覆盆子中4种成分含量测定及其酒炙工艺优化

Determination of four constituents in Rubi fructus and optimization of wine preparation process

关键词:  覆盆子  确定性筛选实验设计  鞣花酸  山柰酚-3-O-芸香糖苷  紫云英苷  椴树苷

Key words:  Rubi fructus  definitive screening design  elleagic acid  kaempferol-3-O-rutinoside  astragalin  tiliroside

摘要:

目的 建立覆盆子中鞣花酸、山柰酚-3-O-芸香糖苷、紫云英苷和椴树苷4种活性成分含量测定的HPLC方法,并对覆盆子的酒炙工艺进行优化。方法 覆盆子中鞣花酸含量测定及3种黄酮含量测定方法所用色谱柱均为Waters CORTECS? C18(4.6×150 mm,2.7 μm)色谱柱,流动相均为0.1%甲酸-水溶液(流动相A)和0.1%甲酸-乙腈(流动相B),梯度洗脱,鞣花酸的紫外检测波长为254 nm,3种黄酮的紫外检测波长为266 nm；经单因素实验确定黄酒用量、闷润时间、炮制时间和炮制温度4个因素的水平范围后,采用确定性筛选实验设计,以鞣花酸、山柰酚-3-O-芸香糖苷、紫云英苷和椴树苷4种成分含量的总评归一值为评价指标,建立各因素与总评归一值之间的函数模型,预测优选最佳的酒炙工艺参数。结果 4种成分在线性范围内线性系数r ≥ 0.999；平均回收率分别为103.7%、97.2%、96.9%和97.4%,RSD < 2% (n=6);优化得到的覆盆子酒炙工艺参数为：黄酒用量19.4%、闷润时间1.8 h、炮制时间10.5 min、炮制温度149 ℃；3次验证实验所得各成分含量的RSD<12%；3批次覆盆子/酒炙覆盆子中鞣花酸含量为2.65 ~ 3.80 mg·g-1 / 3.00 ~ 4.97 mg·g-1,山柰酚-3-O-芸香糖苷的含量为0.344 ~ 0.476 mg·g-1 / 0.316 ~ 0.460 mg·g-1,紫云英苷的含量为0.216 ~ 0.255 mg·g-1 / 0.195 ~ 0.280 mg·g-1,椴树苷的含量为0.470% ~ 0.568 mg·g-1 / 0.559 ~ 0.787 mg·g-1。结论 建立的HPLC方法操作简单,适用性强,可用于(酒)覆盆子中鞣花酸、山柰酚-3-O-芸香糖苷、紫云英苷和椴树苷的含量测定；优选的覆盆子酒炙工艺稳定可靠,所得活性成分含量较高,可为酒炙覆盆子的质量标准制定及临床应用提供一定的科学依据。

Abstract:

OBJECTIVE To develop a HPLC method for determining elleagic acid, kaempferol-3-O-rutinoside, astragalin, and tiliroside in Rubi fructus, and to optimize the wine preparation process of Rubi fructus. METHODS Waters CORTECS? C18 (4.6×150 mm，2.7μm) was used as the chromatography column for the analytical methods for elleagic acid and three flavonoids; Mobile phase A and B were 0.1% formic acid – H2O and 0.1% formic acid – CH3CN, respectively; Gradient elution, the UV detection wavelength for elleagic acid was 254 nm, the UV detection wavelength for three flavonoids was 266 nm; After determining the level ranges of four factors, including yellow rice dosage, damping time, processing time, and processing temperature by using single factor experiments, definitive screening design was employed to design the experiments. The overall normalized value of ellagic acid, kaempferol-3-O-rutinoside, astragalin, and tiliroside was adopted as the evaluation index, which was used to establish the functional model between the factors and the normalized value of the total evaluation was established. And then the optimum process parameters were predicted. RESULTS The correlation coefficient r ≥ 0.999 in linearity ranges of four consitutents; The average recoveries were 103.7%, 97.2%, 96.9%, and 97.4%, respectively, with RSD < 2% (n=6); The optimized process parameters for the wine preparation process of Rubi fructus were as follows: the amount of yellow rice wine is 19.4%, the soaking time is 1.8 hours, the processing time is 10.5 min, and the processing temperature is 149 ℃. The RSD values of contents of four constituents were less than 12% for three verification experiments. The contents of ellagic acid were 2.65 ~ 3.80 mg·g-1 / 3.00 ~ 4.97 mg·g-1, kaempferol-3-O-rutinoside 0.344 ~ 0.476 mg·g-1 / 0.316 ~ 0.460 mg·g-1, astragalin 0.216 ~ 0.255 mg·g-1 / 0.195 ~ 0.280 mg·g-1, and tiliroside 0.470 ~ 0.568 mg·g-1 / 0.559 ~ 0.787 mg·g-1 in the three batches of Rubi fructus/wine prepared Rubi fructus. CONCLUSION The developed HPLC methods were simple, and applicable, which could be used to determine elleagic acid, kaempferol-3-O-rutinoside, astragalin and tiliroside in (wine) Rubi fructus. The optimum preparation process of wine prepared Rubi fructus was stable and reliable, which could obtain high contents of active constituents. The results can provide a scientific basis for the quality standard and clinical application of wine prepared Rubi fructus.