十八反“半蒌贝蔹芨攻乌”物质基础及毒性研究

【作者】 张琦；

【导师】 张大方；

【作者基本信息】 长春中医药大学 ， 中药学， 2010， 博士

【摘要】 目的:在中医药理论指导下,研究川乌头炮制、配伍前后的化学成分变化、药效和毒性变化,阐明川乌头炮制和配伍禁忌的化学物质基础及毒性作用机理,通过对不同病症动物模型的基础毒性研究,揭示中药毒性与机体作用的相关性,进而分析炮制和配伍前后化学成分的变化及对中药药性之毒性的影响,深化对炮制及配伍过程中化学变化的认识,用科学的数据对传统中药“十八反”中与川乌头相关的配伍禁忌进行评价,揭示中药药性之毒性规律。方法:采用高效液相色谱及电喷雾质谱技术对川乌头生品、炮制品及按配伍禁忌配伍前后的单煎液、共煎液的化学成分变化进行物质基础研究;采用Bliss法测定川乌头生品、炮制品及配伍“半蒌贝蔹芨”后共煎液的LD50值,对其基础毒性进行评价和对比研究;通过抗炎实验和急性心衰实验对提取液的药理作用进行研究,考察炮制前后药效和毒性的变化;在配伍禁忌研究中选择毒性较小、临床应用较多的药对,进行正常动物和慢性心力衰竭、佐剂关节炎病症动物模型上的长期毒性实验,根据主要脏器的病理切片、生理生化指标等对其基础毒性进行评价,考察药物对不同病症模型毒性的影响,探讨其毒性机制。结果:1用高效液相色谱方法对川乌头生品、炮制品及其单煎液、配伍后共煎液的主要化学成分---双酯型生物碱含量的变化进行分析,得出:生川乌中的含量>制川乌中的含量;生川乌配伍浙贝母>生川乌配伍瓜蒌皮>生川乌配伍全瓜蒌>生川乌配伍瓜蒌子>生川乌配伍白芨>生川乌配伍白蔹>生川乌配伍生半夏>生川乌单煎液>生川乌配伍川贝母>生川乌配伍法半夏;制川乌配伍浙贝母>制川乌单煎>制川乌配伍白芨>制川乌配伍瓜蒌子>制川乌配伍川贝母>制川乌配伍生半夏>制川乌配伍全瓜蒌>制川乌配伍白蔹>制川乌配伍瓜蒌皮>制川乌配伍法半夏。2用Bliss法测定小鼠经口给药LD50,得出:川乌头生品、炮制品及其单味药单煎液、配伍后共煎液的毒性大小的顺序与其双酯型生物碱含量的多少相一致。说明生川乌炮制后毒性降低;生川乌配伍瓜蒌、浙贝母、白芨、生半夏、白蔹后毒性增大,生川乌配伍法半夏、川贝后毒性降低;制川乌配伍浙贝母后毒性较制川乌单煎液毒性增大,其他配伍均可减低制川乌的毒性。3在炮制前后毒性评价的基础上,结合不同的证型对生川乌、制川乌的药效进行对比研究,分别造成实验动物炎症模型、急性心衰模型,考察炮制减毒后药效、毒性的变化。通过抗炎实验得出:在等量剂量条件下,生川乌提取液抑制大鼠蛋清致足肿作用强于制川乌,但制川乌维持时间略长于生川乌;在等毒剂量条件下,制川乌提取液抑制大鼠蛋清致足肿胀作用强于生川乌,且作用持久。通过对急性心衰大鼠血流动力学考察得出:生川乌、制川乌单煎液均有增强心肌收缩力的功能,生川乌的作用较强,但持续时间短,制川乌的作用较弱,但维持的时间长。4在配伍前后毒性评价的基础上,选择毒性较小、临床应用较多的配伍药对---制川乌配伍全瓜蒌、制川乌配伍法半夏,分别在慢性心力衰竭和佐剂关节炎病理模型基础上进行长期毒性试验,考察川乌头配伍后对正常动物及病症动物模型的毒性影响。?制川乌配伍全瓜蒌对慢性心力衰竭病理模型长期毒性研究结果:(1)制川乌配伍全瓜蒌给药30天观察,正常给药组大鼠出现活动量减少、嗜睡、体重下降等轻微中毒症状,大鼠肝、肾脏脏器指数变化明显,血液学检测结果显示血液粘度有增加,血流动力学检测无明显变化,血中甘油三酯、谷丙转氨酶、钾离子含量增高、白蛋白含量降低,肌酐、尿素氮、钠离子含量增多,HE染色显示肝脏、肾脏有轻度的病理损伤。心力衰竭模型大鼠出现呼吸困难、水肿、紫绀、食欲下降、嗜睡等较为明显的心衰及中毒症状,大鼠心脏、肝脏、肾脏、肾上腺、睾丸脏器指数有明显变化,血液中红细胞、血红蛋白、血小板、红细胞比容、红细胞平均血红蛋白量及红细胞平均血红蛋白浓度均有明显升高,全血粘度增加,血清中谷丙转氨酶、肌酐、尿素氮、钾离子含量升高、总蛋白、氯离子、钠离子含量减低,血流动力学显示模型给药组与模型对照组比较左室收缩压峰值升高、左室舒张末期压减低,心率降低,流式细胞仪检测心肌细胞凋亡指数减低,免疫组化染色检测心肌细胞Bcl-2蛋白表达增加,Bax蛋白表达减少,Bcl-2/Bax比值升高,具有统计学意义。(2)制川乌配伍全瓜蒌停药15天可逆性观察,正常给药组中毒症状消失,体重、进食量、活动量逐渐回升,血液生化指标趋于正常。模型给药组大鼠心衰症状明显,大鼠脏器指数、血液学检测、血生化检测与给药前相比变化不明显,血流动力学检测与模型对照组无显著性差异,心肌细胞凋亡指数增加,Bcl-2蛋白表达减弱,Bax蛋白表达上调,Bcl-2/Bax比值下降。制川乌配伍法半夏对佐剂关节炎动物模型长期毒性研究结果显示:(1)制川乌配伍法半夏给药30天观察,正常给药组大鼠出现皮毛发黄、腹泻、体重增长缓慢等轻度中毒症状,大鼠心、肝、肾脏器指数变化明显,大鼠肝、肾、脾、胃脏器有明显的病理变化,血液学指标无明显变化,血清中谷丙转氨酶、碱性磷酸酶、钾离子含量升高,白蛋白、尿素氮含量减低。模型给药组大鼠行动困难、进食量减少、体重减低明显、右后足及继发病变关节红肿明显、足垫增厚,大鼠心、肝、肾、脾、胸腺有明显的病理变化,血液中白细胞增多、血小板减少、血液粘度低切变率增加,红细胞聚集指数升高,血清中谷丙转氨酶、肌酐、碱性磷酸酶、磷酸肌酸激酶、钾离子含量升高,白蛋白、尿素氮、氯离子含量降低,与模型对照组比较足趾肿胀程度有消退,关节炎指数明显减低。(2)制川乌配伍法半夏停药15天可逆性观察,正常给药组毛色光泽、活动自如中毒症状消失,体重逐渐回升,脏器指数正常,血液生化指标显示无明显的病理变化。模型给药组大鼠血液生化检测趋于正常,与模型对照组相比大鼠进食量增加,关节肿胀较轻,脾、胸腺脏器指数下降明显。结论:1川乌炮制前后毒性大小与其主要成分双酯型生物碱的含量多少有关,即双酯型生物碱含量越高其毒性也越大,炮制减毒的机理是由于毒性成分---双酯型生物碱在炮制过程中发生了几种化学反应,生成了毒性很低的热解、水解或酯化反应产物。同时这种化学成分的变化也引起了药效的变化,说明有毒中药的“毒”与“效”是辩证统一的,炮制发挥了对中药“减毒存效”的作用,进而影响药性的变化。2十八反“半蒌贝蔹芨攻乌”不是绝对的配伍禁忌。从化学成分分析方面看,法半夏可促进生川乌配伍法半夏共煎液中双酯型生物碱的转化与分解,川贝母配伍生川乌共煎液中双酯型生物碱的含量较生川乌单煎液中的含量低;除制川乌配伍浙贝母外,其他配伍后的共煎液中双酯型生物碱含量均降低。从急性毒性考察方面看,生川乌配伍法半夏共煎液的毒性较生川乌单煎液配伍的毒性小;制川乌配伍白蔹、制川乌配伍瓜蒌皮、制川乌配伍法半夏后共煎液的毒性较制川乌单煎液的毒性低,有些甚至检测不到毒性。从长期毒性考察方面看,制川乌配伍全瓜蒌共煎液对正常大鼠具有一定可逆的肝肾毒性,制川乌配伍全瓜蒌对慢性心衰病证模型大鼠具有一定的强心功能,其强心作用与改善Bcl-2与Bax表达失衡,抑制大鼠心肌细胞的凋亡有关。制川乌配伍法半夏共煎液对正常大鼠有轻微的神经毒性,停药后即可消失。对佐剂关节炎模型大鼠有一定可逆的心、肝、肾毒性,同时制川乌配伍法半夏可抑制大鼠的足趾肿胀,其抗炎作用可能与制川乌配伍法半夏抑制炎症组织PGE2的合成或释放有关。3中药的炮制和配伍均可影响其毒性、改变其药效,毒性的大小与模型动物的证候类型密切相关,初步证实了中药药性是与机体和环境相互作用表现出来的基本属性。更多还原

