Isolation and purification of alkaloids from lotus leaves by ionic‐liquid‐modified high‐speed countercurrent chromatography

An effective high‐speed countercurrent chromatography method was successfully established by using ionic liquids as the modifier of the two‐phase solvent system. Adding a small amount of ionic liquids significantly shortens the separation time and improves the separation efficiency. The conditions of ionic‐liquid‐modified high‐speed countercurrent chromatography including solvent systems, types and content of added ionic liquids, and ionic liquids posttreatment were investigated. The established method was successfully applied to separate alkaloids from lotus leaves using a two‐phase solvent system composed of petroleum ether/ethyl acetate/methanol/water/[C₄mim][BF₄] (1:5:1:5:0.15, v/v/v/v/v). Four alkaloids pronuciferine (1.7 mg), N‐nornuciferine (4.3 mg), nuciferine (3.1 mg), and roemerine (2.1 mg) were obtained with the purities of 90.53, 92.25, 99.86, and 98.63%, respectively, from 100 mg crude extract of lotus leaves. The results indicated that the ionic‐liquid‐modified high‐speed countercurrent chromatography method was suitable for alkaloid separation from lotus leaves and would be a promising method for the separation of alkaloids from other natural products.     

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.     

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.     

Separation and purification of hydrolyzable tannin from Geranium wilfordii Maxim by reversed‐phase and normal‐phase high‐speed counter‐current chromatography

Three hydrolyzable tannins, geraniin, corilagin and gallic acid, main active components of Geranium wilfordii Maxim, have been separated and purified in one‐step by both reversed‐phase and normal‐phase high‐speed counter‐current chromatography. Gallic acid, corilagin and geraniin were purified from 70% aqueous acetone extract of G. wilfordii Maxim with solvent system n‐hexane–ethyl acetate–methanol–acetic acid–water (1:10:0.2:0.2:20) by reversed‐phase high‐speed counter‐current chromatography at purities of 94.2, 91.0 and 91.3%, at yields of 89.3, 82.9 and 91.7%, respectively. Gallic acid, corilagin and geraniin were purified with solvent system n‐hexane–ethyl acetate–methanol–acetic acid–water (0.2:10:2:1:5) by normal‐phase high‐speed counter‐current chromatography at purities of 85.9, 92.2 and 87.6%, at yields of 87.4, 94.6 and 94.3%, respectively. It was successful for both reversed‐phase and normal‐phase high‐speed counter‐current chromatography to separate high‐polarity of low‐molecular‐weight substances.     

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.     

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.     

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.     

Ultra‐high‐pressure‐assisted extraction of wedelolactone and isodemethylwedelolactone from Ecliptae Herba and purification by high‐speed counter‐current chromatography

Ultra‐high‐pressure extraction combined with high‐speed counter‐current chromatography was employed to extract and purify wedelolactone and isodemethylwedelolactone from Ecliptae Herba. The operating conditions of ultra‐high‐pressure extraction were optimized using an orthogonal experimental design. The optimal conditions were 80% aqueous methanol solvent, 200 MPa pressure, 3 min extraction time and 1:20 (g/mL) solid–liquid ratio for extraction of wedelolactone and isodemethylwedelolactone. After extraction by ultra‐high pressure, the extraction solution was concentrated and subsequently extracted with ethyl acetate; a total of 2.1 g of crude sample was obtained from 100 g of Ecliptae Herba. A two‐phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (3:7:5:5, v/v) was used for high‐speed counter‐current chromatography separation, by which 23.5 mg wedelolactone, 6.8 mg isodemethylwedelolactone and 5.5 mg luteolin with purities >95% were purified from 300 mg crude sample in a one‐step separation. This research demonstrated that ultra‐high‐pressure extraction combined with high‐speed counter‐current chromatography was an efficient technique for the extraction and purification of coumestans from plant material.     

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.     

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 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.     

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.     

