Boka Vasiliki-Ioanna, Argyropoulou Aikaterini, Gikas Evangelos, Angelis Apostolis, Aligiannis Nektarios, Skaltsounis Alexios-Leandros
Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens, Greece.
Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens, Greece.
Planta Med. 2015 Nov;81(17):1628-35. doi: 10.1055/s-0035-1558140. Epub 2015 Nov 18.
A high-performance thin-layer chromatographic methodology was developed and validated for the isolation and quantitative determination of oleuropein in two extracts of Olea europaea leaves. OLE_A was a crude acetone extract, while OLE_AA was its defatted residue. Initially, high-performance thin-layer chromatography was employed for the purification process of oleuropein with fast centrifugal partition chromatography, replacing high-performance liquid-chromatography, in the stage of the determination of the distribution coefficient and the retention volume. A densitometric method was developed for the determination of the distribution coefficients, KC = CS/CM. The total concentrations of the target compound in the stationary phase (CS) and in the mobile phase (CM) were calculated by the area measured in the high-performance thin-layer chromatogram. The estimated Kc was also used for the calculation of the retention volume, VR, with a chromatographic retention equation. The obtained data were successfully applied for the purification of oleuropein and the experimental results confirmed the theoretical predictions, indicating that high-performance thin-layer chromatography could be an important counterpart in the phytochemical study of natural products. The isolated oleuropein (purity > 95%) was subsequently used for the estimation of its content in each extract with a simple, sensitive and accurate high-performance thin-layer chromatography method. The best fit calibration curve from 1.0 µg/track to 6.0 µg/track of oleuropein was polynomial and the quantification was achieved by UV detection at λ 240 nm. The method was validated giving rise to an efficient and high-throughput procedure, with the relative standard deviation % of repeatability and intermediate precision not exceeding 4.9% and accuracy between 92% and 98% (recovery rates). Moreover, the method was validated for robustness, limit of quantitation, and limit of detection. The amount of oleuropein for OLE_A, OLE_AA, and an aqueous extract of olive leaves was estimated to be 35.5% ± 2.7, 51.5% ± 1.4, and 12.5% ± 0.12, respectively. Statistical analysis proved that the method is repeatable and selective, and can be effectively applied for the estimation of oleuropein in olive leaves' extracts, and could potentially replace high-performance liquid chromatography methodologies developed so far. Thus, the phytochemical investigation of oleuropein could be based on high-performance thin-layer chromatography coupled with separation processes, such as fast centrifugal partition chromatography, showing efficacy and credibility.
开发并验证了一种高效薄层色谱方法，用于分离和定量测定油橄榄叶两种提取物中的橄榄苦苷。OLE_A是粗丙酮提取物，而OLE_AA是其脱脂残渣。最初，在测定分配系数和保留体积阶段，采用高效薄层色谱结合快速离心分配色谱法进行橄榄苦苷的纯化过程，取代了高效液相色谱法。开发了一种密度测定法来测定分配系数KC = CS/CM。通过高效薄层色谱图中测得的面积计算固定相（CS）和流动相（CM）中目标化合物的总浓度。估算的Kc也用于通过色谱保留方程计算保留体积VR。所得数据成功应用于橄榄苦苷的纯化，实验结果证实了理论预测，表明高效薄层色谱可能是天然产物植物化学研究中的重要方法。随后，使用一种简单、灵敏且准确的高效薄层色谱法，对分离得到的橄榄苦苷（纯度>95%）在每种提取物中的含量进行估算。橄榄苦苷从1.0μg/点到6.0μg/点的最佳拟合校准曲线为多项式，通过在λ240nm处的紫外检测进行定量。该方法经过验证，产生了一种高效且高通量的程序，重复性和中间精密度的相对标准偏差%不超过4.9%，准确度在92%至98%（回收率）之间。此外，该方法还针对稳健性、定量限和检测限进行了验证。OLE_A、OLE_AA和橄榄叶水提取物中橄榄苦苷的含量估计分别为35.5%±2.7、51.5%±1.4和12.5%±0.12。统计分析证明该方法具有可重复性和选择性，可有效应用于橄榄叶提取物中橄榄苦苷的估算，并可能取代迄今为止开发的高效液相色谱方法。因此，橄榄苦苷的植物化学研究可以基于高效薄层色谱结合快速离心分配色谱等分离过程，显示出有效性和可信度。
