Direct purification of tanshinones from Salvia miltiorrhiza Bunge by high‐speed counter‐current chromatography without presaturation of the two‐phase solvent mixture

Tanshen, the rhizome of Salvia miltiorrhiza Bunge, is a famous Traditional Chinese Medicine for multiple therapeutic remedies. This work presents the isolation and purification of tanshinone I and tanshinone IIA from the extract of the rhizome of S. miltiorrhiza by using high‐speed counter‐current chromatography (CCC) without presaturation of the two‐phase solvent mixture. The CCC method combines the results of CCC solvent system selection and components analyses of solvent mixture by GC, and thus it is possible to add accurately each individual solvent to prepare single saturated solvent phase without presaturation. The optimum CCC solvent system is a system of hexane–ethyl acetate–ethanol–water (8:2:7:3, v/v), which has been determined by usual solvent system selection and CCC runs. As a result, over 98% pure tanshinone IIA and over 94% pure tanshinone I have been obtained by using less solvent volume. Their structures have been identified by ESI‐MS, NMR spectra.     

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.     

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.     

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.     

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 flavonoids from Millettia griffithii with high‐performance counter‐current chromatography guided by anti‐inflammatory activity

Millettia griffithii is a unique Chinese plant located in the southern part of Yunnan Province. Up to now, there is no report about its phytochemical or related bioactivity research. In our previous study, the n‐hexane crude extract of Millettia griffithii revealed significant anti‐inflammatory activity at 100 μg/mL, inspiring us to explore the anti‐inflammatory constituents. Four fractions (I, II, III, and A) were fractionated from n‐hexane crude extract by high‐performance counter‐current chromatography with solvent system composed of n‐hexane/ethyl acetate/methanol/water (8:9:8:9, v/v) and then were investigated for the potent anti‐inflammatory activity. Fraction A, with the most potent inhibitory activity was further separated to give another four fractions (IV, V, VI, and B) with solvent system composed of n‐hexane/ethyl acetate/methanol/water (8:4:8:4, v/v). Compound V and fraction B exhibited remarkable anti‐inflammatory activity with nitric oxide inhibitory rate of 80 and 65%, which was worth further fractionation. Then, three fractions (VII, VIII, and IX) were separated from fraction B with a solvent system composed of n‐hexane/ethyl acetate/methanol/water (8:1:8:1, v/v), with compound VIII demonstrating the most potent inhibitory activity (80%). Finally, the IC₅₀ values of compound V and VIII were tested as 38.2 and 14.9 μM. The structures were identified by electrospray ionization mass spectrometry and¹H and ¹³C NMR spectroscopy.     

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.     

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.     

Application of high‐speed counter‐current chromatography and HPLC to separate and purify of three polyacetylenes from Platycodon grandiflorum

Three polyacetylenes were isolated and purified from Platycodon grandiflorum A. DC for the first time by high‐speed counter‐current chromatography using a two‐phase solvent system composed of hexane/ethyl acetate/methanol/water (1:31:1:31, v/v/v/v) and high‐performance liquid chromatography with an Agilent ZORBAX® SB‐C18 column (4.6 mm × 150 mm, 5 μm). After separation by high‐speed counter‐current chromatography and high‐performance liquid chromatography, we obtained 3.5 mg of platetyolin A, 4.1 mg of platetyolin B, and 18.1 mg of lobetyolin with purities of 97.2, 96.7, and 96.9%, respectively. The purity of each compound was assessed by high‐performance liquid chromatography and the chemical structures were evaluated by high‐resolution electrospray ionization time‐of‐flight mass spectrometry and one‐ and two‐dimensional NMR spectroscopy. Among the isolated compounds, platetyolin A and platetyolin B are newly reported compounds.     

Separation and purification of antioxidants from Ampelopsis heterophylla by counter‐current chromatography

In this study, bioactive components from Ampelopsis heterophylla were separated by counter‐current chromatography (CCC). The antioxidant activity of the crude extract was initially evaluated by an online HPLC method. Five compounds in the crude extract exhibited good antioxidant activities, namely, hyperoside ( 1 ), isoquercitrin ( 2 ), rutin ( 3 ), kaempferol‐3‐rutinoside ( 4 ), and quercetin ( 5 ). These compounds were further separated by CCC with biphasic solvent systems and their structures were identified by MS and NMR spectroscopy. All the compounds exhibited significant 1,1‐diphenyl‐2‐picryl‐hydrazyl radical scavenging activities with IC₅₀ values at 18.2 ± 1.3, 17.0 ± 1.4, 24.2 ± 1.2, 38.1 ± 1.7, and 9.0 ± 1.2 μM, respectively. The scavenging ratios of the compounds against hydroxyl radicals were 65 ± 5, 68 ± 4, 96 ± 2, 70 ± 4, and 98 ± 2%, respectively.     

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.     

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.     

