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.     

Extraction and purification of five terpenoids from olibanum by ultrahigh pressure technique and high‐speed countercurrent chromatography

Five terpenoids, including two new ones, 3,7‐dioxo‐tirucalla‐8,24‐dien‐21‐oic acid ( 2 ) and 3α‐acetoxyl‐7‐oxo‐tirucalla‐8,24‐dien‐21‐oic acid ( 3 ), and three known ones, boscartol A ( 1 ), 11‐keto‐β‐boswellic acid ( 4 ), and acetyl‐11‐keto‐boswellic acid ( 5 ), have been extracted by the ultrapressure extraction and purified by pH‐zone‐refining countercurrent chromatography and high‐speed countercurrent chromatography from olibanum. For ultrapressure extraction, the optimal condition including 200 MPa of extraction pressure, ethyl acetate of extraction solvent, 1:20 (g/mL) of solid/liquid ratio, and 2 min of extraction time were obtained. For the separation, from 1.5 g of the terpenoid extract, 220.1 mg of 4 , 255.5 mg of 5 , and 212.3 mg of the mixture of 1 , 2 , and 3 were obtained by pH‐zone‐refining countercurrent chromatography under the solvent system of chloroform/ethyl acetate/methanol/water (3:1:3:2, v/v/v/v) with aqueous ammonia and trifluoroacetic acid as retention and eluter agents. The enriched mixture (210 mg) was further separated by conventional high‐speed countercurrent chromatography with petroleum ether/ethyl acetate/methanol/water (1:0.8:1.1:0.6, v/v/v/v), yielding 30.1 mg of 1 , 35.5 mg of 2 , 12.3 mg of 3 . The structures of these five terpenoids were elucidated by extensive spectroscopic methods.     

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.     

Effective on‐line high‐speed shear dispersing emulsifier technique coupled with high‐performance countercurrent chromatography method for simultaneous extraction and isolation of carotenoids from Lycium barbarum L. fruits

An efficient combination strategy based on high‐speed shear dispersing emulsifier technique and high‐performance countercurrent chromatography was developed for on‐line extraction and isolation of carotenoids from the fruits of Lycium barbarum. In this work, the high‐speed shear dispersing emulsifier technique has been employed to extract crude extracts using the upper phase of high‐performance countercurrent chromatography solvent system composed of n‐hexane?dichloromethane?acetonitrile (10:4:6.5, v/v) as the extraction solvent. At the separation stage, the high‐performance counter‐current chromatography process adopts elution–extrusion mode and the upper phase of the solvent system as stationary phase (reverse‐phase mode). As a result, three compounds including zeaxanthin, zeaxanthin monopalmitate, and zeaxanthin dipalmitate with purities of 89, 90, and 93% were successfully obtained in one extraction‐separation operation within 120 min. The targeted compounds were analyzed and identified by high‐performance liquid chromatography, mass spectrometry, and NMR spectroscopy. The results indicated that the present on‐line combination method could serve as a simple, rapid, and effective way to achieve weak polar and unstable compounds from natural products.     

Preparative separation of five flavones from flowers of Polygonum cuspidatum by high‐speed countercurrent chromatography

A preparative high‐speed countercurrent chromatography method was successfully used for the isolation of five minor flavones from Polygonum cuspidatum flowers. Among them, three compounds were obtained from P. cuspidatum for the first time. A twin two‐phase solvent system composed of n‐hexane/ethyl acetate/ethanol/water (1:6:3:6, v/v/v/v) and petroleum ether/ethyl acetate/methanol/water (2:4:3:3, v/v/v/v) was developed. Compounds were obtained from the fraction B and fraction C prepurified by silica gel column chromatography. Five minor compositions, 6.8 mg of hesperidin, 11.2 mg of phloridzin, 4.9 mg of luteolin, 5.3 mg of hyperin, and 3.7 mg of luteoloside were obtained from 140 mg of the fraction B and 110 mg of fraction C with a purity of 95.3, 96.4, 98.0, 96.8, and 95.3%, respectively, as determined by high‐performance liquid chromatography. The structures of these compounds were identified by ¹H and ¹³C NMR spectroscopy.     

