liver fibrosis role as a hepatoprotective, anti-inflammatory, and antiviral agent. Clinical material demonstrated that glycyrrhizin and matrine combined intravenous drip for chronic hepatitis B treatment effect is good. But chronic diseases require long-term medication. Intravenous infusion for patient compliance is poor. Therefore, we chose glycyrrhizin and matrine combine oral drug treatment for liver fibrosis. Both effective active ingredient combination drugs, it was named for GanKu oral liquid. In order to research GanKu oral liquid of liver fibrosis effect, the subject first optimization the proportion of glycyrrhizin and matrine. Then choose the best ratio and prepared to oral liquid. Methodology investigation to develop an HPLC method for the determination of glycyrrhizin and matrine in GanKu oral liquid, and methodology was also inspected. Observe the physical stability of GanKu oral liquid. Observe the various environment appearance. PH value, the relative density and content of the change of the GanKu oral liquid. We use acute toxicity test examine the safety of GanKu oral liquid. Finally we design animal experiment of resisting hepatic fibrosis言言Methods:'Is1. The carbon tetrachloride (CC14), sulfur and ethyl amide (TAA) and xenogenic pig serum three ways, to SD rats to medicine made mode, the simulation model of liver fibrosis. And the three kinds of animal model of serum ALT, AST, HA and liver homogenate SOD and MDA test, take liver tissue for HE and Masson dyeing, liver tissue were observed the damage degree of collagen hyperplasia and the condition of the comparison.2. Optimized prescription was screened out, adopting uniform design with2-factor5-leveltable and uniform design3.0analysis, inducing rats liver fibrosis with thioacetamide (TAA), and taking the rats liver content of Hyp as the screen index. To compare and verify the efficacy of the optimized prescription, a further study was conducted on the former model by setting four groups(normal control group, TAA group, colchicines group and the optimized prescription group), and observing the changes of Hyp content, serum HA, ALT, AST, ALB and fibrosis pathology. Then choose the best ratio and prepared to GanKu oral liquid.3. To develop a method of detecting the content of GanKu oral liquid and test methodology. The test sample was evaluated the clarity, PH value and relative density by stability tests.4. According to the preliminary results, we set6groups of10mice between the maximum dose of840mg/kg and minimum dose of141.3mg/kg. After one-time irrigation the mice stomach, we observe14days and record the result of the experiment. We dissect death of mice’s heart, liver, kidney, brain and other major organs, do pathological HE dyeing observation. Statistical analysis use Bliss statistical software and the Probit regression calculation GanKu oral liquid’s LD50in mice.5. Rats were randomly divided into normal group, model group, In matrine calculation, Ganku Oral Liquid high-dose group (140mg/kg), medium-dose group (70mg/kg), low-dose group (35mg/kg), medium-dose prevention group (70mg/kg), and Stronger Neo-Minophagen C positive control group (70mg/kg). Except the normal group, other groups were given model preparation method intraperitoneal injection with TAA; the medium-dose prevention group was given mixture at the same time. Four weeks after, the model group and normal group were filled stomach with normal saline; the other five groups were respectively administered. Fasting after the eighth weekend dose, the next day abdominal aortic blood in anesthetized rats, take serum and liver, do the following tests:1) Serum biochemical indicators of liver function:ALT, AST, ALB and G.2) Hepatic fibrosis markers:HA, LN and PC-Ⅲ. 3) The cytokines:TNF-α, TGF-β1and IL-13.4) Fresh liver homogenates MDA, SOD, Hyp.5) HE staining, Masson collagen staining.6) Immunohistochemical staining of collagen types Ⅰ and Ⅲ, α-SMA, TIMP-1.Statistically:The measurement data were expressed as mean±standard deviation (X±s). Handling of missing values:removed the analysis of the variables with missing values in the observation unit. We use SPSS13.0software for data analysis. The diversity of the design of random data of single factor analysis of variance (One-way ANOVA) multiple comparison method, choose the LSD and SNK method; Variance not neat choose when approximate F test Welch method, multiple comparison method select Dunnett’s T3method. P<0.05was considered differences in statistics (science). Comparing the weight difference of group selected analysis of covariance.Grade material adopts the parameters rank and inspection (Kruskal-Wallis H test), P<0.05was considered statistical (learning) differences. The uniform design use uniform design software3.0processing data, regression analysis of each group of Hyp to obtain the regression equation and derive theoretically the best prescription. Nasty poison use of statistical software package Bliss of statistical analysis, the probit regression calculations stand by the oral liquid on mice LD50.Results:1. During the rat model, blank group without death. There was one rat died in