Study On The Compatibility Mechanism Of Chinese And Western Medicine In Zhenjiang 's Antihypertensive Tablets

Zhenju Jiangya tablets, consisting of 3 traditional Chinese medicine extracts (TCM:flos chrysanthemi indici, pearl toffee, and rutin) and 2 western medicines (WM:hydrochlorothiazide and clonidine hydrochloride), is widely used as an antihypertensive agent in the clinical practice in china. The irrational use of the combined preparation of TCM and WM decreasing the clinical effects or inducing toxicity were reported frequently in recent years. And the effectiveness and safety of the combined preparation of TCM and WM are more and more concerned. But most of the the combined preparation are lack of sufficient fundamental research. Therefore, it is hard to provide convincing experimental data to prove those kind of combination improved effectiveness, or still keep their own original properties, while lowering their toxicity. Based on these facts, we propose to conduct more systematic investigation on the activity, target organs and objective evaluation of safety, by employing modern techniques of experimental pharmacology, the methods of safety evaluation, and gene chips. Meanwhile, we also propose to investigate the synergistic effect and toxicity lowering effect by using so called’Component Split’method. The objectives of this study are:1. to seek rational and experimental evidences for designing a "safe and effective" Zhenju Jiangya Tablets combination; 2. To provide scientific evidences to guide clinical application of the preparation; 3. to provide initial trial for R&D of other combination preparations of TCM and WM.This thesis includes seven studies as listed in the following:1、Effect of the combination of TCM and WM in Zhenju Jiangya Tablets on blood pressure in spontaneously hypertensive ratsSpontaneously hypertensive rats (SHR) were administered the combined preparation of Zhenju Jiangya Tablets at 133,200, and 300 mg/kg, and the corresponding amount of TCM and WM, respectively, by oral gavage.The influence on blood pressures of single and multiple administrations of different active ingredients were observed and compared during the study.The results indicated that single or multiple administration of Zhenju Jiangya Tablets showed significant blood pressure lowering effect in SHR, and there was a sensible dose-effect correlation, and the effect onset time depends on the dosage. The blood pressure lowering effect of Zhenju Jiangya Tablets is originated from the WM components. However, when TCM were added, the effect initiation dose can be reduced, the onset time can be shortened, the effect lasting time period can be prolonged, the extent of blood pressure lowering effect can be enhanced. Which indicated that the combination of TCM and WM in Zhenju Jiangya Tablets has synergistic effect, compared to WM only.2、Effect of the combination of TCM and WM in Zhenju Jiangya Tablets on the expression of candidate gene linked to hypertensionICR mice were administered the combined preparation of Zhenju Jiangya Tablets at a dose of 280 mg/kg, and the corresponding amount of TCM and WM,respectively, by oral gavage once per day for 7 days. Blood samples were taken at 2-hour post dosing. The serum which contains the active ingredients were separated and used to incubate ECV-304 human umbilical vein endothelial cells (HUVEC) for 24 hrs to obtain HUVEC at various cell cycles. Flow cytometer was used to measure the changes in cell cycle of HUVEC. The RNA was extracted and used for Affimatrix expression spectrum by using gene chip.The results revealed that the action mechanisms of Zhenju Jiangya Tablets were to adjust the expression of candidate gene linked to hypertension. It was also found from the change of the gene expression that multi-target effects were exerted. The combined preparation group showed different gene targets, compared to those in the TCM and WM groups. This finding suggested that combination of TCM and WM may exert different effects compared to those of TCM only or WM only.3、Effect of the combination of TCM and WM in Zhenju Jiangya Tablets on central nervous systemICR mice were administered the combined preparation of Zhenju Jiangya Tablets at 280,1120, and 4480mg/kg, and the corresponding amount of TCM and WM, respectively, by oral gavage. The influence on central nervous system were observed and measured.The results indicated that oral administration of Zhenju Jiangya Tablets at high dose can decrease spontaneous activity and showed synergistic effect on hypnosis that induced by co-administering pentobarbital sodium at sub-threshold dose, along with prolonged sleep span. Therefore Zhenju Jiangya Tablets possesses notable sedation effect. These effects were mainly ascribed to the WM components. The addition of TCM has no significant impact.4、Effect of the combination of TCM and WM in Zhenju Jiangya Tablets on cardiovascular and respiratory system Beagle dogs were administered the combined preparation of Zhenju Jiangya Tablets at 60,240, and 960mg/kg, and the corresponding amount of TCM and WM, respectively, by oral gavage. The influence on blood