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.     

Combination of supercritical fluid extraction with counter‐current chromatography to isolate anthocyanidins from the petals of Chaenomeles sinensis based on mathematical calculations

Supercritical fluid extraction (SFE) coupled with high‐speed counter‐current chromatography (HSCCC) was successfully used for the extraction and on‐line isolation of the anthocyanidins from the petals of Chaenomeles sinensis in two stages. The SFE parameters were optimized by an orthogonal test, and the solvent systems of SFE and HSCCC were calculated and optimized with the help of a multiexponential function model. In the first stage, the lower phase of the solvent system of n‐butanol/tert‐butyl methyl ether/acetonitrile/0.1% aqueous TFA (0.715:1.0:0.134:1.592, v/v/v/v) was used as both the SFE modifier and the HSCCC stationary phase, after extraction, the extractants were pumped into HSCCC column, and then eluted with the corresponding upper phase to isolate the moderately hydrophobic compounds. In the second stage, the upper phase of the solvent system of n‐butanol/ethyl acetate/acetonitrile/0.1% aqueous TFA (1.348:1.0:0.605:2.156, v/v/v/v) was used as both the SFE modifier and the HSCCC stationary phase, followed by elution with the corresponding lower phase to separate the hydrophobic compounds. With the help of two‐stage SFE/HSCCC, six compounds including delphinidin‐3‐O‐glucoside (Dp3G), cyanidin‐3‐O‐glucoside (Cy3G), peonidin‐3‐O‐glucoside (Pn3G), delphinidin (Dp), peonidin (Pn), and malvidin (Mv) were successfully separated within 300 min. The targeted compounds were identified by UV spectrophotometry, MS, and NMR spectroscopy. This research has opened up great prospects for the industrial application of SFE–HSCCC for the automatic extraction and separation of unstable compounds.     

Application of supercritical fluid extraction coupled with counter–current chromatography for extraction and online isolation of unstable chemical components from Rosa damascena

Supercritical fluid extraction (SFE) coupled with high‐speed counter‐current chromatography (HSCCC) was successfully used for the extraction and online isolation of the unstable compounds from Rosa damascene in a single extraction and separation operation in two stages. The solvent systems of SFE/HSCCC were optimized with the help of multiexponential function model. At the first stage, the upper phase of the solvent system of n‐butanol–tert‐butyl methyl ether–acetonitrile–0.1% aqueous TFA (1.7:1.0:0.8:4.0, v/v/v/v) was used as both the SFE entrainer and the HSCCC stationary phase, and the target compounds were eluted with the corresponding lower phase to separate the hydrophobic compounds. At the second stage, the upper phase of the solvent system of n‐hexane–ethyl acetate–methanol–water (3.2:1.0:2.8:2.6, v/v/v/v) was used as both the SFE entrainer and the HSCCC stationary phase, followed by elution with the corresponding lower phase to separate the moderate hydrophobic compounds. Six compounds including formononetin, delphinidin, cyaniding, 5,6,4′‐trihydroxy‐7,8‐dimethoxy flavone, 5,3′‐dihydroxy‐7,8‐dimethoxy flavone, and 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxy flavone were successfully separated in one extraction–separation operation within 300 min. The targeted compounds were identified by MS and NMR spectroscopy. This research has opened up great prospects for industrial application of SFE/HSCCC to the extraction and separation of unstable compounds.     

Application of an efficient strategy based on MAE,HPLC‐DAD‐MS/MS and HSCCC for the rapid extraction,identification, separation and purification of flavonoids from Fructus Aurantii Immaturus

This study presents an efficient strategy based on microwave‐assisted extraction (MAE), HPLC‐DAD‐MS/MS and high‐speed counter‐current chromatography (HSCCC) for the rapid extraction, identification, separation and purification of active components from the traditional Chinese medicine Fructus Aurantii Immaturus. An LC‐DAD‐MS/MS method was applied for the screening and structural identification of main components in crude extract, and five components were preliminarily identified as neoeriocitrin, narirutin, naringin, hesperidin and neohesperidin according to their UV and mass spectra. An efficient MAE method for the extraction of the three most abundant components (narirutin, naringin and neohesperidin) was optimized by the combination of univariate and multivariate approaches. The crude extract was then separated and purified by HSCCC and a total of 61.6 mg of narirutin, 207.3 mg of naringin and 159.5 mg of neohesperidin at high purities of 98.1, 97.2 and 99.5%, respectively, were obtained from 1.42 g of crude extract. The recoveries of these compounds were 86, 93 and 89%, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

