Development of an efficient method for isolation and purification of bioactive Compounds from panax ginseng and rhodiola rosea using high performance counter current chromatography

Abstract

Traditional Chinese medicine has developed for several thousands of years and accumulated an abundant amount of human pharmacological information and experience as a large potential resource in drug discovery and development. Panax ginseng C. A. Meyer and Rhodiola rosea are both popular herbs worldwide and have many potential pharmacological effects including adaptogenic, antistress, and vasodilating, anticancer and anti-inflammatory. To improve the isolation and separation effect of the two traditional Chinese herbs for quantity and quality control herbal products in general, Separation and purification techniques using high performance counter-current chromatography (HPCCC) are widely applied for this purpose. High performance counter-current chromatography (HPCCC) is a form of liquid liquid chromatography with the higher “g” ” field, which the Brunel Institute for Bioengineering (BIB) team have developed to process scale. It would provide not only more rapid and high throughout isolation and purification process of crude sample, but also relatively simply volumetric and linear scale-up between all scales instruments. A rapid and convenient method for the separation and purification of five ginsenosides from crude sample of Panax ginseng by high-performance counter-current chromatography was successfully developed. One gradient method in normal phase mode was applied for the first separation step for the isolation of ginsenosides using a Spectrum CCC (73 ml coil volume, 1.6 mm bore) with an EtOAc/BuOH/aqueous 5mM ammonium acetate solvent system. The composition ratio of mobile phase changed from 3.5:0.5:4 (v/v) to 2.5:1.5:4 (v/v). Ginsenosides Rd, Rg1 and Rb1 were separated in less than 120 minutes with purities of 96.3% and 98.6% yield, 88.5% and 95.8% yield, 93.7% and 97.4% yield respectively and ginsenosides Rb2 and Rb3, Re and Rc were co-eluted. The final retention of stationary phase was 77.6%. Methylene chloride/methanol/aqueous 5mM ammonium acetate/isopropanol (6:3:4:3, v/v/v) solvent system was used for the isocratic separation of ginsenosides Re and Rc in the second separation step. The ginsenosides Rb2 and Rb3 have similar structures and properties as they are isomers and their aglycone moieties both belong to the same 20 (S) – protopanaxadiol. They were difficult to isolate using this solvent system. The purity of ginsenosides Re and Rc was assessed by HPLC–DAD to be 97.5% with 98.2% recovery and 92.6% with 96.3% recovery. These purified ginsenosides was identified using commercial reference standard on the HPLC and MS.A simple and efficient high-performance counter-current chromatography method for the simultaneous separation and purification for quantity and quality of the two biological compounds salidroside and tyrosol from Rhodiola rosea extract has been developed and scale up for the first time. The experiment were conducted initially by sample concentration, sample loading volume and flow rate study at analytical scale using a Mini HPCCC (17.7 ml coil, 0.8 mm bore) with a new MTBE/butanol /ACN/water (4:2:4:10 v/v/v) solvent system. Then linearly scaled up to the Midi-HPCCC (923 ml coil volume, 4.0 mm bore). Midi-HPCCC runs produced 28.2mg (purity 93%) of salidroside and 13.1mg (purity 96.5%) of tyrosol from the 1000mg crude Rhodiola rosea extract. with the throughput scaled up 50 times. The results demonstrate that HPCCC operating at the high flow and high “g” field is a reliable strategy for linear scale-up from analytical-scale high-throughput screening to preparative-scale.