pressure, cardiac rate, electrocardiogram, and breath were observed and measured.The results indicated that oral administration of Zhenju Jiangya Tablets at high dose can decrease systolic and diastolic pressures, heart rate, and breathe rate. These effects were mainly ascribed to the WM components. The addition of TCM can significantly enhance the blood pressure lowering effect, prolong the duration of hypotensive effect, suppress the prolongation of PR-interval.5、Acute toxicity study of the combination of TCM and WM in Zhenju Jiangya Tablets (maximum dose determination)ICR mice were administered 35%of the combined preparation at a single dose of 40ml/kg by oral gavage. The corresponding amount of TCM and WM were also administered to ICR mice, respectively, in the same treatment. The acute toxic reactions were observed.The results indicated that the maximum dose of Zhenju Jiangya Tablets for mice by oral gavage is 14 g/kg, equivalent to 140 times of the daily dose for clinical human application (calculated based on body surface area). No noticeable toxic reaction was observed. Similarly, no toxic reaction was observed in the groups administered the equivalent amount of TCM and WM.6、Long term toxicity study of the combination of TCM and WM in Zhenju Jiangya TabletsWistar Rats were administered the combined preparation of Zhenju Jiangya Tablets at 0.2,0.8, and 3.2 mg/kg, and the corresponding amount of TCM and WM, respectively, by oral gavage once per day for consecutive 26 weeks. The rats were observed for 8 weeks posting dosing. The general activity of the rats was observed daily, the body weight and food consumption were measured once per week. One third of the total number of rats with half male and half female were sacrificed at 13-and 26-week of dosing and 8-week post dosing. Blood samples were collected for hematological tests; the serum was separated for biochemical analyses of serum; the urine samples were collected for urinary biochemical analyses; the organs were taken for general observation; the organ wet weights and organ coefficients were measured. Histomorphological exams were also conducted on the selected key organs by microscopy. The results revealed that oral administration of Zhenju Jiangya Tablets at high dose for long term can induce disorders in lipid metabolism and ion transports, mild histological and functional damages on organs, such as liver and kidney, decline the natural weight gain and depress the function of central nervous system of the experimental rats. The influences were positively related to the dose and dosing period. However, the functional damage to liver and kidney and the metabolic disorders were reversible. The symptoms gradually disappeared after termination of drug administration. The safe oral dose of Zhenju Jiangya Tablets is 0.2 g/kg, which is 2.8 times of daily dose for clinical human application. The side effects that induced by long term, high dose administration of Zhenju Jiangya Tablets were mainly ascribed to the WM components. The addition of TCM can dramatically counteract or partially improve the side effects induced by the WM components.7. The molecular mechanisms of nephrotoxicity of the combination of TCM and WM in Zhenju Jiangya TabletsThe kidneys of Wistar Rats that orally administered Zhenju Jiangya Tablets for consecutive 13 weeks in the long term toxicity study, were selected to investigate the change of gene expression spectrum with different treatments by gene chip technology.The results indicated that Zhenju Jiangya Tablets can induce changes in gene expression in the kidneys. The changes in gene expressions, including apoptosis related gene Casp3, Calcium ion regulatory protein gene Calm2, and mitogen-activated protein kinase (MAPK) for signal transduction, suggested that long term administration of high dose Zhenju Jiangya Tablets can induce damage in kidneys, changes in renal calcium ion levels, activation of MAPK signal transduction path or hypertrophy of renal cells, accumulation of extracellular tissues. These lead to glomeruloscerosis and renal tubulointerstial fibrosis. The influence of Zhenju Jiangya Tablets on kidney gene expression changes mainly originated from the WM components, in a positive dose-effect correlation. The addition of TCM, to certain extent, decreased the changes of gene expression that induced by WM, and maintained the renal gene expression spectrum with long term treatment of Zhenju Jiangya Tablets. This protective effect of TCM depended on the dose of WM in the combined preparation of Zhenju Jiangya Tablets.Through the pharmacodynamics, toxicology,and mechanism study, the results indicated that Zhenju Jiangya Tablets is safe and effective, and its effects and adverse reactions mainly originated from the WM components. However, the addition of TCM, enhanced its pharmacological effects and reduced adverse reactions. The results demonstrate the TCM and WM in Zhenju Jiangya Tablets play their own characteristics and advantages,and the combination of TCM and WM is rational,and also show the superiority and good prospects for development of the combined preparation of TCM and WM.