木薯叶黄酮醇检测方法优化及其含量比较分析

Optimization of Detection Methods for Flavonols in Cassava Leaves

木薯叶富含丰富的黄酮醇类物质,高效的提取和分析方法可为获取木薯叶黄酮醇提供至关重要的评价技术。本研究旨在优化木薯叶中4种黄酮醇物质(杨梅苷、芦丁、烟花苷和水仙苷)的提取和检测方法,以及分析不同木薯品种、采收期和成熟度对这些黄酮醇含量的影响。结果表明:使用50%乙醇水溶液,料液比为1∶50(g/mL),超声提取温度50℃,超声提取时间60 min可有效提取木薯叶中的4种黄酮醇;不同的C18色谱柱配备HPLC-DAD能有效分离木薯叶中的4种黄酮醇;方法验证结果显示,4种黄酮醇在一定浓度范围内线性相关性良好,R^(2)分别达到0.9999、0.9999、0.9999和0.9998;检出限在6.0~10.0 mg/kg之间;定量限在20.0~32.0 mg/kg之间;检测方法系统适应性较好,保留时间和峰面积变化相对标准偏差(RSD)均小于1%;样品日内、日间和月内稳定性较好,含量变化RSD在0.44%~3.57%之间,平均加标回收率在92.68%~109.14%之间,RSD均小于6.0%。利用建立的提取和分析方法,分析了30个木薯种质中4种黄酮醇含量,芦丁和烟花苷含量占4种黄酮醇总量的93.50%~99.30%,其含量高低由芦丁和烟花苷决定,但品种间决定黄酮醇总含量高低的相关性顺序为芦丁>水仙苷>烟花苷>杨梅苷;木薯种质的不同采收期和不同成熟度叶片中黄酮醇分析表明,在多数木薯种质中,第270天采收的芦丁、烟花苷和水仙苷含量高于第180天,杨梅苷因品种而异;不同木薯种质(除SC09外)幼叶期的杨梅苷、芦丁和烟花苷含量均高于嫩叶期和成熟期;成熟期(除花叶木薯)水仙苷含量均高于幼叶期和嫩叶期。本研究结果可为木薯叶黄酮醇物质的开发利用(原料的选择、质量控制等方面)提供评价技术和依据,也为揭示木薯叶黄酮醇物质积累规律奠定基础。

Cassava leaves are rich in abundant flavonols,and efficient extraction and analysis methods are crucial for the evaluation of flavonol content in the cassava leaves.This study aims to optimize the extraction and detection methods for four flavonols(myricetin,rutin,nicotiflorin,narcissoside)in cassava leaves and to analyze the influence of different cassava varieties,harvesting periods,and maturity on the content of these flavonols.The results indicated that using a 50%ethanol-water solution,a liquid-to-material ratio of 1∶50(g/mL),an ultrasonic extraction temperature of 50℃,and an ultrasonic extraction time of 60 minutes could effectively extract the four flavonols in the cassava leaves.The combination of different C18 chromatographic columns in HPLC-DAD effectively separated the four flavonoids present in the cassava leaves.The results of the method validation showed that the four flavonols exhibited good linear correla-tions within a certain concentration range,with R^(2)values of 0.9999,0.9999,0.9999 and 0.9998,respectively.The de-tection limits ranged from 6.0 mg/kg to 10.0 mg/kg,and the quantification limits ranged from 20.0 mg/kg to 32.0 mg/kg.The detection method demonstrates good system adaptability,with retention time and peak area relative standard devia-tions(RSD)being less than 1%.The samples showed good stability in terms of intra-day,inter-day,and intra-month variations,with RSD in content variations ranging from 0.44%to 3.57%.The average recovery rates of the method ranged from 92.68%to 109.14%,all with RSD values less than 6.0%.Utilizing the established extraction and analysis methods,the study analyzed the content of the four flavonols in 30 cassava germplasm resources.Rutin and nicotiflorin maked up 93.50%to 99.30%of the total flavonol content in the cassava leaves,with the levels primarily determining the total flavonol content.However,the correlation order for the total flavonol content among different varieties was ru-tin>narcissoside>nicotiflorin>myricetin.Analysis of flavonol content in the cassava leaves from different harvest times and maturity levels of cassava germplasm revealed that in most cases,the levels of rutin,nicotiflorin and narcissoside were higher in the leaves harvested at 270 days compared to those harvested at 180 days,with myricetin varying by variety.Except for SC09,the levels of myricetin,rutin and nicotiflorin were higher in the young leaves compared to the tender and mature leaves in different cassava germplasms.The content of narcissoside in mature leaves(except for flower leaf cassava)was higher than that in young leaves and tender leaves.The results would provide an evaluation basis for the development and utilization of cassava flavonols in the selection of raw materials and quality control,and lay a foundation for revealing the accumulation rules of cassava flavonols.