A rapid and convenient method for preparative separation of three indissolvable polyphenols from Euphorbia pekinensis by the flexible application of solvent extraction combined with counter‐current chromatography

A rapid and convenient method was established to preparatively isolate the three ellagic acid types of compounds, which were the main polyphenols in Euphorbia pekinensis, by flexibly applying solvent extraction combined with counter‐current chromatography (CCC). The total extract (extracted using 95% ethanol) of E. pekinensis was pretreated by two simple steps before CCC isolation, following the procedure: the total extract was extracted by classical solvent extraction using petroleum ether and ethyl acetate, respectively, and then the ethyl acetate extract was suspended using 95% ethanol, after being allowed to stand overnight, the sediment was obtained. Partial sediment (100 mg) was then directly separated by CCC with a two‐phase solvent system composed of chloroform‐95% ethanol‐water‐85% formic acid (50:50:50:5, v/v/v/v). About 22 mg of 3,3′‐dimethoxy ellagic acid (1), 12 mg of 3,3′‐di‐O‐methyl‐4‐O‐(β‐d ‐xylopyranosyl)ellagic acid (2), and 35 mg of ellagic acid (3) with purities of 96.0, 95.2, and 95.4% were obtained respectively in one step within 4 h. After being purified by washing with methanol, the purities of the three compounds obtained were all above 98%. The purities were determined by HPLC and their chemical structures were further identified by ¹H and ¹³C NMR spectroscopy. The recoveries were calculated as 84.6, 85.7, and 89.5%, respectively. The result demonstrated that the present isolation method was rapid, economical and efficient for the preparative separation of polyphenols from E. pekinensis.     

A consecutive preparation method based upon accelerated solvent extraction and high‐speed counter‐current chromatography for isolation of aesculin from Cortex fraxinus

A consecutive preparation method based upon accelerated solvent extraction (ASE) coupled with high‐speed counter‐current chromatography (HSCCC) was presented and aesculin was obtained from Cortex fraxinus. The extraction condition of ASE was optimized with response surface methodology; some significant parameters such as the solvent system and its stability, the amount of loading sample in HSCCC were also investigated. The original sample was first extracted with methanol at 105°C and 104 bar for 7 min using ASE, then the extracts were consecutively introduced into the HSCCC system and separated and purified with the same ethyl acetate/n‐butanol/water (7:3:10, v/v/v) solvent system for five times without further exchange and equilibrium. About 3.1 ± 0.2 mg/g in each time and total of 15.4 mg/g aesculin with purity over 95% was isolated from Cortex fraxinus. The results demonstrated that the consecutive preparation method was time and solvent saving and high throughput, it was suitable for isolation of aesculin from Cortex fraxinus, and also has good potential on the separation and purification of effective compounds from natural product.     

Preparative isolation and purification of antioxidative stilbene oligomers from Vitis chunganeniss using high‐speed counter‐current chromatography in stepwise elution mode

Preparative high‐speed counter‐current chromatography (HSCCC) was successfully applied to the isolation and purification of three stilbene oligomers from Vitis chunganeniss using stepwise elution with a pair of two‐phase solvent systems composed of n‐hexane–ethyl acetate–methanol–water at (2:5:2:5, v/v) and (1:2:1:2, v/v). The preparative HSCCC separation was performed on 800 mg of crude sample yielding hopeaphenol (21.1 mg), amurensin G (37.2 mg) and vitisin A (95.6 mg) in a one‐step separation, with purities over 95% as determined by HPLC. The structures of these three compounds were identified by MS, ¹H NMR and ¹³C NMR. In addition, their antioxidant activities were screened by DPPH assay, where vitisin A showed strong antioxidant activity. Further EPR experiments with spin‐trapping technique demonstrated that vitisin A is a potent and selective singlet oxygen quencher, which may be used in singlet oxygen‐mediated diseases as a pharmacological agent.     