CCl4group at4th week and8th weeks respectively. There was one rat died in TAA Group at7th weeks. There was one died in the pig serum group at the second week. Compared with the blank group, there was a significant difference (P=0.000) for weight gain in CCI4group, the TAA group and the pig serum group. Compared with CCl4group and the TAA group, the pig serum group increase weight was significantly different (P=0.000). Compared with the blank group of the ALT values, there was a significant difference (P=0.001, P=0.000) in CCl4group and TAA group. Compared with blank control group of AST values, there was a significant difference in the each group (P=0.000). HA value each group and blank group significantly by difference (P=0.000), the comparison between groups, and a significant difference (P<0.01). CCl4group, the TAA group of MDA value and blank group compared with significant difference (P=0.000). SOD value CCl4group, the TAA group and in the blank group compared with significant difference (P=0.015, P=0.000). The pathology of normal liver cells observed blank no lesions. CCl4group, TAA group, pig serum group, liver cell lesions, the degree of order of CCl4group> TAA group> pig serum group. The SSS CCl4group score higher than TAA group (P=0.000), and TAA group of SSS score higher than pig serum group (P=0.001).2. Prescription screening the results showed that the uniform design3.0software stepwise regression analysis, the regression equation: Y=456-0.575X1-0.02X12×X2, Multiple correlation coefficient R=0.9986, coefficient of determination R2=0.9971, the best ratio for bitter and alkali and liquorice sweet grain are70mg/kg. Verify the experimental results, HE dyeing a microscope model group liver cell changes obviously, a lot of false flocculus to form; colchicine group and optimize prescription group derangement of liver cells, the basic integrity of lobular architecture, portal area there are a small number of microscopic tissue, but also varying degrees of degeneration and necrosis, but a significant improvement compared with the model group, both compared to no significant difference. Hyp, HA content delivery was significantly lower than model group (P<0.01). ALB, ALT, AST values for medicine group and model group has more difference (P<0.05). Preparations stand and then verify the results of uniform design and pharmacodynamic oral solution is pale yellow clear liquid, sweet, and slightly astringent.25℃, pH value of7.2, the relative density of1.005.3. Contents determination method of glycyrrhizin and matrine was developed by using HPLC. Chromatographic condition of glycyrrhizin:DIKMA, Diamonsil5u C18column (250×4.6mm), column temperature was30℃,and mobile phase was acetonitrile:0.05%phosphoric acid solution (45:55PH=3) with flow rate of1ml/min, ultraviolet wavelength detection was254nm. Chromatographic condition of matrine:DIKMA, Diamonsil5u C18column (250×4.6mm), column temperature was30℃, and mobile phase was acetonitrile:0.05%phosphoric acid solution (35:65PH value was fit to8by using triethylamine) with flow rate of1ml/min, Ultraviolet wavelength detection was220nm. Methods of specificity, linear relation, accuracy, precision, stability was good. Stability test results, in the glare, high temperature condition and the relevant material has the change, PH value and relative density are changed, content is also lower, but there was no change in appearance. Low temperature conditions have crystallization precipitation, normal temperature and to become clear.4. According to the calculation method of Bliss, we get regression equation y (Probit)=-5.3694+4.2167Log (D). LD50=287.84mg/kg, LD50(Feiller correction)95%confidence limit=224.08-360.08mg/kg, LD5=117.23mg/kg, LD95=706.72mg/kg. Gross anatomy observation results show that liver has a light yellow with increased by concentration. The heart, liver, kidney and compared with the control group are not obvious change. The HE dyeing results show that, heart myocardial interstitial vascular filling (silting) blood, and myocardial cell mild atrophy, degeneration, myocardial cells visible fat invasion. Cerebral tissue’s neurons disorder and brain’s neural cells have degeneration phenomenon change. A few nerve cells appear solid shrinkage, suggesting that the nervous system has degeneration. 5. Anti-liver fibrosis pharmacodynamic findings of Ganku Oral Liquid:ALT, AST, G, MDA and Hyp in each group of Ganku Oral Liquid were a significant difference with Stronger Neo-Minophagen C positive control group and model group (P<0.01or P<0.05). Except the low-dose group (35mg/kg), ALB, HA and TGF-β1in Ganku Oral Liquid group were a significant difference with model group (P<0.01or P<0.05). LN and PC-Ⅲ in each group of Ganku Oral Liquid were a significant difference with Stronger Neo-Minophagen C positive control group and model group (P<0.01or P<0.05). Except the high-dose group (140mg/kg), TNF-alpha, IL-13and SOD in Ganku Oral Liquid group were a significant difference with model group (P <0.01or P<0.05). HE staining and Masson staining in the low-dose group (35mg/kg) were no significant difference with model group. Liver cell