Preparative isolation of neomangiferin and mangiferin from Rhizoma anemarrhenae by high‐speed countercurrent chromatography using ionic liquids as a two‐phase solvent system modifier

A preparative high‐speed countercurrent chromatography method for isolation and purification of neomangiferin and mangiferin from Rhizoma anemarrhenae was successfully established by using ionic liquids as the modifier of the two‐phase solvent system. Neomangiferin and mangiferin were purified from the crude extract of R. anemarrhenae by using ethyl acetate‐water‐[C₄mim][PF₆] (5:5:0.2 v/v) as two‐phase solvent system. In total, 22.5 mg of neomangiferin and 70.6 mg of mangiferin were obtained from 150 mg of the crude extract. The purities of neomangiferin and mangiferin were 97.2 and 98.1%, respectively, as determined by HPLC. The chemical structures of the isolated compounds were identified by ¹H‐NMR and ¹³C‐NMR.     

An efficient strategy for the extraction and purification of lignans from Schisandra chinensis by a combination of supercritical fluid extraction and high‐speed counter‐current chromatography

An efficient strategy for extracting and separating five lignans from Schisandra chinensis (Turcz.) Baill has been developed using supercritical fluid extraction (SFE) and high‐speed counter‐current chromatography (HSCCC) in the present study. First, the extraction was performed by a preparative SFE system under 15 MPa of pressure at 36°C for 4 h. Then, the SFE extract was successfully separated and purified by HSCCC with a two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water (6:4:5:5, 6:4:6:4, 6:4:8:2, v/v) in a stepwise elution mode. The fractions were analyzed by HPLC, and the chemical structures of the products were identified by ESI‐MS and ¹H NMR spectroscopy. As a result, a total of 12.5 mg of schisandrin at 98.0% purity, 7.1 mg of gomisin A at 98.1% purity, 1.8 mg of schisantherin B at 93.3% purity, 4.4 mg of deoxyschisandrin at 92.9% purity, and 6.8 mg of γ‐schisandrin at 89.1% purity were obtained from 300 mg crude extract in a one‐step purification.     

Preparative separation and purification of two highly polar alkaloids derived from Semen Strychni extracted with dichloromethane by high‐speed countercurrent chromatography

Brucine chloromethochloride and strychnine chloromethochloride, the two chloromethochloride derivatives formed during the extraction of Semen Strychni in which dichloromethane was used as the extracting solvent, were isolated and purified by high‐speed countercurrent chromatography for the first time. The two‐phase solvent system composed of chloroform/methanol/0.3 mol/L hydrochloric acid (4:3:2, v/v/v) was selected for separation. From 300 mg of the crude extracts, 56.2 mg of brucine chloromethochloride and 60.2 mg of strychnine chloromethochloride were obtained with the purity of 99.78 and 96.99%, respectively, and the structures were confirmed by mass spectrometry, ¹H, ¹³C, and two‐dimensional NMR spectroscopy. The results indicated that the present method is a powerful technology for large‐scale isolation of alkaloids from Semen Strychni.     

Separation and purification of two taxanes and one xylosyl‐containing taxane from Taxus wallichiana Zucc.: A comparison between high‐speed countercurrent chromatography and reversed‐phase flash chromatography

10‐Deacetylbaccatin III, an important semisynthetic precursor of paclitaxel and docetaxel, can be extracted from Taxus wallichiana Zucc. A process for the isolation and purification of 10‐deacetylbaccatin III ( 1 ), baccatin III ( 2 ), and 7β‐xylosyl‐10‐deacetyltaxol ( 3 ) from the leaves and branches of Taxus wallichiana Zucc. via macroporous resin column chromatography combined with high‐speed countercurrent chromatography or reversed‐phase flash chromatography was developed in this study. After fractionation by macroporous resin column chromatography, 80% methanol fraction was selected based on high‐performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis. A solvent system composed of n‐hexane, ethyl acetate, methanol, and water (1.6:2.5:1.6:2.5, v/v/v/v) was used for the high‐speed countercurrent chromatography separation at a flow rate of 2.5 mL/min. The reversed‐phase flash chromatography separation was performed using methanol/water as the mobile phase at a flow rate of 3 mL/min. The high‐speed countercurrent chromatography separation produced compounds 1 (10.2 mg, 94.4%), 2 (2.1 mg, 98.0%), and 3 (4.6 mg, 98.8%) from 100 mg of sample within 110 min, while the reversed‐phase flash chromatography separation purified compounds 1 (9.8 mg, 95.6%) and 3 (4.9 mg, 97.9%) from 100 mg of sample within 120 min.     

