Preparative isolation and purification of two isoflavones from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography

Two isoflavones, calycosin-7-O-beta-D-glycoside and formononetin-7-O-beta-D-glycoside, were separated from n-butanol extract of the root of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography in two steps using two different solvent systems composed of ethyl acetate-ethanol-n-butanol-water (30:10:6:50, v/v) and ethyl acetate-ethanol-water (5:1:5, v/v). From 200 mg of crude extract, calycosin-7-O-beta-D-glycoside (12 mg) and formononetin-7-O-beta-D-glycoside (10 mg) were isolated at over 95% purity by HPLC analyses, and their structures were identified by MS, 1H NMR and 13C NMR.     

Preparative isolation and purification of isoflavan and pterocarpan glycosides from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography

(3R)-(-)-7,2'-Dihydroxy-3',4'-dimethyl isoflavan-7-O-beta-D-glucopyranoside and (6aR, 11aR) 9,10-di-methoxypterocarpan-3-O-beta-D-glucopyranoside were separated from the ethyl acetate extract of the root of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography (HSCCC). A two-phase system composed of ethyl acetate-ethanol-acetic acid-water (4:1:0.25:5, v/v) was selected by analytical HSCCC. Preparative HSCCC yielded, from 100 mg of the partially purified extract, 50 mg of isoflavan glycoside and 10 mg of pterocarpan glycoside each at over 95% purity by high-performance liquid chromatography (HPLC) analysis. Their structures were identified by MS, 1H NMR and 13C NMR.     

Determination of five toxic alkaloids in two common herbal medicines with capillary electrophoresis

Calycosin was purified from an ethyl acetate extract of the root of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography. The separation was performed in two steps with a two-phase solvent system composed of n-hexane-chloroform-methanol-water (1:3:3:2, v/v). From 200 mg of the crude extract, 14.8 mg of calycosin was obtained at over 99% purity as determined by HPLC analysis, and its chemical structure was confirmed by MS, 1H and 13C nuclear magnetic resonance.     

Isolation of three sesquiterpene lactones from the roots of Cichorium glandulosum Boiss. et Huet. by high-speed counter-current chromatography

Because of the skeletal complexity and similarity of the polarity, little research was reported on the isolation of sesquiterpene lactones by high-speed counter-current chromatography (HSCCC). Herein, three sesquiterpene lactones were successfully purified from the ethyl acetate extract of the roots of the traditional Uyghur medicinal plant Cichorium glandulosum Boiss. et Huet. by HSCCC. The separation was performed in two steps with two solvent systems: n-hexane-ethyl acetate-methanol-water (1.5:5:2.75:5, v/v/v/v) and ethyl acetate-methanol-water (20:1:20, v/v/v). From 166 mg of the ethyl acetate extract, 19 mg of lactucopicrin was isolated with the first solvent system and 10 mg of 11beta,13-dihydrolactucin and 16 mg of lactucin were obtained with the second solvent system. All purified compounds were over 94% purity as determined by HPLC analysis, and these chemical structures were confirmed by (1)H NMR and (13)C NMR.     

Preparative isolation and purification of flavone compounds from sophora japonica L. by high-speed counter-current chromatography combined with macroporous resin column separation

High-speed counter-current chromatography combined with macroporous resin column separation was applied to the isolation and purification of genistein-7,4'-di-O-beta-D-glucoside (I), genistein-7-O-beta-D-glucopyranoside-4'-O-[(alpha-L-rhamnopyransoyl)-(1-2)-beta-D-glucopyranoside] (II), kaempferol-3-O-beta-D-sophoroside(III), quercetin-3-O-beta-L-ramnopyranosyl-(1 - 6)-beta-D-glucopyranoside (IV), genistein-4'-beta-L-rhamnopyransoyl-(1 - 2)-alpha-D-glucopyranoside (V), and kaempferol-3-O-beta-L-ramnopyranosyl-(1 - 6)-beta-D-glucopyranoside (VI) from the Chinese medicinal herb Sophora japonica L. The crude extracts from the pericarps of Sophora japonica L. were pre-separated on a D-101 macroporous resin column and divided into two parts as sample 1 and sample 2. An 80-mg portion of sample 1 was separated by using n-butanol-acetic acid (1%) (5:5, v/v) as the two-phase solvent system and yielded 30.1 mg of compound I, 23.3 mg of compound II. A 120 mg portion of sample 2 was separated by using ethyl acetate-n-butanol-acetic acid (1%) (5:0.8:5, v/v) as the two-phase solvent system and yielded 5.5 mg of compound III, 31.7 mg of compound IV, 37.4 mg of compound V, and 6.2 mg of compound VI. The purities of compounds I, II, III, IV, V, and VI were 98.7, 98.2, 97.8, 98.5, 99.3, and 98.9%, respectively, as determined by HPLC. The chemical structures of these components were identified by 1H-NMR and 13C-NMR.     

