摘要
目的:
探讨研究苦参、黄芪药对以苦参中槐果碱、苦参碱、氧化槐果碱、槐定碱、氧化苦参碱、黄芪中黄芪甲苷和以增强小鼠免疫功能为指标确定最佳提取方法和最佳配伍比例;研究苦参、黄芪药对配伍后对苦参中氧化苦参碱、氧化槐果碱在大鼠体内血药浓度影响;苦参、黄芪及苦参、黄芪药对提取物体外抗CVB_3病毒及体外对感染CVB_3病毒SD大鼠心肌细胞的保护对比实验研究;研究苦参、黄芪药对提取物对CVB_3诱导Balb/c小鼠心肌炎的治疗作用;研究苦参、黄芪药对提取物对CVB_3诱导Balb/c小鼠心肌炎治疗的可能机制。
材料与方法:
1、苦参、黄芪药对的最佳提取方法、比例的确定:苦参、黄芪药对用水分提合并、水合提取、醇分提合并、醇合提取四种提取方法,苦参、黄芪药对按生药量比例1:1、1:2、1:3、1:4、1:5、2:1、3:1、4:1、5:1混合,通过紫外分光光度法测定总生物碱、高效液相色谱法测定苦参中槐果碱、苦参碱、氧化槐果碱、槐定碱、氧化苦参碱、黄芪中黄芪甲苷指标性成分和增强小鼠免疫功能影响的药效研究,确定了苦参、黄芪药对最佳提取方法、最佳配伍比例。
2、苦参、黄芪药对提取物、单味药苦参提取物中氧化苦参碱、氧化槐果碱在大鼠血浆中的药代动力学研究:分别取健康大鼠6只,雌、雄各半,取空白血浆0.5 mL后,分别灌胃给药(黄芪、苦参药对提取物和苦参提取物),于给药后0.5、1、2、3、4、6、8、10、12、24 h,各从大鼠眼眶后静脉丛取血0.5 mL,进行血浆内苦参、黄芪药对提取物、苦参提取物中有效成分氧化苦参碱、氧化槐果碱的药代动力学分析。
3、苦参、黄芪及苦参、黄芪药对提取物体外抗CVB_3病毒及体外对感染CVB_3病毒SD大鼠心肌细胞的保护对比实验研究:在体外CVB_3病毒感染的培养心肌细胞模型成功建立的基础上,分病毒对照组、正常细胞对照组、阳性对照药干扰素组,苦参提取物高、中、低剂量组,黄芪提取物高、中、低剂量组,苦参、黄芪药对提取物高、中、低剂量组,分别于第2、5d时间点,在倒置显微镜下测定每孔细胞搏动速率,并进行形态学观察,吸取上清液测定肌酸激酶同工酶(CK-MB)的含量和测定药物抑制病毒滴度。
4、苦参、黄芪药对提取物对CVB_3感染的Balb/c小鼠心肌炎治疗作用实验研究:纯系雄性Balb/c小鼠,4周龄(体重为12g左右),设苦参、黄芪药对提取物高、中、低剂量组,另外设三个对照组:空白对照组、病毒对照组、玉丹荣心丸阳性对照组,每组二十只小鼠,统计小鼠死亡率,分别在第7d、14d无菌条件下取心脏,用HE染色方法观察小鼠心肌损伤、测定心肌病毒滴度。
5、苦参、黄芪药对提取物对CVB_3感染的Balb/C小鼠心肌炎治疗作用机制研究:纯系雄性Balb/c小鼠,4周龄(体重为12g左右),设苦参、黄芪药对提取物高、中、低剂量组,另外设三个对照组:空白对照组、病毒对照组、玉丹荣心丸阳性对照组,给药14d后通过免疫组化、原位杂交方法和原位末端标记法对组织进行凋亡形态学分析,观察心肌颗粒酶B的蛋白、BCL-2基因表达,Caspase3的蛋白、基因表达,及心肌细胞凋亡情况,探索苦参、黄芪药对提取物治疗CVB_3感染心肌炎的可能机制。
结果:
1.苦参、黄芪药对的最佳提取方法、最佳配伍比例的确定
苦参、黄芪药对经乙醇合提取,用高效液相色谱法测定苦参中槐果碱、苦参碱、氧化槐果碱、槐定碱、氧化苦参碱五种生物碱的含量最高,分别为1.0128、2.9458、1.0803、0.71790、3.0960mg·g~(-1),黄芪甲苷含量0.11920mg·g~(-1),用分光光度法总生物碱含量3.1880 mg·g~(-1),通过对小鼠免疫功能影响的研究,苦参、黄芪药对经乙醇合提对小鼠免疫功能影响明显优于与其它提取方法对小鼠的免疫功能影响。
苦参、黄芪药对4:1配伍用高效液相色谱法测定苦参中槐果碱、苦参碱、氧化槐果碱、槐定碱、氧化苦参碱5种生物碱的含量较高高,分别为3.9020、10.740、2.4293、2.2400、6.9530mg·g~(-1),苦参、黄芪2:1配伍黄芪甲苷含量最高为0.13060mg·g~(-1),用分光光度法测总生物碱苦参、黄芪5:1配伍含量最高为3.4740 mg·g~(-1),通过对小鼠免疫功能影响的研究,苦参、黄芪药对2:1配伍比例对小鼠免疫功能影响明显优于与其它配伍比例对小鼠的免疫功能影响。在中药配伍的运用中,我们不仅要考虑单味药物的剂量,更要注意药物用量之间的比例。通过配伍,既能增强原药物的作用,更能调和偏性,取长补短,发挥药物配伍的长处,使各具特征的药物形成一个新的有机整体,以适应复杂病症的治疗需要。在本实验中,以中医药基础理论为指导依据,以药理疗效结果为主要依据,有效成分含量为辅助指标,最终确定苦参、黄芪按生药量配伍的最佳比例为2:1。
2、苦参、黄芪药对提取物、单味药苦参提取物中氧化苦参碱、氧化槐果碱在大鼠血浆中的药代动力学研究
