Comprehensive separation and identification of chemical constituents from Apocynum venetum leaves by high-performance counter-current chromatography and high performance liquid chromatography coupled with mass spectrometry

High-performance counter-current chromatography (HPCCC) and high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) was efficiently utilized for the separation and identification of the chemical components with a wide range of polarity from the mixed extract of Chinese medicinal herb Apocynum venetum. For HPCCC separation, four sets of solvent systems, n-hexane-ethyl acetate-acetonitrile-water (1.5:3.5:2:4.5, v:v:v:v), ethyl acetate-methanol-water (5:2:5, v:v:v) and n-butanol-methanol-water (5:1:5, v:v:v) were used for the one-step separation by four stages. The HPCCC separation was initiated by filling the column with the lower phase of n-hexane-ethyl acetate-acetonitrile-water (1.5:3.5:2:5, v:v:v:v) as a stationary phase followed by elution with the upper phase of n-hexane-ethyl acetate-acetonitrile-water (1.5:3.5:2:5, v:v:v:v) to separate the hydrophobic compounds (tail to head). Then the mobile phase was switched to the upper phase of ethyl acetate-acetonitrile-water (5:3:7, v:v:v) to eluted the moderate hydrophobic compounds, then the mobile phase was switched to the upper phase of ethyl acetate-methanol-water (5:2:5, v:v:v) to eluted the moderate hydrophilic compounds, and finally the hydrophilic compounds still retained in the column was eluted by the upper phase of n-butanol-methanol-water (5:1:5, v:v:v). A total of 16 named compounds including adhyperforin, hyperforin, amentoflavone, biapigenin, quercetin, avicularin, acetylated isoquercetin, acetylated hyperoside, astragalin, trifolin, isoquercetin, hyperoside, querciturone, rutin, chlorogenic acid and quercetin-3-O-β-D-glucosyl-β-D-glucopyranoside were successfully separated via the four sets of solvent systems in one step operation for 130 min. The compounds separated by HPCCC were identified by comparing with mixed standards data of HPLC-MS as well as NMR data.     

Predictable and linear scale-up of four phenolic alkaloids separation from the roots of Menispermum dauricum using high-performance counter-current chromatography

This paper describes how distribution ratios were used for prediction of peak elution in analytical high-performance counter-current chromatography (HPCCC) to explore the method for separation and purification of bioactive compounds from the roots of Menispermum dauricum. Then important parameters related to HPCCC separations including solvent systems, sample concentration, sample loading volume and flow rate were optimized on an analytical Mini-DE HPCCC and finally linearly scaled up to a preparative Midi-DE HPCCC with nearly the same resolutions and separation time. Four phenolic alkaloids were for the first time obtained by HPCCC separation with a two-phase solvent system composed of petroleum ether–ethyl acetate–ethanol–water (1:2:1:2, v/v). This process produced 131.3 mg daurisolin, 197.1 mg dauricine, 32.4 mg daurinoline and 14.7 mg dauricicoline with the purity of 97.6%, 96.4%, 97.2% and 98.3%, respectively from 500 mg crude extract of the roots of M. dauricum in a one-step separation. The purities of compounds were determined by high-performance liquid chromatography (HPLC). Their structures were identified by electrospray ionization mass spectrometer (ESI-MS) and nuclear magnetic resonance (NMR).