【Abstract】 Purpose: Under the guidance of TCM theory, Research the chemical composition, efficacy and toxicity changes about Radix aconitum processing and compatibility. Clarify the chemical fundamental substances and toxicity mechanism of action about Radix aconitum processing and compatibility. By the studies toxicity of different diseases animal models, to reveal the role of traditional Chinese medicine toxicity associated with the body, analysis the impact of Changes in chemical composition of toxic, Deepen the understanding of chemical change in the course of processing and compatibility, Using scientific data evaluation the traditional Chinese medicines Aconitum associated with "Eighteen Incompatible Pairs", To revealed the rules of toxicity about traditional Chinese medicine properties.Method: Study the material basis of chemical composition changes in Aconitum hygiene products, processed products and decoction of compatibility using HPLC and LC-MS techniques. Determination the LD50 values about aconiti hygiene products, processed products and the decoction of compatibility with“Pinellia tuber, Trichosanthes fructus, Fritillaria, Ampelosis Radix, Common Bletilla Tuber”using bliss techniques in order to evaluate and study of their toxicity. Through the study of pharmacological effects about decoction with anti-inflammatory and acute heart failure experiment, review the changes of pharmacological effects and toxicity about processing before and after. Selective less toxic and more clinical compatibilities on long-term toxicity experiments, carried out on normal animals and chronic heart failure animal model and adjuvant arthritis animal symptoms. Evaluate the toxicity of main organs according to pathological, physiological and biochemical. Study the effects of its toxic about animal models with different disease and syndrome, to explore the mechanism of its toxicity.Result: 1 The analysis of contents of diester type alkaloids---the main chemical ingredient, conclusion: the contents of Radix Aconiti more than Radix Aconiti Preparata; Radix Aconiti compatibility with Fritillaria thunbergii Miq higher than Radix Aconiti compatibility with Trichosanthes Fructus Pericarpium higher than Radix Aconiti compatibility with Trichosanthes Fructus higher than Radix Aconiti compatibility with Trichosanthes Fructus Semen higher than Radix Aconiti compatibility with Common Bletilla Tuber higher than Radix Aconiti compatibility with Ampelosis Radix higher than Radix Aconiti compatibility with Pinellia Tuber higher than Radix Aconiti higher than Radix Aconiti compatibility with Fritillaria cirrhosa D. Don higher than Radix Aconiti compatibility with Pinellia Tuber Preparata. Radix Aconiti Preparata compatibility with Fritillaria