One-step High-speed Counter-current Chromatography Research Articles

Characterization, Large-Scale HSCCC Separation and Neuroprotective Effects of Polyphenols from Moringa oleifera Leaves

Moringa oleifera leaves have been widely used for the treatment of inflammation, diabetes, high blood pressure, and other diseases, due to being rich in polyphenols. The main objective of this work was to largely separate the main polyphenols from Moringa oleifera leaves using the technique of high-speed counter-current chromatography (HSCCC). The phenolic composition in Moringa oleifera leaves was first analyzed qualitatively and quantitatively by UPLC-Q-Exactive Orbitrap/MS and UPLC-QqQ/MS, respectively, indicating that quercetin and kaempferol derivatives, phenolic acid and apigenin are the main polyphenols in Moringa oleifera leaves, with quercetin and kaempferol derivatives predominating. Furthermore, the conditions of HSCCC for large-scale separation of polyphenols from Moringa oleifera leaves were optimized, which included the selection of the solvent system, flow rate and the sample load. Only by one-step HSCCC separation (within 120 min) under the optimized conditions, six quercetin and kaempferol derivatives, a phenolic acid and an apigenin could be individually isolated at a large scale (yield from 10% to 98%), each of which possessed high purity. Finally, the isolated polyphenols and phenolic extract from Moringa oleifera leaves (MLPE) were verified to have strong neuroprotective activities against H2O2-induced oxidative stress in PC-12 cells, suggesting that these compounds would contribute to the main beneficial effects of Moringa oleifera leaves.

Rapid large‐scale preparation of polysaccharides from jackfruit peel waste by high‐speed countercurrent chromatography and their antioxidant and hypoglycemic activities

Polysaccharides with antioxidant and hypoglycemic activities were first isolated from jackfruit (Artocarpus heterophyllus Lam.) peel through the one-step high-speed countercurrent chromatography. The separation process was completed using the polymer two-phase aqueous system constituted by PEG1000-K2 HPO4 -KH2 PO4 -H2 O (0.8:1.25:1.25:6.5, w/w). For every separation process, two main polysaccharides, namely, fraction-1 and fraction-2 (165 and 225mg, respectively) were obtained from a 2.0g crude sample. As suggested by high-performance gel permeation chromatography, jackfruit peel polysaccharides had the mean molecular weight values of 113.3 and 174.3kDa, separately. Physicochemical analysis suggested that two polysaccharides were dominant in galacturonic acid, galactose, rhamnose, arabinose, glucose, mannose, as well as fucose, which were highly esterified. Biological activity analysis showed that fraction-1 exhibited stronger antioxidant activity in vitro and hypoglycemic activity in streptozotocin-induced diabetic mice compared with fraction-2. The results suggest that polysaccharide fraction-1 may be developed as a potential functional food supplement.

Purification of Four Caffeoylquinic Acid Derivatives from the Flowers of Gynura Procumbens by HSCCC.

Four caffeoylquinic acid derivatives from the Gunura procumbens flowers (GPF) were successfully isolated and purified by high-speed counter-current chromatography (HSCCC). Ethyl acetate-methanol-water (3:1:3, v/v/v) was the optimum biphasic solvent system, which was selected by high-performance liquid chromatography (HPLC) and run on a preparative scale where the lower aqueous phase was used as the mobile phase with a head-to-tail elution mode. Chlorogenic acid (3.83 mg), Isochlorogenic acid A (6.51 mg), Isochlorogenic acid B (4.38 mg) and Isochlorogenic acid C (4.47 mg) were obtained for the first time in an one-step HSCCC separation from 800 mg of the crude extracts. The purities of four compounds were determined to be >95% by HPLC. Chemical structures of each isolated compounds were identified by nuclear magnetic resonance and electrospray ionization mass spectrometry methods. It is worth noting that all the four compounds were isolated here for the first time from GPF and this work confirms the effectiveness of HSCCC for the separation of compounds contained in complex samples, and provides a foundation for further exploitation of G. procumbens.

Novel flavan-3-ol-glutathione conjugates from the degradation of proanthocyanidins as highly bioactive antioxidants

Synthesis, preparation and antioxidant capacity evaluation of flavan-3-ol-glutathione conjugates.