大鼠灌胃苦参、黄芪药对提取物后的氧化苦参碱在大鼠体内的最大血药浓度为2.010μg·mL~(-1),达峰时间为6小时,药物分子在体内0-24小时的平均停留时间为12.91小时, 0-24小时药时曲线下面积为22.61mg/L*h;苦参药材中的氧化苦参碱在大鼠体内的最大血药浓度为6.526μg·mL~(-1),达峰时间为4小时,药物分子在体内0-24小时的平均停留时间为15.70小时,0-24小时药时曲线下面积为46.50mg/L*h。将药对提取物和苦参药材提取物处理结果相比较,可得知,黄芪、苦参药对配伍后达峰时间有所延后,除MRT(药物分子在大鼠体内的平均停留时间)较苦参药材提取物大,其余统计据参数均小于苦参药材提取物。
大鼠灌胃苦参、黄芪药对提取物后的氧化槐果碱在大鼠体内的最大血药浓度为3.954μg·mL~(-1),达峰时间为6小时,药物分子在体内0-24小时的平均停留时间为8.679小时, 0-24小时药时曲线下面积为43.31mg/L*h;苦参药材中的氧化槐果碱在大鼠体内的最大血药浓度为2.202μg·mL~(-1),达峰时间为4小时,药物分子在体内0-24小时的平均停留时间为9.385小时, 0-24小时药时曲线下面积为26.11mg/L*h。从统计据参数结果分析,给苦参药材的血药达峰时间为4小时,而给药对的为6小时,苦参药材明显比药对的时间短,一般会认为苦参中的氧化槐果碱吸收的较快,更易被大鼠吸收,但是从大鼠体内的最大血药浓度分析,给苦参的血药最大浓度为2.202μg·mL~(-1),而给药对的则是3.954μg·mL~(-1),药对明显高于苦参,说明药对中的氧化槐果碱比较容易被大鼠吸收。此外从AUC(药时曲线下峰面积)来看,苦参、黄芪药对明显高于苦参药材,认为苦参、黄芪药对提取物能促进氧化槐果碱的吸收。
3、苦参、黄芪及苦参、黄芪药对提取物体外抗CVB_3病毒及体外对感染CVB_3病毒SD大鼠心肌细胞的保护对比实验研究
在第2d时,苦参提取物高剂量组,苦参、黄芪药对提取物高、中剂量组与病毒对照组有显著性差异(P<0.05),但是苦芪的高、中剂量组与苦参高剂量组没有显著性差异(P>0.05),但在第5d天时,苦参高、中剂量组和苦参、黄芪药对提取物高、中、低剂量组与病毒对照组有显著性差异(P<0.05),并且苦参、黄芪药对提取物高、中剂量组与苦参高、中剂量组有显著性差异(P<0.05)
4、苦参、黄芪药对提取物对CVB_3感染的Balb/C小鼠心肌炎治疗作用实验研究
通过死亡率统计、病毒滴定分析、病理切片结果分析在7d时,只有高剂量苦参、黄芪药对提取物组有降低死亡率、对病毒的有抑制及保护心肌作用。在第14d时苦参、黄芪药对提取物高、中、低均能对病毒的有抑制及保护心肌作用,并且有良好的剂量-效用关系。
5、苦参、黄芪药对提取物对CVB_3感染的Balb/C小鼠心肌炎治疗作用机制研究
给药14d后通过免疫组化、原位杂交方法和原位末端标记法对心肌组织进行凋亡形态学分析,苦参、黄芪药对提取物能够降低心肌颗粒酶B的蛋白、Caspase3的蛋白、基因及心肌细胞凋亡情况表达,上调BCL-2基因表达,苦参、黄芪药对提取物的高、中、低剂量对蛋白、基因表达水平有显著性差异。
结论:
1、苦参、黄芪药对最佳提取方法为乙醇合提取,最佳配伍比例为2:1。
2、苦参、黄芪药对提取物中黄芪能够促进苦参中氧化槐果碱吸收,促进氧化苦参碱转化为苦参碱而被吸收。
3、苦参、黄芪药对提取物对CVB_3感染的心肌细胞的保护及抗病毒作用优于单味药苦参、黄芪。
4、苦参、黄芪药对提取物对CVB_3感染的Balb/c小鼠心肌炎有治疗作用。
5、苦参、黄芪药对提取物可能是通过降低心肌颗粒酶B的蛋白、Caspase3的蛋白、基因表达,及心肌细胞凋亡情况表达,上调BCL-2基因表达而对病毒性心肌炎起到一定的治疗作用。
Objection:
1.To determine the proportion of the optimal compatibility and the optimal extracting methods of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines through studying the enhancing immune function of mice.
2.To compare the rats’plasma levels of Oxymatrine and Oxysophorcarpine after administering the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines with those only administering Sophorae Flavescentis Radix .
3. To study of Antivirus effects against coxsackie virus B_3 of the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines and its protective effects on myocardial cells infected by Coxsackie virus B_3 in vitro.
