复方“二神丸”中药物炮制增效机理研究

Decoction pieces processing、Chinese herbal compound used as medicines are the important characteristics of clinical medication in TCM.In recent years，the study on the mechanisms of the preparation of the processing of Chinese Herbal Medicine increased gradually，but researched on a single drug-oriented mostly and was seldom brought into research of Chinese herbal compound，leading to mutually detachment of the study on the processing of Chinese Herbal Medicine and clinical compound medication in TCM,and finally did not reveal significance of the processing of Chinese Herbal Medicine comprehensively..Research group under the financial aid of the National's 11th five-year science and technology supporting plan，have completed the salt processing、quality standards 、differences of pharmacodynamics before and after salt processing、the election for the active fractions and effective component in vivo changes before and after salt processing of monarch drug -Malaytea Scurfpea Fruit in"ershen pill(ESP) "established animal models on syndromes in Chinese medicine of the spleen and kidney-yang deficiency diarrhea and so on，and proved that Malaytea Scurfpea Fruit after salt processing in summary of clinical medication of physicians of past generations enhanced the correctness of warming kidney -spleen and checking diarrhea.This topic on the basis of preparatory work，taking the ancient formula "ershen pill(ESP) "for representative compound ，used composing prescriptions of decoction pieces before and after processing，by"efficacy-fingerprint of effective parts-vivo process of effective components-studying its mechanism with pathological model consideration to'a temporary solution 'and' a permanent cure '"，explored the mechanism of synergism of Malaytea Scurfpea Fruit、Myristica fragrans Houtt processing in"ershen pill(ESP)"with multi-angle and multi-level， researched the scientific connotation of processing synergism of Traditional Chinese Medicine with compound-angle， regarding the practice of clinical medication in TCM as the starting point，and provided a research model and thought for processing mechanism in compound.

饮片炮制、复方入药是中医临床用药重要特色，近年关于中药炮制机理研究逐渐增多，但多以单味药为主，很少有将其纳入复方中研究，导致中药炮制的研究与中医临床复方用药实际相脱离，不能全面揭示中药炮制的意义。课题组在国家十一五科技支撑计划资助下，已完成"二神丸"中君药补骨脂的盐炙工艺、质量标准、盐炙前后的药效学差异、有效部位筛选及盐炙前后有效组分体内变化，建立脾肾阳虚泄泻中医证侯动物模型等工作，证明历代医家临证用药总结的补骨脂盐炙后增强温肾暖脾止泻的正确性。.本课题在前期工作基础上，以古方"二神丸"为代表方，采用炮制前后的饮片组方，以"药效-有效部位指纹图谱-有效成分体内过程-'标''本'兼顾的病理模型研究其作用机理"，多角度、多层次探讨补骨脂、肉豆蔻炮制在二神丸中增效的机理，从复方角度研究药物炮制增效的科学内涵。以中医临证用药实际为出发点，为复方中炮制机理研究提供一种研究模式和思路。

[项目背景]二神丸源于《普济本事方》，具有温肾暖脾、涩肠止泻之功，是治疗脾肾阳虚泄泻基础方。原方中补骨脂炒香，肉豆蔻生用。现代研究表明，补骨脂多盐炙，以增强对肾经作用，减弱其燥毒性；肉豆蔻是否需要炮制则存在分歧，故二神丸中药物炮制与否及其炮制机理还需深入研究。本项目在国家自然科学基金支持下将补骨脂和肉豆蔻炮制前后分别纳入复方二神丸中，采用炮制前后“化学成分-药效-指纹图谱-体内代谢排泄过程-作用机理”对比研究，多角度、多层次地探讨补骨脂、肉豆蔻炮制在复方中增效的机理。.[研究内容]主要进行了补骨脂、肉豆蔻炮制前后在二神丸中涩肠止泻药效对比、有效部位/成分群筛选及谱效关系建立、有效部位-药效-药代动力学研究、有效部位“温肾暖脾-涩肠止泻”作用机理研究。在完成任务书内容同时，超额进行了代谢组学以及基于二神丸的“盐炙入肾-肾司二便”的研究。.[研究结果]二神丸四个组合指纹图谱和气质联用总离子流图色谱峰保留时间和峰面积都有一定差异；盐炙补骨脂和麸煨肉豆蔻组合的二神丸可明显降低急性腹泻小鼠的腹泻指数及健康小鼠的小肠推进率和胃排空率，产生药效的主要为极性较小的化学成分，石油醚部位效果最明显。采用复合造模建立脾肾阳虚泄泻大鼠模型，发现经“腺嘌呤+饮食失节+劳倦过度+苦寒泻下”处理的大鼠，尿Ｄ-木糖排泄率降低、NOS活性和cGMP增高，基础代谢率及能量代谢相关酶活性改变，蛋白表达水平降低，脾脏、肾脏脏器指数增大，且肾脏有明显病理特征，符合脾肾阳虚泄泻症候。盐炙补骨脂和麸煨肉豆蔻组方的二神丸，可显著改善模型大鼠异常脏器指数，修复肾脏病理状态，调节基础代谢和能量代谢异常，并通过增高血清中CD3+，CD3+CD4+及降低CD3+CD8+百分比含量进而调节Ｔ淋巴细胞亚群水平，最终达到“温肾暖脾”作用；能显著调节模型大鼠异常胃肠激素水平，通过扶植肠道正常菌群生长和减低致病菌群来调整肠道菌群的失调，通过增加结肠AQP3表达量来增强大鼠结肠对水的吸收，通过抗H1受体兴奋来抑制胃肠平滑肌的收缩运动，最终达到“涩肠止泻”作用。药动学表明二神丸中药物炮制后增效作用可能与减慢补骨脂素、异补骨脂素和补骨脂在体内的消除和分布过程，延长体内滞留时间，并减低补骨脂酚总排泄率，提高生物利用度有关。代谢组学研究表明，二神丸炮制增效机制可能在于抑制过氧化、改善糖脂、氨基酸及能量代谢，调节肠道菌群等多层面协同发挥作用。