Separation of nine compounds from Salvia plebeia R.Br. using two‐step high‐speed counter‐current chromatography with different elution modes

Nine compounds were successfully separated from Salvia plebeia R.Br. using two‐step high‐speed counter‐current chromatography with three elution modes. Elution–extrusion counter‐current chromatography was applied in the first step, while classical counter‐current chromatography and recycling counter‐current chromatography were used in the second step. Three solvent systems, n‐hexane/ethyl acetate/ethanol/water (4:6.5:3:7, v/v), methyl tert‐butyl ether/ethyl acetate/n‐butanol/methanol/water (6:4:1:2:8, v/v) and n‐hexane/ethyl acetate/methanol/water (5:5.5:5:5, v/v) were screened and optimized for the two‐step separation. The separation yielded nine compounds, including caffeic acid ( 1 ), 6‐hydroxyluteuolin‐7‐glucoside ( 2 ), 5,7,3′,4′‐tetrahydroxy‐6‐methoxyflavanone‐7‐glucoside ( 3 ), nepitrin ( 4 ), rosmarinic acid ( 5 ), homoplantaginin ( 6 ), nepetin ( 7 ), hispidulin ( 8 ), and 5,6,7,4′‐tertrahydroxyflavone ( 9 ). To the best of our knowledge, 5,7,3′,4′‐tetrahydroxy‐6‐methoxyflavanone‐7‐glucoside and 5,6,7,4′‐tertrahydroxyflavone have been separated from Salvia plebeia R.Br. for the first time. The purities and structures of these compounds were identified by high‐performance liquid chromatography, electrospray ionization mass spectrometry, ¹H and ¹³C NMR spectroscopy. This study demonstrates that high‐speed counter‐current chromatography is a useful and flexible tool for the separation of components from a complex sample.     

Separation of phenolic acids from sugarcane rind by online solid‐phase extraction with high‐speed counter‐current chromatography

Sugarcane rind contains some functional phenolic acids. The separation of these compounds from sugarcane rind is able to realize the integrated utilization of the crop and reduce environment pollution. In this paper, a novel protocol based on interfacing online solid‐phase extraction with high‐speed counter‐current chromatography (HSCCC) was established, aiming at improving and simplifying the process of phenolic acids separation from sugarcane rind. The conditions of online solid‐phase extraction with HSCCC involving solvent system, flow rate of mobile phase as well as saturated extent of absorption of solid‐phase extraction were optimized to improve extraction efficiency and reduce separation time. The separation of phenolic acids was performed with a two‐phase solvent system composed of butanol/acetic acid/water at a volume ratio of 4:1:5, and the developed online solid‐phase extraction with HSCCC method was validated and successfully applied for sugarcane rind, and three phenolic acids including 6.73 mg of gallic acid, 10.85 mg of p‐coumaric acid, and 2.78 mg of ferulic acid with purities of 60.2, 95.4, and 84%, respectively, were obtained from 150 mg sugarcane rind crude extracts. In addition, the three different elution methods of phenolic acids purification including HSCCC, elution–extrusion counter‐current chromatography and back‐extrusion counter‐current chromatography were compared.     

Separation and purification of five phenylpropanoid glycosides from Lamiophlomis rotata (Benth.) Kudo by a macroporous resin column combined with high‐speed counter‐current chromatography

Five phenylethanoid glycosides (PhGs), forsythoside B, verbascoside, alyssonoside, isoverbascoside, and leucosceptoside B, were isolated and purified from Lamiophlomis rotata (Benth.) Kudo by high‐speed counter‐current chromatography (HSCCC) combined with macroporous resin (MR) column separation. In the present study, the two‐phase solvent system composed of ethyl acetate/n‐butanol/water (13:3:10, v/v/v) was used for HSCCC separation. A total of 27 mg of forsythoside B, 41 mg of verbascoside, 29 mg of alyssonoside, 23 mg of isoverbascoside, and 13 mg of leucosceptoside B with purities of 97.7, 99.2, 99.5, 99.3, and 97.3%, respectively, were obtained in a one‐step separation within 4 h from 150 mg of crude extract. The recoveries of the five PhGs after MR‐HSCCC separation were 74.5, 76.5, 72.5, 76.4, and 77.0%, respectively. The chemical structures of all five compounds were identified by ¹H and ¹³C NMR spectroscopy.     