Direct from solid natural products to pure compounds in a single step: Coupling online extraction with high‐speed counter‐current chromatography

Extraction is the most important step in the purification of bioactive compounds from natural products. This study introduces a simple online extraction strategy coupled with high‐speed counter‐current chromatography for efficient extraction and purification of bioactive components from solid natural products. For online extraction strategy, 1.0 g of ground Mangnolia officinalis or Piper nigrum was loaded into a guard column, which was then positioned on the manual injection valve instead of the sample loop. Bioactive components were directly extracted by the mobile phase of high‐speed counter‐current chromatography, and then transferred into high‐speed counter‐current chromatography for purification. In addition, the compatibility of the developed methodology for direct purification of bioactive components from fresh M. officinalis was successfully demonstrated. Obviously, in comparison with traditional offline heat‐reflux extraction, online extraction avoided the instrument, time, solvent, and energy consumption, and purified two phenolic compounds (honokiol and magnolol) from M. officinalis and three alkaloids (piperyline, piperine, and piperanine) from P. nigrum with high extraction efficiency. The superiority of the developed methodology is to establish an easy, rapid, and efficient technique for the purification of a wide variety of bioactive components from solid natural products.     

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.     

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 and purification of five alkaloids from Aconitum duclouxii by counter‐current chromatography

C₁₉‐diterpenoid alkaloids are the main components of Aconitum duclouxii Levl. The process of separation and purification of these compounds in previous studies was tedious and time consuming, requiring multiple chromatographic steps, thus resulted in low recovery and high cost. In the present work, five C₁₉‐diterpenoid alkaloids, namely, benzoylaconine ( 1 ), N‐deethylaconitine ( 2 ), aconitine ( 3 ), deoxyaconitine ( 4 ), and ducloudine A ( 5 ), were efficiently prepared from A. duclouxii Levl (Aconitum L.) by ethyl acetate extraction followed with counter‐current chromatography. In the process of separation, the critical conditions of counter‐current chromatography were optimized. The two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water/NH₃·H₂O (25%) (1:1:1:1:0.1, v/v) was selected and 148.2 mg of 1 , 24.1 mg of 2 , 250.6 mg of 3 , 73.9 mg of 4, and 31.4 mg of 5 were obtained from 1 g total Aconitum alkaloids extract, respectively, in a single run within 4 h. Their purities were found to be 98.4, 97.2, 98.2, 96.8, and 96.6%, respectively, by ultra‐high performance liquid chromatography analysis. The presented separation and purification method was simple, fast, and efficient, and the obtained highly pure alkaloids are suitable for biochemical and toxicological investigation.     

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.     

Optimization of microwave‐assisted extraction of protopine and allocryptopine from stems of Macleaya cordata (Willd) R. Br. using response surface methodology

The purpose of the research was to investigate the multiple response optimizations for the extraction of protopine and allocryptopine from the stems of Macleaya cordata (Willd) R. Br. by using microwave‐assisted extraction (MAE). A three‐level, three‐factor Box–Behnken design of response surface methodology was used to develop response model, and desirability function was employed to optimize the effects of main extraction parameters. Three variables, ethanol concentration (20–80%, v/v), extraction temperature (30–70°C) and solvent/solid ratio (10:1 to 30:1, mL/g), were investigated in this study. The results showed that the optimum parameters of MAE were ethanol concentration of 45.2 % (v/v), extraction temperature of 54.7°C and solvent/solid ratio of 20.4:1 (mL/g). Under these conditions, the extraction yields of protopine and allocryptopine were 89.4 and 102.0%, respectively, and the extracta sicca yield was 12.5%. The combination use of response surface methodology, Box‐Behnken design and the appropriate desirability function could provide an insight into a lab‐scale MAE process, and help to develop procedures for commercial production of active ingredients from medical plants.     

Semi‐preparative high‐speed counter‐current chromatography separation of alkaloids from embryo of the seed of Nelumbo nucifera Gaertn by pH‐gradient elution

A new high‐speed counter‐current chromatography method for semi‐preparative separation and purification of alkaloids from embryo of the seed of Nelumbo nucifera Gaertn was developed by using pH‐gradient elution mode. Diethyl ether was used as the stationary phase of the two‐phase solvent system and Na₂HPO₄/NaH₂PO₄ buffer solution with pH values of 7.5 and 7.2 in gradient mode as the mobile phase. Consequently, 33 mg of liensinine, 42 mg of isoliensinine, and 67 mg of neferine were obtained from 200 mg of crude extracts. The purities of them were all over 98% as determined by HPLC area normalization method, and the structures were identified by 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.