Preparative isolation and purification of four compounds from Cistanches deserticola Y.C. Ma by high-speed counter-current chromatography

Following a constituent enrichment step on a silica gel column, four phenyl-ethanoid glycosides were successfully isolated from Cistanches deserticola and purified by preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of ethyl acetate-n-butanol-ethanol-water (40:6:6:50, v/v/v/v). A total of 30.9 mg acteoside, 13.0 mg isoacteoside, 12.5 mg syringalide A 3'-α-L-rhamnopyranoside and 7.2 mg 2'-acetylacteoside with purity of higher than 95%, as determined by HPLC-ELSD, were obtained in one-step separation from 297 mg of Cistanche deserticola extract, respectively. Their structures were identified by HR-MS, 1H-NMR and 13C-NMR.     

Rapid separation of three glucosylated resveratrol analogues from the invasive plant Polygonum cuspidatum by high‐speed countercurrent chromatography

Three glucosylated resveratrol analogues (piceid, piceatannol glucoside, resveratroloside) were successfully isolated from the crude MeOH extract of the invasive plant species Polygonum cuspidatum by semi‐preparative high‐speed countercurrent chromatography with a two‐phase solvent system composed of cyclohexane‐ethyl acetate‐methanol‐water (1:5:1:5, v/v/v/v). Piceid (23 mg), resveratroloside (17 mg), piceatannol glucoside (15 mg) of purities over 80% were isolated from 500 mg crude MeOH extract in one step. Subsequent passage over a SPE column was used to quickly bring their purities to over 90%. The purities were determined by HPLC analysis and their structures were elucidated by proton nuclear magnetic resonance (¹H‐NMR), HMBC, ESI‐MS and HR‐MS.     

高速逆流色谱法分离纯化黄芪中的芒柄花素和毛蕊异黄酮

采用高速逆流色谱法(HSCCC),以正己烷-氯仿-甲醇-水组成二相系统作为固定相与流动相,对黄芪的乙酸乙酯粗提 物进行了分离纯化。 结果发现:以正己烷-氯仿-甲醇-水(体积比为1.5∶3∶3∶2)组成的系统可以从黄芪的乙酸乙酯粗 提物中分离出毛蕊异黄酮,纯度可达95％以上,并可以初步纯化芒柄花素;接着用正己烷-氯仿-甲醇-水(体积比为4∶4∶5 ∶4)组成的系统进一步纯化芒柄花素,其纯度达95％以上。利用该方法,可以对中药黄芪中的异黄酮进行快速的分离和纯 化。     

Preparative isolation of flavonoid compounds from Oroxylum indicum by high‐speed counter‐current chromatography by using ionic liquids as the modifier of two‐phase solvent system

A preparative high‐speed counter‐current chromatography method for isolation and purification of flavonoid compounds from Oroxylum indicum was successfully established by using ionic liquids as the modifier of the two‐phase solvent system. Two flavonoid compounds including baicalein‐7‐O‐diglucoside and baicalein‐7‐O‐glucoside were purified from the crude extract of O. indicum by using ethyl acetate–water–[C₄mim][PF₆] (5:5:0.2, v/v) as two‐phase solvent system. 36.4 mg of baicalein‐7‐O‐diglucoside and 60.5 mg of baicalein‐7‐O‐glucoside were obtained from 120 mg of the crude extract. Their purities were 98.7 and 99.1%, respectively, as determined by HPLC area normalization method. The chemical structures of the isolated compounds were identified by ¹H‐NMR and ¹³C‐NMR.     