Antioxidant and anticomplement compounds isolated from Nitraria sibirica fruit by high-speed counter-current chromatography

Background: Nitraria sibirica fruit is a kind of folk medicine widely used in Northwest China. It contains diverse kinds of bioactive constituents such as flavonoids, alkaloids, and phenols. Objectives: The research was performed to develop an efficient separation method for the bioactive constituents of N. sibirica fruit and evaluate the isolated compounds bioactivities. Materials and Methods: An optimized method of high-speed counter-current chromatography (HSCCC) combined with preparative high-performance liquid chromatography (HPLC) was established and applied to prepare the bioactive compounds from the ethyl acetate extract of N. sibirica fruit. The antioxidant and anticomplement activities of the isolated fractions and compounds were evaluated in vitro. Results: Ethyl acetate extract was the predominant antioxidant and anticomplement fraction of N. sibirica fruit, which yielded 18 compounds including six phenols, three flavonoids, and five alkaloids. Seven compounds (2, 4, 5, 9, 10, 12, and 17) were first isolated from genus Nitraria. Furthermore, under the optimized chromatography conditions, one anticomplement alkaloid (1) could be achieved directly by one-step HSCCC with high purity (92.34%), the other seventeen compounds were further purified with preparative HPLC. Ten compounds (3, 6, 7, 8, 9, 10, 11, 12, 13, and 14) exhibited significant antioxidant activities. Five compounds (1, 2, 7, 10, and 16) showed anticomplement activities and targeted different components in the complement system. Conclusion: The study provided an efficient method to prepare antioxidant and anticomplement compounds and the activities of the compounds were deeply investigated. Abbreviations used: HSCCC: High-speed counter-current chromatography; DPPH: 1,1-diphenyl-2-picrylhydrazyl; FRAP: Ferric reducing antioxidant power; ABTS: 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid); BBS: Barbiturate buffer solution; ASEA: Anti-sheep erythrocyte antibody; NHS: Normal human serum; 2% SRBC: 2% sheep red blood cells.

Isolation and purification of two tyrosinase inhibitors from camellia pollen by high-speed counter-current chromatography

Bee pollen is a health food with a wide range of nutritional and therapeutic properties. However, the bioactive compounds of bee pollen have not been extensively revealed due to low efficacy in separation. High-speed counter-current chromatography (HSCCC) and solvent extraction were applied to separate tyrosinase inhibitors from camellia pollen in this study. The camellia pollen extracts prepared with petroleum ether, ethyl acetate, and n-BuOH have tyrosinase inhibitory activity. Acidic hydrolysis could promote the tyrosinase inhibitory activity of crude sample. Three fractions with tyrosinase inhibitory activity were separated from the hydrolysate by a one-step HSCCC procedure. Among the fractions, two chemicals were sufficiently purified and identified to be levulinic acid (LA) and 5-hydroxymethylfurfural (5-HMF). The recovery was 0.80 g kg−1 pollen for LA and 1.75 g kg−1 pollen for 5-HMF; and their purity was all over 98%. The study demonstrates that HSCCC method is powerful for preparative separation of tyrosinase inhibitors from camellia pollen.

Purification of Four Flavonoid Glycosides from Lotus (Nelumbo nucifera Gaertn) plumule by Macroporous Resin Combined with HSCCC.

High-speed counter-current chromatography (HSCCC) combined with macroporous resin (MR) column was successfully applied to the isolation and purification of four flavonoid glycosides from the medicinal herb Lotus plumule (LP). A polar two-phase solvent system composed of ethyl acetate-n-butanol-water (1:2:3, v/v/v) was selected by high-performance liquid chromatography (HPLC) and run on a preparative scale where the lower aqueous phase was used as the mobile phase with a head-to-tail elution mode. Quercetin-3-O-β-D-glucopyranoside (15 mg), isorhamnetin-3-O-β-D-glucopyranoside (13 mg), apigenin 6-C-β-D-glucopyranosyl-8-C-α-L-arabinopyranoside (18 mg) and apigenin 6,8-di-C-β-D-glucopyranoside (48 mg) were obtained in a one-step HSCCC separation from 240 mg of the sample. The purity of each compound was over 95% as determined by HPLC. Chemical structures of the isolated compounds were identified by electrospray ionization mass spectrometry (ESI-MS-MS) and nuclear magnetic resonance (NMR) methods. Moreover, the four compounds were isolated from LP for the first time.

