Zhang Xinxin, Liang Jinru, Zhang Yongmin, Liu Jianli, Sun Wenji, Ito Yoichiro
Biomedicine Key Laboratory of Shaanxi Province, Northwest University, 229 Taibai North Road, Xi'an, Shaanxi 710069, China.
Institut Parisien de Chimie Moléculaire, Université Pierre et Marie Curie-Paris 6, 4 place Jussieu, 75005 Paris, France.
J Steroids Horm Sci. 2015 Mar;6(1). doi: 10.4172/2157-7536.1000.150. Epub 2015 Feb 25.
Steroid saponins from C.H.Wright were separated for the first time using two chromatographic methods for comparison: counter-current chromatography (CCC) coupled with evaporative light scattering detector (ELSD) and preparative reversed phase high-performance liquid chromatography (RP-HPLC) with an ultraviolet detector. Ethyl acetate-n-butanol-methanol-water (4:1:2:4, v/v) was chosen as the two-phase solvent system for CCC, while the acetonitrile-water (25:75 for the first step and15:85 for the second step, v/v) was used as the mobile phase in the preparative RP-HPLC. The following five steroid saponins were purified by theses two chromatographic methods, in one-step operation by CCC and by two-step operation in preparative RP-HPLC: 1) 26-O-β-D- glucopyranosyl-(25R)-furost-5-en-3β, 22ζ, 26-triol-3-O-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (), 2) 26-O-β-D-glucopyranosyl-(25R)-furost-5-en-3β, 22ζ, 4) 26-triol-3-O-[β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (), 3) 26-O-β-D-glucopyranosyl-(25R)-furost-5-en-3β, 22ζ, 26-triol-3-O-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside (), 4) 26-O-β-D-glucopyranosyl-(25R)-furost-5, 20(22)-diene-3β, 26-diol-3-O-{α-L-rhamnopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→2)]}-β-D-glucopyranoside () and 5) 26-O-β-D-glucopyranosyl-(25R)-furost-5, 20(22)-diene-3β, 26-diol-3-O-[β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosy-(1→2)]-β-D-glucopyranoside (). The purities of these five steroid saponins separated by both methods were over 95%, and structural identification of these compounds was performed by ESI-MS, and C NMR. Comparison of these two established approaches revealed that CCC required a longer separation time but with less solvent consumption, whereas preparative RP-HPLC gave a shorter separation time but with higher solvent consumption. These results demonstrated that either of these two methods of different separation mechanism is feasible, economical and efficient for rapid preparative isolation and purification of steroid saponins from C.H.Wright.
首次采用两种色谱方法对C.H. Wright中的甾体皂苷进行分离比较：逆流色谱（CCC）结合蒸发光散射检测器（ELSD）以及带有紫外检测器的制备型反相高效液相色谱（RP-HPLC）。乙酸乙酯-正丁醇-甲醇-水（4:1:2:4，v/v）被选为CCC的两相溶剂系统，而乙腈-水（第一步为25:75，第二步为15:85，v/v）用作制备型RP-HPLC的流动相。通过这两种色谱方法纯化得到了以下五种甾体皂苷，CCC一步操作，制备型RP-HPLC两步操作：1）26-O-β-D-吡喃葡萄糖基-(25R)-呋甾-5-烯-3β,22ζ,26-三醇-3-O-[β-D-吡喃葡萄糖基-(1→3)-β-D-吡喃葡萄糖基-(1→4)-α-L-鼠李糖基-(1→2)]-β-D-吡喃葡萄糖苷（），2）26-O-β-D-吡喃葡萄糖基-(25R)-呋甾-5-烯-3β,22ζ,4）26-三醇-3-O-[β-D-吡喃葡萄糖基-(1→3)-α-L-鼠李糖基-(1→2)]-β-D-吡喃葡萄糖苷（），3）26-O-β-D-吡喃葡萄糖基-(25R)-呋甾-5-烯-3β,22ζ,26-三醇-3-O-[α-L-鼠李糖基-(1→4)]-β-D-吡喃葡萄糖苷（），4）26-O-β-D-吡喃葡萄糖基-(25R)-呋甾-5,20(22)-二烯-3β,26-二醇-3-O-{α-L-鼠李糖基-(1→4)-[β-D-吡喃葡萄糖基-(1→3)-β-D-吡喃葡萄糖基-(1→2)]}-β-D-吡喃葡萄糖苷（）和5）26-O-β-D-吡喃葡萄糖基-(25R)-呋甾-5,20(22)-二烯-3β,26-二醇-3-O-[β-D-吡喃葡萄糖基-(1→4)-α-L-鼠李糖基-(1→2)]-β-D-吡喃葡萄糖苷（）。两种方法分离得到的这五种甾体皂苷纯度均超过95%，并通过电喷雾电离质谱（ESI-MS）和碳核磁共振（(^{13})C NMR）对这些化合物进行了结构鉴定。对这两种既定方法的比较表明，CCC分离时间较长但溶剂消耗较少，而制备型RP-HPLC分离时间较短但溶剂消耗较高。这些结果表明，这两种分离机制不同的方法对于从C.H. Wright中快速制备分离和纯化甾体皂苷都是可行、经济且高效的。