thunbergii Miq higher than Radix Aconiti Preparata compatibility with higher than Radix Aconiti Preparata higher than Radix Aconiti Preparata compatibility with Common Bletilla Tuber higher than Radix Aconiti Preparata compatibility with Trichosanthes Fructus Semen higher than Radix Aconiti Preparata compatibility with Fritillaria cirrhosa D. Don higher than Radix Aconiti Preparata compatibility with Pinellia Tuber higher than Radix Aconiti Preparata compatibility with Trichosanthes Fructus higher than Radix Aconiti Preparata compatibility with Ampelosis Radix higher than Radix Aconiti Preparata compatibility with Trichosanthes Fructus Pericarpium higher than Radix Aconiti Preparata compatibility with Pinellia Tuber Preparata.2 The results of acute toxicity indicate that the degree of toxicity is in direct proportion to the contents of diester type alkaloids. Show: Processing attenuated the toxin of Radix Aconiti, Radix Aconiti compatibility with Trichosanthes fructus, Fritillaria thunbergii Miq, Ampelosis Radix, Common Bletilla Tuber make accreted their toxicity. Radix Aconiti compatibility with Pinellia tuber, Fritillaria cirrhosa D. Don reduced their toxicity. Radix Aconiti Preparata compatibility with Fritillaria thunbergii Miq enhance its toxicity. Others compatibility lessened their toxicity.3 The results of experiment on the pharmacological effects of Radix Aconiti and Radix Aconiti Preparata. By the anti-inflammatory experiments conclude: In the equivalent dose of conditions, the function of inhibition of foot swelling, Radix Aconiti was stronger than the Radix Aconiti Preparata, but the duration of efficacy, Radix Aconiti was shorter than Radix Aconiti Preparata. By the acute heart failure experiments obtained: both Radix Aconiti and Radix Aconiti Preparata have the functions of enhance contractility. Radix Aconiti was stronger than the Radix Aconiti Preparata, but the duration of efficacy, Radix Aconiti Preparata was longer than Radix Aconiti.4 The results of long-term toxicity on the model of chronic heart failure indicate: (1) Results of administration Radix Aconiti Preparata compatibility with Trichosanthes fructus for 30 days observation. The normal administration group rats appeared mild symptoms of poisoning, such as less active, lethargy, weight loss, etc. the organ index of liver and kidney were significantly increased, no significant changes in hematology testing, blood viscosity increased, hemodynamics detected no significant changes, in blood serum triglyceride levels,glutamic pyruvic transaminase levels and Kalium ion increased, albumin reduced, creatinine, blood urea nitrogen, Natrium ion content decreased, HE staining showed liver, kidney mild pathological damage. Model administration group rats breathing difficulties, edema, cyanosis, loss of appetite, lethargy and other more obvious symptoms of heart failure and poisoning, the organ index of heart, liver, kidney, thymus, adrenal