甘苦口服液的研制及其抗肝纤维化药效学的实验研究
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摘要
研究背景和目的
各种肝病所致的肝纤维化严重危害人类健康,给家庭、社会带来沉重的负担,研究开发治疗肝纤维化的药物、特别是中药具有重要的社会、经济意义。将中医药理论与现代药理学研究成果结合起来,结合古今临床实践,筛选出能治疗肝纤维化的中药,提取活性成分将之组合成方,然后进行实验研究及临床试验的方法,是研究开发中药新药的一种行之有效的手段。因此,在这种思想的指导下,笔者考察了大量的中医古代文献、现代研究文献及国内外现代药理学研究成果,筛选出甘草、苦参两味中药。甘草补脾益气,清热解毒,祛痰止咳,缓急止痛,调和诸药。用于脾胃虚弱,倦怠乏力,心悸气短,咳嗽痰多,脘腹、四肢挛急疼痛,痈肿疮毒,缓解药物毒性、烈性,有效成分为甘草甜素；苦参清热燥湿,杀虫,利尿,用于热痢,便血,黄疸,尿闭,有效成分为苦参碱。现代药理研究表明二者皆有保肝、抗炎,抗病毒等治疗肝纤维化的作用。临床资料显示,甘草甜素联合苦参碱静脉滴注对于慢性乙型肝炎治疗效果良好。但是慢性疾病需要长期服用药物,静脉滴注,患者顺应性差。因此笔者选择研究甘草甜素和苦参碱联合口服给药治疗肝纤维化,将二者有效活性成分组合成方,命名为甘苦口服液。为了研究甘苦口服液抗肝纤维化的作用效果,本课题首先对两者的配伍进行筛选,选择出最佳配比,制备成口服液。进行HPLC测定甘苦口服液中甘草酸铵和苦参碱的含量进行方法学考察,确定检测条件；对甘苦口服液的稳定性进行考察,观察其各种环境下外观、PH值、相对密度和含量的改变；采用急毒试验考察甘苦口服液的安全性。最后,对甘苦口服液进行抗肝纤维化的药理效应及作用机理进行研究,为以后的临床试验奠定实验基础。
方法
1.采用四氯化碳(CCl4)、硫代乙酰胺(TAA)和异体猪血清三种方式,对SD大鼠腹腔注射给药,模拟肝纤维化模型。并对这三种动物模型的血清ALT, AST,HA和肝匀浆SOD和MDA检测,取肝组织进行HE染色,分别观察肝组织的损伤程度和胶原增生的情况等进行比较。
2.运用TAA诱导大鼠肝纤维化模型,采用均匀设计法(二因素五水平)分组实验,并设有正常组,模型组,以大鼠肝组织Hyp含量作为检测指标,经均匀设计软件3.0处理分析得最佳处方；运用相同方法诱导大鼠肝纤维化模型,设正常组,模型组,秋水仙碱组,最佳处方组,通过观察肝组织羟脯氨酸含量,大鼠血清HA, ALT, AST, ALB含量以及肝组织纤维化变化,对于最佳处方的疗效进行比较和验证实验。找到最佳处方后,按照最佳处方制备成甘苦口服液。
3.建立HPLC法测定甘苦口服液的含量,进行方法学考察。稳定性实验法,按各法分别检测样品的澄明度,PH值,相对密度等,并采用HPLC法测定复方中甘草甜素和苦参碱含量。
4.以预实验结果,在最大剂量840mg/kg,最小剂量141.3mg/kg之间,设置6组,每组10只小鼠,一次性灌胃后观察14天,记录实验结果。解剖观察死亡小鼠的心、肝、肾和脑等主要器官,做病理切片HE染色观察。采用Bliss统计软件包进行统计分析,采用Probit回归计算甘苦口服液对小鼠的LD50。
5.大鼠设正常组,模型组,以苦参碱计,甘苦口服液高(140mg/kg),中(70mg/kg),低(35mg/kg)剂量组,中剂量(70mg/kg)预防组,阳性对照美能(70mg/kg)组。除正常组外,其他按模型制备方法腹腔注射TAA,中剂量预防组同时给予合剂,四周后模型组与正常组生理盐水灌胃,其他五组分别给药。第八周末次给药后,禁食,第二天麻醉大鼠,腹主动脉采血,取血清,并取肝脏,做以下检测项：