Preparative separation and purification of bufadienolides from Chinese traditional medicine of ChanSu using high‐speed counter‐current chromatography

A preparative high‐speed counter‐current chromatography method for isolation and purification of bufadienolides from ChanSu was developed by using a stepwise elution with two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water at the ratios of 4:6:2:4 v/v, 4:6:2.5:4 v/v and 4:6:3.2:4 v/v. A total of 3.8 mg of gamabufotalin (1), 7.2 mg of arenobufagin (2), 3.4 mg of telocinobufagin (3), 5.3 mg of bufotalin (4), 8.5 mg of cinobufotalin (5) and 8 mg of bufalin (6) were obtained in one‐step separation from 80 mg of the crude extract with purity of 92.7, 96.7, 87.2, 97.3, 94.9 and 99.4%, respectively. Their chemical structures were identified on the basis of ¹H‐NMR and ¹³C‐NMR technology.     

Isolation and purification of macrocyclic components from Penicillium fermentation broth by high‐speed counter‐current chromatography

In this paper, high‐speed counter‐current chromatography (HSCCC), assisted with ESI‐MS, was first successfully applied to the preparative separation of three macrolide antibiotics, brefeldin A (12.6 mg, 99.0%), 7′‐O‐formylbrefeldin A (6.5 mg, 95.0%) and 7′‐O‐acetylbrefeldin A (5.0 mg, 92.3%) from the crude extract of the microbe Penicillium SHZK‐15. Considering the chemical nature and partition coefficient (K) values of the three target compounds, a two‐step HSCCC isolation protocol was developed in order to obtain products with high purity. In the two‐step method, the crude ethyl acetate extract was first fractionated and resulted in two peak fractions by HSCCC using solvent system n‐hexane/ethyl acetate/methanol/water (HEMWat) (3:7:5:5 v/v/v/v), then purified using solvent systems HEMWat (3:5:3:5 v/v/v/v) and HEMWat (7:3:5:5 v/v/v/v) for each fraction. The purities and structures of the isolated compounds were determined by HPLC, X‐ray crystallography, ESI‐MS and NMR. The results demonstrated that HSCCC is a fast and efficient technique for systematic isolation of bioactive compounds from the microbes.     

Accelerated solvent extraction and pH‐zone‐refining counter‐current chromatographic purification of yunaconitine and 8‐deacetylyunaconitine from Aconitum vilmorinianum Kom

This study aimed to seek an efficient method to extract and purify yunaconitine and 8‐deacetylyunaconitine from Aconitum vilmorinianum Kom. by accelerated solvent extraction combined with pH‐zone‐refining counter‐current chromatography. The major extraction parameters for accelerated solvent extraction were optimized by an orthogonal test design L₉ (3)⁴. Then a separation and purification method was established using pH‐zone‐refining counter‐current chromatography with a two‐phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (5:5:2:8, v/v) with 10 mM triethylamine in the upper phase and 10 mM HCl in the lower phase. From 2 g crude extract, 224 mg of 8‐deacetylyunaconitine (I) and 841 mg of yunaconitine (II) were obtained with a purity of over 98.0%. The chemical structures were identified by ESI‐MS and ¹H and ¹³C NMR spectroscopy.     

An Efficient Method for Extraction, Separation and Purification of Naphthoquinone Pigments from Lithospermum erythrorhizon Sieb. et Zucc. by SFE and HSCCC

Supercritical fluid extraction was used to extract naphthoquinone pigments from Lithospermum erythrorhizon Sieb. et Zucc. The crude extracts were separated and purified by high-speed counter-current chromatography with light petroleum–ethyl acetate–methanol–water (5:5:8:2, v/v) as the two-phase solvent system. Three kinds of naphthoquinone pigments including 17.6 mg of β-hydroxyisovalerylshikonin (I), 17.6 mg of acetylshikonin (II), and 19.7 mg of isobutyrylshikonin (III) were obtained from 150 mg crude sample. The purity of these compounds was 96.7, 99.3 and 95.5%, respectively, as determined by liquid chromatograph. Their structures were identified by ¹H NMR and ¹³C NMR.     