gland, testis have significant change, in the blood red blood cells, hemoglobin, platelet, hematocrit, mean corpuscular hemoglobin volume and mean corpuscular hemoglobin concentration were significantly increased, whole blood viscosity increased, in blood serum glutamic pyruvic transaminase, creatinine, Kalium ion levels increased, albumin,Chlorine ion,Natrium ion contents reduced. The test of hemodynamics display Model administered group left ventricular systolic pressure increased, left ventricular end-diastolic pressure reduction, heart rate reduced. Flow cytometer dectect cardiomyocyte apoptosis- index reduced, immune histochemical staining show the expression of Bcl-2 protein increased, Bax protein expression was decreased, Bcl-2/Bax ratio increased. (2) The results of stop administration Radix Aconiti Preparata compatibility with Trichosanthes fructus withdrawal for 15 days observation, the normal administration group symptoms of poisoning disappeared, body weight, food intake, activity levels rose gradually, blood biochemical parameters of normalcy. Model administration group with heart failure symptoms, rat organ indices, hematology testing, Blood serum tests have no change significantly, compared with the previous administration, hemodynamics testing was no significant difference, the cardiomyocyte apoptosis- index increased, Bcl-2 protein expression decreased, Bax protein expression increased, Bcl-2/Bax ratio decreased.The results of long-term toxicity on the model of adjuvant arthritis indicate that: (1) Results of administration Radix Aconiti Preparata compatibility with Pinellia Tuber Preparata for 30 days observation. The normal administration group rats appeared mild symptoms of poisoning, the organ index of heart, liver and kidney were significantly increased, no significant changes in hematology testing, in blood serum triglyceride levels,glutamic pyruvic transaminase, alkaline phosphatase and Kalium ion levels increased, albumin and blood urea nitrogen reduced, Model administration group rats action difficulties, reduced food intake, body weight significantly reduced, the secondary lesions of joint swelling obviously, foot pad thickness, the organ of heart, liver, kidney, spleen, thymus gland have significant changes, in the blood white blood cells increased, platelet reduced, blood viscosity high shear rat increased, red blood cell aggregation index increased, in blood serum glutamic pyruvic transaminase, creatinine, alkaline phosphatase, creatine phosphokinase, Kalium ion levels increased, albumin, blood urea nitrogen, Chlorine ion, contents reduced. Toe swelling subsided, arthritis index was reduced compared with the model control group. (2) The results of stop administration Radix Aconiti Preparata compatibility with Pinellia Tuber Preparata withdrawal for 15 days observation, the normal administration group symptoms of poisoning disappeared, body weight rose gradually, organ indices