基于炮制化学-药效评价-代谢组学技术的酒苁蓉补肾阳作用增效机制研究

The processing of traditional Chinese medicine is the character of TCM clinical syndrome differentiation. It is the core content of the processing research of traditional Chinese medicine to elucidate the principle of material foundation change before and after the processing of traditional Chinese medicine. The raw Cistanche deserticola was partial to the bowel movement, while the wine-processed material was better for invigorating the kidney. The chemical basis and mechanism of synergistic effect are not yet clear. In this project, the effective parts of the kidney-replenishing will be screened, then systematically separating for the chemical components of the effective parts were needed, to get the difference of chemical composition between raw and processed products, in addition, to find the chemical marker using plant metabolomics methods. The changes of endogenous metabolic biomarkers in rat model were investigated, also found the chemicals that really go into the blood of Cistanche deserticola. Meanwhile we investigate the adjustment effect of Cistanche deserticola for treating the hypothalamus-pituitary-target gland axis dysfunction. Ultimately, we will establish the correlation between metabolites and metabolic pathways with the blood constituents in the treatment of kidney-yang deficiency; find out the direct action substance and treatment mechanism. It provides the basis for the clinical application of different processed products of Cistanche deserticola.

中药炮制是中医临床辨证用药的特色，阐释中药炮制前后物质基础变化规律和作用机制是中药炮制研究的核心内容。肉苁蓉生品偏于润下通便，酒制后补肾壮阳作用增强，酒苁蓉增效的物质基础和机制尚不明确。采用炮制化学-药效评价-代谢组学相结合技术，对肉苁蓉炮制前后的体内外化学成分变化规律进行深入研究，评价肉苁蓉生、制品对下丘脑-垂体-靶腺轴功能紊乱的调节作用，分析肉苁蓉不同炮制品干预大鼠肾阳虚模型内源性代谢生物标记物的变化趋势，探索肉苁蓉炮制前后对肾阳虚大鼠的入血成分差异，建立与治疗肾阳虚证过程的相关代谢物及代谢通路的相关性，挖掘酒苁蓉发挥治疗作用的体内直接作用物质及增效机制，探究肉苁蓉酒制的科学内涵，整体评价肉苁蓉炮制的合理性和科学性。为肉苁蓉不同炮制品临床应用提供依据，为中药传统炮制理论研究提供新思路。

本课题主要研究肉苁蓉的产地加工炮制一体化工艺、肉苁蓉炮制前后体内外化学成分变化规律及补肾壮阳作用增强的作用机制。确定最佳产地加工工艺为趁鲜切片，水焯后60℃减压干燥，其干燥速率和有效成分含量均为最高。建立了酒苁蓉的高压蒸制工艺，肉苁蓉加30%黄酒闷润4 h后，于0.025 MPa压力下蒸制3 h。明确了肉苁蓉酒蒸前后化学成分的变化规律，发现肉苁蓉酒蒸后8-O-β-D-glucopyranosyl 部位具有4'-O-caffeoyl 基团的苯乙醇苷类成分，如acteoside, cistanoside C, campneoside II, osmanthuside 含量下降，而8-O-β-D-glucopyranosyl 部位的4'-O-caffeoyl 部位的苯乙醇苷类成分如，isoacetoside, isocistanoside C, isocampneoside I, isomartynosideA 含量增加。对肉苁蓉中的苯乙醇苷单体成分在不同pH值，不同温度下的热稳定性进行研究，发现松果菊苷、毛蕊花糖苷和异毛蕊花糖苷粉末状态均比较稳定，在黄酒溶液中热稳定性明显好于水溶液、甲醇溶液。采用血清药物化学的方法，鉴定肉苁蓉生、制品有效成分在大鼠体内代谢产物，在血液、尿液、粪便样品中共检测出54种成分，炮制会影响体内代谢产物的浓度，II相代谢过程是肉从蓉及其炮制品体内代谢的主要过程。比较研究肉苁蓉及其炮制品（高压蒸制酒苁蓉）对肾阳虚大鼠HPAT轴的调节作用，酒苁蓉的补肾壮阳作用优于生品。富集得到肉苁蓉总多糖、总寡糖、总苷，经对肾阳虚大鼠治疗后，确定补肾壮阳有效部位为总苷和总多糖部分。研究肉苁蓉生、制品总苷对肾阳虚大鼠HPT轴及能量代谢调节作用，发现制品高剂量总苷的活性优于生品总苷。基于代谢组学-16S rRNA测序技术联合分析研究肉苁蓉总苷对肾阳虚大鼠的干预作用机制，发现肉苁蓉生、制总苷可通过调节鞘脂代谢、类固醇生物合成、初级胆汁酸生物合成、类固醇激素生物合成、不饱和脂肪酸等代谢通路，干预肾阳虚证；通过调节紫单胞菌属（Porphyromonadaceae）等，改善肾阳虚大鼠的肠道菌群多样性。通过相关性分析发现吡哆胺与大多数菌群呈显著负相关，与双歧杆菌属（Bifidobacterium）存在显著正相关。牛磺酸、尿苷、胆固醇与多种有益菌呈现正相关，与脂质代谢通路关系密切。