PH-Zone-refining counter-current chromatography for two new lipo-alkaloids separated from refined alkaline extraction of Kusnezoff monkshood root.

Jiadi Zhao,Xiaowen Sun,Zhong Zheng,Zhiqiang Liu,Limei Duan,Wuliji Ao,Jinghai Liu,Peihe Li,Liang Xu,Zifeng Pi

doi:10.1002/jssc.201901224

Abstract

An efficient and refined method for the separation of six aconitine‐type alkaloids from the alkaline prepared “Kusnezoff monkshood root” was established. It is the first study that two new lipo‐alkaloids were successfully isolated from refined sample by pH‐zone‐refining counter‐current chromatography rather than synthetic method. It was of interest that a great deal of lipo‐alkaloids was produced in crude extract from the alkalization of “Kusnezoff monkshood root.” A refined sample method was proposed to enrich two types of alkaloids by liquid–liquid extraction, i.e. lipo‐alkaloids and monoester‐diterpenoid alkaloids. The pH‐zone‐refining counter‐current chromatography was performed with an optimized two‐phase solvent system composed of n‐hexane‐ethyl acetate–methanol–water (3:5:4:5, v/v), where upper organic phase was added to 3 mmol/L triethylamine as a retainer and lower aqueous mobile phase was added to 3 mmol/L hydrochloric acid as an eluter. As a result, six aconitum alkaloids, including two lipo‐alkaloids (8‐lino‐14‐benzoylaconine, 8‐pal‐14‐benzoylaconine), three monoester‐diterpenoid alkaloids (14‐benzoylmesaconine, 14‐benzoylaconine, beyzoyldeoxyaconine), and one aconine alkaloid (neoline) were acquired from the plant at the same time. The anti‐inflammatory activities of the two new lipo‐alkaloids were compared to the six alkaloids in vitro, in cyclo‐oxygen‐ase‐2 inhibition assays. The separation mechanism of six alkaloids by pH‐zone‐refining counter‐current chromatography was illustrated.

Highlights

Aconitum kusnezoffii Reichb (Ranunculaceae, Chinese name: Cao Wu), a perennial herb that is widely distributed in the northern China, is one of the most centuries-old Chinese herbs possessing biological activities [1,2]
Six compounds were identified using high definition MS (HDMS) and the area normalization method was used to determine the purity of each component
During the extraction of crude alkaloids from alkaline prepared Kusnezoff monkshood root, we found that the components of alkaloids were monoester-diterpenoid alkaloids and lipo-alkaloids without poisonous diester-diterpenoid alkaloids

Summary

Introduction

Aconitum kusnezoffii Reichb (Ranunculaceae, Chinese name: Cao Wu), a perennial herb that is widely distributed in the northern China, is one of the most centuries-old Chinese herbs possessing biological activities [1,2]. The main bioactive constituents are aconitum alkaloids that have anti-inflammatory activity. These alkaloids contain three types: monoesterditerpenoid alkaloids, diester-diterpenoid alkaloids, and lipoalkaloids [3–5], which possess important biological activities and are difficult to obtain. Developing an efficient and rapid method to obtain and separate these alkaloids is of great importance elucidate their biological activities [6–8]. Many traditional separation methods are used to isolate alkaloids They usually require multiple steps, leading to time-consuming, expensive, and active constituent decomposition [9–13]. Compared with the conventional methods, high-speed counter-current chromatography can overcome the disadvantages caused by large sample loading and irreversible absorption in solid support matrix [14–17]. The high-speed counter-current chromatography is widely used to separate isomeric compounds and natural and synthetic products [10,18–21]. The acid–base equilibrium mechanisms of several target compounds can be elucidated [22–25]

Methods

Results

Discussion

Conclusion