| ObjectiveThe present study focus on improving and optimizing the BuyangHuanwu Decoction Compound Granules preparation process,and conducting pharmacodynamics tests which was composed of several herbal active ingredients, by detecting the content of Platelet factor 4, the β-Thromboglobulin(β-TG),Dynorphin and Neuropeptide Y in plasma of ischemic cerebral stroke rats, observing the drug’s regulated effects on hemorheological disorders and neurons histopathological changes,Naoxintong capsules used for positive control drug, to observe and verify the curative effects of Buyang Huanwu Decoction Compound Granules. At the same time, perform an acute toxicity experiment in Kunming mice to explore whether the Buyang Huanwu Decoction Compound Granules produced toxic and side effects, so as to provide safe and scientific basis for better ischemic cerebral stroke treatment and prevention in clinic.Methods(1) Quality control of several herbal active ingredients from the drugs in Buyang Huanwu Decoction and Compound Granules preparationIn according to the Huangqi, Dangguiwei, Chuanxiong, Chishao, Taoren, Honghua and Dilong in Buyang Huanwu Decoction, each ingredient has its special physical and chemical properties, used corresponding methods and solvents to extract and purify given the drugs powder, then by the High Performance Liquid Chromatography(HPLC) controlling the quality, these herbal powder were mixed in accordance with the prescription principles of Monarch, Minister, Assistant and Guide, forming the BYHWDCG.(2) Animal induction of ischemic cerebral stroke modelTwo hundred male adult Sprague-Dawley rats (weighing 280~300 g), were acclimatized to the facilities for two weeks, weight and mark down the weigh, randomly picked eight rats grouped as control rats, then the four-vessel occlusion (4-VO) method with some modifications was used to subjected to the acute ischemic cerebral stroke animal model.(3) Evaluation on the efficacy of Buyang Huanwu Decoction compound granules coronary ischemic cerebral strokeAfter the surgery, animals with a failed operation were excluded, these successfully operational rats were randomly divided into four groups with 10-12 rats in each group for the next experimental procedures, including:(3) Ischemic cerebral stroke(CIS) rats; (4) Ischemic cerebral stroke rats orally pretreated with BYHWDCG preparation (30.4g/kg, high-dose);(5) Ischemic cerebral stroke rats orally pretreated with BYHWDCG preparation(15.2g/kg, middle-dose); (6) Ischemic cerebral stroke rats orally pretreated with Naoxintong at a dose of 0.4g/kg, seting up the (1) Control rats(without operation) and (2) Sham-Operated rats (manipulation without vertebral arteries and carotid arteries occlusion)separately.These rats were administrated by gavage from the second day after operation, at the end day, all were anesthetized by intraperitoneal injection of 10% chloral hydrate (3.5 ml/kg body weight), collected blood samples fromabdominal aorta,2.5 ml fresh blood sample of ischemic cerebral stroke rats was put into the glass test tubes containing heparin (20 U/ml) as an anticoagulant, plasma for testing of the whole blood viscosity and the whole blood reduced viscosity, erythrocyte sedimentation rate and erythrocyte’s deformability; 2.0 ml fresh blood were collected into glass test tubes containing 3.8% anticoagulant sodium citrate solution (citrate/blood:1/9, v/v) to measure the levels of plasma fibrinogen and platelet factor 4,the β-Thromboglobulin(β-TG),Dynorphin and Neuropeptide Y in plasma of ischemic cerebral stroke rats. The rest blood were determined by blood routine examination. After finished blood collection, the rats were sacrificed by rapid decapitation under deep anesthesia, the whole brain was rapidly removed and fixed in 10% formaldehyde solution, stained to get pathological sections. Structural changes in cell morphology of neurons in hippocampus and cerebral cortical in ischemic cerebral stroke rats were observed under a microscope.