1)肝功能血清生化指标ALT、AST、ALB、G。
2)肝纤维化指标：HA、LN、PC-Ⅲ。
3)细胞因子：TNF-α、TGF-β1、IL-13。
4)新鲜肝组织匀浆MDA、SOD、Hyp。
5)HE染色,Masson胶原染色。
6)Ⅰ型、Ⅲ型胶原蛋白、a-SMA、TIMP-1免疫组织化学染色。
统计学处理
计量资料以均数±标准差(X±s)表示。缺失值的处理：剔除分析所涉及变量中带缺失值的观察单位。用SPSS13.0软件进行数据分析。对随机设计的多样本资料进行单因素方差分析(One-way ANOVA)多重比较方法,选择LSD和SNK法；方差不齐时选择近似F检验Welch法,多重比较方法选择Dunnett'sT3法。P<0.05认为有统计(学)差异。比较各组的增重差异采用协方差分析。等级资料采用多组秩和检验(Kruskal-Wallis H test),P<0.05认为有统计(学)差异。均匀设计采用均匀设计软件3.0处理数据,对各组Hyp含量进行回归分析,获得回归方程,得出理论上的最佳处方。急毒试验采用Bliss统计软件包进行统计分析,Probit回归计算甘苦口服液对小鼠LD50。
结果
1.空白组在造模期间无大鼠死亡,CCL4组在第4,8周各死亡1只。TAA组在第7周时死亡1只,猪血清组在第2周时死亡1只。CCL4组、TAA组、猪血清组与空白组相比增重有显著差异(P均为0.000),猪血清组与CCL4组、TAA组相比增重有显著差异(P均为0.000)。ALT值CCL4组、TAA组与空白组相比有显著差异(P=0.001,P=0.000)。AST值各组与空白组相比有显著差异(P均为0.000)。HA值各组与空白组相比均有显著差异(P均为0.000),各组组间比较,也有显著差异(P<0.01)。MDA值CCL4组、TAA组与空白组相比有显著差异(P均为0.000)。SOD值CCL4组、TAA组与空白组相比有显著差异(P=0.015,P=0.000)。各组病理学观察空白组肝细胞正常无病变。CCl4组、TAA组、猪血清组肝细胞病变明显,程度依次为CCl4组>TAA组>猪血清组。CCl4组的SSS计分高于TAA组(P=0.000),而TAA组的SSS计分高于猪血清组(P=0.001)。
2.处方筛选结果显示,经均匀设计3.0软件逐步回归分析,得回归方程：Y=456-0.575X1-0.02X12×X2,复相关系数R=0.9986,决定系数R2=0.9971,最佳配比为苦参碱和甘草甜素均为70mg/kg。验证实验结果,HE染色镜下观察模型组肝细胞病变明显,大量假小叶形成；秋水仙碱组与优化处方组肝细胞排列紊乱,肝小叶结构基本完整,汇管区有少量显微组织增生,也有不同程度的变性,坏死,但较模型组有明显改善,二者相比无明显差异。Hyp、HA含量给药组显著低于模型组(P<0.01)。ALB,ALT, AST值给药组与模型组比较有差异(P<0.05)。根据均匀设计与药效学再验证结果制备甘苦口服液为淡黄色澄清液体,味甜,微涩。25℃时PH值为7.2,相对密度1.005。
3.建立了HPLC法测定甘苦口服液中的甘草甜素和苦参碱含量。甘草甜素色谱条件：色谱柱：DIKMA, Diamonsil5u C18色谱柱(250x4.6mm),柱温30℃,流动相：乙腈：0.05%磷酸水溶液=45：55(PH=3)流速：1ml/min,紫外检测波长254iun。苦参碱色谱条件：色谱柱：DIKMA, Diamonsil5u C18色谱柱(250x4.6mm),柱温30℃,流动相：乙腈：0.1%磷酸水溶液(三乙胺调PH至8)=35：65,流速：lml/min,紫外检测波长220nm。方法的专属性,线性关系,准确度,精密度,稳定性均良好。稳定性试验结果,在强光照射,高温条件下其有关物质有变化,PH值和相对密度均有改变,含量也降低,但外观无变化。低温条件下有结晶析出,恢复正常温度又可重新变澄清。其他性状和内在质量等各项指标均无明显变化。
