Peng Aihua, Li Rui, Hu Jia, Chen Lijuan, Zhao Xia, Luo Houding, Ye Haoyu, Yuan Yuan, Wei Yuquan
State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Gaopeng Street, Keyuan Road 4, Chengdu 610041, China.
J Chromatogr A. 2008 Jul 25;1200(2):129-35. doi: 10.1016/j.chroma.2008.05.041. Epub 2008 May 23.
In this paper, high-speed counter-current chromatography (HSCCC) instruments with different gravitational forces were applied for the separation of bioactive compounds from Triperygium wilfordii Hook.f. The critical parameters including sample concentration, sample volume and flow rate were first optimized on an analytical Mini-DE HSCCC system, and then scaled up to a preparative TBE 300A HSCCC system. Although this scale-up process was performed using different CCC instruments with different centrifuges and gravitational forces, the same resolutions were obtained and the elution time could be predictable. Five diterpenoid compounds and one unknown compound were separated from Triperygium wilfordii Hook.f. by HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (HEMW) (3:2:3:2, v/v/v/v). This one-step flow gradient separation produced triptonide (25 mg), isoneotriptophenolide (77 mg), hypolide (83 mg), unknown compound (1 mg), triptophenolide (42 mg), triptonoterpene methyl ether VI (37 mg) from 320 mg crude extract with purities of 98.2%, 96.6%, 98.1%, 95.3%, 95.1%, and 96.5%, respectively. Their purities and structures were identified by high-performance liquid chromatography, mass spectrometry and NMR. This paper demonstrates that analytical CCC plays an important role in optimizing parameters and scale-up process when analytical CCC and preparative CCC are supplied by different manufacturers with different gravitational forces, and the scale-up process from analytical CCC to preparative CCC is still predictable.
本文采用不同重力的高速逆流色谱（HSCCC）仪器从雷公藤中分离生物活性化合物。首先在分析型Mini-DE HSCCC系统上对包括样品浓度、样品体积和流速在内的关键参数进行优化，然后放大至制备型TBE 300A HSCCC系统。尽管这种放大过程是使用配备不同离心机和重力的不同CCC仪器进行的，但仍获得了相同的分离度，且洗脱时间可预测。采用正己烷 - 乙酸乙酯 - 甲醇 - 水（HEMW）（3:2:3:2，v/v/v/v）组成的两相溶剂系统，通过HSCCC从雷公藤中分离出5种二萜类化合物和1种未知化合物。这种一步流动梯度分离法从320 mg粗提物中制备得到雷公藤内酯醇（25 mg）、异新雷公藤酚内酯（77 mg）、hypolide（83 mg）、未知化合物（1 mg）、雷公藤酚内酯（42 mg）、雷公藤三萜甲醚VI（37 mg），纯度分别为98.2%、96.6%、98.1%、95.3%、95.1%和96.5%。通过高效液相色谱、质谱和核磁共振对它们的纯度和结构进行了鉴定。本文表明，当分析型CCC和制备型CCC由不同制造商提供且具有不同重力时，分析型CCC在优化参数和放大过程中起着重要作用，并且从分析型CCC到制备型CCC的放大过程仍然是可预测的。
