【作者】 李文兵；

【导师】 胡昌江；

【作者基本信息】 成都中医药大学 ， 中药学， 2013， 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,是中医临床常用的一味中药。在国家“十一五”期间,基于益智仁“生品入脾,盐炙入肾,生熟有别”的传统认识,我们对其展开了系统深入的研究,初步阐明了益智仁“生品入脾·止泻,盐炙入肾·缩尿”中药炮制理论的科学内涵。目的本课题在国家自然科学基金面上项目“益智仁‘盐炙入肾-温肾缩尿’的物质基础和作用机理研究”(30973941)的支持下,对益智仁温肾缩尿的物质基础和作用机理进行研究。前期以小鼠水负荷多尿模型为基础,已完成盐益智仁缩尿有效部位筛选和部分机理研究。本文在前期基础上,建立两种大鼠多尿模型对有效部位缩尿作用进行验证和评价,并按照中医标本兼治的用药原则,设计“温肾”为本,“缩尿”为标药理指标,探讨其作用机理。方法采用文献学、药理学和化学相结合的手段开展益智仁盐炙“温肾缩尿”作用机理研究。1采用文献学方法,整理回顾益智仁、多尿动物模型的现代研究进展,为实验研究提供理论依据。2采用药理学方法,建立两种大鼠多尿模型,对盐益智仁有效部位缩尿作用进行验证和评价,并比较盐炙前后作用差异。3以肾阳虚多尿模型为基础,从脏器指数、病理形态变化、血清BUN、Cr含量和全血CD3/CD4/CD8表达等方面探讨益智仁盐炙“温肾”作用；从“抗利尿激素-环磷酸腺苷-水通道蛋白”信号途径研究“缩尿”作用机制。4采用血清药物化学方法,对盐益智仁有效部位“入血成分”进行分离鉴别,并以其为指标,比较益智仁盐炙前后药代动力学变化,从体内过程变化探讨益智仁“生熟有别”科学内涵。结果1对水负荷多尿模型大鼠,益智仁石油醚部位在9g·kg-1剂量时能显著降低其0～1h段尿量(P<0.01),延长大鼠的首次排尿时间(P<0.05)；盐炙后在4.5g·kg-1时就有显著缩尿作用(P<0.01),盐炙前后在18g·kg-1剂量时均能显著降低水负荷大鼠6h排泄率(P<0.05)。对肾阳虚多尿模型大鼠,益智仁石油醚部位在9g·kg’剂量时能显著降低其24h尿量(P<0.05),盐炙前后无显著差异。2益智仁石油醚部位对肾阳虚多尿模型大鼠肾脏、脾脏、胸腺和睾丸指数的改变有显著抑制作用(P<0.05-0.01),对模型大鼠肾脏和睾丸病理损伤有一定程度的改善,并且可以降低其血清BUN、Cr含量,增加外周血CD3+、CD3+CD4+相对量,降低CD3+CD8+相对量,对肾脏指数和病理改变的治疗作用以盐炙品为佳；益智仁石油醚部位能显著提高肾阳虚多尿模型大鼠血浆AVP和cAMP含量,上调AQP-2mRNA和AVPR-V2mRNA生肾髓质的表达,提高AQP-2和AVPR-V2蛋白的表达,且盐炙后作用均明显增强,辅料盐起到协同增效作用。3采用血清药物化学方法和手段,分离鉴定出盐益智仁石油醚部位的“入血成分”11,12-二羟基圆柚酮。盐炙能加快机体对11,12-二羟基圆柚酮的吸收,增加其生物利用度。结论益智仁温肾缩尿作用的物质基础是石油醚部位,辅料盐起到协同增效作用。该部位可通过改善肾脏病理损伤程度,缓和肾脏排泄功能障碍,提高机体免疫力而达到“温肾”作用；能促进肾阳虚多尿模型大鼠分泌和释放AVP,使机体合成cAMP量增加,从而使肾脏集合管AQP-2蛋白的表达量上升,对水的通透性增强达到“缩尿”作用。益智仁盐炙后“温肾缩尿”作用增强,可能与盐炙能加快“入血成分”11,12-二羟基圆柚酮的吸收,增加其生物利用度有关。创新点本文在中医药理论指导下,运用现代科学技术和方法,开展益智仁盐炙前后温肾缩尿作用的研究,其创新性如下：1从中医整体观出发,设计“温肾”为本,“缩尿”为标的药理指标,充分体现中医“标本兼治”的用药原则,为中药及复方的研究提供一种思路。2从AQP-2基因和蛋白水平进一步揭示了益智仁盐炙“缩尿”作用机制。3从体内过程角度阐明益智仁“生熟有别”,进一步完善了益智仁“盐炙入肾”科学内涵。4在小鼠饲养笼基础上进行适当改进,获得操作方便、经济实惠的大鼠代谢笼装置,并申请实用新型专利。更多还原