(4) Acute toxicity experimentAdult female and male Kunming mice, weight between 18~22 g, were acclimatized to the facilities for two weeks, with free access to water but not to food for a 4~6h period prior to the experiment.The limit test acute toxicity studies were performed in mice of both sexes to proved up whether the maximum tolerated dose or LD50 should be measured.The formal experiment is based on the results of the pre-experiment, the exterior signs, the colour and appearance of the vital organs (heart, liver, spleen, lung, kidney, brain and thymus) were observed carefully, then the weight (absolute and relative to body weight) were recorded. For the histopathological evaluation of these organs, after that these samples were fixed in 10% paraformaldehyde and processed for histological studyResults(1) Quality control of drugs extractsAccording to the extraction protocols in the study, Huangqi was extracted and pured to given crud total saponins and flavonoids, the percentage is 2.83%。Analyzed by HPLC to give astragaloside regression equation:y hat= 1.3741x+2.9379, (R2=0.9995, r=0.9997), the percentage of astragaloside in extraction product is 6.39%; the calycosin regression equation:y hat= 3476.8x-4375.7, (R2=0.9996, r=0.9998), the percentage of calycosin in extraction product is 1.34%。(2) The study of ischemic cerebral stroke animal modelThe Male SD rats were reproduced by the four-vessel occlusion (4-VO) method with some modifications to give the ischemic cerebral stroke model. After surgery, rats manifested dark eyes, narrow eyes fission and tears; spirits drooping, appetite lost, dull looking hair, slowness, lags in response, hemidysesthesia with gained the body weight slowly and other symptoms similar to ischemic cerebral stroke patients in clinic, suggesting that the model is successful. The hemoglobin and mean corpuscular hemoglobin concentration decreased;the platelet count, the whole blood viscosity, whole blood reduced viscosity, the total leucocyte and neuropeptide substance in plasma increased significantly, compared with that of Control rats. In addition, significant histopathological changes were observed in the CA1 hippocampus of operated rats.Therefore, the method we used could established the ischemic cerebral stroke model successfully.(3) Efficacy criterions of Buyang Huanwu Decoction compound granules against ischemic cerebral strokeThe operated rats were lavaged with Buyang Huanwu Decoction compound granules, their exterior signs and mental states have improved;the elevated platelet count, plasma fibrinogen, the whole blood viscosity, whole blood reduced viscosity, the total leucocyte and neuropeptide substance in plasma significantly decreased, when compared with the ischemic cerebral stroke rats.Furthermore, rats treated with Buyang Huanwu Decoction compound granules, neurons in the CA1 hippocampus aligned neatly and closely, the edges and gaps of which were clearly.(4) Study about the Buyang Huanwu Decoction compound granules acute toxicityThe pre-experiment acute oral toxicity of Buyang Huanwu Decoction compound granules was assessed by "The limit test" in mice of both sexes, when at the dose of 6g/kg, still no mice died,so the LD50 could not be estimated, the compound granules could consider be practically nontoxic, the maximum tolerance dose in mice should be estimated. In the formal experiment, Buyang Huanwu Decoction compound granules by oral administration maximum tolerance dose above 6. Og/kg. which is equivalent to 82times of adult. No statistically significant differences in the hepatic index, cardiac index, Lung index, spleen index,thymus index, when compared with the control mice. The study suggesting that Buyang Huanwu Decoction compound granules by oral administration is nontoxic, so its security is extensive.ConclusionThe four-vessel occlusion (4-VO) method with some modifications used in this subject established the ischemic cerebral stroke animal model successfully, the Buyang Huanwu Decoction compound granules which still in the research could regulate the hemorheological disorders and improve abnormal biochemical markers levels, in addition, regenerate and restore the cerebral neurons, Thus play a role in the treatment of ischemic stroke. Meanwhile, the drug has no obvious acute toxicity, make sure the safety of medication.The present study provides a theory basis for developing a safer, low toxicity, available and convenient Chinese medicine, lays the foundation of more scientific guidance for clinical medication. |
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