normally, blood biochemical indicators showed no significant pathological changes. The rats of model administration group blood biochemistry test treatment group tended to be normal, as compared with the model control group rats food intake increased, joint swelling and lighter, spleen, thymus organ index has fallen markedly.Conclusion: 1 Contents of diester type alkaloids are directly proportional to the degree of Radix Aconiti and Radix Aconiti Preparata’s toxicity. Namely the greater content of diester type alkaloids the higher the toxicity, processing attenuated mechanism is due to toxic components witch are diester type alkaloids changes during the processing, produce low toxic pyrolysis, hydrolysis, or esterification reaction products. At the same time the chemical composition changes also caused changes in efficacy, indicating "poison" and "efficiency" of Chinese medicines is a dialectical unity. Processing has played an effective role in keeping attenuated, thereby affecting the changes in potency.2 The Eighteen Incompatible Pairs about“Pinellia tuber, Trichosanthes fructus, Fritillaria, Ampelosis Radix, Common Bletilla Tuber attack Radix Aconiti”is not absolute incompatibility. View from the chemical composition analysis, the contents of diester type alkaloids witch are included in Radix Aconiti compatibility with Pinellia Tuber Preparata and Fritillaria cirrhosa D. Don are lessen. The contents of diester type alkaloids are reduced Except Radix Aconiti Preparata compatibility with Fritillaria thunbergii Miq. View from the acute toxicity study, the toxic of Radix Aconiti compatibility with Pinellia Tuber Preparata are less than Radix Aconiti. The toxic of Radix Aconiti Preparata compatibility with Ampelosis Radix and Trichosanthes Fructus Pericarpium and Pinellia Tuber Preparata are less than Radix Aconiti. View from the long-term toxicity study, The drugs witch are Radix Aconiti Preparata compatibility with Trichosanthes fructus have toxin on liver and kidney, but this toxicity is reversible after discontinuation could be removed. Drugs have function of improve the role of cardiac function, its cardiac effects may be related to improve the Bcl-2 and Bax expression imbalance, inhibit apoptosis of cardiomyocyte. The drugs witch are Radix Aconiti Preparata compatibility with Pinellia Tuber Preparata have toxin on heart, liver, kidney. The toxicity would disappear immediately after withdrawal. Aconiti Preparata compatibility with Pinellia Tuber Preparata would control toe swelling. The mechanism of anti-inflammatory may be relate to Aconiti Preparata compatibility with Pinellia Tuber Preparata suppress PGE2 synthesis or release.3 The processing and compatibility of Chinese medicine can affect its toxicity, change their efficacy. Toxicity of drugs closely related with the symptoms of animal model. Confirmed the primary attribute of potency were taken out by environment interact with the body.更多还原