4. To investigate the curing effects of the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines on experimental coxsackievirus B_3 myocarditis in vivo.
5. To study the underlying molecular mechanisms of the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines on murine virus myocarditis model induced by Coxsackievirus B_3.
Material and method:
1. Four methods of extracting were used: Water-extracting respectively and then combined the extracting-liquid together, and then fried; combined the two medicines together and then Water-extracting and then fried; ethanol-extracting respectively and then combined the extracting - liquid together, and then fried; combined the two medicines together and then ethanol-extracting and then fried. The dose ratio of the two herbs are 1:1、1:2、1:3、1:4、1:5、2:1、3:1、4:1、5:1. The contents of the total alkaloids of the extracts have been analyzed by ultraviolet spectrophotometry and the contents of Sophocarpin、Matrine、Oxysophoridine、Sophoridine、Oxymatrine and AstragalosideⅣwere analyzed by high performance liquid chromatography. The mice immune functions were studied after administered the components extracted from Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines. Using orthogonal design method, the optimal extraction process of the two herbs was determined.
2. To investigate and compare with the Oxymatrine and Oxysophorcarpine pharmacokinetic parameters after giving the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines with those only giving the extracting components of Sophorae Flavescentis Radix using the internal standard method. There are two groups of mice, each six animals: one group administered the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines intragastrically, the other administered the extracting components of Sophorae Flavescentis Radix intragastrically. At 0.5 hours before the administration and at 0.5、1、2、3、4、6、8、10、12、24 h after the administration,0.5ml of the vein blood was sampled to observe the contents of Oxymatrine and Oxysophorcarpine in the blood serum.
