Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 183, 41125 Modena, Italy.
J Pharm Biomed Anal. 2013 Jul-Aug;81-82:126-32. doi: 10.1016/j.jpba.2013.04.003. Epub 2013 Apr 11.
A closed-vessel microwave-assisted extraction (MAE) technique was optimized for the first time for the extraction of polyphenols from raw propolis. The results obtained by means of response surface experimental design methodology showed that the best global response was reached when the extraction temperature was set at 106 °C, the solvent composition close to EtOH-H2O 80:20 (v/v), with an extraction time of 15 min. In comparison with other techniques, such as maceration, heat reflux extraction (HRE) and ultrasound-assisted extraction (UAE), the extraction with MAE was improved by shorter extraction time and lower volume of solvent needed. The HPLC analyses of propolis extracts were carried out on a fused-core Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 μm), with a gradient mobile phase composed by 0.1% formic acid in water and acetonitrile. Detection was performed by DAD and MS. The method validation indicated that the correlation coefficients were >0.999; the limit of detection was in the range 0.5-0.8 μg/ml for phenolic acids and 1.2-3.0 μg/ml for flavonoids; the recovery range was 95.3-98.1% for phenolic acids and 94.1-101.3% for flavonoids; the intra- and inter-day %RSD values for retention times and peak areas were ≤ 0.3 and 2.2%, respectively. The quali- and quantitative analysis of polyphenols in Italian samples of raw propolis was performed with the validated method. Total phenolic acids ranged from 5.0 to 120.8 mg/g and total flavonoids from 2.5 to 168.0mg/g. The proposed MAE procedure and HPLC method can be considered reliable and useful tools for the comprehensive multi-component analysis of polyphenols in propolis extracts to be used in apitherapy.
首次采用密闭微波辅助提取（MAE）技术从天然蜂胶中提取多酚。通过响应面实验设计方法得到的结果表明，当提取温度设置为 106°C、溶剂组成为接近 80:20（v/v）的 EtOH-H2O、提取时间为 15min 时，可获得最佳的整体响应。与浸渍、热回流提取（HRE）和超声辅助提取（UAE）等其他技术相比，MAE 提取具有提取时间短、所需溶剂体积少的优点。采用梯度洗脱程序，以 0.1%甲酸水溶液和乙腈为流动相，在 Ascentis Express C18 柱（150mm×3.0mmID，2.7μm）上对蜂胶提取物进行高效液相色谱（HPLC）分析。采用 DAD 和 MS 进行检测。方法验证表明，相关系数均>0.999；酚酸的检测限为 0.5-0.8μg/ml，黄酮类的检测限为 1.2-3.0μg/ml；酚酸的回收率为 95.3-98.1%，黄酮类的回收率为 94.1-101.3%；保留时间和峰面积的日内和日间%RSD 值均≤0.3 和 2.2%。采用验证后的方法对意大利天然蜂胶样品中的多酚进行了定性和定量分析。总酚酸含量为 5.0-120.8mg/g，总黄酮含量为 2.5-168.0mg/g。该 MAE 工艺和 HPLC 方法可作为蜂胶提取物中多酚进行综合多组分分析的可靠、有用工具，可用于蜂疗。
