Qizhen Du

Isolation of dammarane saponins from Panax notoginseng by high-speed counter-current chromatography

The Chinese phytomedicinal formulation Sanqi Zongdai Pian, traditionally prepared from crude extracts from roots of Panax notoginseng (Araliaceae), contains highly polar dammarane saponins which were separated at a preparative scale using high-speed counter-current chromatography (HSCCC). In each operation, 283 mg methanolic extract of five tablets was separated and yielded pure 157, 17, 13 and 56 mg of ginsenoside-Rb1, notoginsenoside-R1, ginsenoside-Re and ginsenoside-Rg1, respectively, n-hexane-n-butanol-water (3:4:7, v/v/v) was used for the two-phase solvent system of the HSCCC separation. The chemical structures of three ginsenosides and one notoginsenoside were elaborated by means of electrospray ionization MS-MS and NMR analysis.

Separation of andrographolide and neoandrographolide from the leaves of Andrographis paniculata using high-speed counter-current chromatography

The bioactive diterpenes andrographolide and neoandrographolide from the leaves of Andrographis paniculata NEES (Acanthaceae) were successfully separated by counter-current chromatography. A single 280-min separation yielded 189 mg of 99.9% andrographolide and 9.5 mg of 98.5% neoandrographolide applying water-methanol-ethyl acetate-n-hexane (2.5:2.5:4:1) solvent system. Structure confirmation was done by electrospray MS, one-dimensional NMR experiments, circular dichroism, optical rotation dispersion, and specific optical rotation [alpha]D.

Purification of (+)-dihydromyricetin from leaves extract of Ampelopsis grossedentata using high-speed countercurrent chromatograph with scale-up triple columns

Purification of (+)-dihydromyricetin from an extract (16 g) of leaves of Ampelopsis grossedentata was performed using a preparative triple-column countercurrent chromatograph. With a solvent system composed of n-hexane-ethyl acetate-methanol-water (1:3:2:4, v/v) 11.3 g of (+)-dihydromyricetin was obtained at a high purity of over 99% by HPLC at 254 nm in 9 h.

Isolation of a polysaccharide with anticancer activity from Auricularia polytricha using high-speed countercurrent chromatography with an aqueous two-phase system.

Polysaccharides from a crude extract of Auricularia polytricha were separated by high-speed countercurrent chromatography (HSCCC). The separation was performed with an aqueous two-phase system of PEG1000-K2HPO4-KH2PO4-H2O (0.5:1.25:1.25:7.0, w/w). The crude sample (2.0 g) was successfully separated into three polysaccharide components of AAPS-1 (192 mg), AAPS-2 (137 mg), and AAPS-3 (98 mg) with molecular weights of 162, 259, and 483 kDa, respectively. These compounds were tested for growth inhibition of transplanted S180 sarcoma in mice. AAPS-2 had an inhibition rate of 40.4%. The structure of AAPS-2 was elucidated from partial hydrolysis, periodate oxidation, acetylation, methylation analysis, and NMR spectroscopy (1H, 13C). These results showed AAPS-2 is a polysaccharide with a backbone of (1-->3)-linked-beta-d-glucopyranosyl and (1-->3, 6)-linked-beta-D-glucopyranosyl residues in a 2:1 ratio, and has one terminal (1-->)-beta-D-glucopyranosyl at the O-6 position of (1-->3, 6)-linked-beta-D-glucopyranosyl of the main chain.

Preparation of Ursane Triterpenoids from Centella asiatica Using High Speed Countercurrent Chromatography with Step‐Gradient Elution

Abstract Pentacyclic triterpene aglycones and glycosides of the ursane type were successfully separated from 600 mg of a polar extract from Centella asiatica (Apiaceae) by high speed countercurrent chromatography (HSCCC), applying a mobile phase gradient with a step‐wise increase of elution strength. The lower phase of the biphasic liquid system composed of n‐hexane–n‐butanol–0.05 M NaOH (5:1:6, v/v/v) was used as the stationary phase, the upper phase was used as the initial mobile phase. The mobile phase was changed systematically into 1:1, 1:2 and 1:4 consisting of n‐hexane–n‐butanol saturated by 0.05 M NaOH. The separation mainly yielded five fractions with asiatic acid (18 mg), madecassic acid (13 mg), asiaticoside (140 mg), and madecassoside (75 mg). The chemical structures of the four compounds were confirmed by means of electrospray ionization ion trap multiple mass spectrometry (ESI–MS–MS) and NMR analysis.

