School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203, China.
Research Department, Zhejiang Wolwo Natural Medicine Co. Ltd., 926 East Changhong Street, Deqing 313200, Zhejiang, China.
J Chromatogr A. 2022 Aug 2;1676:463205. doi: 10.1016/j.chroma.2022.463205. Epub 2022 Jun 9.
Chromatography is an essential method for separating natural products. In this study, we proposed the concept of 'relayed chromatography', based on the strategy of combining different chromatography with relayed resolution by in-situ concentration technique. The following chromatographic methods were used: high-speed countercurrent chromatography (HSCCC), silica gel liquid chromatography (silica gel LC), and reverse phase liquid chromatography (reverse phase LC). The proposed strategy was effectively applied to the preparative separation of naturally existing naphthaquinones. After the first separation stage (silica gel LC), acetylalkannin (1) was directly collected, while fractions 1, 4 and 5 were collected and respectively subjected to recycling CCC separation after concentration. Thus, deoxyshikonin (2), 8-O-methyl-11-O-acetylshikonin (6), β-acetoxyisovalerylalkannin (7) and alkannin (8) were collected. Fraction 2 was concentrated and injected in reverse phase LC separation. After collection of isobutyrylalkannin (3), the remaining effluent from reverse phase LC retained the peak resolution (R=0.45) and was injected into a recycling CCC elution. Finally, β, β-dimethylacrylalkannin (4), and isovalerylalkannin (5) were collected with sufficient resolution (R=1.25). Eight naturally occurring naphthaquinones were thus isolated from Arnebia euchroma. The purities of all the compounds were determined by HPLC to be > 90%, and the chemical structures were determined by spectral method. Among the aforementioned compounds, 8-O-methyl-11-O-acetylshikonin (6) was separated as a new compound from A. euchroma. In conclusion, the relayed strategy that retains the resolution of the previous chromatographic stage can improve CCC separation efficiency, which may expand the range of application of CCC combined with different chromatography to the separation of natural products.
色谱法是分离天然产物的重要方法。本研究基于结合不同色谱法和原位浓缩技术接力拆分的策略，提出了“接力色谱法”的概念。使用了以下色谱方法：高速逆流色谱（HSCCC）、硅胶液相等度色谱（silica gel LC）和反相液相色谱（reverse phase LC）。该策略有效地应用于天然存在的萘醌的制备分离。在第一分离阶段（硅胶 LC）后，直接收集乙酰基紫堇宁（1），而将馏分 1、4 和 5 收集并分别浓缩后进行循环 CCC 分离。因此，收集到去氧紫草素（2）、8-O-甲基-11-O-乙酰基紫草素（6）、β-乙酰氧基异戊酰基紫堇宁（7）和紫堇宁（8）。将馏分 2 浓缩并注入反相 LC 分离。收集异丁酰基紫堇宁（3）后，从反相 LC 流出物中保留峰分辨率（R=0.45）并注入循环 CCC 洗脱。最后，β，β-二甲基丙烯酰基紫堇宁（4）和异戊酰基紫堇宁（5）以足够的分辨率（R=1.25）收集。从Arnebia euchroma 中分离出八种天然萘醌。所有化合物的纯度均通过 HPLC 测定>90%，并通过光谱法确定其化学结构。在所提到的化合物中，8-O-甲基-11-O-乙酰基紫草素（6）是从 A. euchroma 中分离出的新化合物。总之，保留前一色谱阶段分辨率的接力策略可以提高 CCC 分离效率，这可能会扩大 CCC 与不同色谱法相结合应用于天然产物分离的范围。
