Abstract
The traditional methods used in natural product separation primarily target the major components and the minor components may thus be lost during the separation procedure. Consequently, it’s necessary to develop efficient methods for the preparative separation and purification of relatively minor bioactive components. In this paper, a LC/MS method was applied to guide the separation of crude extract of lotus (Nelumbo nucifera Gaertn.) leaves whereby a minor component was identified in the LC/MS analysis. Afterwards, an optimized pH-zone-refining CCC method was performed to isolate this product, identified as N-demethylarmepavine. The separation procedure was carried out with a biphasic solvent system composed of hexane-ethyl acetate-methyl alcohol-water (1:6:1:6, v/v) with triethylamine (10 mM) added to the upper organic phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase eluent. Two structurally similar compounds – nuciferine and roemerine – were also obtained from the crude lotus leaves extract. In total 500 mg of crude extract furnished 7.4 mg of N-demethylarmepavine, 45.3 mg of nuciferine and 26.6 mg of roemerine with purities of 90%, 92% and 96%, respectively. Their structures were further identified by HPLC/ESI-MSn, FTICR/MS and the comparison with reference compounds.
Highlights
Medicinal plants are viewed as important sources of new drugs since they contain numerous compounds with significant pharmacological potential, many of which may serve as lead compounds in the development of new drugs
Counter-current chromatography (CCC) is a solid support-free liquid-liquid partition chromatography technique [4] that eliminates the complications resulting from the use of a solid support matrix, such as irreversible adsorptive sample loss and deactivation, tailing of solute peaks, and contamination
CCC has been widely used as a preparative separation method in natural products chemistry [5,6,7]
Summary
Medicinal plants are viewed as important sources of new drugs since they contain numerous compounds with significant pharmacological potential, many of which may serve as lead compounds in the development of new drugs. Liquid chromatography tandem mass spectrometry (LC/MS) has been widely used to analyze complex mixtures, such as biological samples [1,2,3] This protocol is very effective at identifying a large number of constituents in a sample, especially when looking for minor components. Developed by Ito in the 1990s [8,9], pH-zone-refining CCC enables separation of organic acids and bases into a succession of highly concentrated rectangular peaks that elute according to their corresponding pKa values and hydrophobicities This method has been applied successfully to the analysis and separation of various natural and synthetic products, especially alkaloids and organic acids [10,11,12,13]. It has many important advantages compared with traditional CCC, including an over 10-fold increase in sample loading capacity, high concentration of fractions, concentration of minor impurities, etc
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