Purification of icariin from the extract of Epimedium segittatum using high-speed counter-current chromatography

Icariin was purified from the extract of Epimedium segittatum by high-speed counter-current chromatography with a two-phase solvent system composed of n-hexane-n-butanol-methanol-water (1:4:2:6, v/v). We used two multilayer coil planet centrifuges with different capacities. A 300 mg amount of the extract was separated using a semipreparative instrument equipped with a 230-ml capacity column to yield 103 mg of icariin at 86.2% purity. An 8 g amount of the extract was separated with a large preparative instrument equipped with a 2460-ml capacity column to produce 2.45 g of icariin at 85.7% purity. From these fractions over 98% pure icariin was obtained by recrystallization with water.

Isolation and identification of phenolic compounds in the fruit of Benincasa hispida by HSCCC

Abstract Separation of phenolic components from the fruit Benincasa hispida was conducted using high‐speed countercurrent chromatography (HSCCC) with two solvent systems, n‐hexane–n–butanol–methanol–water (10:16:5:20, v/v) and n‐hexane–ethyl acetate–methanol–water (1:1:1:1, v/v). Three compounds, astilbin (120 mg), catechin (103 mg), and naringenin (79 mg) were obtained from the separation of 2 g of crude phenolic component extract of the fruit. Chemical structures of these three compounds were confirmed by electrospray ionization ion trap multiple mass spectrometry (ESI‐MS) and NMR.

Preparation of Three Flavonoids from the Bark of Salix alba by High‐Speed Countercurrent Chromatographic Separation

Abstract The main flavonoids from the bark extract of Salix alba (Salicaceae) were separated on preparative scale using high‐speed countercurrent chromatography (HSCCC). In each separation, 1.0 g crude extract was applied to yield pure eriodictyol (120 mg), 5,7‐dihydroxychromen‐4‐one (29.5 mg), and naringenin (50 mg), respectively, while water–methanol–ethyl acetate–n‐hexane (3:2:2:2, v/v) was used for a two‐phase solvent system. The chemical structures of three flavonoids were elucidated by means of electrospray ionization ion trap multiple mass spectrometry (ESI‐MS–MS), as well as 1H‐, 13C‐, and DEPT‐NMR spectroscopy.

Preparation of Solanesol from a Tobacco Leaf Extract Using High Speed Countercurrent Chromatography

Abstract Separation of solanesol from the tobacco leaves extract was performed by high speed countercurrent chromatography using a solvent system composed of petroleum ether‐ethanol‐methanol (200∶1∶100, v/v). In each separation, one gram of the tobacco leaves extract was injected to the multilayer coil separation column to yield 121 mg of solanesol with a purity of 90.7%. The obtained solanesol was identified by ESI‐MS, 1H, and 13C‐NMR.

Large Convoluted Tubing for Scale‐Up of Slow Rotary Countercurrent Chromatograph

Abstract The chromatographic performance of 1.5 cm I.D. convoluted teflon tubing was studied at low rotary speeds ranging from 34 to 105 rpm. Results were as follows: (i) many solvent systems showed excellent retention of the stationary phase even at a high flow rate of the mobile phase; (ii) a good mixing of the two phases gave efficient solute partitioning resulting in high peak resolution; (iii) increasing the sample size caused only minor peak broadening; and (iv) increase in peak width and resolution using longer columns is predictable. Our data show that it is feasible to scale up a slow rotary countercurrent chromatograph with 1.5 cm I.D. convoluted tubing for industrial use.