Extraction and isolation of diphenylheptanes and flavonoids from Alpinia officinarum Hance using supercritical fluid extraction followed by supercritical fluid chromatography

In this paper, an off-line combination method of supercritical fluid extraction and supercritical fluid chromatography was developed for the selective extraction and isolation of diphenylheptanes and flavonoids from Alpinia officinarum Hance. The enrichment of target components was successfully achieved using supercritical fluid extraction with the following conditions (8% ethanol as co-solvent at 45°C and 30 MPa for 30 min). Taking full advantage of the complementarity of supercritical fluid chromatography stationary phases, a two-step preparative supercritical fluid chromatography strategy was constructed. The extract was firstly divided into seven fractions on a Diol column (250 × 20 mm internal diameter, 10 μm) within 8 min by gradient elution increasing from 5% to 20% modifier (methanol) at 55 ml/min and 15 MPa. Then the seven fractions were separated by using a 1-AA or a DEA column (250 × 19 mm internal diameter, 5 μm) at 50 ml/min and 13.5 MPa. This two-step strategy showed superior separation ability for structural analogs. As a result, seven compounds, including four diphenylheptanes and three flavonoids with high purity, were successfully obtained. The developed method is also helpful for the extraction and isolation of other structural analogs of traditional Chinese medicines.     

Evaluation of gardenia yellow using crocetin from alkaline hydrolysis based on ultra high performance liquid chromatography and high‐speed countercurrent chromatography

Gardenia yellow is globally the most valuable spice and food color. It is generally a mixture of water‐soluble carotenoid glycosyl esters which consist of crocetin bis(gentiobiosyl) ester as the main component. Crocetin is a natural carotenoid dicarboxylic acid that may be a candidate drug for pharmaceutical development, however, it is either present in trace amounts or is absent in natural gardenia yellow products. We here propose that crocetin produced by alkaline hydrolysis can be used to qualitatively evaluate gardenia yellow products using an ultra high performance liquid chromatographic assay. A useful and efficient isolation technique for isolating high‐purity crocetin from gardenia yellow using high‐speed countercurrent chromatography is described. High‐speed countercurrent chromatographic fractionation followed by an ultra high performance liquid chromatographic assay showed that trans‐crocetin is easily converted to about 15% cis‐crocetin (85% trans‐crocetin). Crocetin in gardenia yellow was quantitatively evaluated. Our approach is based on the hydrolysis process for converting crocetin glycosyl esters to crocetin before evaluation and isolation using the ultra high performance liquid chromatographic and high‐speed countercurrent chromatographic methods. The combination of hydrolysis and chromatographic methods allows evaluation of the purity and quantity of crocetin in gardenia yellow.     

Determination of major aromatic constituents in vanilla using an on‐line supercritical fluid extraction coupled with supercritical fluid chromatography

An on‐line supercritical fluid extraction coupled with supercritical fluid chromatography method was developed for the determination of four major aromatic constituents in vanilla. The parameters of supercritical fluid extraction were systematically investigated using single factor optimization experiments and response surface methodology by a Box–Behnken design. The modifier ratio, split ratio, and the extraction temperature and pressure were the major parameters which have significant effects on the extraction. While the static extraction time, dynamic extraction time, and recycle time had little influence on the compounds with low polarity. Under the optimized conditions, the relative extraction efficiencies of all the constituents reached 89.0–95.1%. The limits of quantification were in the range of 1.123–4.747 μg. The limits of detection were in the range of 0.3368–1.424 μg. The recoveries of the four analytes were in the range of 76.1–88.9%. The relative standard deviations of intra‐ and interday precision ranged from 4.2 to 7.6%. Compared with other off‐line methods, the present method obtained higher extraction yields for all four aromatic constituents. Finally, this method has been applied to the analysis of vanilla from different sources. On the basis of the results, the on‐line supercritical fluid extraction‐supercritical fluid chromatography method shows great promise in the analysis of aromatic constituents in natural products.     

