Separation of caffeoylquinic acids and flavonoids from Asteris souliei by high‐performance counter‐current chromatography and their anti‐inflammatory activity in vitro

Eleven compounds were successfully separated from Asteris souliei by using a two‐step high‐performance counter‐current chromatography method. The first step involved a reversed phase isocratic counter‐current chromatography separation using hexane/ethyl acetate/methanol/water (1:0.8:1:1 v/v/v/v), which produced three fractions, the first two of which were mixtures. The second step used step‐gradient reversed‐phase counter‐current chromatography with hexane/butanol/ethyl acetate/methanol/water (1:0.5:3.5:1:4 v/v/v/v/v) initially followed by hexane/ethyl acetate/methanol/water (1:2:1:2 v/v/v/v) to separate Fraction 1 into seven compounds; and hexane/ethyl acetate/methanol/water (1:1:1:1.2 v/v/v/v) to separate Fraction 2 into three further compounds. The chemical structures of the separated compounds were identified by ESI‐MS and NMR spectroscopy (¹H and ¹³C). Baicalin ( 5 ), eriodictyol ( 7 ), apigenin‐7‐glycoside ( 8 ), quercetin ( 9 ), luteolin ( 10 ), and apigenin ( 11 ) showed obvious inhibitory effects on lipopolysaccharide‐induced nitric oxide production in RAW264.7 cells at a concentration of 10 μg/mL.     

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.     

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.     

Combinative application of pH‐zone‐refining and conventional high‐speed counter‐current chromatography for preparative separation of caged polyprenylated xanthones from gamboge

An efficient method for the preparative separation of four structurally similar caged xanthones from the crude extracts of gamboge was established, which involves the combination of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography for the first time. pH‐zone‐refining counter‐current chromatography was performed with the solvent system composed of n‐hexane/ethyl acetate/methanol/water (7:3:8:2, v/v/v/v), where 0.1% trifluoroacetic acid was added to the upper organic stationary phase as a retainer and 0.03% triethylamine was added to the aqueous mobile phase as an eluter. From 3.157 g of the crude extract, 1.134 g of gambogic acid, 180.5 mg of gambogenic acid and 572.9 mg of a mixture of two other caged polyprenylated xanthones were obtained. The mixture was further separated by conventional high‐speed counter‐current chromatography with a solvent system composed of n‐hexane/ethyl acetate/methanol/water (5:5:10:5, v/v/v/v) and n‐hexane/methyl tert‐butyl ether/acetonitrile/water (8:2:6:4,v/v/v/v), yielding 11.6 mg of isogambogenic acid and 10.4 mg of β‐morellic acid from 218.0 mg of the mixture, respectively. The purities of all four of the compounds were over 95%, as determined by high‐performance liquid chromatography, and the chemical structures of the four compounds were confirmed by electrospray ionization mass spectrometry and NMR spectroscopy. The combinative application of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography shows great advantages in isolating and enriching the caged polyprenylated xanthones.     

Novel linear and step‐gradient counter‐current chromatography for bio‐guided isolation and purification of cytotoxic podophyllotoxins from Dysosma versipellis (Hance)

Dysosma versipellis (Hance) is a famous traditional Chinese medicine for the treatment of snakebite, weakness, condyloma accuminata, lymphadenopathy, and tumors for thousands of years. In this work, four podophyllotoxin‐like lignans including 4′‐demethylpodophyllotoxin (1), α‐peltatin (2), podophyllotoxin (3), β‐peltatin (4) as major cytotoxic principles of D. versipellis were successfully isolated and purified by several novel linear and step gradient counter‐current chromatography methods using the systems of hexane/ethyl acetate/methanol/water (4:6:3:7 and 4:6:4:6, v/v/v/v). Compared with isocratic elution, linear and step‐gradient elution can provide better resolution and save more time for the separation of photophyllotoxin and its congeners. Their cytotoxicities were further evaluated and their structures were validated by high‐resolution electrospray TOF MS and nuclear magnetic resonance spectra. All components showed potent anticancer activity against human hepatoma cells HepG2.     

Separation of α‐amylase inhibitors from Abelmoschus esculentus (L).Moench by on‐line two‐dimensional high‐speed counter‐current chromatography target‐guided by ultrafiltration‐HPLC

In this study, an on‐line two‐dimensional high‐speed counter‐current chromatography system based on a six‐port valve was developed. Target‐guided by ultrafiltration with high‐performance liquid chromatography, the one‐step isolation of three potential α‐amylase inhibitors from Abelmoschus esculentus (L).Moench was achieved by employing the developed orthogonal system and extrusion elution mode. The purities of three potential α‐amylase inhibitors were all over 95% as determined by high‐performance liquid chromatography. Furthermore, UV, mass spectrometry and ¹H NMR spectroscopy were applied to the structural identification of the isolated three target compounds, their structures were assigned as quercetin‐3‐O‐sophoroside (i), 5,7,3′,4′‐tetrahydroxy flavonol‐3‐O‐[β‐d ‐rhamnopyranosil‐(1→2)]‐β‐d ‐glucopyranoside (ii ) and isoquercitrin (iii), respectively. The Results demonstrated that the proposed method was highly efficient to screen and isolate enzyme inhibitors from complex natural products extracts, and on‐line two‐dimensional high‐speed counter‐current chromatography can effectively increase the peak resolution of target compounds.     