4.根据Bliss法计算,得到回归方程y(Probit)=-5.3694+4.2167Log(D)。半数致死量LD50=287.84mg/kg, LD50(Feiller校正)95%的可信限=224.08--360.08mg/kg, LD5=117.23mg/kg, LD95=706.72mg/kg。大体解剖观察结果显示肝脏有淡黄色随浓度加大而加深,其他心、肝、和肾脏与对照组相比,均无明显变化。HE染色结果,心脏心肌间质血管充(淤)血,心肌细胞轻度萎缩、变性,心肌细胞间可见脂肪侵润。脑组织神经元紊乱,有神经细胞退变现象。少数神经细胞出现固缩,提示神经系统有退行性变。
5.甘苦口服液抗肝纤维化药效学研究结果：ALT、AST、G、MDA、Hyp值甘苦口服液各组、美能组与模型组比较均有差异(P<0.01或P<0.05),ALB、 HA、TGF-β1值除低剂量组(35mg/kg)外与模型组比较均有差异(P<0.01或P<0.05)。LN值、PC-Ⅲ值甘苦口服液各组、美能组与模型组比较有差异(P<0.01或P<0.05)。TNF-α、IL-13、SOD值除高剂量组(140mg/kg)外与模型组比较均有差异(P<0.01或P<0.05)。HE染色、Masson染色结果低剂量组(35mg/kg)同模型组相比,未见明显差异,高剂量组(140mg/kg)同模型组相比,肝细胞坏死程度有所减轻。中剂量组(70mg/kg)、中剂量预防组(70mg/kg)、美能组同模型组相比,肝细胞坏死程度明显减轻,三组差异不算明显。SSS评分中剂量组(70mg/kg)、中剂量预防组(70mg/kg)、美能组与模型组比较有差异(P<0.01或P<0.05)。肝纤维化分期比较,甘苦口服液各剂量组与模型组有差异(P<0.05)。Ⅰ型、Ⅲ型胶原阳性细胞免疫组化染色弱于模型组(P<0.01),α-SMA、TIMP-1的表达明显降低(P<0.01)。
结论
1. CCl4、TAA、猪血清三种方法均可诱导肝纤维化模型。猪血清诱导的免疫性肝纤维化的特征为胶原纤维间隔纤细,缺乏明显的炎症反应和显著的肝细胞损伤,效果差于其他两个实验组。CCl4组大鼠在诱导期间一般情况差,且并发症较其他组大鼠多。TAA成功地诱导出肝纤维化模型,其腹腔注射更易于控制大鼠的摄入量,组内差异小,组间差异较易于量化控制,重复性较好。利用TAA腹腔注射制备肝纤维化模型,是比较理想的实验肝纤维化模型制备方法,值得笔者在接下来的实验中采用。
2.均匀设计软件处理得到最佳配比为苦参碱和甘草甜素均为70mg/kg。药效学再验证,病理学观察显示均匀设计法筛选出的最佳处方能明显改善TAA造模大鼠肝纤维化的程度,修复受损的肝细胞,能显著降低TAA造模大鼠的HA,Hyp含量,能显著改变TAA造模大鼠的血清ALB, ALT, AST值。
3.本实验采用的HPLC法测定甘草酸铵和苦参碱的方法适合甘苦口服液中甘草酸铵和苦参碱的含量测定以及药物代谢实验血药浓度的测定。回收率实验的结果表明本方法准确性较好,精密度实验和稳定性实验结果表明本方法稳定性较好,符合定量测定要求。稳定性实验结果表明3批样品在一年内性状稳定,pH值及相对密度都在合格范围内,样品中甘草甜素和苦参碱含量在一年内均比较稳定,因此可认为甘苦口服液剂的质量在一年内是稳定可靠的。
