益智仁盐炙“缩尿”作用的研究

【作者】 黄勤挽；

【导师】 谢秀琼； 胡昌江；

【作者基本信息】 成都中医药大学 ， 中药学， 2008， 博士

【摘要】 益智为姜科山姜属植物益智Alpinia oxyphylla Miq.的干燥成熟果实,药用部位为种仁。生品辛温而燥,主要归脾经,以温脾止泻、收摄涎唾力胜,多用于腹痛吐泻、口涎自流;盐炙可缓和辛燥之性,主要归肾经,专行下焦,长于固精、缩尿,用于肾气虚寒的遗精、早泄、尿频、遗尿。目的本课题在国家科技部十一五支撑计划“炮制共性技术—盐炙法”的支持下,选用临床疗效确切的中药益智仁为研究对象,以益智仁盐炙前后功效和临床应用迥异为切入点,拟初步阐明盐益智仁“缩尿”作用的机理和物质基础,进而解释益智仁“盐制入肾—缩尿”理论的科学内涵,并为研究盐制共性技术的其它药物提供一种思路。方法重点观察益智仁盐炙前后的“缩尿”作用有无差异,采用药效学和化学结合的手段开展研究。1.在尿频、尿多动物模型的国内外研究现状基础上,研究小鼠代谢笼,对三种尿频、尿多动物模型进行制作与观察。2.在固定药材来源、饮片炮制方法的基础上,研究益智仁生品和盐炙品的急性毒性和对正常及水负荷小鼠尿量影响,研究盐益智仁“缩尿”作用的关键因素。3.“缩尿”作用机理研究:建立小鼠水负荷尿多模型,进行尿液电解质含量、血浆醛固酮含量、血浆抗利尿激素含量测定;并开展对离体膀胱平滑肌活动影响的实验研究。4.“缩尿”作用物质基础研究:对益智仁生品和盐炙品的化学成分进行预试验研究;对盐益智仁“缩尿”有效浸出物进行筛选和制备,研究该有效浸出物的急性毒性和量效关系;对有效浸出物进行化学成分预试验、总萜类含量测定和GC-MS分析,并用指纹图谱技术控制有效浸出物质量;对盐益智仁中食盐含量限度进行研究。结果1.研制出适合小鼠尿液收集的代谢笼（已申请专利）,建立了稳定、可控的小鼠水负荷尿多模型。2.急性毒性试验,益智仁生品和盐炙品对小鼠的最大给药量为临床成人用量的924.4倍和888.8倍。3.益智仁生品和盐炙品在成人用量80倍时,均可显著降低正常小鼠尿量。对于水负荷小鼠尿多模型,益智仁盐炙品起效的关键因素在于药物本身,但食盐起到协同增效作用;益智仁生品用量在成人日服剂量120倍起效,而盐炙品用量在成人日服剂量80倍即可起效。4.益智仁生品可显著降低水负荷小鼠血浆醛固酮浓度,具有保钠排钾的作用,但盐炙后该作用却降低;盐炙品能显著增加水负荷小鼠血浆抗利尿激素浓度;生品和盐炙品均随剂量增大而拮抗乙酰胆碱引起的豚鼠离体膀胱平滑肌兴奋,且盐炙后效果增强。5.盐益智仁“缩尿”作用物质基础是“75%醇-石油醚浸出物+食盐”,该浸出物的LD₅₀为成人日服剂量的378倍,具有一定的神经毒性,起效剂量为临床成人日服剂量的100倍;暂定75%醇-石油醚浸出物中总萜类成分以努特卡酮（Nootkatone）计,不得少于15.1%;建立该有效浸出物的指纹图谱,并用GC-MS技术鉴定出其中的16个化学成分;暂定盐益智仁中食盐含量限度以氯离子（Cl^-）计为1.5%～1.8%。结论益智仁盐炙前后的“缩尿”作用具有明显的差异,小鼠实验中生品的起效剂量为临床成人日服剂量的120倍,而盐炙后可将起效剂量降低到80倍,说明益智仁临床用于治疗尿频、尿多疾病需盐炙的科学合理性,也初步证明了益智仁“盐制入肾·缩尿”中医药理论的正确性。盐益智仁的“缩尿”作用是药物和食盐协同增效的结果,其物质基础为“益智仁75%醇-石油醚浸出物+食盐”;其“缩尿”作用的机理在于增加抗利尿激素的分泌而增加水液的重吸收,以及产生类似M受体抑制剂的作用,作用于膀胱平滑肌的M受体,降低膀胱平滑肌的收缩。创新点1.研究辅料食盐与益智仁的相关性,初步阐明两者在“缩尿”方面的协同增效作用,为“盐制入肾”的炮制理论找到一定的科学依据。2.初步阐明盐益智仁“缩尿”作用的物质基础和机理。3.设计创制了小鼠代谢笼（收集尿液的装置）,并申请实用新型专利。更多还原

