槟榔炒焦“长于消食导滞”的物质基础与作用机理研究

On the basis of this project is a former National Natural Science Fund of "The material basis of the biological effects of dynamic variation of Areca seed in the frying process and related studies". We found that the alkaloid content of Areca seed which is the main active ingredient significantly reduced after Chaojiao, the content of tannin and amino acid is also reduced, but the pharmacodynamics is not down. At the same time, some new components(5-HMF, etc.) have been observed in this procedure.Therefore, we propose a working hypothesis of the project: The Areca seed's chemical composition changed significantly after Chaojiao, the efficacy is obvious different, the effect of specializes in "Xiaoshidaozhi" is due to the synergy of saved alkaloids and generated new components (Maillard reaction products, etc.) in the frying process, they cooperate with each other through a variety of channels to promote gastrointestinal digestive function, such as the regulation of gastrointestinal smooth muscle contraction, increasing gastrointestinal hormone secretion, regulation of intestinal flora, etc. Based on the "spectrum - effective" research ideas, we use new technology such as GC, MS, UPLC, HPCE, etc, combination of the in vitro and in vivo efficacy experiments to explore the relationship between the chemical composition and pharmacodynamics. Its purpose is to reveal material basis and mechanism of action of the effect of specializes in "Xiaoshidaozhi". Our project can provide the basis to clarify scientific connotation of the principle of Areca seed Chaojiao and the new ideas and technical means of researching Chinese medicine Chaojiao common material foundation and action mechanism. Undoubtly, this helps to accelerate elaborating the scientific of Chaojiao, to rich and develop this traditional processing theory.

本项目系在前一个国家自然基金课题"槟榔炒制过程中物质基础与生物效应动态变化规律研究"及相关研究的基础上，发现槟榔炒焦后主要有效成分生物碱含量明显降低，鞣质、氨基酸等含量也降低，但药效不降，观察到产生新成分（5-HMF等）。故提出本项目的工作假说：槟榔炒焦后化学成分发生明显改变，药效明显差异，其中"长于消食导滞"功效方面的作用是保存的生物碱与产生的新成分（美拉德反应产物等），通过调节胃肠平滑肌收缩、增加胃肠激素分泌、调节肠道菌群等多途径协同作用，促进胃肠消化功能。基于多维"谱-效"等研究思路，采用GC、MS、UPLC、HPCE等新技术，结合体内外药效实验，探讨化学成分与药效的关系，旨在揭示槟榔炒焦"长于消食导滞"功效的物质基础与作用机理，为阐明槟榔炒焦原理的科学内涵提供依据，为中药炒焦共性物质基础及作用机制的研究提供新的思路与技术手段，有助于加快阐明炒焦的科学性，丰富并发展炒焦传统炮制理论。

本项目立足于槟榔炒焦“长于消食导滞”的功效变化，对槟榔炒焦的功效变化的物质基础和作用机制进行了研究。首先，通过制备HPLC以及NMR技术分离并鉴定了槟榔炒焦后新生成的3个化合物，分别鉴定为2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone（DDMP），5-Hydroxymethyl-2-furfural （5-HMF）和3-hydroxy-2-methyl-γ-pyrone（Maltol）；进一步通过研究槟榔炒焦前后二阶导数红外法、A420、pH、还原糖、氨基酸以及5-HMF的含量变化，证明了槟榔炒焦前后成分变化与美拉德反应有关；建立了16批槟榔炒制样品的HPLC指纹图谱，并基于HPLC指纹图谱和肠平滑肌张力，采用双变量相关法分析16批槟榔炒制样品“HPLC谱-促胃肠动力”谱-效关系。结果表明槟榔炒制样品有5个共有特征峰与肠平滑肌张力均呈现正相关且具有统计学意义，特别是3个美拉德反应产物（DDMP、5-HMF和 Maltol）与十二指肠平滑肌张力密切相关。其次，通过建立慢性应激性大鼠模型，观察焦槟榔和生槟榔对正常和慢性应激性大鼠胃肠运动、“脑肠肽”以及大鼠离体胃肠平滑肌收缩的影响。结果表明，焦槟榔与生槟榔相比，可更显著地促进正常大鼠和慢性应激大鼠胃肠运动，提高正常大鼠和慢性应激大鼠血清MTL、SP含量和抑制慢性应激大鼠小肠和下丘脑CCK mRNA 的表达，从而增加大鼠胃肠平滑肌张力，此为焦槟榔“长于消食导滞”作用机制之一。最后，采用“敲除”“敲入”法的思路，采用离体平滑肌张力实验评价，结合流式细胞术、细胞DAPI染色、western blot法等技术，研究确定了焦槟榔中最佳的槟榔碱含量约为0.12%，此时焦槟榔能够保留原有药效并毒性降低。本项目对槟榔炒焦的“长于消食导滞”的物质基础、作用机制及槟榔碱的最佳含量等的研究，为阐明槟榔炒焦原理提供了依据，并为中药炒焦共性物质基础及作用机制的研究提供新的思路与技术手段。