4.解剖观察结果显示肝脏有淡黄色随浓度加大而加深,可能是由于肝脏血流量大,肝脏中浓度比较好,其他脏器因本身组织颜色较深,也没有肝脏中血液丰富,所以直观上没有肝脏明显。甘苦口服液的毒性主要来自于苦参碱,急毒试验结果初步判断甘苦口服液可能通过影响小鼠神经系统和心脏导致死亡。能使小鼠神经系统发生退行性变,表明神经系统是其毒性靶器官；心脏血管充血和细胞有病变,表明其导致小鼠死亡的原因还可能是心肌充血。提示临床应用时要特别注意其毒性。本研究检测甘苦口服液小鼠口服灌胃给药LD50为287.84mg/kg,95%可信区间为(224.08～360.08)mg/kg,为甘苦口服液临床实验用药范围提供了参考。
5.药效学试验结果表明甘苦口服液各组能降低TAA造模大鼠血清ALT,AST, G, HA, LN, PC-Ⅲ值,升高ALB值,中剂量和中剂量预防组(70mg/kg)效果更好,提示甘苦口服液抗肝纤维化机制可能是调节血清中的ALT, AST, G, HA, LN, PC-Ⅲ值。细胞因子的结果显示甘苦口服液各组能降低TAA造模大鼠TNF-α、IL-13、TGF-β1,提示其抗肝纤维化机制还可能是调节这些细胞因子。甘苦口服液各组能降低TAA造模大鼠肝匀浆中Hyp, MDA值,升高SOD值,中剂量预防组(70mg/kg)效果最好,提示其抗肝纤维化机制还包括调节Hyp,MDA值,SOD值。HE染色、Masson染色结果、SSS评分与肝纤维化分期比较,甘苦口服液各剂量组均与模型组有差异,提示中剂量组(70mg/kg)抗肝纤维化效果最好。Ⅰ型、Ⅲ型胶原、α-SMA、TIMP-1阳性细胞免疫组化染色结果甘苦口服液各组明显降低,表明其能减少纤维胶原的合成从而抗肝纤维化。
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:搒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.
各种肝病所致的肝纤维化严重危害人类健康,给家庭、社会带来沉重的负担,研究开发治疗肝纤维化的药物、特别是中药具有重要的社会、经济意义。将中医药理论与现代药理学研究成果结合起来,结合古今临床实践,筛选出能治疗肝纤维化的中药,提取活性成分将之组合成方,然后进行实验研究及临床试验的方法,是研究开发中药新药的一种行之有效的手段。因此,在这种思想的指导下,笔者考察了大量的中医古代文献、现代研究文献及国内外现代药理学研究成果,筛选出甘草、苦参两味中药。甘草补脾益气,清热解毒,祛痰止咳,缓急止痛,调和诸药。用于脾胃虚弱,倦怠乏力,心悸气短,咳嗽痰多,脘腹、四肢挛急疼痛,痈肿疮毒,缓解药物毒性、烈性,有效成分为甘草甜素；苦参清热燥湿,杀虫,利尿,用于热痢,便血,黄疸,尿闭,有效成分为苦参碱。现代药理研究表明二者皆有保肝、抗炎,抗病毒等治疗肝纤维化的作用。临床资料显示,甘草甜素联合苦参碱静脉滴注对于慢性乙型肝炎治疗效果良好。但是慢性疾病需要长期服用药物,静脉滴注,患者顺应性差。因此笔者选择研究甘草甜素和苦参碱联合口服给药治疗肝纤维化,将二者有效活性成分组合成方,命名为甘苦口服液。为了研究甘苦口服液抗肝纤维化的作用效果,本课题首先对两者的配伍进行筛选,选择出最佳配比,制备成口服液。进行HPLC测定甘苦口服液中甘草酸铵和苦参碱的含量进行方法学考察,确定检测条件；对甘苦口服液的稳定性进行考察,观察其各种环境下外观、PH值、相对密度和含量的改变；采用急毒试验考察甘苦口服液的安全性。最后,对甘苦口服液进行抗肝纤维化的药理效应及作用机理进行研究,为以后的临床试验奠定实验基础。
方法
1.采用四氯化碳(CCl4)、硫代乙酰胺(TAA)和异体猪血清三种方式,对SD大鼠腹腔注射给药,模拟肝纤维化模型。并对这三种动物模型的血清ALT, AST,HA和肝匀浆SOD和MDA检测,取肝组织进行HE染色,分别观察肝组织的损伤程度和胶原增生的情况等进行比较。
