基于细胞共培养人体皮肤类似物模型研究挥发油对中药成分促透规律及其作用机制

There is currently no system information available about the effect of the essential oil in external preparatons of Chinese medicine on the transdermal permeation of the penetrants with different lipophilicity, especially the enhancement properties and its mechanisms of action, which limits the application of these herbal drugs in external perparations as a combination of drug and excipient. In this study, the excised animal skin, cell co-culture analogues of the skin and the living animal were employed,and the Zanthoxylum bungeanum and Mentha haplocalyx with different TCM property recognition were chosen as representative drugs to investigate their transdermal enhancement properties and mechanisms of action. Based on the results of the in vitro and in vivo evaluation, the mathematical relationship between the oil-water partition coefficiency and the enhancement activity would been established to summarise the transdermal enhancement properties of the essential oil on complex composition system of TCM. Meanwhile, these techniques, including the CLSM, molecular modeling,FT-IR，Raman, doppler blood flowmeter, were employed to investigate the effect of the essential oils on thermodynamic activity of drugs, interaction of stratum corneum and the skin biochemical barrier. The investigations of transdermal enhancement mechanisms of the essential oils would be contributed to explain the application characteristic of the essential oil in TCM as combination of drug and excipient, conduct their compatibility application in external preparation of Chinese medicine, and supply the data support for the development of natural transdermal penetration enhancers.

中药外治组方中具"通经开窍"之引药功效挥发油对复方中不同成分促透规律及其作用机制研究的缺乏，限制了该类药味在外用制剂中"药辅合一"作用的充分发挥及其作为天然促透剂的推广应用。本课题拟选择具有温性及凉性的药味花椒、薄荷为代表，在细胞共培养人类皮肤类似物，并结合离体动物皮肤以及整体动物多种模型下，研究其挥发油对系列不同理化性质中药药效成分的促透特征，并建立药物油水分配系数与促透效果的数学关系式，总结其对中药复杂成分系统的促透规律。以激光扫描共聚焦显微镜、分子模型、FT-IR、拉曼光谱、多谱勒血流仪等技术为工具，结合挥发油对药物有效成分热力学活性、与皮肤角质层间相互作用及挥发油药理效应对皮肤通透性的影响，阐明其对不同有效成分的促透机理，有助于说明其在处方中"药辅合一"的应用特色，对中药外用制剂中挥发油的配伍应用及新型中药透皮促进剂的开发具有指导意义。

为阐明挥发油对中药外治组方中复杂中药成分的经皮促透规律及其作用机制，指导中药外用制剂的组方配伍及开发中药经皮促透剂。本课题首先建立了细胞共培养皮肤类似物模型，其宏观形态结构和超微结构与人类皮肤相似，其屏障功能趋势与国外市售皮肤类似物一致，但与动物皮肤仍有差距，对亲脂性化合物呈现出相对较高的屏障作用，接近小鼠；而对亲水性化合物的屏障作用则相对减弱，48h亲水性化合物的累积透过量约为动物皮肤的38倍。此外，本课题以系列不同logP值有效成分的经皮渗透特征总结并推测了花椒、薄荷挥发油的经皮促透规律，结果显示：花椒、薄荷挥发油对不同logP值模型药均具有良好经皮促透作用，并随着挥发油浓度增加促透效果增强，呈浓度依赖性；挥发油作用下药物促透倍数与药物logP值相关性分析结果提示，花椒挥发油表现出对logP值越低的药物似乎表现出更高的促透倍数，即对强亲水性有效成分具有更佳的促透效果，而薄荷挥发油与常用的化学促透剂氮酮具有相似的促透特征，呈现抛物线关系式，即对适度亲水性药物（logP值约为-0.5）具有更佳的促透倍数。并且通过体内经皮微透析试验进一步印证体外透皮试验可以大致反映其体内促透结果。最后基于药物经皮转运过程，从“药物热力学活性-角质层结构-皮肤活性表皮”多层次解释了花椒、薄荷挥发油的经皮促透作用机制，即（1）花椒、薄荷挥发油可以通过促进药物向角质层中分配，从而利于药物从给药介质向皮肤表皮层的转运；（2）花椒挥发油中约17.29%成分、薄荷挥发油中约62.48%成分可与角质层脂质的神经酰胺形成稳定的分子间作用力，一定程度上影响角质层脂质间相互作用力，从而影响脂质间有序排列结构，降低皮肤屏障功能；（3）花椒、薄荷挥发油可改变角质层脂质分子结构并具直接抽提脂质作用，降低皮肤的屏障作用；（4）花椒、薄荷挥发油可能通过改变角质细胞内Ca2+平衡而影响细胞膜流动性等细胞功能，有利于药物透过皮肤活性表皮层。综上，本课题的研究成果为指导中药外用制剂的组方配伍及中药经皮促透剂的开发提供了实验依据，对中药外用制剂的发展有积极的促进作用。