【Abstract】 Alpiniae Oxyphyllae（Alpinia Roxb,Zingiberaceae） is the dry ripe fruit of Alpinia oxyphylla Miq.with kernal as its medicinal part.The crude Alpiniae Oxyphyllae Fructus（CAOF） is pungent in flavour,warm and dry in property,acting on the pleen channel,and good at wanning the pleen and stopping diarrhea,astringing and preserving the droll-spittle,usually being used to treat abdominal pain,vomiting and diarrhoea,uncontrollable drolling.The stir-heated Alpiniae Oxyphyllae Fructus （SAOF） with salt soltuion can moderate the pungent,warm property,act on the kidney channel and the lower jiao exclusively,and specialize in conserving essence and arresting polyuria,usually being used for emissions,premature ejaculation, frequent mieturition and enuresis resulted from kidney qi deficiency cold.Aims:Being a branch of "technical commonness in the processing medthod of stir-heating with salt solution"under the State Ministry of Science and Technology 11th Five-Year Plan Support,our study aimed at the traditioanl medicinal herb Alpiniae OxyphyUae Fructus having affirmative clinical therapeutic effect,focused on the differences between the effects and clinical applications respectively before and after processing,expecting to elucidate the mechnism and active components for the arresting polyuria effect of SAOF,further interpreting the scientific connotation of the theory for "The stir-heated drug with salt solution acts on kidney,and the arresting polyuria effect",and then providing a new idea for the investigations on the drags processed by common techniques in stir-heating with salt solution.Methods:The differences in the arresting polyuria effect of Alpiniae Oxyphyllae Fructus before and after stir-heating with salt solution were focused by both of pharmacodynamical and chemical means.1.According to the present study situations of the animal models of frequent micturition and polyurine at home and abroad,the mouse metabolic cage was employed to perform empirical study of three types of animal models of frequent micturition and polyurine. 2.With the fixed herbal sources and fixed processing methods,the acute toxicity and the influences on the noraml and water-loading mouse of CAOF and SAOF were studied,and the crucial factors of SAOF for arresting polyuria were investigated.3.In the course of urine production,using water-loading mouse model of polyurine as the study object,the mechanism was investigated through the quantification of electrolyte,plasma aldosterone and plasma antidiuretic hormone;in the course of urine excretion,the mechnism was investigated on the effects on the ex vivo detrusor of bladder.4.Prelimianry tests were carried out to study the chemical compositions of CAOF and SAOF.The acitve fraction with the arresting polyuria effect of SAOF was screened and prepared,of which the acute toxicity and dose-effect relationship were studied.The initial investigations were carried out on the chemicial compositions of that fraction,such as the prelimianry tests,assaying of total terpenoid and G-C-MS analysis. The salt content limitation of SAOF was considered as well.Results:1.The metabolic cage suitable for the mouse urine collection was prepared,and the stable and controllable water-loading mouse model for polyurine was developed.2.The maximum dosages of CAOF and SAOF for the acute-term toxicity test were 924.4 times and 888.8 times of the clinical adult dosage,respectively.3.With 80 times adult dosage,both of them showed the ability to reduce the urinary volume of normal mouse significantly;for the water-loading mouse model of polyurine,the crucial working substance of SAOF was the drug itself,and the salt played a synergistic role.CAOF took effect at 120 times adult dosage,while SAOF at 80 times.4.CAOF significantly reduced the plasma aldosterone concentration of water-loading mouse and showed the effect of guaranteeing a sodium row of potassium,whereas this effect decreased after stir-heaing with salt solution;SAOF significantly increased the plasma aldosterone concentration of water-loading mouse; both of CAOF and SAOF stimulated the ex vivo bladder of Cavia procellus via acetylcholine antagonism with an increasing dosage,and SAOF showed a stronger effect. 5.The acitve components of SAOF for arresting polyuria were"petroleum benzine fraction + salt",namely active group.With LD₅₀ of 375 times adult dosage this group showed some neurotoxicity;the working dosage is 100 times adult daytime dosage.The limitation of total terpenoid was not less than 15.1%referenced to Nootkatone in petroleum benzine fraction,and to establish the chromatographic fingerprint.16 chemical compositions of petroleum benzine fraction were identified using GC-MS.The limitation of salt content was tentatively set as 1.5%-1.8% referenced to chioridion.Conclusions:The effect of arresting polyuria of Alpiniae Oxyphyllae Fructus before and after stir-heating with salt solution are evidently different.The working dosage of CAOF was 120 times adult dosage while that of SAOF was 80 times,which indicates the rationality of the clinical applications of SAOF in the treatment of frequent urination and polyurine,intiaUy proving the TCM theory for "Stir-heating drug with salt solution acts on kidney channel,and kidney governs water".The effect of arresting urtrination of Alpiniae Oxyphyllae Fructus after stir-heating with salt solution attributes to the synergistic actions of the drug and salt,with "petroleum benzine fraction of Alpiniae Oxyphyllae Fructus + salt"as the active components.The action mechnism lies in the increased water reabsorption due to the increased ntidiuretic hormon secretion and its influence on the M-receptor of bladder smooth muscle as M-receptor inhibitor thus decreasing the construction of bladder smooth muscle.Innovations:1.Elucidating the synergistic actions of Alpiniae OxyphyUae Fructus and salt for arresting polyuria initially,such can certify the theory about "the stir-heated drug with salt solution acts on kidney,and the arresting polyuria effect".2.Elucidating the active components and mechnisms for arresting polyuria initially.3.Developing the water-loading mouse model for polyurine,and applying for patent of utility model.更多还原