金银花腺毛形态特征及内含物与药材品质的相关性研究

Glandular trichomes are the epidermal hairs of plants, known as "Biosynthesis Factory". Glandular trichomes of Lonicerae Japonicae flos are the main characteristics of microscopic identification. At the early stage, we found that the quality of Lonicerae japonicae flos from three main producing areas was obviously different, and the morphology and quantity of glandular trichomes were also significantly different. In addition, the color of glandular trichomes changed from yellow to dark brown before and after the opening of flower buds, which was consistent with the medical use of flower buds. Therefore, we speculated that the morphological characteristics and inclusions of glandular trichomes were closely related to the quality of the medicinal materials. However, due to the obstruction of glandular tissues separation technology, there is no research report on the inclusions of glandular trichomes of Lonicerae Japonicae flos. In the early stage, the author used laser microdissection (LMD) to successfully separate the glandular tissues of Lonicerae Japonicae flos. Based on our previous researches, in this study we plan to use Lonicerae Japonicae flos from different developmental periods and origins as materials, ①Using the microscope to observe the morphological characteristics of glandular trichomes and the synthesis and distribution of inclusions; ②LMD, HPLC-MS, GC-MS Techniques were used to establish qualitative and quantitative analysis methods for the inclusions of glandular trichomes; ③HPLC-MS and GC-MS were used to determine the contents of active constituents of medicinal materials. Through correlation analysis and grey correlation Analysis, we intend to provide ideas and basis for the new variety breeding and quality evaluation of Lonicerae japonicae flos by discussing the relationship between morphological characteristics of glandular trichomes and types and contents of the inclusions and the quality of the medicinal materials of Lonicerae Japonicae flos.

腺毛是植物的表皮毛，被誉为“生物合成工厂”。金银花腺毛是其显微鉴别的主要特征。前期发现金银花三大主产区药材质量明显不同，其腺毛的形态与数量也有显著差异；且花蕾开放前后腺毛色泽由黄润变深褐，与花蕾药用一致，因此我们推测腺毛形态特征及内含物与药材品质密切相关。但由于腺毛分离技术阻碍，目前还未有金银花腺毛内含物研究报道，笔者前期利用激光显微切割仪（LMD）成功分离了金银花腺毛组织。本项目拟在此基础上，以不同产地、发育时期的金银花为研究对象，①利用显微镜观察腺毛的形态特征及内含物合成分布；②利用LMD、HPLC-MS、GC-MS联用技术建立腺毛内含物定性定量分析方法；③利用HPLC-MS、GC-MS测定药材中有效成分含量，通过相关性分析、灰色关联度分析，探讨金银花腺毛形态特征及内含物种类、含量与药材品质的关系，揭示腺毛对金银花药材品质影响的内涵，为中药材质量评价、新品种选育及道地性研究提供新依据。

腺毛是植物的表皮毛，被誉为“生物合成工厂”，与中药材品质密切相关。金银花腺毛是其显微鉴别的主要特征。本研究以金银花腺毛为研究对象，采用石蜡切片、光学显微镜等技术，观察发现金银花腺毛有倒圆锥形和类圆形或略扁圆形两种类型，不同发育时期两种类型腺毛均有，但分布密度不同；腺毛原始细胞起源于幼嫩花蕾表皮细胞，经一系列平周与垂周分裂发育成完整腺毛；腺毛在花蕾长约1mm时开始出现，随着花蕾发育，花蕾表面腺毛密度和大小呈增长趋势，腺头颜色逐渐加深。本研究确定了激光捕获显微切割（LMD）是分离及纯化富集腺毛的最优方法，并创新性的发明了一种快速分离表皮毛，制取无表皮毛花蕾的方法—低温冷冻涡旋法。本项目采用HPLC-TOF/MS技术对花蕾、去表皮毛花蕾及腺毛进行定性分析，在花蕾、去表皮毛花蕾、腺毛中分别鉴定出40种、34种和38种成分，有32种共有成分。相对于花蕾，去表皮毛花蕾减少了6种黄酮类成分，腺毛减少了2种酚酸类成分，提示腺毛中的次生代谢物与花蕾其他部位有所不同；HPLC-MS/MS技术对金银花花蕾和去表皮毛花蕾中非挥发性成分进行定量分析，通过计算表皮毛在花蕾中的重量占比，实现对花蕾表皮毛中成分定量分析。结果发现花蕾中7种酚酸类成分、9种黄酮类成分主要分布在去表皮毛花蕾；有4种黄酮类成分主要分布在表皮毛中，结合定性研究结果，推断金银花花蕾腺毛不是酚酸类成分合成积累的主要场所，而是某些黄酮类成分，尤其是黄酮苷元类成分合成积累的主要场所。利用GC-MS技术在花蕾、去表皮毛花蕾、腺毛中分别鉴定出40、37、14种挥发性成分，去表皮毛花蕾与花蕾中挥发性成分的数量及出峰时间高度一致，说明腺毛不是花蕾挥发性成分合成积累的唯一场所，对花蕾挥发性成分起主要贡献的是去表皮毛花蕾。上述结果说明，腺毛可合成积累酚酸类和黄酮类等多种次生代谢物，是某些黄酮类成分，尤其是黄酮苷元类成分合成积累的主要场所，腺毛的密度、发育程度与药材品质形成具有密切关系，可考虑作为评价金银花药材质量的性状指标，为金银花药材道地性研究、品质评价及新品种选育提供新的思路和方法。