多伞阿魏的冰冻切片方法建立及挥发油、香豆素类成分组织化学定位分析

Objective: To establish a method for frozen sectioning of Ferula ferulaeoides and conduct a histochemical localization study on the volatile oils and coumarin components in its different organs. Methods: Using the roots, stems, petioles and leaves of Ferula ferulaeoides as materials, the influencing factors such as the mass fraction of sucrose protectant, the quick-freezing time in liquid nitrogen, the embedding conditions, the slice thickness, the freezing temperature and time, and the post-treatment methods were investigated. The integrity, microscopic effect, stretching and curling degree and clarity of the frozen sections were compared to screen the optimal sectioning conditions. The Sudan Ⅳ staining method and fluorescence microscope technology were used respectively to localize the volatile oils and coumarin components of Ferula ferulaeoides. Results: The optimal conditions for the frozen sections of the roots, stems, petioles and leaves of Ferula ferulaeoides were as follows: for the roots, sucrose with mass fractions of 10%, 15% and 20% was used as a protectant in a gradient; for the stems and petioles, sucrose with mass fractions of 10%, 20% and 30% was used as a protectant in a gradient; for the leaves, sucrose with mass fractions of 20%, 25% and 30% was used as a protectant in a gradient; glue - water (2∶1) was used as an embedding agent; quick-freezing in liquid nitrogen for 20 s and rewarming at -25 °C for 30 min; sectioning at -20 °C with a slice thickness of 25 μm; after mounting, rinsing with a sucrose solution of the same mass fraction, allowing the slices to stand on an ice pack for a period of time and storing them under normal temperature conditions. Among them, the mass fraction of the sucrose protectant was the most important influencing factor. The results of histochemical localization showed that the volatile oils and coumarin components in the four organs of Ferula ferulaeoides were mainly distributed in the resin ducts. Conclusion: This study established for the first time a method for frozen sectioning of Ferula ferulaeoides, which has a high rate of slice formation and relatively simplified steps; the volatile oils and coumarin components of Ferula ferulaeoides are mainly accumulated in the resin ducts.

目的:建立多伞阿魏的冰冻切片方法,并对其不同器官中挥发油和香豆素类成分进行组织化学定位研究。方法:以多伞阿魏根、茎、叶柄、叶为材料,考察蔗糖保护剂质量分数、液氮速冻时间、包埋条件、切片厚度、冷冻温度和时间、后处理方式等影响因素,对比冰冻切片的完整度、显微效果、伸卷度和清晰度,进而筛选最佳切片条件。分别利用苏丹Ⅳ染色法和荧光显微镜技术定位多伞阿魏的挥发油和香豆素类成分。结果:多伞阿魏根、茎、叶柄、叶冰冻切片的最佳条件为根以质量分数10%、15%、20%梯度蔗糖作为保护剂;茎和叶柄以质量分数10%、20%、30%梯度蔗糖作为保护剂;叶以质量分数20%、25%、 30%梯度蔗糖作为保护剂;以胶水-水(2∶1)作为包埋剂;液氮速冻20 s,-25 ℃回温30 min;-20 ℃下切片,切片厚度25 μm;贴片后用相同质量分数的蔗糖溶液冲洗,将切片在冰袋上静置一段时间,于常温条件下保存,其中蔗糖保护剂质量分数是最主要的影响因素。组织化学定位结果显示,多伞阿魏4种器官中挥发油和香豆素类成分均主要分布于树脂道。结论:该研究首次建立了多伞阿魏冰冻切片方法,该方法成片率高、步骤相对简化;多伞阿魏的挥发油和香豆素类成分主要积累在树脂道中。