Isolation and purification of prenylated phenolics from Amorpha fruticosa by high‐speed counter‐current chromatography

Prenylated phenolics such as amorfrutins are recently identified potent anti‐inflammatory and antidiabetic natural products. In this work, high‐speed counter‐current chromatography was investigated for the isolation and purification of prenylated phenolics from the fruits of Amorpha fruticosa by using a two‐phase solvent system composed of n‐hexane/ethanol/water (5:4:1, v/v). As a result, 14.2 mg of 5,7‐dihydroxy‐8‐geranylflavanone, 10.7 mg of amorfrutin A and 17.4 mg of amorfrutin B were obtained from 200 mg of n‐hexane‐soluble crude extract in one step within 250 min. The purities of 5,7‐dihydroxy‐8‐geranylflavanone, amorfrutins A and B were 95.2, 96.7 and 97.1%, respectively, as determined by ultra high performance liquid chromatography. The structural identification was performed by mass spectrometry and ¹H and ¹³C NMR spectroscopy. The results indicated that the established method is an efficient and convenient way to purified prenylated phenolics from A. fruticosa extract.     

Screening and separation of α‐amylase inhibitors from Solanum nigrum with amylase‐functionalized magnetic graphene oxide combined with high‐speed counter‐current chromatography

A screening method using α‐amylase‐functionalized magnetic graphene oxide combined with high‐speed counter‐current chromatography was proposed and utilized to screen and separate α‐amylase inhibitors from extract of Solanum nigrum . The α‐amylase‐functionalized magnetic graphene oxide was characterized and found to demonstrate satisfactory structure, magnetic response (24.5 emu/g), and reusability (retained 90% of initial activity after five cycles). The conditions for the screening with α‐amylase functionalized magnetic graphene oxide were optimized and set at pH 7.0 and 25°C. As a result, two potent flavonoid compounds, apigenin‐7‐O‐glucuronide ( 1 ) and astragalin ( 2 ), were separated and collected through high‐speed counter‐current chromatography and subjected to high‐performance liquid chromatography analysis with purity higher than 90% (according to HPLC data), which were identified as α‐amylase inhibitors. These results suggested that utilization of α‐amylase functionalized magnetic graphene oxide in the rapid screening and isolation bioactive compounds from complex natural products is a feasible and environmentally friendly method.     

An interesting two‐phase solvent system and its use in preparative isolation of aconitines from aconite roots by counter‐current chromatography

Two‐phase solvent system plays crucial role in successful separation of organic compounds using counter‐current chromatography (CCC). An interesting two‐phase solvent system, composed of chloroform/ethyl acetate/methanol/water, is reported here, in which both phases contain sufficient organic solvents to balance their dissolving capacities. Adjusting the solvent system to get satisfactory partition coefficients (K values) for target compounds becomes relatively simple. This solvent system succeeded in sample preparation of aconitine (8.07 mg, 93.69%), hypaconitine (7.74 mg, 93.17%), mesaconitine (1.95 mg, 94.52%) from raw aconite roots (102.24 mg, crude extract), benzoylmesaconine (34.79 mg, 98.67%) from processed aconite roots (400.01 mg, crude extract), and yunaconitine (253.59 mg, 98.65%) from a crude extract of Aconitum forrestii (326.69 mg, crude extract).     

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.     

Isolation of terpenoids from Pimpinella anisum essential oil by high‐performance counter‐current chromatography

High‐performance counter‐current chromatography was successfully used for the isolation and purification of terpenoid compounds from the essential oil of Pimpinella anisum L. A two‐phase solvent system composed of n‐heptane/methanol/ethyl acetate/water (5:2:5:2, v/v/v/v) was suitable for the purification of linalool, terpinen‐4‐ol, α‐terpineol, p‐anisaldehyde, while n‐heptane/methanol (1:1, v/v) was used for the isolation of anethole and foeniculin. A scale‐up process from analytical to preparative was developed. Additionally, a stepwise gradient elution was applied and instead of two different runs, 40 min each, one 80 min separation was performed; although the time of separation remains the same, it was possible to repeat the efficiency even if the water‐containing mobile phase was changed to a nonaqueous system. The obtained essential oil, as well as purified compounds, was analyzed by GC. A total of 0.64 mg of linalool, 0.52 mg of terpinen‐4‐ol, 0.10 mg of α‐terpineol, 0.62 mg of p‐anisaldehyde, 15 mg of anethole, and 2.12 mg of foeniculin were obtained from 210 mg of the essential oil of P. anisum L. in a short time with purities of 99, 98, 94, 93.54, 93, and 93.6%, respectively.     

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.     

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.     

Effect of inorganic salt on partition of high‐polarity parishins in two‐phase solvent systems and separation by high‐speed counter‐current chromatography from Gastrodia elata Blume

Parishins are high‐polarity and major bioactive constituents in Gastrodia elata Blume. In this study, the effect of several inorganic salts on the partition of parishins in two‐phase solvent systems was investigated. Adding ammonium sulfate, which has a higher solubility in water, was found to significantly promote the partition of parishins in the upper organic polar solvents. Based on the results, a two‐phase solvent system composed of butyl alcohol/acetonitrile/near‐saturated ammonium sulfate solution/water (1.5:0.5:1.2:1, v/v/v/v) was used for the purification of parishins by high‐speed counter‐current chromatography. Fractions obtained from high‐speed counter‐current chromatography were subjected to semi‐preparative high‐performance liquid chromatography to remove salt and impurities. As a result, parishin E (6.0 mg), parishin B (7.8 mg), parishin C (3.2 mg), gastrodin (15.3 mg), and parishin A (7.3 mg) were isolated from water extract of Gastrodia elata Blume (400 mg). These results demonstrated that adding inorganic salt that has high solubility in water to the two‐phase solvent system in high‐speed counter‐current chromatography was a suitable approach for the purification of high‐polarity compounds.