Separation and purification of four compounds from Desmodium styracifolium using off‐line two‐dimensional high‐speed counter‐current chromatography

An off‐line 2D high‐speed counter‐current chromatography technique in preparative scale has been successfully applied to separate and purify the main compounds from the ethyl acetate extract of Desmodium styracifolium. A two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water at an optimized volume ratio of 1:2:1:2 v/v/v/v was used. Conventional high‐speed counter‐current chromatography was used as the first dimension, and the upper phase of the solvent system was used as the stationary phase in the head‐to‐tail elution mode at a flow rate of 2.0 mL/min and a rotation speed of 900 rpm. Recycling high‐speed counter‐current chromatography served as the second dimension to separate an impure fraction of the first dimension. A total of four well‐separated substances including vanillic acid ( 1 ), β‐sitosterol ( 2 ), formononetin ( 3 ), and aromadendrin ( 4 ) were obtained, and their purities and structures were identified by HPLC–MS and ¹H NMR spectroscopy. The results illustrated that off‐line 2D high‐speed counter‐current chromatography is an effective way to isolate compounds in complex samples.     

Preparative isolation and purification of 12 main antioxidants from the roots of Polygonum multiflorum Thunb. using high‐speed countercurrent chromatography and preparative HPLC guided by 1,1′‐diphenyl‐2‐picrylhydrazyl‐HPLC

A hyphenated strategy by off‐line coupling of 1,1′‐diphenyl‐2‐picrylhydrazyl‐high‐performance liquid chromatography, high‐speed countercurrent chromatography, and preparative high‐performance liquid chromatography was established to screen and separate antioxidants from ethyl acetate fraction of the roots of Polygonum multiflorum. Under the targeted guidance of 1,1′‐diphenyl‐2‐picrylhydrazyl‐high‐performance liquid chromatography experiment, 12 compounds were identified as potential antioxidants and readily isolated by high‐speed counter‐current chromatography and preparative high‐performance liquid chromatography. Ultraviolet spectroscopy, mass spectrometry, and ¹H NMR spectroscopy were employed to identify their structures, which were assigned as gallic acid ( 1 , 6.2 mg, 98.28%), catechin ( 2 , 8.8 mg, 90.69%), epicatechin ( 3 , 4.1 mg, 96.71%), polydatin ( 4 , 5.3 mg, 94.91%), 2,3,5,4′‐tetrahydroxy stilbene‐2‐Ο‐β‐D‐glucoside ( 5 , 20.2 mg, 95.23%), piceatannol ( 6 , 5.3 mg, 96.85%), rutin ( 7 , 5.4 mg, 97.92%), resveratrol ( 8 , 5.2 mg, 96.94%), isorhapontigenin ( 9 , 11.4 mg, 94.81%), hyperoside ( 10 , 9.7 mg, 98.52%), rhein ( 11 , 4.9 mg, 97.46%), and emodin ( 12 , 8.2 mg, 95.74%). Notably, compounds 6 and 9 were isolated from Polygonum multiflorum for the first time. In addition, antioxidant activity of compounds 1–12 were evaluated, and compounds 1–8 and 10 exhibited stronger antioxidant activity than ascorbic acid (positive control). These results indicated that the proposed method is a highly efficient strategy to screen and isolate antioxidants from complex natural products.     

Separation of five diketopiperazines from the marine fungus Alternaria alternate HK‐25 by high‐speed counter‐current chromatography

High‐speed counter‐current chromatography was applied to the separation of five diketoperazines from the marine Alternaria alternate HK‐25 for the first time using one‐step elution method with a pair of two‐phase solvent systems composed of petroleum ether/ethyl acetate/methanol/water (5.5:11:5:7, v/v). Where 151.6 mg of crude sample yielded five diketoperazines, 12,13‐dihydroxy‐fumitremorgin C ( 1 ), gliotoxin ( 2 ), demethoxyfum itremorgin C ( 3 ), bisdethiobis(methylthio)gliotoxin ( 4 ), fumitremorgin C ( 5 ), and the purities of all compounds were above 94% as determined by high‐performance liquid chromatography. The structures of these compounds were identified by ¹H and ¹³C NMR spectroscopy. These results showed that high‐speed counter‐current chromatography can provide a feasible way for highly effective preparation of marine natural products, which ensured the supple of numerous samples for drug development.     

