啤酒花中8-异戊烯基柚皮素提取及含量测定方法的优化及验证

Optimization and verification of a method for extraction and determination of content of 8-prenylnaringenin in hops

目的优化啤酒花中8-异戊烯基柚皮素(8-prenylnaringenin,8-PN)的提取及含量测定方法,并进行验证。方法采用L_8(4~1×2~2)正交试验,料液比为1∶15,利用回流法提取啤酒花中的8-PN,反相高效液相色谱法测定啤酒花中8-PN的含量[色谱分离条件:ODS C18反相柱(4.6 mm×250 mm,0.45μm),用乙腈、1%甲酸进行梯度洗脱,流速:1.2 ml/min,检测波长:288 nm,柱温:30℃]。探讨提取溶剂(甲醇、乙醇、正丁醇、乙酸乙酯)、提取时间(40、60 min)、提取温度(40、60℃)对啤酒花中8-PN提取含量的影响。结果以正丁醇为提取溶剂,料液比为1∶15,利用回流提取,提取温度40℃,时间40 min,测得啤酒花中8-PN的最高提取含量为79.1 mg/kg。该方法的标准曲线相关系数R^2=0.999 8,线性范围为0.001~0.1 mg/ml,检出限为0.122 mg/L;检测浓度为0.03 mg/ml标准液的保留时间和峰面积的相对标准偏差(relative standard deviation,RSD)均小于1%,加标回收率在98%~102%之间。结论优化的方法可简便、准确地测定啤酒花中8-PN含量,为啤酒花中8-PN的进一步分离、纯化提供了实验依据。

Objective To optimize and verify a method for extraction and determination of content of 8-prenylnaringenin（8-PN）in hops. Methods The L8（4~1 × 2~2）orthogonal test was adopted, serving the solid-liquid ratio as 1 ∶ 15. The 8-PN in hops was extracted by reflux extraction, and determined for content by reverse phase HPLC under the following condition： ODS C18 reverse column（4. 6 mm × 250 mm, 0. 45 μm）was adopted, serving acetonitrile-1% formic acid as an eluent for gradient elution at a flow rate of 1. 2 ml/min. The detection wavelength was 288 nm, while the column temperature was 30 ℃. The effects of extraction solvent（methanol, ethanol, n-butanol and ethyl acetate）, extraction time（40 and 60 min）and extraction temperature（40 and 60 ℃）on the content of 8-PN extracted from hops were evaluated.Results The method for extraction of 8-PN in hops was optimized as follows： reflux extraction was performed serving nbutanol as solvent and the solid-liquid ratio as 1 ∶ 15. The optimal temperature and time for extraction were 40 ℃ and 40 min respectively. The maximum extraction rate of 8-PN was 79. 1 mg/kg. The correlation coefficient（R^2 value） of standard curve of the method was 0. 999 8, while the linear range was 0. 001 ~ 0. 1 mg/ml, and the detection limit was 0. 122 mg/L. The RSDs of retention time and peak area for determination of standard solution at a concentration of0. 03 mg/ml were less than 1%, while the spike recovery was 98% ~ 102%. Conclusion The optimized method was suitable for simple and accurate determination of 8-PN content in hops, which provided an experimental basis for further separation and purification of 8-PN in hops.