Separation of polysaccharides from Spirulina platensis by HSCCC with ethanol-ammonium sulfate ATPS and their antioxidant activities

Three separation methods for water-soluble polysaccharides from Spirulina platensis (PSP) were compared, including sevage deproteinization and column chromatography, ethanol-ammonium sulfate aqueous two-phase system (ATPS) and column chromatography, and one-step high speed counter-current chromatography (HSCCC) with ethanol-ammonium sulfate ATPS. ATPS was confirmed as an efficient alternative method of protein removal for PSP purification. For the HSCCC with ethanol-ammonium sulfate ATPS, the stationary retention reached to 50.8% at the optimized rotation speed and flow rate. Moreover, the yield of PSP purified by one-step HSCCC rose nearly five times to 12.45mg/g(dry algae powder), it was higher than PSP yield by two-step column chromatography separation methods. PSP purified by HSCCC has the same purity as PSP obtained by traditional methods, which was proved by a single symmetrical peak of purified PSP with molecular weight of 12.33kDa through gel chromatography. Purified PSP was an α-acidic polysaccharide, composed of major glucose, slight rhamnose and mannose, which were detected within GC and FT-IR spectra. The antioxidation activity experiment showed that HSCCC-purified PSP had strong scavenging effects on hydroxyl free radical and DPPH free radical.

Separation and purification of polyphenols from red wine extracts using high speed counter current chromatography.

Polyphenols are important compounds of red wine owing to their contribution to sensory properties and antioxidant activities. In this study, high-speed counter-current chromatography (HSCCC) coupled with semi-preparative HPLC was used for large-scale separation and purification of polyphenols from red wine extracts. With the solvent system of hexane-ethyl acetate-water (1-50-50), various oligomeric procyanidins including monomer catechin, epicatechin, dimers B1, B2; phenolic acids including coutaric acid, caftaric acid and other type of polyphenols were largely separated within 370min and most of these compounds presented high yields (0.97mg to 13.79mg) with high purity (90.34% to 98.91%) after the semi-preparative HPLC isolation. Using the solvent system of Methyl tert-Butyl Ether (MTBE) - n-butyl alcohol- acetonitrile-water (1-40-1-50, acidified with 0.01% trifluoroacetic acid (TFA)) by one-step HSCCC of 100mg of the red wine extracts, the major anthocyanins, i.e., malvidin-3-O-glucoside, delphinidin-3-O-glucoside and peonidin-3-O-glucoside, as well as two polymeric proanthocyanidin fractions were successfully separated one another within 320min. The yields of malvidin-3-O-glucoside, delphinidin-3-O-glucoside and peonidin-3-O-glucoside were 12.12mg, 1.78mg and 11.57mg with the purity of 92.74%, 91.03% and 91.21%, respectively. Thiolysis-UPLC analysis indicated that the two polymeric proanthocyanidin fractions presented high purity, with mean degree of polymerization of 7.66±0.12 and 6.20±0.09, respectively. The further experiments on the antioxidant activities by DPPH radical test, FRAP assay and ABTS method showed that all of the isolated procyandins and anthocyanins and the two polymeric proanthocyanidin fractions, with exception of phenolic acids possessed much greater antioxidant activities compared to standard Trolox andl-ascorbic acid (2-14 times).

Identifying Natural syNergist from Pongamia pinnata Using High-Speed Counter-Current Chromatography Combined with Isobolographic Analysis.

For identifying the synergistic compounds from Pongamia pinnata, an approach based on high-speed counter-current chromatography (HSCCC) combined with isobolographic analysis was designed to detect the synergistic effects in the complex mixture. In the approach, the complex mixture was considered as the combination of two individual samples for isobolographic analysis: the target compound and the mixture with complete removal of the target compound (subtracted residue). The two samples were prepared by HSCCC, and were used for the calculation of the expected effect of their combination. Using this approach, three compounds representing the major peaks in the HPLC of the brine shrimp toxic extract from P. pinnata (brine shrimp lethality test (BST) LC50 36.5 μg/mL), pinnatin (1), 3,7-Dimethoxy-3′,4′-methylenedioxy flavone (2), and karanjin (3), were prepared from the extract, and were tested for their synergistic potency by BST. The two-phase solvent system containing n-hexane-ethyl acetate–MeOH–water (14:7:10:10, v/v/v/v) was selected for the one-step HSCCC separation according to the partition coefficient values (K). The extract was separated into seven fractions (Fr1–7) by HSCCC with a total mass recovery of 96.3%. Fr2, 4, and 6 were the peak fractions corresponding to compounds 3, 2, and 1, respectively. The purities and recoveries of the target compounds after the chromatographic analysis were 95.9%–97.5% and 92.2%–96.1%, respectively. The subtracted residue of each target compound was performed by recombining all HSCCC fractions except the fraction containing the target compound. Isobolographic analysis disclosed a significant synergistic effect between karanjin and its subtracted residue (potency ratio of 0.47), which gave clear evidence that the toxicity of the extract results from synergistic interactions, and karanjin was one of the synergists participating in the interaction. The other two compounds were excluded from the synergism because these two compounds showed additive effects with their subtracted residues. Karanjin was the first synergistic compound identified from P. pinnata.