Preparative mass‐spectrometry profiling of minor concentrated metabolites in Salicornia gaudichaudiana Moq by high‐speed countercurrent chromatography and off‐line electrospray mass‐spectrometry injection

Salicornia species have just been introduced to the European market as a vegetable named ‘samphire’, ‘green asparagus’, or ‘sea asparagus’. Due to its increasing attention, and associated value, minor compounds of Salicornia gaudichaudiana Moq were investigated. The use of countercurrent chromatography and mass spectrometry enabled the search for known, as well as potentially novel natural products. Their identification was achieved based on molecular weights and mass‐spectrometric fragmentation data. Low detection limits enabled the visualization of all compounds with their identification in almost real time close to the preparative countercurrent chromatography experiment. A list of known natural products from Salicornia genus guided the identification process of compounds occurring in Salicornia gaudichaudiana Moq by tandem mass spectrometry fragment comparison. The natural product classes were divided into four groups: chlorogenic acid derivatives; flavonoid derivatives; pentacyclic triterpenoid saponins; and other compounds.     

Separation of phenylsuccinic acid enantiomers using biphasic chiral recognition high‐speed countercurrent chromatography

High‐speed countercurrent chromatography (HSCCC) combined with biphasic chiral recognition was successfully applied to the resolution of phenylsuccinic acid enantiomers. d ‐Isobutyl tartrate and hydroxypropyl‐β‐cyclodextrin were employed as lipophilic and hydrophilic selectors dissolved in the organic stationary phase and aqueous mobile phase, respectively. The two‐phase solvent system was made up of n‐hexane/methyl tert‐butyl ether/water (0.5:1.5:2, v/v/v). Impacts of the type and concentration of chiral selectors, the pH value of the aqueous phase solution as well as the temperature on the separation efficiency were investigated. By means of preparative HSCCC, pure enantiomer was obtained by separating 810 mg of racemate with a purity >99.5% and a recovery rate between 82 and 85%. The experimental results indicate that biphasic recognition HSCCC provide a promising means for efficient separation of racemates.     

Separation of acteoside and linarin from Buddlejae Flos by high‐speed countercurrent chromatography and their anti‐inflammatory activities

Buddleja officinalis Maxim., a deciduous, flowering shrub, is used as a traditional Chinese medicine; the bioactivity of B. officinalis is primarily due to flavonoids and phenylethanoid glycosides. In the study, acteoside and linarin were successfully isolated from B. officinalis by high‐speed countercurrent chromatography with a two‐phase solvent system composed of ethyl acetate: n‐butanol: water (5:0.8:5, v/v/v). The purities of acteoside and linarin were determined to be 97.3 and 98.2%, respectively, using one‐step high‐speed countercurrent chromatography separation. The chemical structures of the two compounds were identified by electrospray ionization‐mass spectrometry and nuclear magnetic resonance. After separation, the anti‐inflammatory effects of the two compounds were evaluated using lipopolysaccharide‐induced human umbilical vein endothelial cells. Acteoside and linarin inhibited the expression of nitric oxide, tumor necrosis factor α and interleukin 1β, which demonstrated that acteoside and linarin possessed anti‐inflammatory activity.     