2.运用TAA诱导大鼠肝纤维化模型,采用均匀设计法(二因素五水平)分组实验,并设有正常组,模型组,以大鼠肝组织Hyp含量作为检测指标,经均匀设计软件3.0处理分析得最佳处方；运用相同方法诱导大鼠肝纤维化模型,设正常组,模型组,秋水仙碱组,最佳处方组,通过观察肝组织羟脯氨酸含量,大鼠血清HA, ALT, AST, ALB含量以及肝组织纤维化变化,对于最佳处方的疗效进行比较和验证实验。找到最佳处方后,按照最佳处方制备成甘苦口服液。
3.建立HPLC法测定甘苦口服液的含量,进行方法学考察。稳定性实验法,按各法分别检测样品的澄明度,PH值,相对密度等,并采用HPLC法测定复方中甘草甜素和苦参碱含量。
4.以预实验结果,在最大剂量840mg/kg,最小剂量141.3mg/kg之间,设置6组,每组10只小鼠,一次性灌胃后观察14天,记录实验结果。解剖观察死亡小鼠的心、肝、肾和脑等主要器官,做病理切片HE染色观察。采用Bliss统计软件包进行统计分析,采用Probit回归计算甘苦口服液对小鼠的LD50。
5.大鼠设正常组,模型组,以苦参碱计,甘苦口服液高(140mg/kg),中(70mg/kg),低(35mg/kg)剂量组,中剂量(70mg/kg)预防组,阳性对照美能(70mg/kg)组。除正常组外,其他按模型制备方法腹腔注射TAA,中剂量预防组同时给予合剂,四周后模型组与正常组生理盐水灌胃,其他五组分别给药。第八周末次给药后,禁食,第二天麻醉大鼠,腹主动脉采血,取血清,并取肝脏,做以下检测项：
1)肝功能血清生化指标ALT、AST、ALB、G。
2)肝纤维化指标：HA、LN、PC-Ⅲ。
3)细胞因子：TNF-α、TGF-β1、IL-13。
4)新鲜肝组织匀浆MDA、SOD、Hyp。
5)HE染色,Masson胶原染色。
6)Ⅰ型、Ⅲ型胶原蛋白、a-SMA、TIMP-1免疫组织化学染色。
统计学处理
计量资料以均数±标准差(X±s)表示。缺失值的处理：剔除分析所涉及变量中带缺失值的观察单位。用SPSS13.0软件进行数据分析。对随机设计的多样本资料进行单因素方差分析(One-way ANOVA)多重比较方法,选择LSD和SNK法；方差不齐时选择近似F检验Welch法,多重比较方法选择Dunnett'sT3法。P<0.05认为有统计(学)差异。比较各组的增重差异采用协方差分析。等级资料采用多组秩和检验(Kruskal-Wallis H test),P<0.05认为有统计(学)差异。均匀设计采用均匀设计软件3.0处理数据,对各组Hyp含量进行回归分析,获得回归方程,得出理论上的最佳处方。急毒试验采用Bliss统计软件包进行统计分析,Probit回归计算甘苦口服液对小鼠LD50。
结果
1.空白组在造模期间无大鼠死亡,CCL4组在第4,8周各死亡1只。TAA组在第7周时死亡1只,猪血清组在第2周时死亡1只。CCL4组、TAA组、猪血清组与空白组相比增重有显著差异(P均为0.000),猪血清组与CCL4组、TAA组相比增重有显著差异(P均为0.000)。ALT值CCL4组、TAA组与空白组相比有显著差异(P=0.001,P=0.000)。AST值各组与空白组相比有显著差异(P均为0.000)。HA值各组与空白组相比均有显著差异(P均为0.000),各组组间比较,也有显著差异(P<0.01)。MDA值CCL4组、TAA组与空白组相比有显著差异(P均为0.000)。SOD值CCL4组、TAA组与空白组相比有显著差异(P=0.015,P=0.000)。各组病理学观察空白组肝细胞正常无病变。CCl4组、TAA组、猪血清组肝细胞病变明显,程度依次为CCl4组>TAA组>猪血清组。CCl4组的SSS计分高于TAA组(P=0.000),而TAA组的SSS计分高于猪血清组(P=0.001)。