An effective method based on medium‐pressure liquid chromatography and recycling high‐speed counter‐current chromatography for enrichment and separation of three minor components with similar polarity from Dracocephalum tanguticum

The separation of minor compounds, especially those with similar polarities from a complex sample, remains challenging. In the proposed study, an effective method based on medium‐pressure liquid chromatography and recycling high‐speed counter‐current chromatography was developed for the enrichment and separation of three minor components from Dracocephalum tanguticum. The crude extract was directly introduced to medium‐pressure liquid chromatography for the enrichment of the three minor components. Based on high‐performance liquid chromatography analysis, the total content of these three compounds increased from 0.48% in the crude extract to 85.3% in the medium‐pressure liquid chromatography fraction. In addition, high‐speed counter‐current chromatography was employed to separate the enriched compounds using the solvent system hexane/ethyl acetate/methanol/water (1.18:8.82:1.18:8.82, v/v/v/v). As a result, compound 3 and a mixture of compounds 1 and 2 were obtained. In order to improve the resolution of compounds 1 and 2 while saving separation time, a recycling and heart‐cut mode was used. Finally, compounds 1 and 2 were obtained after five cycles. These compounds were identified as 3‐phenylethyl β‐d ‐glucopyranoside ( 1 ), tazettoside E ( 2 ), and cirsiliol‐4′‐glucoside ( 3 ). Compounds 1 and 2 were primarily separated from D. tanguticum. Moreover, the developed method provided a reference for the separation of minor components from the complex sample.     

Rapid preparative separation of six bioactive compounds from Gentiana crassicaulis Duthie ex Burk. using microwave‐assisted extraction coupled with high‐speed counter‐current chromatography

A rapid method combining microwave‐assisted extraction (MAE) and high‐speed counter‐current chromatography (HSCCC) was applied for preparative separation of six bioactive compounds including loganic acid ( I ), isoorientin‐4′‐O‐glucoside ( II ), 6′‐O‐β‐d ‐glucopyranosyl gentiopicroside ( III ), swertiamarin ( IV ), gentiopicroside ( V ), sweroside ( VI ) from traditional Tibetan medicine Gentiana crassicaulis Duthie ex Burk. MAE parameters were predicted by central composite design response surface methodology. That is, 5.0 g dried roots of G. crassicaulis were extracted with 50 mL 57.5% aqueous ethanol under 630 W for 3.39 min. The extract (gentian total glycosides) was separated by HSCCC with n‐butanol/ethyl acetate/methanol/1% acetic acid water (7.5:0.5:0.5:3.5, v/v/v/v) using upper phase mobile in tail‐to‐head elution mode. 16.3, 8.8, 12., 25.1, 40.7, and 21.8 mg of compounds I–VI were obtained with high purities in one run from 500 mg of original sample. The purities and identities of separated components were confirmed using HPLC with photo diode array detection and quadrupole TOF‐MS and NMR spectroscopy. The study reveals that response surface methodology is convenient and highly predictive for optimizing extraction process, MAE coupled with HSCCC could be an expeditious method for extraction and separation of phytochemicals from ethnomedicine.     

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.     

Enrichment and separation of antitumor triterpene acids from the epidermis of Poria cocos by pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography

Triterpene acids were extracted from the epidermis of Poria cocos (Schw.) Wolf. These acids were found to inhibit the growth of lung cancer cells in vitro and in vivo. An efficient method for the preparative separation of antitumor triterpene acids was established that involves the combination of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography. We used pH‐zone‐refining counter‐current chromatography to concentrate the triterpene acids using a two‐phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (3:7:5:5, v/v/v/v), trifluoroacetic acid (10 mM) was added to the upper phase as a retainer, and ammonia (10 mM) was added to the lower phase as an eluter. As a result, 200 mg concentrate of triterpene acids was obtained from 1.0 g of crude extract. The concentrate was further separated by conventional high‐speed counter‐current chromatography using a solvent system composed of petroleum ether/ethyl acetate/methanol/water (0.8:1.2:1.2:0.9, v/v), yielding 50 mg of poricoic acid A and 5 mg of poricoic acid B from 120 mg concentrate, respectively. The inhibitory activity of the major compound on lung A549 cells was examined and poricoic acid A was found to significantly inhibit the growth of A 549 cells.     

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.     

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.     