necrosis was alleviated in the high-dose group (140mg/kg) compared with model group. The degree of necrosis of liver cells was significantly reduced in the medium-dose group (70mg/kg), medium-dose prevention group (70mg/kg) and Stronger Neo-Minophagen C group compared with the model group, but no significant difference in these three groups. SSS score in medium-dose group (70mg/kg) was a significant difference with medium-dose prevention group (70mg/kg), Stronger Neo-Minophagen C group and model group (P<0.01or P<0.05). Comparison in liver fibrosis stage, each group of Ganku Oral Liquid were a significant difference with model group (P<0.05). Type I and III collagen-positive cells by immunohistochemical staining were weaker than model group (P<0.01), the expression of a-SMA and TIMP-1was significantly lower (P<0.01).Conclusions:1. CCl4, TAA, pig serum three methods are induced liver fibrosis model. The pig serum induction of hepatic fibrosis immunity characteristics for collagen fiber interval slender, the lack of obvious inflammatory reaction and significant liver cell damage, the effect is poorer than the other two. CCl4rats induced in general situation during the poor, and complications than other rats. TAA successfully induced liver fibrosis model, the intraperitoneal injection of intake is easier to control rats, and the small differences within the group, the difference between the groups are easier to quantify the control, with good reproducibility. Use TAA intraperitoneal injection of preparation of liver fibrosis model, is an ideal experimental liver fibrosis model preparation methods, is worth us in the next in the experiments.2. Even design software process to get the best ratio for matrine and glycyrrhizin are70mg/kg. Pharmacodynamics revalidation, pathology observation shows uniform design method of selected the best prescription can obviously improve the TAA model rat liver fibrosis made the degree, repair damaged liver cell, can significantly change the TAA model rat serum ALB, ALT and AST values.3. The contents determination of ammonium glycyrrhizinate and matrine by HPLC used in our study is suitable for both contents determination of GanKu oral liquid and drug concentration in blood with drug metabolism experiments. The accuracy and stability of this study was proved meeting the quantitative of determination requirements by the results of recovery experiment, precision experiment and stability testing. Stability test results of3samples show that PH value and relative density were in qualified range in a year, the contents of glycyrrhizin and matrine were relatively stable in one year, so that GanKu oral liquid’s quality is stable and reliable in one year.4. Gross anatomy observation results show that liver has a light yellow with increased by concentration. May be due to the flow of blood to the liver, the liver in density is good, other organs because of its organization darker, no blood rich in the liver, so intuitively without liver obvious. GanKu oral liquid toxic mainly comes from matrine. According to acute toxicity test, preliminary judgment GanKu oral liquid may affect the mice nervous system and heart to lead it to death. Can make mice occur nervous system degeneration; show that neural system is its toxicity target organs. The cardiovascular congestion and cells have lesions; show that the mice in the cause of death might also be myocardial hyperemia. It shows that clinical application must especially careful in its toxicity. This study testing GanKu oral liquid oral irrigation medicine to mice stomach LD50is287.84mg/kg,95%confidence interval is224.08-360.08mg/kg. It provides information purpose to GanKu oral liquids limits using drug clinical trials.5. The pharmacodynamic results shows that each group of Ganku Oral Liquid can reduce ALT, AST, G, HA, LN, PC-III and elevate ALB in serum of the TAA model rats. Better result in the medium-dose group (70mg/kg) and medium-dose prevention group (70mg/kg). The results suggest that anti-liver fibrosis mechanisms of Ganku Oral Liquid may be the regulation of ALT, AST, HA, LN, PC-III in serum. Result of cytokines shows that each group of Ganku Oral Liquid can reduce TNF-alpha, IL-13and TGF-β1of the TAA model rats. It suggests that its anti-hepatic fibrosis mechanisms may also be the regulation of these cytokines. Each group of Ganku Oral Liquid can reduce Hyp, MDA and improve SOD of liver homogenate in the TAA model rat. Better result in the medium-dose group (70mg/kg). The results suggest that its anti-hepatic fibrosis mechanisms include the regulation of Hyp, MDA and SOD. Each group of Ganku Oral Liquid were a difference with model group with comparison in HE staining, Masson staining, SSS score and liver fibrosis staging. It suggests that the medium-dose group (70mg/kg) with best result in anti-hepatic fibrosis. Each group of Ganku Oral Liquid significantly reduced type Ⅰ,Ⅲ collagen, alpha-SMA, TIMP-1positive cells by immunohistochemical staining results, indicating that it can reduce the fibrillar collagen synthesis to be anti-liver fibrosis. |
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