Preparative isolation of ganoderic acid S,ganoderic acid T and ganoderol B from Ganoderma lucidum mycelia by high‐speed counter‐current chromatography

Ganoderic acid S, ganoderic acid T and ganoderal B are the main bioactive triterpenes of Ganoderma lucidum. In this study, mycelia of G. lucidum were obtained by two‐stage fermentation and then extracted by ethanol and petroleum ether sequentially to obtain crude triterpenes. The crude sample was further purified by recycling high‐speed counter‐current chromatography with n‐hexane–ethyl acetate–methanol–water (7:12:11:5, v/v/v/v) as the optimized two‐phase solvent system. A 16.4 mg aliquot of ganoderol B with a purity of 90.4% was separated from 300 mg of the crude sample in a single run. After employing the recycling elution mode of HSCCC with n‐hexane–ethyl acetate–methanol–water (6:10:8:4.5, v/v/v/v) for five cycles, 25.7 mg ganoderic acid T and 3.7 mg ganoderic acid S with purities of 97.8 and 83.0%, respectively, were obtained. The purities of three compounds were determined by high‐performance liquid chromatography and their chemical structures were identified by NMR and MS data.     

Separation of three phenolic high‐molecular‐weight compounds from the crude extract of Terminalia Chebula Retz. by ultrasound‐assisted extraction and high‐speed counter‐current chromatography

This study presents an efficient strategy for separation of three phenolic compounds with high molecular weight from the crude extract of Terminalia chebula Retz. by ultrasound‐assisted extraction and high‐speed counter‐current chromatography. The ultrasound‐assisted extraction conditions were optimized by response surface methodology and the results showed the target compounds could be well enriched under the optimized extraction conditions. Then the crude extract was directly separated by high‐speed counter‐current chromatography without any pretreatment using n‐hexane/ethyl acetate/methanol/water (1:7:0.5:3, v/v/v/v) as the solvent system. In 180 min, 13 mg of A, 18 mg of B, and 9 mg of C were obtained from 200 mg of crude sample. Their structures were identified as Chebulagic acid (A, 954 Da), Chebulinic acid (B, 956 Da), and Ellagic acid (C) by ¹H NMR spectroscopy.     

Preparative separation of capsaicin and dihydrocapsaicin from Capsicum frutescens by high‐speed counter‐current chromatography

Capsaicin and dihydrocapsaicin are two main bioactive components of Capsicum frutescens and are widely used as food additives and drugs in China and India. Due to their similarity in structures, isolation of capsaicin and dihydrocapsaicin with traditional methods such as silica gel column chromatography, normal‐phase thin‐layer chromatography (TLC) becomes difficult. This study involves separating capsaicin and dihydrocapsaicin with sufficient purity and recovery using high‐speed counter‐current chromatography (HSCCC) with a solvent system composed of n‐hexane–ethyl acetate–methanol–water–acetic acid (20:20:20:20:2, v/v/v/v/v). Separation parameters such as sample volume, and sample concentration were first optimized on analytical HSCCC, and then scaled up to preparative HSCCC. 0.65 g capsaicin and 0.28 g dihydrocapsaicin were obtained from 1.2 g crude extract and their purities were 98.5 and 97.8%, respectively. The recoveries of the two compounds were 86.3 and 85.4%, respectively. The purity of the isolated compounds was analyzed by high‐performance liquid chromatography (HPLC) and their structures were identified by ¹H nuclear magnetic resonance (NMR) and ¹³C NMR analysis.     

Efficient methods for isolating five phytochemicals from Gentiana macrophylla using high‐performance countercurrent chromatography