Preparative isolation and purification of syringin and edgeworoside C from Edgeworthia chrysantha Lindl by high-speed counter-current chromatography

The bioactive compound syringin along with edgeworoside C were separated from the n-butanol extract of the stems and barks of Edgeworthia chrysantha Lindl (E. papyrifera) by high-speed counter-current chromatography (HSCCC) while it was difficult to purify each compound by silica gel column chromatography. Syringin was isolated from this plant for the first time. The two-phase solvent system used was composed of ethyl acetate-ethanol-water at an optimized volume ratio of 15:1:15 (v/v/v). Preparative HSCCC yielded, from 110mg of the partially purified extract, 28mg of syringin and 45 mg edgeworoside C each at over 96% purity by high-performance liquid chromatography analysis. Their structures were identified by electron impact ionization MS, 1H NMR and 13C NMR.     

One step purification of flavanone glycosides from Poncirus trifoliata by centrifugal partition chromatography

Flavanone glycosides were successfully separated from the crude extract of Poncirus trifoliata by preparative centrifugal partition chromatography with a two-phase solvent system composed of ethyl acetate-acetonitrile-water (3:2:5, v/v/v). Naringin (50.0 mg), neoponcirin (16.8 mg), and poncirin (71.9 mg) were purified from the 524 mg crude extract in only one step. The purities of the isolated compounds were determined to be over 90% by HPLC analysis and their structures were elucidated by (1)H NMR, (13)C NMR, and ESI-MS.     

Separation and purification of furanocoumarins from Toddalia asiatica (L.) Lam. using microwave-assisted extraction coupled with high-speed counter-current chromatography

A method of microwave-assisted extraction coupled with high-speed counter-current chromatography was established for separation and purification of isopimpinellin, pimpinellin and phellopterin from Toddalia asiatica (L.) Lam. The conditions of MAE including the extraction solvent, size of sample, solid/liquid ratio, extraction temperature and extraction time were optimized by a mono-factor test. That is, 2.0 g dried powder of T. asiatica (L.) Lam of 0.30-0.15 mm size was extracted with 20 mL (solid/liquid ratio of 1:10, g/mL) methanol under 50 °C for 1 min. The crude extract was separated and purified by high-speed counter-current chromatography with hexane-ethyl acetate-methanol-water (5:5:5.5:4.5, v/v/v/v) solvent system. 0.85 mg/g of isopimpinellin, 2.55 mg/g of pimpinellin and 0.95 mg/g of phellopterin were obtained from original sample in one-step within 240 min, the purity determined by high performance liquid chromatography was 95.0%, 99.1% and 96.4%, respectively. Their chemical structures were further identified by mass spectroscopy and nuclear magnetic resonance spectroscopy. The results demonstrated that microwave-assisted extraction coupled with high-speed counter-current chromatography was a feasible, economical and efficient technique for rapid extraction, separation and purification of effective compounds from natural products.     

Simultaneous analysis of seven astragalosides in Radix Astragali and related preparations by liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry

A method has been developed for the qualitative and quantitative analysis of pharmacologically active astragalosides isolated from several species of the genus Astragalus by high performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry. Seven astragalosides in Radix Astragali and their commercial pharmaceutical preparations were analyzed using the developed method. The extracted ion current chromatograms were obtained from the total ion current chromatogram using the m/z of [M+Na]+ ions produced by target compounds for peak determination. The limits of detection and limits of quantification were in the range of 0.10-0.22 ng and 0.22-0.52 ng in full scan mode, respectively. All calibration curves showed good linear regression (r2 > or = 0.9965) within the test range. The overall intra- and inter-day precision was less than 2.86% for peak area and the accuracy was higher than 92.9% on using ginsenoside I as internal standard. The assay was successfully utilized to analyze the major biologically active astragalosides in six samples of Astragalus membranaceus (Fisch.) Bge var. mongholicus (Bge.) Hsiao. and eight commercial preparations. The overall results demonstrate that this method is simple, selective, and suitable for the quality control of Chinese medicine and their preparation in the low nanogram range.     