【Abstract】 Alpiniae Oxyphyllae (Alpinia Roxb,Zingiberaceae),as the dry ripe fruit of Alpinia oxyphylla Miq,is commonly used in clinical practice of TCM.During the national11th five-year plan,systematic and thorough research on Alpiniae Oxyphyllae has been completed on the basis of the traditional understanding"the crude acting on the spleen,the salt-stiring product acting on the the kidney,the crude and the processed product being different",and it has clarified the scientific meaning that "the crude Alpiniae Oxyphyllae act on spleen and stop diarrhea,whilehe salt-stiring product act on the kidney and ruduce urine".ObjectivesUnder the support of NSFC surface project "studies on the Seed Alpiniae Oxyphyllae’s material basis and mechanism of’salt processing into kidney-warming the kidney and reducing urine’"(30973941),we set about doing research on it. In early stage, we have finished the election for the effective fractions and some mechanism study, based on the urine volume of the water loading rats with the polyuria model.Based on the previous research, this article have built two kinds of polyuria models to test and evaluate the effective fractions. According to TCM medication principle " looking into both its roots cause and symptoms ", we devise the pharmacology index that warming the kidney for basis and reducing urine for manifestation to discuss its mechanism of action.MethodsStudies on the mechanism of Seed Alpiniae Oxyphyllae’s function of warming the kidney and reducing urine" after stir-heating with salt-water can be complished by adopting the combination of philology pharmacology and chemical means.1.By adopting philological method,the modern research progress of Seed Alpiniae Oxyphyllae and polyuria animal model can be generalized to lay a foundation for experimental studies.2.By adopting pathological method,two kinds of polyuria rat model can be made to prove and value the function of reducing urine of Seed Alpiniae Oxyphyllae’s effective part after after stir-heating with salt-water.and then compare the difference of the crude and the processed product.3.Based on the polyuria rat model of kidney-yang asthenia,the Alpiniae Oxyphyllae’ s function of warming the kidney after stir-heating with salt-water can be studied by checking the differences of organ index,pathological morphological changes,blood uria nitrogen,the content of Cr and the expression of CD3/CD4/CD8in whole blood.The mechanism of the function of reducing urine has been researched by "vasopressin-ring adenosine phosphate-water channel protein" signal pathway.4.By adopting serum drug chemical methods/the components entering into blood of the effective part in Seed Alpiniae Oxyphyllae after stir-heating with salt-water can be seperated and identified,and it can be the indicator to compare the pharmacokinetic changes before and after stir-heating with salt-water.Then the scientific meaning of "the difference of the crude and the processed"can be studied in the body.Results1.To the water loading rats with the polyuria model, at the9g·kg-1dose, the petroleum ether fraction extracted from Seed Alpiniae Oxyphyllae can significantly reduce urine volume during0～1h section (P<0.01), lengthen rats’first average urination time (P<0.05).After salt processing, it can significantly reduce urine volume at the4.5g·kg-1dose. Both after and before can significantly reduce excretion rate in6h towards the water loading rats with the polyuria model (P<0.05).For the polyuria model of kidney-yang asthenia, the petroleum ether fraction extracted from Seed Alpiniae Oxyphyllae can significantly reduce urine volume24h at the9g·kg-1dose (P<0.05),in addition, after and before salt processing have no significant difference.2.For the polyuria model of kidney-yang asthenia, the petroleum ether fraction extracted from Seed Alpiniae Oxyphyllae can significantly inhibit the changes of kidney,spleen, thymus and testis indexes (P<0.05-0.01).and improve the pathological injury of kidney and testis to some extent, also it can reduce the content of BUN and Cr in serum and increase relative amount of CD3+、CD3+CD4+in PB.For therapeutic action of kidney index and pathologic change, the salt broiled product is better. The petroleum ether fraction can significantly increase the content of AVP and cAMP in rat plasma, up regulate the expression of AQP-2mRNA and AVPR-V2mRNA in medulla nephrica, enhance the protein expression of AQP-2and AVPR-V2. As well, it enhances obviously after salt processing and the excipient salt shows synergistic interaction.3.Adopted ways and means of serum pharmacochemistry, we have isolated and identified the constituent migrating to blood of the petroleum ether fraction extracted from Seed Alpiniae Oxyphyllae,11,12-Dihydroxynootkatone. Salt processing can accelerate organism’s absorption and elimination of11,12-Dihydroxynootkatone, and affect action time in vivo and increase its bioavailability.ConclusionsMaterial basis of Seed Alpiniae Oxyphyllae’s function of "warming the kidney and reducing urine" is the petroleum ether fraction, the excipient salt shows synergistic interaction. This fraction can ameliorate the degree of kidney’s injury, and mitigate excretory dysfunction of kidney, and also enhance immunity of the organism achieving the function of warming the kidney. It is able to promote secreting and releasing AVP for the polyuria model of kidney-yang asthenia, and make organism increase the content of cAMP, thus make the AQP-2protein’s expression quantit of kidney collecting tube go up and strengthen the permeability for water to achieve the function of reducing urine. Seed Alpiniae Oxyphyllae’s function of warming the kidney and reducing urine enhances after salt processing. It may related to accelerating the absorption of " the constituent migrating to blood ",11,12-Dihydroxynootkatone, and increasing bioavailability.InovationsIn this paper,under the guidance of TCM theory,modern scientific techonology and methods have been used to research Seed Alpiniae Oxyphyllae’s function of warming the kidney and reducing urine before and after stir-heating with salt-water.The inovations are showed as follws.1.Based on holism of TCM,pharmacological indicators that warming the kidney is the root cause and reducing urine is the symptom have been designed.And the medication principle of addressing both symptoms and root causes has been fully reflected.This experiment can provide a train of thought for the research of traditional Chinese medicine and prescriptions.2.The mechanism of the function of reducing urine can be further identified from the AQP-2gene and protein levels.3.The crude and the processed Seed Alpiniae Oxyphyllae with different functions can be clarified in the body process.and the scientific meaning of "the product by stir-heating with salt-water acting on the kidney"can be further imprved.4.Appropriate improvement can be made on the basis of mouse cage in order to make convenient and economical rat cage device,and then file an utility model patent application.更多还原