Preparative high-speed counter-current chromatography separation of grape seed proanthocyanidins according to degree of polymerization.

Separation of plant proanthocyanidins remains a major challenge for scientists due to the structural diversity and complexity. In this work, a new and effective method was developed for preparative separation of grape seed proanthocyanidins (GSPs) according to degree of polymerization (DP) by high-speed counter-current chromatography (HSCCC). Under the optimized HSCCC conditions, GSPs could be separated into seven distinct fractions (F1-F7) with mean degree of polymerization in increasing order, from 1.44 to 6.95. High yields for these fractions (53.7, 12.2, 29.5, 30.2, 11.2, 50.8 and 169.8mg, respectively) were achieved by only one-step HSCCC of 400mg of GSPs. Further, seventeen individual proanthocyanidins, most of which are commercially not available, were efficiently isolated by re-chromatography on HSCCC or prep-HPLC; each of the isolated compounds presented high yields (7.1-78.9mg) and high purity (70.0-95.7%). The positive correlation was observed between DP and antioxidant activity of proanthocyanidins.

Separation and Purification of Four Stilbenes from Vitis vinifera L. cv. Cabernet Sauvignon Roots Through High-speed Counter-current Chromatography

A method for preparative separation and purification of trans-resveratrol, δ-viniferin, e-viniferin and trans-vitisin B from the roots of Vitis vinifera L. cv. Cabernet Sauvignon was successfully established and is reported on in this paper. The four important stilbenes were purified by high-speed counter-current chromatography (HSCCC) with a suitable quaternary solvent system composed of chloroform–methanol–n-butanol–water (4:3:0.05:2, v/v). A total of 7.1 mg ± 0.2 mg of trans-resveratrol, 1.1 mg ± 0.1 mg of δ-viniferin, 18.7 mg ± 0.5 mg of e-viniferin, and 12.2 mg ± 0.2 mg of trans-vitisin B, with purities of 97.89%, 90.61%, 94.37% and 78.38% respectively, were obtained from 241 mg of crude sample in a one-step HSCCC separation. The chemical structures of trans-resveratrol and δ-viniferin were further confirmed with the retention time using the method of standard addition, while the structural identification of e-viniferin and trans-vitisin B was performed with LC-ESI/MS, 1H-NMR, and 13C-NMR.

Isolation and Purification of Isochlorogenic Acid A fromLonicera japonicaThunb Using High-Speed Counter-Current Chromatography

A high-speed counter-current chromatography (HSCCC) method was established for the isolation and purification of isochlorogenic acid A from Lonicera japonica Thunb. The two-phase solvent system was composed of n-hexane:ethyl acetate: isopropanol:water (2:3:2:5, v/v/v/v). From 150 mg of the ethyl acetate fraction of L. japonica Thunb, 19.65 mg of isochlorogenic acid A was obtained in a one-step HSCCC separation, with a purity of 99.1%, as determined by high-performance liquid chromatography (HPLC). The structure was further identified by ultraviolet (UV), mass spectrometry (MS) and nuclear magnetic resonance (NMR).

One-step separation of antioxidant compounds from Erythrina variegata by high speed counter-current chromatography.

