山慈菇活性成分微波提取工艺优化、指纹图谱与抗植物病菌活性分析

余怡欣; 陈龙惠; 曹丹; 王栋; 刘泓杏; 唐伟卓

doi:10.13386/j.issn1002-0306.2022060225

山慈菇活性成分微波提取工艺优化、指纹图谱与抗植物病菌活性分析

Optimization of Microwave Extraction of Active Ingredients Technology, Fingerprint Analysis and Anti-phytopathogenic Activity of Cremastra appendiculata (D. Don) Makino

摘要

摘要: 本文综合运用单因素和Box-Behnken响应面试验，以山慈菇提取率和其中3种主要化学成分：双4-（葡萄糖氧）苄基-2-异丁基苹果酸酯-2-葡萄糖苷（dactylorhin A）、双4-(葡萄糖氧)苄基-2-异丁基苹果酸酯(militarine)和秋水仙碱的含量为综合权重指标，对其微波提取工艺进行考察。同时，利用高效液相色谱技术建立13批不同产地山慈菇的指纹图谱，明确其主要成分含量分布情况。最后，运用滤纸片扩散法，对制备的山慈菇提取物进行抗菌活性评价。结果表明：山慈菇的最佳微波提取工艺条件：乙醇浓度55%、料液比1:30 g/mL、提取时间17 min。在此条件下，山慈菇的提取率为12.83%。指纹图谱结果表明，山慈菇中3种化学成分的含量分布趋势一致，其中militarine为13批山慈菇中的共有化学成分，而dactylorhin A和秋水仙碱分别在1个产地样品中未检出，表明不同产地山慈菇中主要化学成分分布具有差异性。抗菌实验结果显示，山慈菇提取物对胶孢炭疽菌、辣椒疫霉菌、灰葡萄孢菌、茄链格孢菌、荧光假单胞菌、水稻白叶枯菌和禾谷镰孢菌均没有显示抗菌活性（抑菌圈小于6 mm），对雷尔氏菌具有较好的抑制活性（抑菌圈8.5 mm），表现出一定的抗菌选择性。本研究确定了微波提取山慈菇的最佳工艺，建立的山慈菇中3种成分同步含量分析方法稳定可行，研究结果可为山慈菇药材的质量标准建立和综合开发利用提供参考。

Abstract: In this present paper, the optimization of microwave extraction (ME) of Cremastra appendiculata (D. Don) Makino was performed using the single factor design and response surface methodology. The ME extraction yield along with the contents of three major components of C. appendiculata Makino, including militarine, dactylorhin A, and colchicine was selected as the comprehensive indicators to evaluate the extraction process. Subsequently, the HPLC fingerprint of the above three components from 13 batches of C. appendiculata Makino was investigated. Finally, the anti-fungal activity of C. appendiculata Makino extract was evaluated by conducting a disk-diffusion method. The results demonstrated that the optimal ME technologies of C. appendiculata Makino were the ethanol concentration of 55%, the solid-liquid ratio of 1:30 g/mL, and the extraction time of 17 min. Under the above conditions, the extract yield was 12.83%. HPLC fingerprint analysis results showed that the contents of three major components displayed a consistent trend in all 13 tested samples. Militarine was found existed in 13 batches of C. appendiculata Makino, while dactylorhin A and colchicine were not detected in one sample. The results indicated a content difference in three active ingredients in C. appendiculata Makino from different areas. The anti-phytopathogenic activity results showed that the C. appendiculata Makino extract did not shown inhibitory activity against Colletotrichum gloeosporioides, Phytophthora capsica, Botrytis cinerea, Alternaria solani, Pseudomonas fluorescens, Xanthomnas oryzae, and Fusarium graminearum with their inhibition zones were less than 6 mm. However, the C. appendiculata Makino extract exhibited inhibitory activity toward to Ralstonia solanacearum with the diameter of the inhibition zone was 8.5 mm, showed antibacterial selectivity. In this study, the optimal ME technologies of C. appendiculata Makino was determined, and the established method the contents of three major components of C. appendiculata Makino was stable and feasible.These results can provide the basis for the quality control and comprehen-sive utilization of C. appendiculata Makinos.