Separation of betacyanins from flowers of Amaranthus cruentus L. in a polar solvent system by high‐speed counter‐current chromatography

Betacyanin extract of Amaranthus cruentus L. flowers was fractionated by semi‐preparative high‐speed counter‐current chromatography in a highly polar solvent system: propan‐1‐ol/acetonitrile/(NH₄)₂SO_{4satd. soln}/H₂O (1.0:0.5:1.2:1.0, v/v/v/v) in tail‐to‐head mode with 76% retention of the stationary phase. The crude extract as well as the fractions containing betacyanins were analyzed by liquid chromatography with tandem mass spectrometry as well as by high‐resolution ion‐trap time‐of‐flight mass spectrometry detection technique for the molecular formulae and multi‐step fragmentation pattern elucidation. Four betacyanins; namely, amaranthin, betanin, 6′‐O‐formyl‐amaranthin, and 6′‐O‐malonyl‐amaranthin as well as their diastereomeric forms differing in the configuration of the C‐15 carbon atom were identified in the fractions. Amaranthin was the dominant pigment in the extract and was additionally analyzed by nuclear magnetic resonance correlation techniques after the counter‐current chromatographic and high‐performance liquid chromatographic isolation. Betacyanins were highly enriched during a single high‐speed counter‐current chromatographic step; therefore, the tentative identification of new compounds for the whole Amaranthaceae family, 6′‐O‐formyl‐amaranthin and 6′‐O‐malonyl‐amaranthin was possible. Different elution profiles of the pigments observed in the counter‐current chromatographic system in comparison to high‐performance liquid chromatography system confirm a complementarity of both the techniques especially in the separation of diastereomeric pairs of betacyanins.     

Enantioseparation of chiral aromatic acids by multiple dual mode counter‐current chromatography using hydroxypropyl‐β‐cyclodextrin as chiral selector

This work concentrates on extending the utilization of multiple dual mode (MDM) counter‐current chromatography in chiral separations. Two aromatic acids, 2‐(6‐methoxy‐2‐naphthyl)propionic acid (NAP) and 2‐phenylpropionic acid (2‐PPA), were enantioseparated by MDM counter‐current chromatography using hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as chiral selector. The two‐phase solvent systems consisting of n‐hexane/ethyl acetate 0.1 mol/L phosphate buffer pH 2.67 containing 0.1 mol/L HP‐β‐CD (7.5:2.5:10 for NAP and 7:3:10 for 2‐PPA, v/v/v) were used. Conventional MDM and modified MDM were compared according to peak resolution under current separation mechanism. The influence of elution time after the first‐phase inversion and number of cycles for MDM were investigated. Peak resolution of NAP and 2‐PPA increased from 0.62 to 1.05 and 0.72 to 0.84, respectively, using optimized MDM conditions. Being an alternative elution method for counter‐current chromatography, MDM elution greatly improved peak resolution in chiral separations.     

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 arctigenin from Fructus Arctii by enzymatic hydrolysis combined with high‐speed counter‐current chromatography

Enzymatic hydrolysis pretreatment combined with high‐speed counter‐current chromatography for the transformation and isolation of arctigenin from Fructus Arctii was successfully developed. In the first step, the extract solution of Fructus Arctii was enzymatic hydrolyzed by β‐glucosidase. The optimal hydrolysis conditions were 40°C, pH 5.0, 24 h of hydrolysis time, and 1.25 mg/mL β‐glucosidase concentration. Under these conditions, the content of arctigenin was transformed from 2.60 to 12.59 mg/g. In the second step, arctigenin in the hydrolysis products was separated and purified by high‐speed counter‐current chromatography with a two‐phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (10:25:15:20, v/v), and the fraction was analyzed by HPLC, ESI‐MS, and ¹H NMR spectroscopy. Finally, 102 mg of arctigenin with a purity of 98.9% was obtained in a one‐step separation from 200 mg of hydrolyzed sample.     

Efficient isolation and purification of five products from microbial biotransformation of cinobufagin by high‐speed counter‐current chromatography

An efficient separation method of using high‐speed counter‐current chromatography was successfully established to directly purify cytotoxic transformed products of cinobufagin by Cordyceps militaris. The two‐phase solvent system composed of n‐hexane–ethyl acetate–methanol–water (4:6:3:4, v/v) was used in high‐speed counter‐current chromatography. A total of 9 mg of 4β,12α‐dihydroxyl‐cinobufagin ( 1 ), 15 mg of 12β‐hydroxyl‐cinobufagin ( 2 ), 8 mg of 5β‐hydroxyl‐cinobufagin ( 3 ), 12 mg of deacetylcinobufagin ( 4 ) and 6 mg of 3‐keto‐cinobufagin ( 5 ) were obtained in a one‐step separation from 400 mg of the crude extract with purity of 98.7, 97.2, 90.6, 99.1 and 99.4%, respectively, as determined by HPLC. Their chemical structures were identified on the basis of ¹H‐NMR and ¹³C‐NMR technology. All products ( 1 – 5 ) showed the potent activities against human carcinoma cervicis (Hela) and malignant melanoma (A375) cells in vitro.