3. Construct VMC model in vitro by infecting myocardiocytes of newborn Wistar rat. Groups: negative control(NC),virus group(VK),Ribavirin (RBV); Sophorae Flavescentis Radix high、medium、lower does treatment, Astragalus membranaceus high, medium、lower does treatment, the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines high、medium、lower does treatment, At 2,5 d after administering medicine, respectively, the cell beating rate of each hole are determined under the inverted microscope. The morphological changes of the cells were also observed under the inverted microscope. Cell survival rate was measured by MTT, and observe the protection from cytopathic effects (CPE),percentage of beat cells(PBC)and creatine kinase isoenzyme MB(CK-MB)
4. Experimental research of the treatment of the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines on VMC model infected by CVB_3. Animals: pure male Balb/C mice, 4 weeks of age (weight about 12g). Groups: the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines high, medium and low dose group, control group, virus control group, Yu Dan Rong Xin Pill positive control group, 20 mice each group. Each group was divided into two sub-groups: one group sacrificed at 7d, the other at 14d. The survival rates, TCID50, histopathology changes were detected and compared.
5. Using immunohistochemistry, in situ hybridization and in situ end labeling, Granzyme B protein, BCL-2 gene expression, Caspase3 protein, gene expression, and myocardial apoptosis were observed of each group sacrificed at 14d.
Results:
1. The optimal compatibility ratio of the Sophorae Flavescentis Radix and Astragalus membranaceus is 2:1. The optimal method of extracting is combining the two medicines together and then ethanol-extracting. The contents of the total alkaloids of the extracts have been analyzed using high performance liquid chromatography. The five highest contents of alkaloids were sophocarpine, matrine, oxysophocarpine, sophoridine and oxymatrine, And their contents 1.0128, 2.9458, 1.0803, 0.71790,
3.0960 mg·g~(-1) respectively. The astragaloside is 0.1192 mg·g~(-1), the total alkaloid content 3.188 mg·g~(-1) by spectrophotometry.
However when the ratio of the two herbs 4:1 , using the method combining the two medicines together and then ethanol–extracting, the five highest content of alkaloids were 3.9020, 10.470, 2.4293, 2.2400, 6.9530 mg·g~(-1), respectively. The astragaloside was up to 0.1306mg·g~(-1) the total alkaloid content was 3.474 mg·g~(-1).
Through the study of the effects on immune function research, we found the method combining the two medicines (Sophorae Flavescentis Radix and Astragalus membranaceus ratio 2:1) together and then ethanol–extracting was better than other extraction methods on enhancing the immune function of mice. When the Chinese herbal medicines were used, we should not only consider the dose of the single herbs, but also pay attention to the ratio between the amounts of herbs. By compatibility, not only the role of the original herbs, but also their co-effect should be considered. Side effects and therapy effects play the same functions to the strengths of drug compatibility, so that the characteristics of the drugs with the formation of a new organic whole, to meet the needs of complex treatment of disease. In this experiment, with the guidance of the basic theory of Chinese medicine based on the results with pharmacological effect is mainly based on secondary indicators of active ingredient content, finalize flavescens, Astragalus dosage according to the compatibility of Health and the optimum ratio of 2:1.
