The Active Ingredient In Traditional Chinese Medicine In The Application Of High-speed Countercurrent Chromatography And Ms Structure Research

High-speed counter-current chromatography (HSCCC) is a new and high efficiency form of liquid-liquid partition chromatography, which was developed by the Dr Yoichiro Ito of national sanitation academe of USA in 1982. Compared with conventional chromatographic methods, HSCCC does not use any solid matrix.Therefore it eliminates irreversible adsorption, decomposition, contamination and many other disadvantages owing to chemical reactions of samples with solid support. In addition, it has many advantages, such as wide-range, high speed, high loading capacity and high recovery and so on. So HSCCC have been used in the fields of biology, medicament, foodstuff, material, environment protection and so on. Especially in the aspect of separation and purification of effective compounds.With the history of more than 2000 years, Chinese traditional medicine(CTM) is the quintessence of China and help Chinese to prevent and cure disease all along. But since China enter WTO, CTM is difficult to achieve the modernization of Chinese traditional medicine because it have complex compounds and lacks the quality control,clear standard of traditional Chinese herbs. In order to put the axe in the helve, we must extract, isolate and identify of the active components from CTM.Electrospray ionization mass spectrometry (ESI-MSn) is a soft ionization technique that can be used to produce ions even from thermally labile, non-volatile, polar compounds. Because it can provides a direct means for measurement of molecular weight, so it has been widely used for the analysis of natural products in either positive or negative ion mode.In the present paper, under the optimized experiment conditions and difficult two-phase solvent systems, active components from the ethyl acetate or n-butanol crude extract of common traditional Chinese herbs(Exocarpium Citri Grandis,Hypericum japonicum Thumb,Belamcanda chinensis)and the Shi-Zhen academe of SUN YAT-SEN University were separated and purified. The purity of the obtained compounds are all above 90% determined by HPLC analysis and their chemical structures were ESI-MSn.This also prove HSCCC have many advantages over traditional chromatographic methods such as fast, convenience, simple, high purity compound and so on, in the aspect of separation and purification of effective compounds.A two-phase solvent system composed of Hex-BuOH-MeOH-0.5% acetic acid (1:3:1:4, v/v) was used as the two-phase solvent system, stationary phase as upper organic phase, mobile phase as lower aqueous phase;at a flow-rate of 1.5 mL·min^-1, while the preparative HSCCC instrument was run at a revolution speed of 800 rpm·min^-1.From 50 mg of n-butanol crude extracts of Exocarpium Citri Grandis 28.8 mg of naringin, 1.4 mg of rhoifolin, and 5.7 mg of an unknown compound were obtained with the purity of 97.1%, 95.5 %, and 97.5%, respectively, in a single run of 550min.A two-phase solvent system composed of n-Hex-EtOAc-MeOH-0.5% acetic acid at the volume ratio of 0.25:5:0.25:5(v/v) was used,stationary phase as upper organic phase, mobile phase as lower aqueous phase;at a flow-rate of 2 mL·min^-1 during 0～185min and of 3 mL·min^-1 during185～400min, while the preparative HSCCC instrument was run at a revolution speed of 800 rpm.minl.And the effluent was detected at 254nm. From 50 mg of ethyl-acetate crude extracts of Hypericum japonicum Thumb, 4.2 mg of isoquercitrin, 5.2 mg of quercitrin, 2.5 mg of Taxfolin -7-O-rhamnose, and 0.8 mg unknown compound 1, 0.2mg unknown compound 2 were obtained with the purity of 99.1%,97.5%,90.3 %,93.5%and 95.3% respectively, in a single run.A two-phase solvent system composed of n-Hex-EtOAc-MeOH-0.5% acetic acid (4:5:4:5, v/v) was used as the two,phase solvent system, stationary phase as upper organic phase, mobile phase as lower aqueous phase;at a flow-rate of 2 mL.min1, while the preparative HSCCC instrument was run at a revolution speed of 800 rpm·min^-1.From 50 mg of non-polar ethyl-acetate crude extracts of Belamcanda chinensis, 1.3 mg of Irigenin and 1.1 mg of irilin D were obtained with the purity of 94.6%, and 93.2%, respectively, in a single run of 250mim. A two-phase solvent system composed of n-Hex-EtOAc-MeOH-0.5% acetic acid (0.5:5:5, v/v) was used as the two-phase solvent system, stationary phase as upper organic phase, mobile phase as lower aqueous phase;at a flow-rate of 2 mL·min^-1, while the preparative HSCCC instrument was run at a revolution speed of 800 rpm·min^-1.From 50 mg of polar ethyl-acetate crude extracts of Belamcanda chinensis, 0.8 mg of 3'-hydroxytectoridin, 0.3 mg and 2.6mg of two unknown compounds were obtained with the purity of 94.6%, and 93.2%, respectively, in a single run of 190mim.A two-phase solvent system composed of n-Hexane-n-butanol-Methanol-Water(1:5.1:5, v/v) was used as the two-phase solvent system, stationary phase as upper organic phase, mobile phase as lower aqueous phase;at a flow-rate of 2 mL·min^-1, while the preparative HSCCC instrument was run at a revolution speed of 800 rpm·min^-1.From 50 mg of n-butanol crude extracts of Fructus aurantii, 0.5 mg and 0.8mg of two unknown compounds were obtained with the purity of 97.8% and 93.1%, respectively, in a single run of 250mim.A two-phase solvent system composed of CC14-CHC13-MeOH-0.5% acetic acid (2.5:1.5:4:2, v/v) was used as the two-phase solvent system, stationary phase as upper organic phase, mobile phase as lower aqueous phase;at a flow-rate of 1 mL·min^-1, while the analytical HSCCC instrument was run at a revolution speed of 1400 rpm·min^-1.From 5 mg of the sample of Shi-Zhen academe of SUN YAT-SEN University, 1.8 mg,0.6 mg and 0.2mg of three unknown compounds were obtained with the purity of 88.2 %, 83.6 % and 99% respectively, in a single run of 140mim.