Efficient new method for extraction and isolation of three flavonoids from Patrinia villosa Juss. by supercritical fluid extraction and high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of orotinin, orotinin-5-methyl ether and licoagrochalcone B from Patrinia villosa was performed. The optimization of parameters including pressure, temperature, modifier and sample particle size on yield was carried out using an analytical-scale SFE system. The process was then scaled up by 100 times using a preparative SFE system under the optimized conditions of 25 MPa, 45 degrees C, a sample particle size 40-60 mesh and modified CO2 with 20% methanol. The yield of the preparative SFE was 2.82% (crude extract I) and the combined yield of orotinin, orotinin-5-methyl ether and licoagrochalcone B was 0.82 mg/g of dry sample mass. Then the crude extract I was re-dissolved in methanol and methanol soluble fraction (crude extract II, 0.17%) was obtained, which was successfully isolated and separated by a preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:6:6:6, v/v/v/v) by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml/min after 3 h. The target compounds isolated and purified by HSCCC were analyzed by high performance liquid chromatography. The separation produced total of 38.2 mg of orotinin at 99.2% purity, 19.8 mg of orotinin-5-methyl ether at 98.5% purity and 21.5 mg of licoagrochalcone B at 97.6% purity from 400 mg of the crude extract in a one-step separation. The recoveries of orotinin, orotinin-5-methyl ether and licoagrochalcone B were 91.1, 91.6 and 90.3%, respectively, and the chemical structure identification was carried out by UV, IR, MS, 1H NMR and 13C NMR.     

Counter‐current chromatographic method for preparative scale isolation of picrosides from traditional Chinese medicine Picrorhiza scrophulariiflora

Apocynin, androsin, together with picroside I, II and III from crude extracts of Picrorhiza scrophulariiflora were isolated by means of high‐speed counter‐current chromatography (CCC) combining elution‐extrusion (EE) and cycling‐elution (CE) approach. The EECCC took full advantages of the liquid nature of the stationary phase for a complete sample recovery and extended the solute hydrophobicity window, while CECCC showed its unique advantage in achieving effective separation of special compounds through preventing stationary phase loss. In the present work, the biphasic liquid system composed of n‐hexane/ethyl acetate/methanol/water (1:2:1:2, v/v/v/v) was used for separation of apocynin and androsin, ethyl acetate/n‐butanol/water/formic acid (4:1:5:0.005, v/v/v/v) for picroside I, II and III. However, due to the extremely similar K values (K₁/K₂≈1.2), picroside I and III were always eluted together by several biphasic solvent systems. In this case, the CECCC exhibited great superiority and baseline separated in the sixth cycle using ethyl acetate/water (1:1, v/v) as biphasic liquid system. Each fraction was analyzed by UPLC‐UV and ESI‐MS analysis, and identified by comparing with the data of reference substances. Compared with classical elution, the combination of EE and CE approach exhibits strong separation efficiency and great potential to be a high‐throughput separation technique in the case of complex samples.     

Preparative isolation and purification of antioxidative diarylheptanoid derivatives from Alnus japonica by high-speed counter-current chromatography

This study employed the online HPLC-2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)(+) bioassay to rapidly determine the antioxidant compounds occurring in the crude extract of Alnus japonica. The negative peaks of the ABTS(+) radical scavenging detection system, which indicated the presence of antioxidant activity, were monitored by measuring the decrease in absorbance at 734 nm. The ABTS(+)-based antioxidant activity profile showed that three negative peaks exhibited antioxidant activity. High-speed counter-current chromatography (HSCCC) was used for preparative scale separation of the three active peaks from the extract. The purity of the isolated compounds was analyzed by HPLC and their structures were identified by (1)H- and (13)C-nuclear magnetic resonance spectrometry (NMR), heteronuclear multiple bond correlation (HMBC), and heteronuclear single quantum correlation (HSQC). Two solvent systems composed of n-hexane/ethylacetate/methanol/water (4:6:4:6, v/v) and of ethyl acetate/methanol/water (1:0.1:1, v/v) were performed in high-speed counter-current chromatography. Consequently, a total of 527 mg of hirsutanonol 5-O-β-D-glucopyranoside, 80.04 mg of 3-deoxohirsutenonol 5-O-β-D-glucopyranoside, and 91.0 mg of hirsutenone were obtained with purity of 94.7, 90.5, and 98.6%, respectively.