Screening and isolation of cyclooxygenase‐2 inhibitors from Trifolium pratense L. via ultrafiltration,enzyme‐immobilized magnetic beads,semi‐preparative high‐performance liquid chromatography and high‐speed counter‐current chromatography

Nonsteroidal anti‐inflammatory drugs reportedly reduce the risk of developing cancer. One mechanism by which they reduce carcinogenesis involves the inhibition of the activity of cyclooxygenase‐2, an enzyme that is overexpressed in various cancer tissues. Its overexpression increases cell proliferation and inhibits apoptosis. However, selected cyclooxygenase‐2 inhibitors can also act through cyclooxygenase‐independent mechanisms. In this study, using ultrafiltration, enzyme‐immobilized magnetic beads, high‐performance liquid chromatography, and electrospray‐ionization mass spectrometry, several isoflavonoids in Trifolium pratense L. extracts were screened and identified. Semi‐preparative high‐performance liquid chromatography and high‐speed counter‐current chromatography were then applied to separate the active constituents. Using these methods, seven major compounds were identified in Trifolium pratense L. As cyclooxygenase‐2 inhibitors: rothindin, ononin, daidzein, trifoside, pseudobaptigenin, formononetin, and biochanin A, which were then isolated with >92% purity. This is the first report of the presence of potent cyclooxygenase‐2 inhibitors in Trifolium pratense L. extracts. The results of this study demonstrate that the systematic isolation of bioactive components from Trifolium pratense L., by using ultrafiltration, enzyme‐immobilized magnetic beads, semi‐preparative high‐performance liquid chromatography, and high‐speed counter‐current chromatography, represents a feasible and efficient technique that could be extended for the identification and isolation of other enzyme inhibitors.     

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.     

Separation of three anthraquinone glycosides including two isomers by preparative high‐performance liquid chromatography and high‐speed countercurrent chromatography from Rheum tanguticum Maxim. ex Balf

Anthraquinone glycosides, such as chrysophanol 1‐O‐β‐d‐ glucoside, chrysophanol 8‐O‐β‐d‐ glucoside, and physion 8‐O‐β‐d‐ glucoside, are the accepted important active components of Rheum tanguticum Maxim. ex Balf. due to their pharmacological properties: antifungal, antimicrobial, cytotoxic, and antioxidant activities. However, an effective method for the separation of the above‐mentioned anthraquinone glycosides from this herb is not currently available. Especially, greater difficulty existed in the separation of the two isomers chrysophanol 1‐O‐β‐d‐ glucoside and chrysophanol 8‐O‐β‐d‐ glucoside. This study demonstrated an efficient strategy based on preparative high‐performance liquid chromatography and high‐speed countercurrent chromatography for the separation of the above‐mentioned anthraquinone glycosides from Rheum tanguticum Maxim.ex Balf.     

Online hyphenation of extraction,Sephadex LH‐20 column chromatography,and high‐speed countercurrent chromatography: A highly efficient strategy for the preparative separation of andrographolide from Andrographis paniculata in a single step

A novel isolation strategy, online hyphenation of ultrasonic extraction, Sephadex LH‐20 column chromatography combined with high‐speed countercurrent chromatography, was developed for pure compounds extraction and purification. Andrographolide from Andrographis paniculata was achieved only in a single step purification protocol via the present strategy. The crude powder was ultrasonic extracted and extraction was pumped into Sephadex LH‐20 column directly to cut the nontarget fractions followed by the second‐dimensional high‐speed countercurrent chromatography, hyphenated by a six‐port valve equipped at the post‐end of Sephadex LH‐20 column, for the final purification. The results yielded andrographolide with the amount of 1.02 mg and a purity of 98.5% in a single step, indicating that the present method is effective to harvest target compound from medicinal plant.     

Rapid separation of three glucosylated resveratrol analogues from the invasive plant Polygonum cuspidatum by high‐speed countercurrent chromatography

Three glucosylated resveratrol analogues (piceid, piceatannol glucoside, resveratroloside) were successfully isolated from the crude MeOH extract of the invasive plant species Polygonum cuspidatum by semi‐preparative high‐speed countercurrent chromatography with a two‐phase solvent system composed of cyclohexane‐ethyl acetate‐methanol‐water (1:5:1:5, v/v/v/v). Piceid (23 mg), resveratroloside (17 mg), piceatannol glucoside (15 mg) of purities over 80% were isolated from 500 mg crude MeOH extract in one step. Subsequent passage over a SPE column was used to quickly bring their purities to over 90%. The purities were determined by HPLC analysis and their structures were elucidated by proton nuclear magnetic resonance (¹H‐NMR), HMBC, ESI‐MS and HR‐MS.