2.处方筛选结果显示,经均匀设计3.0软件逐步回归分析,得回归方程：Y=456-0.575X1-0.02X12×X2,复相关系数R=0.9986,决定系数R2=0.9971,最佳配比为苦参碱和甘草甜素均为70mg/kg。验证实验结果,HE染色镜下观察模型组肝细胞病变明显,大量假小叶形成；秋水仙碱组与优化处方组肝细胞排列紊乱,肝小叶结构基本完整,汇管区有少量显微组织增生,也有不同程度的变性,坏死,但较模型组有明显改善,二者相比无明显差异。Hyp、HA含量给药组显著低于模型组(P<0.01)。ALB,ALT, AST值给药组与模型组比较有差异(P<0.05)。根据均匀设计与药效学再验证结果制备甘苦口服液为淡黄色澄清液体,味甜,微涩。25℃时PH值为7.2,相对密度1.005。
3.建立了HPLC法测定甘苦口服液中的甘草甜素和苦参碱含量。甘草甜素色谱条件：色谱柱：DIKMA, Diamonsil5u C18色谱柱(250x4.6mm),柱温30℃,流动相：乙腈：0.05%磷酸水溶液=45：55(PH=3)流速：1ml/min,紫外检测波长254iun。苦参碱色谱条件：色谱柱：DIKMA, Diamonsil5u C18色谱柱(250x4.6mm),柱温30℃,流动相：乙腈：0.1%磷酸水溶液(三乙胺调PH至8)=35：65,流速：lml/min,紫外检测波长220nm。方法的专属性,线性关系,准确度,精密度,稳定性均良好。稳定性试验结果,在强光照射,高温条件下其有关物质有变化,PH值和相对密度均有改变,含量也降低,但外观无变化。低温条件下有结晶析出,恢复正常温度又可重新变澄清。其他性状和内在质量等各项指标均无明显变化。
4.根据Bliss法计算,得到回归方程y(Probit)=-5.3694+4.2167Log(D)。半数致死量LD50=287.84mg/kg, LD50(Feiller校正)95%的可信限=224.08--360.08mg/kg, LD5=117.23mg/kg, LD95=706.72mg/kg。大体解剖观察结果显示肝脏有淡黄色随浓度加大而加深,其他心、肝、和肾脏与对照组相比,均无明显变化。HE染色结果,心脏心肌间质血管充(淤)血,心肌细胞轻度萎缩、变性,心肌细胞间可见脂肪侵润。脑组织神经元紊乱,有神经细胞退变现象。少数神经细胞出现固缩,提示神经系统有退行性变。
5.甘苦口服液抗肝纤维化药效学研究结果：ALT、AST、G、MDA、Hyp值甘苦口服液各组、美能组与模型组比较均有差异(P<0.01或P<0.05),ALB、 HA、TGF-β1值除低剂量组(35mg/kg)外与模型组比较均有差异(P<0.01或P<0.05)。LN值、PC-Ⅲ值甘苦口服液各组、美能组与模型组比较有差异(P<0.01或P<0.05)。TNF-α、IL-13、SOD值除高剂量组(140mg/kg)外与模型组比较均有差异(P<0.01或P<0.05)。HE染色、Masson染色结果低剂量组(35mg/kg)同模型组相比,未见明显差异,高剂量组(140mg/kg)同模型组相比,肝细胞坏死程度有所减轻。中剂量组(70mg/kg)、中剂量预防组(70mg/kg)、美能组同模型组相比,肝细胞坏死程度明显减轻,三组差异不算明显。SSS评分中剂量组(70mg/kg)、中剂量预防组(70mg/kg)、美能组与模型组比较有差异(P<0.01或P<0.05)。肝纤维化分期比较,甘苦口服液各剂量组与模型组有差异(P<0.05)。Ⅰ型、Ⅲ型胶原阳性细胞免疫组化染色弱于模型组(P<0.01),α-SMA、TIMP-1的表达明显降低(P<0.01)。