High speed counter-current chromatography (HSCCC) was used for separation and purification of antioxidant compounds from ethyl acetate fraction of the stem bark of Erythrina variegata. The optimal two-phase solvent system was composed of n-hexane-ethyl acetate-methanol-water (1:4:1:4, v/v/v/v). After one-step HSCCC separation, 75 mg of protocatechuic acid ( 1: ), 32 mg of chlorogenic acid ( 2: ) and 44 mg of caffeic acid ( 3: ) were purified from 420 mg of the ethyl acetate fraction. The purity of isolated compounds was determined up to 99.7% as determined by high-performance liquid chromatography (HPLC). The chemical structures of the three compounds were confirmed by UV, HPLC-MS/MS and (1)H NMR. The IC50 values of scavenging DPPH free radical for the three compounds were 22.5, 41.9 and 20.9 μg/mL, respectively. Protocatechuic acid and chlorogenic acid were obtained from the stem bark of E. variegata for the first time.

One-step Separation and Purification of Two Chromones and One Pyrone from Aloe barbadensis Miller: a Comparison Between Reversed-phase Flash Chromatography and High-speed Counter Current Chromatography

Chromones and pyrones are the major secondary metabolites of Aloe barbadensis Miller. As they are minor components of the plant, an efficient purification procedure for them is of great importance for promoting their pharmacological studies. To develop efficient methods for one-step separation and purification of two chromones (5-((S)-2'-oxo-4'-hydroxypentyl)-2-hydroxymethylchromone (1) and 5-((4E)-2'-oxo-pentenyl)-2-hydroxymethylchromone (3)) and one pyrone (aloenin aglycone (2)) from A. barbadensis via reversed-phase flash chromatography (RP-FC) and high-speed counter current chromatography (HSCCC). The RP-FC separation was performed using methanol:water (26:74, v/v) as the mobile phase at a flow rate of 20 mL/min. A solvent system composed of dichloromethane:methanol:water (3:1.5:1, v/v/v) was used for the HSCCC separation, at a flow rate of 2.0 mL/min. A one-step RP-FC operation within 110 min was successfully used for the purification of compounds 1 (27.9 mg, 96.5%), 2 (32.4 mg, 98.2%) and 3 (4.1 mg, 99.0%) from 129 mg of crude sample, and a one-step HSCCC separation within 95 min was successfully implemented for the purification of compounds 1 (31.1 mg, 97.6%), 2 (35.8 mg, 96.7%) and 3 (2.7 mg, 98.1%) from 134 mg of crude sample. The developed procedures were efficient, with low cost and high yield, which would afford sufficient amounts of high-purity compounds for chromatographic purposes and pharmacological activity screening.

Preparative isolation of aldose reductase inhibitory compounds from Nardostachys chinensis by elution–extrusion counter-current chromatography

A method combining enzyme assay-guided high-performance liquid chromatography (HPLC) micro-fractionation and elution-extrusion counter-current chromatography (EECCC) was developed to screen and separate aldose reductase (AR) inhibitory activities from those of the ethyl acetate (EtOAc) fraction of Nardostachys chinensis. Under the target-guidance of HPLC micro-fractionation, two phenolic compounds, three caffeoylquinic acid derivatives and two sesquiterpene were isolated by high-speed countercurrent chromatography (HSCCC) using elution modes of extrusion-elution. A one-step HSCCC isolation method was developed, which included a solvent system of n-hexane-EtOAc-methanol-water at a ratio of 2:8:3:7 (v/v/v/v). The chemical structures of the isolated compounds were determined using (1)H- and (13)C-nuclear magnetic resonance spectrometry. The compounds inhibiting AR in the EtOAc fraction of 70% ethanol extracts of N. chinensis were identified as chlorogenic acid (2) and 1,5-di-O-caffeoylquinic acid (6). Our results indicate that the combined method of HPLC micro-fractionation and EECCC is fast, efficient, and reproducible for systematically isolating AR inhibitory compounds from complex natural products.

Purification of Two Triterpenoids from Schisandra chinensis by Macroporous Resin Combined with High-Speed Counter-Current Chromatography

A method for preparative purification of corosolic acid and nigranoic acid from Schisandra chinensis (SC) was established using a combination of macroporous absorption resin column separation and high-speed counter-current chromatography (HSCCC). The crude extracts obtained from SC using 70% ethanol were separated on a macroporous resin column and then eluted with a graded ethanol series. The 70% ethanol fraction was used as the sample for separation of the two triterpenoids by HSCCC. The two-phase solvent system used for HSCCC separation was chloroform-n-butanol-methanol-water (10:0.5:7:4, v/v/v/v). The upper phase was used as the stationary phase of HSCCC. Corosolic acid (16.4 mg) of 96.3% purity and nigranoic acid (9.5 mg) of 98.9% purity were obtained in a one-step HSCCC separation from 100 mg of the sample. The structures of corosolic acid and nigranoic acid were identified by (1)H-nuclear magnetic resonance (NMR) and (13)C-NMR.

