Xia Yong-Gang, Yang Bing-You, Liang Jun, Wang Di, Yang Qi, Kuang Hai-Xue
School of Pharmacy, Key Laboratory of Chinese Materia Medica (Heilongjiang University of Chinese Medicine), Ministry of Education, Harbin, 150040, P.R. China.
Pharmacogn Mag. 2014 Jul;10(39):292-303. doi: 10.4103/0973-1296.137370.
The compounds (+)-pinoresinol-β-glucoside (1) forsythiaside, (2) phillyrin (3) and phillygenin (4) were elucidated to be the characteristic constituents for quality control of Forsythiae Fructus extract by chromatographic fingerprint in 2010 edition of Chinese Pharmacopoeia due to their numerous important pharmacological actions. It is of great interest to extract these medicinally active constituents from Forsythiae Fructus simultaneously.
In this study, a new ultrasound-assisted extraction (UAE) method was developed for the simultaneous extraction of biological components 1-4 in Forsythiae Fructus. The quantitative effects of extraction time, ratio of liquid to solid, extraction temperature, and methanol concentration on yield of these four important biological constituents from Forsythiae Fructus were investigated using response surface methodology with Box-Behnken design. The compounds 1-4 extracted by UAE were quantitative analysis by high-performance liquid chromatography-photodiode array detect (HPLC-PAD), and overall desirability (OD), the geometric mean of the contents of four major biological components, was used as a marker to evaluate the extraction efficiency.
By solving the regression equation and analyzing 3-D plots, the optimum condition was at extraction temperature 70°C, time 60 min, ratio of liquid to solid 20, and methanol concentration 76.6%. Under these conditions, extraction yields of compounds 1-4 were 2.92 mg/g, 52.10 mg/g, 0.90 mg/g and 0.57 mg/g, respectively, which were in good agreement with the predicted OD values. In order to achieve a similar yield as UAE, soxhlet extraction required at least 6 h and maceration extraction required much longer time of 24 h. Established UAE method has been successfully applied to sample preparation for the quality control of Forsythiae Fructus. Additionally, a quadrupole time-of-flight mass spectrometry was applied to the structural confirmation of analytes from the complex matrices acquired by UAE.
The results indicated that UAE is an effective alternative method for extracting bioactive constituents, which may facilitate a deeper understanding of the extract of active constituents in Forsythiae Fructus from the raw material to its extract for providing the theoretical references.
（+）-松脂醇-β-葡萄糖苷（1）、连翘酯苷（2）、连翘苷（3）和连翘酯素（4）这几种化合物，因其具有众多重要药理作用，在《中国药典》2010年版中被确定为连翘提取物质量控制的特征性成分。同时从连翘中提取这些具有药用活性的成分具有重要意义。
本研究开发了一种新的超声辅助提取（UAE）方法，用于同时提取连翘中的生物成分1 - 4。采用Box-Behnken设计的响应面法，研究了提取时间、液固比、提取温度和甲醇浓度对连翘中这四种重要生物成分得率的定量影响。采用高效液相色谱-光电二极管阵列检测（HPLC-PAD）对UAE提取的化合物1 - 4进行定量分析，并将综合可取性（OD），即四种主要生物成分含量的几何平均值，作为评价提取效率的指标。
通过求解回归方程并分析三维图，最佳条件为提取温度70℃、时间60分钟、液固比20和甲醇浓度76.6%。在此条件下，化合物1 - 4的提取率分别为2.92 mg/g、52.10 mg/g、0.90 mg/g和0.57 mg/g，与预测的OD值吻合良好。为了获得与UAE相似的得率，索氏提取至少需要6小时，浸渍提取所需时间更长，为24小时。所建立的UAE方法已成功应用于连翘质量控制的样品制备。此外，采用四极杆飞行时间质谱对UAE从复杂基质中获取的分析物进行结构确证。
结果表明，UAE是提取生物活性成分的一种有效替代方法，这可能有助于更深入地了解连翘从原料到提取物中活性成分的提取情况，为提供理论参考。