Efficient high‐performance countercurrent chromatography methods were developed to isolate five typical compounds from the extracts of Gentiana macrophylla. n‐Butanol‐soluble extract of G. macrophylla contained three hydrophilic iridoids, loganic acid ( 1 ), swertiamarin ( 2 ) and gentiopicroside ( 3 ), and a chromene derivative, macrophylloside D ( 4 ) which were successfully isolated by flow rate gradient (1.5 mL/min in 0–60 min, 5.0 mL/min in 60–120 min), and consecutive flow rate gradient HPCCC using n‐butanol/0.1% aqueous trifluoroacetic acid (1:1, v/v, normal phase mode) system. The yields of 1 – 4 were 22, 16, 122, and 6 mg, respectively, with purities over 97% in a flow rate gradient high‐performance countercurrent chromatography, and consecutive flow rate gradient high‐performance countercurrent chromatography gave 1 , 2 , 3 (54, 41, 348 mg, respectively, purities over 97%) and 4 (13 mg, purity at 95%) from 750 mg of sample. The main compound in methylene chloride soluble extract, 2‐methoxyanofinic acid, was successfully separated by n‐hexane/ethyl acetate/methanol/water (4:6:4:6, v/v/v/v, flow‐rate: 4 mL/min, reversed phase mode) condition. The structures of five isolates were elucidated by ¹H, ¹³C NMR and ESI‐Q‐TOF‐MS spectroscopic data which were compared with previously reported values.     

Separation and purification of four flavonol diglucosides from the flower of Meconopsis integrifolia by high‐speed counter‐current chromatography

Flavonoids are the main components of Meconopsis integrifolia (Maxim.) Franch, which is a traditional Tibetan medicine. However, traditional chromatography separation requires a large quantity of raw M. integrifolia and is very time consuming. Herein, we applied high‐speed counter‐current chromatography in the separation and purification of flavonoids from the ethanol extracts of M. integrifolia flower. Ethyl acetate/n‐butanol/water (2:3:5, v/v/v) was selected as the optimum solvent system to purify the four components, namely quercetin‐3‐O‐β‐d‐ glucopyrannosy‐(1→6)‐β‐d‐ glucopyranoside (compound 1 , 60 mg), quercetin 3‐O‐[2’’’‐O‐acetyl‐β‐d‐ glucopyranosyl‐(1→6)‐β‐d‐ glucopyranoside (compound 2 , 40 mg), quercetin 3‐O‐[3’’’‐O‐acetyl‐β‐d‐ glucopyranosyl‐(1→6)‐β‐d‐ glucopyranoside (compound 3 , 11 mg), and quercetin 3‐O‐[6’’’‐O‐acetyl‐β‐d‐ glucopyranosyl‐(1→6)‐β‐d‐ glucopyranoside (compound 4 , 16 mg). Among the four compounds, 3 and 4 were new acetylated flavonol diglucosides. After the high‐speed counter‐current chromatography separation, the purities of the four flavonol diglucosides were 98, 95, 90, and 92%, respectively. The structures of these compounds were identified by mass spectrometry and NMR spectroscopy.     

Separation of three polar compounds from Rheum tanguticum by high‐speed countercurrent chromatography with an ethyl acetate/glacial acetic acid/water system

The separation of polar compounds by high‐speed countercurrent chromatography is still regarded as a challenge. In this study, an efficient strategy for the separation of three polar compounds from Rheum tanguticum has been successfully conducted by using high‐speed countercurrent chromatography. X‐5 macroporous resin chromatography was used for the fast enrichment of the target compounds. Then, the target fraction was directly introduced into high‐speed countercurrent chromatography for separation using ethyl acetate/glacial acetic acid/water (100:1:100, v/v/v) as the solvent system. Consequently, three polar compounds including gallic acid, catechin, and gallic acid 4‐O‐β‐d ‐(6′‐O‐galloyl) glucoside were obtained with purities higher than 98%. The results showed glacial acetic acid could be such an appropriate regulator for the ethyl acetate/water system. This study provides a reference for the separation of polar compounds from natural products by high‐speed countercurrent chromatography.     

Separation and purification of 9,10‐dihydrophenanthrenes and bibenzyls from Pholidota chinensis by high‐speed countercurrent chromatography

