甜叶菊叶中酚酸类成分样品制备方法及含量测定

Study of sample preparation and determination of phenolic acids from stevia leaf

目的 研究甜叶菊叶中酚酸类成分的最佳样品制备方法并进行含量测定.方法 通过正交实验设计法,对甜叶菊叶提取所需的乙醇浓度、提取方法、乙醇用量、提取时间及提取次数进行系统考察,分析比较各个因素及水平对酚酸总含量的影响,从而获得最佳的样品制备方法,并用此方法制备不同产地甜叶菊叶样品,检测其中酚酸成分含量.结果 绿原酸标准曲线的回归方程为Y=58.27X-51.38（r＝0.999 9）,在1.25～25.05 mg/L范围内呈现良好的线性关系.绿原酸精密度试验的峰面积分别为240.55、245.65、241.07、241.58、241.09、237.16,相对标准偏差为1.12%,表明精密度良好.稳定性试验测定得绿原酸峰面积分别为240.55、241.65、242.07、238.53、232.12、231.26,相对标准偏差为2.03%,表明供试品在24 h内基本稳定.没食子酸的标准曲线回归方程A= 8.61c＋0.26（r=0.999 5）,总酚酸含量在0.01～0.16 g/L范围内呈现良好线性关系.甜叶菊叶溶液在1 h内稳定,0、10、20、30、40、50、60 min的吸光度分别为0.940 7、0.951 8、0.938 1、0.931 1、0.938 3、0.928 7、0.916 7,相对标准偏差为1.18%.4种因素影响顺序依次为提取方法＞提取时间＞料液比＞提取次数,最后综合考虑各个因素,确定最佳提取工艺为加入20倍量的50%乙醇,超声提取30 min,超声2次.6次提取方法测定的总酚酸含量分别为12.10%、12.23%、12.18%、11.97%、11.42%和11.52%,相对标准偏差为2.93%,方法稳定可靠.分析检测得到不同产地的甜叶菊中绿原酸含量在0.66%～2.17%,总酚酸含量在8.60%～16.15%,绿原酸占总酚酸的比例在8.7%～16.2%.结论 所建立的方法准确、简便、重复性好,可用于甜叶菊叶中酚酸类成分的含量测定及其质量控制.

Objective To study the best method of sample preparation and determination of the phenolic acids from stevia leaf. Methods With the method of orthogonal design, the ethanol concentration, extraction method, ethanol volume, extraction time and extraction times were investigated. Results The regression equation of chlorogenic acid standard curve was Y=58.27X-51.38（r=0.999 9） with a good linear relationship in the range of 1.25-25.05 mg/L. Chlorogenic acid peak area of precision test were 240.55, 245.65, 241.07, 241.58, 241.09 and 237.16, respectively; the relative standard deviation was 1.12%. The peak area of chlorogenic acid by Stability test was 240.55, 241.65, 242.07, 238.53, 232.12 and 231.26; the relative standard deviation was 2.03%. Stevia leaves solution is stable in 1 h, 0, 10, 20, 30, 40, 50, 60 min absorbance was 0.940 7, 0.951 8, 0.938 1, 0.931 1, 0.938 3, 0.928 7 and 0.916 7; the relative standard deviation was 1.18%. The orthogonal design results showed that the best method for preparing stevia leaf was： liquid ratio of 1∶20 with 50% aqueous ethanol and ultrasonic extraction for 30 min. In Stevias from different origins, contents of chlorogenic acid and total phenolic were from 0.66% to 2.17% and between 8.60% to 16.15%; the proportion of chlorogenic acid in total phenolic ranged from 8.70% to 16.2%. Conclusion The method is accurate and can be used for the determination of phenolic components and the quality control of stevia leaf.