Studies On Separation And Purification Of Active Components From Traditional Chinese Medicinal Herbs (Cortex Fraxinus, Radix Linderae, Etc.) By High-speed Counter-current Chromatography

High-speed counter-current chromatography is a support-free liquid-liquid partitionchromatographic technique. The method provides an advantage over the conventional columnchromatography by eliminating the use of a solid support where an amount of stationaryphase is limited and dangers of irreversible adsorption from the support are inevitably present.In addtion, it has many other advantages such as high efficency, high loading capacity andhigh recovery, so it is very suitable for separation and purification of fragile cpmpounds fromtraditional Chinese herbs and other natural products. It has become one of the mostpreponderant separating and analytical techniques.The constituents are complexive in natural drugs. In order to study and apply the activecomponent in use, the separation and purification of these components are very important.Because the natural drugs contain all kinds of constituents and the content of the compoundsis different, so the separation and purification of these componets is an arduous andmeticulous work. As an advanced separation technique, HSCCC has been widely used for theseparation of active components from traditional Chinese herbs and other natural products inrecent years. Successful application of HSCCC has been reported for the purification offlavones, alkaloids, coumarins, anthraquinones and saponins.The present article developed a HSCCC method for the separation and purification ofcoumarins from Cortex Fraxinus;sesquiterpene lactone compounds from Radix Linderae;mangiferin, neomangiferin, cis-hinkiresinol and monoethyl-cis-hinkiresinol from RhizomaAnemarrhenae;flavones from Oroxylum Iindicum and Sophora japonica L.1. Preparative isolation and purification of coumarin compounds from Cortex Fraxinusby HSCCCHSCCC was successfully used for the isolation and purification of coumarin compoundsfrom Cortex Fraxinus, the Chinese herbal drug, n-butanol-methanol-0.5% acetic acid(5:1.5:5,v/v) was used as the two-phase solvent system. 14.3 mg of fraxin, 26.5 mg ofaesculin, 5.8 mg of fraxetin and 32.4 mg of aesculetin with the purity of 97.6%, 99.5%,97.2% and 98.7% respectively were obtained from 150 mg of crude extracts of cortexfraxinus in a single run. The structures of the isolated compounds were identified by 1H-NMRand 13C-NMR.2. Preparative isolation and purification of linderalactone and lindenenol from RadixLinderae by HSCCCHSCCC method for isolation and purification of sesquiterpene lactone compounds fromChinese traditional medicinal herb Radix Linderae (Wuyao in Chinese) was successfullyestablished by using light petroleum-ethyl acetate-methanol-water (5:5:6:4,v/v) as thetwo-phase solvent system. The upper phase of the two-phase solvent system was used as thestationary phase of HSCCC. 40.2 mg of linderalactone and 64.8 mg of lindenenol wereobtained from 450 mg of crude extracts in one-step separation. The purity of linderalactoneand lindenenol was 99.7% and 98.2%, respectively, as determined by HPLC. The structuresof linderalactone and lindenenol were identified by 1H-NMR and 13C-NMR.3. Preparative isolation and purification of four compounds from the Chinese medicinalherb Rhizoma Anemarrhenae by HSCCCHSCCC was applied to the separation and purification of mangiferin, neomangiferin,cis-hinkiresinol and (-)-4'-O-methylnyasol from the Chinese medicinal herb RhizomaAnemarrhenae. 500 mg of crude extracts were separated by using n-butanol-acetic acid (1%)(1:1,v/v) as the two-phase solvent system and yielded 35.3 mg of neomangiferin and 245.4mg of mangiferin. During this separation, cis-hinkiresinol and (-)-4'-O-methylnyasol werestill maintained in the stationary phase. The stationary phase was collected, evaporated todryness and separated with light petroleum-ethyl acetate-methanol-water (1:1:1.2:0.8,v/v) and1:1:1.4:0.6 (v/v) in gradient elution, which yielded 17.2 mg of cis-hinkiresinol and 12.4 mgof (-)-4'-O-methylnyasol. The purity of mangiferin, neomangiferin, cis-hinkiresinol and(-)-4'-O-methylnyasol was 96.3%, 98.0%, 97.3% and 98.2%, respectively, as determined byHPLC. The chemical structures of these components were identified by 1H-NMR and13C-NMR4. Preparative isolation and purification of baicalein and chrysin from the extracts ofOroxylum indicum by HSCCCHSCCC was successfully used for the isolation and purification of baicalein and chrysinfrom the extracts of Oroxylum indicum. From 300 mg of the crude extracts, 25.5 mg ofbaicalein and 36.6 mg of chrysin were obtained with a purity of 99.2% and 100%,respectively.HSCCC conditions: two-phase solvent system: light petroleum-ethyl acetate-methanol-water (5:5:5:5,v/v) and (5:5:7:3,v/v);solid phase: the upper phase of (5:5:5:5,v/v);mobilephase: lower phase of (5:5:5:5,v/v) and (5:5:7:3,v/v) used in gradient elution mode as follows:0-50 min, the volume ratio of (5:5:5:5,v/v) and (5:5:7:3,v/v) system was continuouslychanged from 100:0 to 0:100;flow rate: 2.0 ml min-1;revolution speed: 850 rpm;detectionwavelength: 254 nm;separation temperature: 25°C.HPLC conditions: Column: YWG ODS C18 column (200×4.6 mm ID. 10 μm);mobilephase: methanol-phosphoric acid (0.1%) used in gradient elution mode as follows: 0-30 min,the volume ratio of methanol and phosphoric acid (0.1%) was continuously changed from40:60 to 70:30;flow rate: 1.0 ml min-1;detection wavelength: 254 nm.5. Preparative isolation and purification of flavone compounds from Sophora japonica L.by HSCCCHSCCC and D-101 macroporous resin column were applied to the separation andpurification of genistein-7,4'-di-O-β-D-glucoside (I), genistein-7-O-β-D-glucopyranosde-4'-O-[(α-L-rhamnopyransoyl)-(1-2)-β-D-glucopyranosede] (II), kaempferol-3-O-β-D-sophoroside (III), quercetin-3-O-β-L-ramnopyranosyl-(1-6)-β-D-glucopyranoside (IV),genistein-4'-β-L-rhamnopyransoyl-(1-2)-α-D-glucopyranoside (V) and kaempferol-3-O-β-L-ramnopyranosyl-(1-6)-β-D-glucopyranoside (VI) from the Chinese medicinal herbSophora japonica L. 35 milligrams of sample 1 were separated by using n-butanol-acetic acid(1%) (1:1.v/v) as the two-phase solvent system and yield 7.7 mg compound I, 4.3 mgcompound II. One hundred milligrams of sample 2 was separated by using ethylacetate-n-butanol-acetic acid (1%) (1:0.16:1,v/v) as the two-phase solvent system and yielded4.5 mg compound III, 2.7 mg compound IV, 6.4 mg compound V and 1.2 mg compound VIwith the purity of 97%, 99%, 98%, 99%, 99% and 90%. The chemical structures of thesecomponents were identified by 1H-NMR and 13C-NMR.