Preparation Of Active Constituent From Qianglitianmaduzhong Capsule And Study On Its Effect Mechanism

Qianglitianmaduzhong capsule as national chinese medicine products is approved by the Ministry of Health and made by Tonghualimin Pharmaceutical Co. Ltd. Qianglitianmaduzhong capsule consisting of Gastrodia elata, Eucommia ulmoides, Aconitum carmichaeli, and so on is used to treat pain, dizziness, walking inconvenience and numbness of extremities caused by apoplexy, because it can disperse wind ,dispel cold and promote blood circulation. Pharmacodynamics studies have fully proved that Qianglitianmaduzhong capsule has obvious effects on pain, cerebral ischemia, blood stasis. Pharmacological studies have confirmed that Qianglitianmaduzhong capsule is effective for improving rat hemorheology, blood stasis, whole blood viscosity, plasma viscosity, erythrosedimentation (ESR), and erythrocyte aggregation index, etc. At present, it is mainly used for clinical treatment of arthritis, hypertension and apoplexy. Owing to be effective, Qianglitianmaduzhong capsule is sold throughout the country in China and exported to the United States, Canada, the United Arab Emirates, Southeast Asian countries and regions. This drug has a good application market. At present, because of inadequate preparation, there are some impurities in the capsule, so the patients have to take the more doses every day, which brings lots of inconvenience to weak and elderly patients and leads to certain restrictions in application; In addition, the working mechanism of Qianglitianmaduzhong capsule is unclear. Therefore, the preparation of active constituent from Qianglitianmaduzhong capsule is a great significance to reduce everyday dosage and improve the practical application of drugs. This study will explain the working mechanism of active constituent from Qianglitianmaduzhong capsule and provide the basis for its future application in market. Deeply studies of the active constituent will further expand market of Qianglitianmaduzhong capsule and bring greater economic benefit.Methods and Results:1. Qianglitianmaduzhong capsule is extracted by distilled water and 70% ethanol solution twice, and then filtered, rotary evaporation, cryodesiccation to get its active constituent. 2g active constituent was extracted from 5g original Qianglitianmaduzhong capsule by repeated experiments, and its recovery ratio is about 40%. The active constituent can be completely dissolved in distilled water and it is brown, whereas Qianglitianmaduzhong capsule can not be completely dissolved in distilled water in a cloud state.2. To study analgesic effect of drugs, we observed the mice licking latter feet with hot plate test. The results showed that compared with the control, original Qianglitianmaduzhong capsule group, the middle-dose and high-dose of the active constituent group obviously delayed the begining time of licking latter feet and reduced the times of licking latter feet. It suggested that they both have analgesic effect compared with the negative control group (p<0.05), and the experiments indicate that the active constituent has still equal analgesic effect with 1/3 original drug dose. Low-dose group of active constituent is almost no effect.3. To further study the analgesic effect of drugs, the mice were injected with 0.9% acetic acid by abdominal cavity. The results showed that Qianglitianmaduzhong capsule group and the middle-dose and high-dose of the active constituent group all could obviously prolong the latent period of pain and reduce writhing times (p<0.05), and the active constituent revealed same effect as the original drug. Low-dose group of active constituent was almost no effect.4. To observe effect of original Qianglitianmaduzhong capsule and active components on T cell proliferation activity, we determined mouse T cell proliferative response by MTT method. The results showed that Qianglitianmaduzhong capsule, the middle-dose and high-dose of active constituent group increased lymphocyte stimulator index after mouse splenic T lymphocyte stimulated with Con A. Active constituent of Qianglitianmaduzhong capsule could significantly promote T lymphocytes proliferation (p<0.05), whereas Qianglitianmaduzhong capsule were less effective on T lymphocytes proliferation (p>0.05). 5. To observe the secretion levels of IL-2, IFN-γ, TNF-αand IL-4 of mouse spleen cells, we cultured spleen cells and harvested supernatant and detected cytokine by ELISA. The results showed that the active constituent of Qianglitianmaduzhong capsule significantly increased levels of IL-2 (p<0.01) in a dose-dependent manner and slightly IL-4 levels (p>0.05), as well as significantly decreased level of TNF-αcompared with that of the control (p<0.05). IFN-γwas no obvious change. It suggestded that the active constituent of Qianglitianmaduzhong capsule can regulate the balance between Th1 and Th2, enhance humoral immunity; combining with the results of lymphocyte preliferation, we found that Qianglitianmaduzhong capsule can not regulate the balance between Th1 and Th2.6. To determine macrophage phagocytosis activity we isolated mouse peritoneal macrophages and used them to phagocytose chicken red blood cell. The results showed that the active constituent of high and middle dose group (p<0.05) significantly but Qianglitianmaduzhong capsule slightly (p>0.05) decreased the phagocytosis percentage compared with negative control group. These results suggested that active constituent of Qianglitianmaduzhong capsule can inhibit the activity of mouse peritoneal macrophages but Qianglitianmaduzhong capsule has less effective.In this study, we successfully prepared the active constituent of Qianglitianmaduzhong capsule and confirmed that active constituent of Qianglitianmaduzhong capsule not only has the same analgesic effect with the original drug, but also its effective dose is decreased. We found that the active constituent of Qianglitianmaduzhong capsule can regulate the balance between Th1 and Th2, enhance humoral immune response, and reduce macrophage activity. It suggested that the active constituent of Qianglitianmaduzhong capsule may have anti-inflammatory effects, therefore exert its analgesic effect. The results of this research provide a good experimental and theoretical basis for improving the practicality of Qianglitianmaduzhong capsule and promoting the market development.