Rapid preparative separation of six bioactive compounds fromGentiana crassicaulisDuthie ex Burk. using microwave-assisted extraction coupled with high-speed counter-current chromatography

Abstract

A rapid method combining microwave-assisted extraction and high-speed counter-current chromatography was applied for preparative separation of six bioactive compounds including loganic acid (I), isoorientin-4'-O-glucoside (II), 6'-O-β-d-glucopyranosyl gentiopicroside (III), swertiamarin (IV), gentiopicroside (V), sweroside (VI) from traditional Tibetan medicine Gentiana crassicaulis Duthie ex Burk. Microwave-assisted extraction parameters were predicted by central composite design-response surface methodology. That is, 5.0 g dried roots of G. Crassicaulis was extracted with 50 mL 57.5% aqueous ethanol under 630 W for 3.39 min. The extract (gentian total glycosides) was separated by high-speed counter-current chromatography with n-butanol/ethyl acetate/methanol/1% acetic acid water (7.5:0.5:0.5:3.5, v/v/v/v) using upper phase mobile in tail-to-head elution mode. 16.3 mg, 8.8 mg, 12.8 mg, 25.1 mg, 40.7 mg and 21.8 mg of compounds I–VI were obtained with high purities in one run from 500 mg of original sample. The purities and identities of separated components were confirmed using HPCL with photo diode array detection and quadrupole time-of-flight MS and NMR spectroscopy. The study reveals that response surface methodology is convenient and highly predictive for optimizing extraction process, microwave-assisted extraction coupled with high-speed counter-current chromatography could be an expeditious method for extraction and separation of phytochemicals from ethnomedicine.

An overview of the two-phase solvent systems used in the countercurrent separation of phenylethanoid glycosides and iridoids and their biological relevance

“…The least polar system t-butyl methyl etheracetonitrile-water (30:30:40) was used for the isolation of non-glycosidic iridoids. Some variations in the mode of operation of the CCC and CPC columns were observed during the survey, including step gradient elution (de Juliao et al 2010;Leitao et al 2015), enhancement of flow rate (Liang et al 2013b;Rho et al 2016), two-step CCS (Lei et al 2001b;Lemus et al 2015;Li et al 2008), elution-extrusion CCS (Chen et al 2011;Liang et al 2018) and recycling CCS (Chen et al 2011).…”

“…(Wu et al 2012). Ethyl acetate-n-butanol-methanol-1% (aq) acetic acid (0.5:7.5:0.5:3.5) was successfully used as solvent system for the separation of five iridoid glycosides (6 0 -O-b-D-glucopyranosyl gentiopicroside, gentiopicroside, loganic acid, sweroside and swertiamarin) from the traditional Tibetan medicine Gentiana crassicaulis Duthie ex Burk in a single run (Liang et al 2013b). Moreover, some acidified systems with nonpolar solvents (n-hexane or dichloromethane) were also employed with the purpose to isolate similar plant secondary metabolites.…”

“…As it can be easily deduced, the composition of the microwave-assisted extraction solvent was not chosen randomly, but after optimization experiments in which parameters such as the effect of ethanol concentration, irradiation time and microwave power on the yield of target compounds were statistically analyzed. Consequently, it is understandable why only one purification step was needed for the isolation of pure iridoids (Liang et al 2013b).…”

Elution–extrusion countercurrent chromatography separation of six pairs of isomeric iridoids from Cornus officinalis Sieb. et Zucc. guided by ion current extraction in mass spectrometry

“…The detection system conventionally adapted to determine target K C values and to monitor CCC running process is a UV detector (LC–UV and CCC–UV) . Inevitably, constituents with low quantity might fail to be detected.…”

Efficient methods for isolating five phytochemicals from Gentiana macrophylla using high‐performance countercurrent chromatography

“…To date, most studies have focused on isolating iridoids from Gentiana species using conventional column chromatography, but a few have explored using CCC methods to isolate iridoids such as gentiopicroside derivatives from G . crassicaulis and G . scabra .…”