甘草“生凉炙温”的科学内涵

The "Sheng Liang Zhi Wen" of Glycyrrhizae radix et rhizom has been proved since ancient times. In clinical, the glycyrrhizae radix and prepared glycyrrhiza could not be replaced by each other, but the scientific connotation of its change in medicinal properties before and after concoction has not been explained. The previous studies showed that compared with glycyrrhizae radix, both of chemical composition and pharmacological effects of prepared glycyrrhizae were changed. It was suggested that the change of the medicinal properties of glycyrrhizae radix et rhizom may be related to the change of both of them. Based of these results, our studies focused on the medicinal properties, liang and wen, before and after concoction to reveal the scientific connotation of "Sheng Liang Zhi Wen" of Glycyrrhizae radix et rhizom from the change of two aspects, material basis and pharmacological action. Firstly, the glycyrrhizae radix and prepared glycyrrhiza will be extracted and isolated without cross components and screened the active components devoted to “cool properties” and “warm properties” by the animal model of heat-clearing and detoxifying and strengthening spleen and nourishing Qi, respectively. Activity difference of the same type of isolating component of the glycyrrhizae radix and prepared glycyrrhiza will be clearly identified through detecting the upstream and downstream proteins of TLR4/NF-κB pathway and cAMP-PKA-CREB pathway. Therefore, the scientific connotation of “Sheng Liang Zhi Wen” will be expounded from the change of the active component and pharmacological effect. The difference components of glycyrrhizae radix and prepared glycyrrhiza are found through plant metabonomics and glycomics,. The difference components will be isolated and identified, and the cell level will be verified. The differentiated components are to be processed through simulation to clarify the mechanism of transformation of main chemical components and to reveal the processing mechanism. The scientific connotation of ‘Sheng Liang Zhi Wen’ will be expounded from the perspective of "quality" and "quantity" of active ingredients. This study could provide some ideas and references for the research on the change of medicinal properties of medicinal materials.

甘草“生凉炙温”自古以来就有论证，临床上二者不可替用，但其炮制前后药性改变的科学内涵尚未诠释。前期研究表明甘草炮制后其化学成分和药理作用均有改变，提示其药性改变可能与二者的变化相关。基于此，本研究针对甘草炮制前后的凉温药性，从物质基础及药理作用两方面的变化揭示其生凉炙温的本质。首先将生、炙甘草进行无交叉组分拆分，通过代表凉性的清热解毒、温性的补脾益气动物模型筛选贡献凉温药性的活性组分，通过检测TLR4/NF-κB、cAMP-PKA-CREB通路上关键蛋白，明确同一类型拆分组分活性差异，从效的变化角度揭示其生凉炙温的本质。再通过植物代谢组学与糖组学技术，寻找贡献凉温药性的差异性成分；分离鉴定差异性成分，并进行细胞水平验证；将差异性成分模拟炮制，明确主要成分转化机制，揭示炮制机理，从活性成分质与量的变化角度揭示甘草生凉炙温的本质。本研究可丰富中医药理论，为其他药材药性改变研究提供借鉴。

本项目明确了生、炙甘草中贡献“凉”、“温”药性的活性组分；比较了活性组分的药理活性差异；确定了活性组分中差异性成分及差异规律，明确了单体化合物炮制转化机制，并通过体外细胞实验验证了差异性成分。具体如下：.1.生、炙甘草中黄酮、皂苷、多糖三类组分达到无交叉拆分，方法重复性好；蜜炙后，多糖类成分显著升高。黄酮、皂苷类成分显著下降，其中黄酮含量比皂苷含量下降更显著。蜜炙后，在黄酮-皂苷体系中，皂苷类成分占比升高。.2.药效学实验表明，甘草黄酮是其“凉”药性的物质基础，皂苷、多糖是其“温”药性的物质基础；黄酮可通过下调TLR4/NF-κB通路蛋白IκB-α、IKK、MyD88表达发挥其清热解毒（凉）作用；皂苷、多糖通过上调cAMP/PKA通路蛋白PKA及p-CREB的表达发挥补脾益气（温）作用。.3.甘草黄酮通过上调5-HT和cGMP的含量，下调乳酸、LDH、丙酮酸、PDH等含量，发挥“凉”药性的作用；甘草皂苷、多糖通过上调乳酸、LDH、丙酮酸、PDH等含量，下调5-HT和cGMP含量，发挥“温药性”作用。生甘草总提物通过上调CYP7A1、Acaca、ACSL3基因表达，增加胆固醇、脂肪酸合成，从而抑制机体物质能量代谢，表现为“凉”药性。炙甘草总提物通过下调Ch25h 基因、上调Pklr、ACAT2基因的表达，减少胆固醇合成、促进糖酵解进而促进物质能量代谢，表现为“温”药性。.4.HPLC-Q-TOF-MS显示，甘草蜜炙后25个峰面积显著变化，4个升高，21个降低。其中刺芒柄花素、甘草皂苷E等升高，甘草素，芹糖甘草苷等含量降低。推测甘草“生凉炙温”药性改变可能与炮制后黄酮-皂苷体系中，皂苷占比升高及多糖含量升高相关。.5.HPLC-Q-TOF/MS分析14个模拟蜜炙后的甘草单体化合物，9个化合物峰面积显著下降。包括甘草查尔酮B、甘草素、甘草苷等。其裂解可能与糖苷键断裂、苷元上羟基等取代基断裂或母核上与羰基连接键断裂相关。.6.甘草炮制前后差异性成分甘草酸、甘草苷、甘草素、新异甘草苷、芒柄花苷对RAW 264.7炎症细胞活力有显著的抑制作用，有较好的抗炎疗效，其中甘草酸的抗炎效果最好。