CONNECTION OF HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY WITH EVAPORATIVE LIGHT SCATTERING DETECTOR BY FLOW INJECTION AND ITS APPLICATION FOR PREPARATIVE ISOLATION AND PURIFICATION OF GINKGOLIDE COMPOUNDS FROM GINKGO BILOBA L.

High-speed counter-current chromatography (HSCCC) and evaporative light scattering detector (ELSD) were connected together by flow injection for the first time. A new method for isolation and purification of ginkgolide compounds from Ginkgo biloba L. was developed. Two pairs of two-phase solvent system, n-hexane-ethyl acetate-methanol-water (4:5:1:5, v/v) and n-hexane-ethyl acetate-methanol-water (4:5:2:5, v/v), were used in HSCCC separation. The stationary phase was the upper phase of n-hexane-ethyl acetate-methanol-water (4:5:1:5, v/v). The mobile phase was the lower phase of n-hexane-ethyl acetate-methanol-water (4:5:1:5, v/v) and n-hexane-ethyl acetate-methanol-water (4:5:2:5, v/v) in gradient elution mode. Ginkgo biloba L. powders were degreased by light petroleum and extracted with 70% ethanol. The extracts were purified with ethyl acetate extraction and acidic alumina oxide column separation, and 2.4 g of purified extracts were obtained from 500 g of Ginkgo biloba L. 107 mg of ginkgolides A, 64 mg of ginkgolides B, 52 mg of ginkgolides C, and 83 mg of bilobalide were obtained in one-step HSCCC separation from 500 mg of the purified extracts. The purities of the obtained compounds were all above 98% as determined by HPLC-ELSD area normalization method and their chemical structures were identified by 1H-NMR and 13C-NMR.

PREPARATIVE ISOLATION AND PURIFICATION OF PHENOLIC ACIDS FROM THE DRIED BUDS OF LONICERA JAPONICA THUNB BY HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY IN GRADIENT ELUTION MODE

A new high speed counter-current chromatography (HSCCC) method for preparative separation and purification of three phenolic acids from the dried buds of Lonicera japonica Thunb was developed by using gradient elution mode. Two pairs of two-phase solvent system composed of n-hexane-ethyl acetate-methanol-0.01 M hydrochloric acid at volume ratios of 1:5:1:5 and 1:5:2:5 were used in HSCCC separation. The upper phase of n-hexane-ethyl acetate-methanol-0.01 M hydrochloric acid (1:5:1:5, v/v) was used as the stationary phase. The lower phase of n-hexane-ethyl acetate-methanol-0.01 M hydrochloric acid (1:5:1:5, v/v) and (1:5:2:5, v/v) were used as the mobile phase in gradient elution mode. From 200 mg of the crude extract 51.6 mg of 3-caffeoylquinic acid, 7.5 mg of caffeic acid, and 42.4 mg of 3,5-O-dicaffeoylquinic acid were obtained in one-step HSCCC separation. The purities of the obtained compounds were all above 95% as determined by HPLC area normalization method, and their chemical structures were identified by 1H-NMR and 13C-NMR.

Preparative Isolation and Purification of Three Sesquiterpenoid Lactones from Eupatorium lindleyanum DC. by High-Speed Counter-Current Chromatography

A high-speed counter-current chromatography (HSCCC) method was established for the preparative separation of three sesquiterpenoid lactones from Eupatorium lindleyanum DC. The two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (1:4:2:3, v/v/v/v) was selected. From 540 mg of the n-butanol fraction of Eupatorium lindleyanum DC., 10.8 mg of 3β-hydroxy-8β-[4'-hydroxy-tigloyloxy]-costunolide, 17.9 mg of eupalinolide A and 19.3 mg of eupalinolide B were obtained in a one-step HSCCC separation, with purities of 91.8%, 97.9% and 97.1%, respectively, as determined by HPLC. Their structures were further identified by ESI-MS and 1H-NMR.