2. The pharmacokinetic results of Oxymatrine, oxysophocarpine after administered the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines and the extracting components of Sophorae Flavescentis Radix intragastrically : After administered the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines, the greatest concentration of Oxymatrine in rats’plasma is 2.01μg·mL~(-1) and the peak time was at 6 hour.The peak time was at 12.91 hours after administering. The mean residence time of drug molecules staying in the body at 0-24 hours 22.61mg/L·h hours, The area under the curve of 0-24 hours was 43.31mg / L·h. After administered the extracting components of Sophorae Flavescentis Radix intragastrically, the greatest concentration of Oxymatrine in rats plasma 6.526μg·mL~(-1), and the peak time was at 4 hours, The mean residence time of drug molecules staying in the body at 0-24 hours was 15.70 hours, The area under the curve of 0-24 hours was46.50mg/L*h. After administered the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines, the greatest concentration of Oxysophocarpine in rats’plasma is 3.954μg·mL~(-1).The peak time was at 6 hours after administering. The mean residence time of drug molecules staying in the body at 0-24 hours 8.679 hours, The area under the curve of 0-24 hours was 43.31mg / L * h. After administered the extracting components of Sophorae Flavescentis Radix intragastrically, the greatest concentration of oxysophocarpine in rats plasma 2.202μg·mL~(-1) , and the peak time was at 4 hours, The mean residence time of drug molecules staying in the body at 0-24 hours was 9.385 hours, The area under the curve of 0-24 hours was 26.11mg / L * h. According to the results the plasma Sophors peak time at 4 hours, after administering the extracting components of couplet medicines, compared with administered the extracting components of Sophorae Flavescentis Radix intragastrically,at 6 hours. The plasma peak time of extracting components of couplet medicines is significantly later than that of the extracting components of Sophorae Flavescentis Radix. The maximum concentration of oxysophocarpine in plasma administering the extracting components of couplet medicines was 2.202μg·mL~(-1), while 3.954μg·mL~(-1)after the administration of the extracting components of Sophorae Flavescentis Radix. These meaned that Oxymatrine and oxysophocarpine was to be more easily absorbed in rats.
3. The replication of virus be reduced after administer the the extracting component of Sophorae Flavescentis Radix, Astragalus membranaceus, the extracting components of couplet medicines in vitro and they can increase PBC, reduce CPE and CK-MB of myocardiocytes. At 2 days, there were significant differences in decreaseing the virus titer (P <0.05),between the high dose group Sophorae Flavescentis Radix, the extracting components of couplet medicines high, middle dose group and virus control group. There was no significant difference (P> 0.05) between these three groups respectively. On day 5, There are significant difference (P <0.05), compared Sophorae Flavescentis Radix high、medium does group and high dose group、couplet medicines high, medium and low dose group with virus control group. And there were significant difference (P <0.05) compare the couplet medicines high, medium group with Sophorae Flavescentis Radix high、medium does group. There was significant inhibition of viral replication in the groups of couplet medicines. The extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines can reduce the cytopathic effect, raise the percentage of cultured myocardial cells and reduce the enzyme CK-MB, can have a protective effect myocardial cells against CVB_3 infection.
4. Experimental results of the extracting components of couplet medicines t on CVB_3 drugs on infection of Balb / C mice with myocarditis: At 7d, only the extracting components of couplet medicines high dose group, can reduce mortality, and protect myocardial function against virus, according to mortality statistics, virus titration analysis, histopathology changes. At the time 14d, in the extracting components of couplet medicines high, medium and low dose group, TCID50 and histopathology changes in VMC mice myocardium were reduced significantly compared with other groups. The extracting components of couplet medicines can protect the myocardial function of VMC Balb / C mice and have a good dose-effect relationship. Yu Jung Dan Pill could also increase the survival rate of sick mice, significantly reduced inflammatory cell infiltration and myocardial necrosis.
5. The result of Investigating the underlying mechanism: at 14d after administration by immunohistochemistry, in situ hybridization and in situ end labeling for apoptotic morphological analysis of the organization, the extracting components of couplet medicines can reduce the Granzyme B protein, BCL-2 gene expression, Caspase3 protein, gene expression, and myocardial expression of apoptosis, and the extracting components of couplet medicines were significantly lower on gene expression levels.
Conclusion:
1. The optimal compatibility ratio of the Sophorae Flavescentis Radix and Astragalus membranaceus is 2:1. The optimal method of extracting is combining the two medicines together and then ethanol-extracting
2. The concentrtions of Oxymatrine and Oxysophorcarpine in the blood serum after administering the extracting components of couplet medicines intragastrically was higher than those of administering of Sophorae Flavescentis Radix intragastrically.
3. The extracting components of couplet medicines can antiviral CVB_3 infection of myocardial cells and protection is better than single Chinese medicine Sophorae Flavescentis Radix and matrine, astragalus.
4. The extracting components of couplet medicines has therapeutic effects. CVB_3 infection in Balb / C mice with myocarditis.
5. The extracting components of couplet medicines may be through the reduction of Granzyme B protein, Caspase3 protein and its gene expression, BCL-2 gene expression up-regulation, and myocardial expression of apoptosis play against some viral treatment.
引文
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