结论
1. CCl4、TAA、猪血清三种方法均可诱导肝纤维化模型。猪血清诱导的免疫性肝纤维化的特征为胶原纤维间隔纤细,缺乏明显的炎症反应和显著的肝细胞损伤,效果差于其他两个实验组。CCl4组大鼠在诱导期间一般情况差,且并发症较其他组大鼠多。TAA成功地诱导出肝纤维化模型,其腹腔注射更易于控制大鼠的摄入量,组内差异小,组间差异较易于量化控制,重复性较好。利用TAA腹腔注射制备肝纤维化模型,是比较理想的实验肝纤维化模型制备方法,值得笔者在接下来的实验中采用。
2.均匀设计软件处理得到最佳配比为苦参碱和甘草甜素均为70mg/kg。药效学再验证,病理学观察显示均匀设计法筛选出的最佳处方能明显改善TAA造模大鼠肝纤维化的程度,修复受损的肝细胞,能显著降低TAA造模大鼠的HA,Hyp含量,能显著改变TAA造模大鼠的血清ALB, ALT, AST值。
3.本实验采用的HPLC法测定甘草酸铵和苦参碱的方法适合甘苦口服液中甘草酸铵和苦参碱的含量测定以及药物代谢实验血药浓度的测定。回收率实验的结果表明本方法准确性较好,精密度实验和稳定性实验结果表明本方法稳定性较好,符合定量测定要求。稳定性实验结果表明3批样品在一年内性状稳定,pH值及相对密度都在合格范围内,样品中甘草甜素和苦参碱含量在一年内均比较稳定,因此可认为甘苦口服液剂的质量在一年内是稳定可靠的。
4.解剖观察结果显示肝脏有淡黄色随浓度加大而加深,可能是由于肝脏血流量大,肝脏中浓度比较好,其他脏器因本身组织颜色较深,也没有肝脏中血液丰富,所以直观上没有肝脏明显。甘苦口服液的毒性主要来自于苦参碱,急毒试验结果初步判断甘苦口服液可能通过影响小鼠神经系统和心脏导致死亡。能使小鼠神经系统发生退行性变,表明神经系统是其毒性靶器官；心脏血管充血和细胞有病变,表明其导致小鼠死亡的原因还可能是心肌充血。提示临床应用时要特别注意其毒性。本研究检测甘苦口服液小鼠口服灌胃给药LD50为287.84mg/kg,95%可信区间为(224.08～360.08)mg/kg,为甘苦口服液临床实验用药范围提供了参考。
5.药效学试验结果表明甘苦口服液各组能降低TAA造模大鼠血清ALT,AST, G, HA, LN, PC-Ⅲ值,升高ALB值,中剂量和中剂量预防组(70mg/kg)效果更好,提示甘苦口服液抗肝纤维化机制可能是调节血清中的ALT, AST, G, HA, LN, PC-Ⅲ值。细胞因子的结果显示甘苦口服液各组能降低TAA造模大鼠TNF-α、IL-13、TGF-β1,提示其抗肝纤维化机制还可能是调节这些细胞因子。甘苦口服液各组能降低TAA造模大鼠肝匀浆中Hyp, MDA值,升高SOD值,中剂量预防组(70mg/kg)效果最好,提示其抗肝纤维化机制还包括调节Hyp,MDA值,SOD值。HE染色、Masson染色结果、SSS评分与肝纤维化分期比较,甘苦口服液各剂量组均与模型组有差异,提示中剂量组(70mg/kg)抗肝纤维化效果最好。Ⅰ型、Ⅲ型胶原、α-SMA、TIMP-1阳性细胞免疫组化染色结果甘苦口服液各组明显降低,表明其能减少纤维胶原的合成从而抗肝纤维化。
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:搒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.
引文
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