Stilbenoids are the main components of leaves and stems of Pholidota chinensis. In the present investigation, high‐speed counter‐current chromatography was used for the separation and purification of two classes of stilbenoids, namely, bibenzyls and 9,10‐dihydrophenanthrenes, on a preparative scale from whole plants of P. chinensis with different solvent systems after silica gel column chromatography fractionation. n‐Hexane/ethyl acetate/methanol/water (1.2:1:1:0.8, v/v/v/v) was selected as the optimum solvent system to purify 1‐(3,4,5‐trimethoxyphenyl)‐1′,2′‐ethanediol ( 1 ), coelonin ( 2 ), 3,4′‐dihydroxy‐5,5′‐dimethoxybibenzyl ( 3 ), and 2,?7‐?dihydroxy‐?3,?4,?6‐?trimethoxy‐?9,?10‐?dihydrophenanthrene ( 4 ). While 2,7‐dihydroxy‐3,4,6‐trimethoxy‐?9,?10‐?dihydrophenanthrene ( 5 ), batatasin III ( 6 ), orchinol ( 7 ), and 3′‐O‐methylbatatasin III ( 8 ) were purified by n‐hexane/ethyl acetate/methanol/water (1.6:0.8:1.2:0.4, v/v/v/v). After the high‐speed counter‐current chromatography isolation procedure, the purity of all compounds was over 94% assayed by ultra high performance liquid chromatography. The chemical structure identification of all compounds was carried out by mass spectrometry and ¹H and ¹³C NMR spectroscopy. To the best of our knowledge, the current investigation is the first study for the separation and purification of bibenzyls and 9,10‐dihydrophenanthrenes by high‐speed counter‐current chromatography from natural resources.     

Preparative isolation of pseudolaric acids A and B,and their glucosides from the root bark of Pseudolarix kaempferi using high‐speed counter‐current chromatography

In order to provide the chemical markers for the quality control of herbal medicines, four diterpenoids, pseudolaric acids A and B (PAA and PAB), and their glucosides were isolated from the methanol extract of the Chinese herb Pseudolarix kaempferi using high‐speed counter‐current chromatography (HSCCC). The diphase solvent system was n‐hexane/EtOAc/MeOH/H₂O which was used at two ratios (5:5:5:5 and 1:9:4:6 by volume) in the separation of pseudolaric acids and their glycosides, respectively. As a result, PAA (14 mg), PAB (129 mg), PAA‐O‐β‐D ‐glucopyranoside (8 mg, PAAG), and PAB‐O‐β‐D ‐glucopyranoside (42 mg, PABG) were obtained from 0.5 g of the crude extract. Their purities were determined to be above 97% by HPLC analysis. Their chemical structures were confirmed by ¹H and ¹³C NMR analysis or HPLC comparison with the reference compounds.     

Preparative isolation and purification of harpagoside and angroside C from the root of Scrophularia ningpoensis Hemsley by high‐speed counter‐current chromatography

In this study, the bioactive component harpagoside and angroside C in the root of Scrophularia ningpoensis Hemsley was simultaneously separated by high‐speed counter‐current chromatography (HSCCC). A two‐phase solvent system containing chloroform/n‐butanol/methanol/water (4:1:3:2, v/v/v/v) was selected following consideration of the partition coefficient of the target compound. The crude extract (200 mg) was loaded onto a 280‐mL HSCCC column and yielded 22 mg harpagoside and 31 mg angroside C with the purity of higher than 98 and 98.5%, respectively. It is feasible to isolate active compounds harpagoside and angroside C from S. ningpoensis using HSCCC.     

Enrichment and isolation of barbigerone from Millettia pachycarpa Benth. using high‐speed counter‐current chromatography and preparative HPLC

Enrichment of the anti‐tumor compound barbigerone along with a rotenoid derivative from Millettia pachycarpa Benth. was performed by a two‐step high‐speed counter‐current chromatography (HSCCC) separation process. In the first step, 155.8 mg of target fraction (Fra6) was obtained from 400 mg ethyl acetate extract of M. pachycarpa Benth. with an increase in barbigerone from 5.1 to 13% via HSCCC using a solvent system of n‐hexane–ethyl acetate–methanol–water (5:4:5:3, v/v) under normal phase head to tail elution. HSCCC was repeated to eliminate the major contaminant in this initial fraction 6. After a separation time of 65 min, 22.1 mg barbigerone of 87.7% purity was obtained from Fra6 with the ternary solvent system of n‐hexane–methanol–water (2:2:1, v/v) under normal phase elution. Finally, preparative HPLC was employed for the further isolation of barbigerone and the rotenoid derivative. The structures were confirmed by ESI‐MS, ¹H NMR and ¹³C NMR.