Isolation and purification of flavonoid glycosides from Trollius ledebouri using high-speed counter-current chromatography by stepwise increasing the flow-rate of the mobile phase

Three flavonoid glycosides including orientin, vitexin, quercetin-3-O-neohesperidoside and one unknown compound were isolated and purified by high-speed counter-current chromatography (HSCCC) and semi-preparative HPLC from Trollius ledebouri Reichb., a traditional Chinese medicine. Preparative HSCCC with a two-phase solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v) was successfully performed by increasing the flow-rate of the mobile phase from 1.5 to 2.5 ml/min after 190 min. Consequently, 95.8 mg orientin, 11.6 mg vitexin, 9.3 mg unknown compound with purities of over 97% and one partially purified peak fraction (contained quercetin-3-O-neohesperidoside at 85.1% purity) were obtained from 500 mg of the crude extract. Then the partially purified fraction was further purified by reversed-phase semi-preparative high-performance liquid chromatography. The structure identification of all pure fractions was carried out by UV, MS, 1H NMR and 13C NMR.     

Application of preparative high-speed counter-current chromatography for the separation of flavonoids from the leaves of Byrsonima crassa Niedenzu (IK)

The methanolic extract of the leaves of the medicinal plant Byrsonima crassa (Malpighiaceae) contain flavonoids with antioxidant activity. They were separated in a preparative scale using high-speed counter-current chromatography. The optimum solvent system used was composed of a mixture of ethyl acetate-n-propanol-water (140:8:80 (v/v/v)) and led to a successful separation between monoglucosilated flavonoids (quercetin-3-O-alpha-L-arabinoside, quercetin-3-O-beta-D-galactoside) and the biflavonoid amentoflavone in only 3.5 h. The purities of quercetin-3-O-alpha-L-arabinoside (95 mg), quercetin-3-O-beta-D-galactoside (16 mg) and the biflavonoid amentoflavone (114 mg) were all isolated at purity over 95%. Identification was performed by 1H NMR, 13C NMR and UV analyses.     

Application of preparative high-speed counter-current chromatography for separation of methyl gallate from Acer truncatum Bunge

Preparative separation of methyl gallate in leaves extract of Acer truncatum Bunge was conducted using high-speed counter-current chromatography (HSCCC) with a solvent system composed of ethyl acetate-ethanol-water at volume ratios of 5:1:5 (v/v/v). In a single operation, 57.5 mg of methyl gallate was obtained from 120 mg of the extract. HPLC analyses of the counter-current chromatography (CCC) fraction revealed that the methyl gallate was having over 97% purity. Its structure was identified by 1H NMR and 13C NMR.     

Isolation of three sesquiterpene lactones from the roots of Cichorium glandulosum Boiss. et Huet. by high-speed counter-current chromatography

Because of the skeletal complexity and similarity of the polarity, little research was reported on the isolation of sesquiterpene lactones by high-speed counter-current chromatography (HSCCC). Herein, three sesquiterpene lactones were successfully purified from the ethyl acetate extract of the roots of the traditional Uyghur medicinal plant Cichorium glandulosum Boiss. et Huet. by HSCCC. The separation was performed in two steps with two solvent systems: n-hexane-ethyl acetate-methanol-water (1.5:5:2.75:5, v/v/v/v) and ethyl acetate-methanol-water (20:1:20, v/v/v). From 166 mg of the ethyl acetate extract, 19 mg of lactucopicrin was isolated with the first solvent system and 10 mg of 11beta,13-dihydrolactucin and 16 mg of lactucin were obtained with the second solvent system. All purified compounds were over 94% purity as determined by HPLC analysis, and these chemical structures were confirmed by (1)H NMR and (13)C NMR.     

硅胶柱色谱结合高速逆流色谱法分离纯化荷花中黄酮类化合物

采用硅胶柱色谱结合高速逆流色谱法分离纯化了荷花中3种黄酮类化合物。荷花粗提物先经过硅胶柱色谱初步分离,得到黄酮含量高的组分,再经过高速逆流色谱分离,以乙酸乙酯-乙醇-水-乙酸(4:1:5:0.025, v/v/v/v)为两相溶剂系统,上相为固定相,下相为流动相,在主机转速800 r/min、流速2.0 mL/min、检测波长254 nm条件下,从150 mg样品中一次性分离制备得到6.1 mg槲皮素-3-O-β-D-葡萄糖醛酸苷(I), 14.8 mg杨梅素-3-O-β-D-葡萄糖苷(II)和20.2 mg紫云英苷(III),经高效液相色谱检测其纯度分别为97.0%、95.4%、96.3%,并通过质谱和核磁共振氢谱、碳谱鉴定各化合物的结构。该方法简便、快速、节省溶剂,可以对荷花中的黄酮类化合物进行快速有效的分离纯化,具有较好的实用价值,为荷花资源的进一步开发应用提供了参考依据。     

Preparative isolation and purification of two isoflavones from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography

Two isoflavones, calycosin-7-O-beta-D-glycoside and formononetin-7-O-beta-D-glycoside, were separated from n-butanol extract of the root of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography in two steps using two different solvent systems composed of ethyl acetate-ethanol-n-butanol-water (30:10:6:50, v/v) and ethyl acetate-ethanol-water (5:1:5, v/v). From 200 mg of crude extract, calycosin-7-O-beta-D-glycoside (12 mg) and formononetin-7-O-beta-D-glycoside (10 mg) were isolated at over 95% purity by HPLC analyses, and their structures were identified by MS, 1H NMR and 13C NMR.     

Preparative isolation and purification of isoflavan and pterocarpan glycosides from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography

(3R)-(-)-7,2'-Dihydroxy-3',4'-dimethyl isoflavan-7-O-beta-D-glucopyranoside and (6aR, 11aR) 9,10-di-methoxypterocarpan-3-O-beta-D-glucopyranoside were separated from the ethyl acetate extract of the root of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography (HSCCC). A two-phase system composed of ethyl acetate-ethanol-acetic acid-water (4:1:0.25:5, v/v) was selected by analytical HSCCC. Preparative HSCCC yielded, from 100 mg of the partially purified extract, 50 mg of isoflavan glycoside and 10 mg of pterocarpan glycoside each at over 95% purity by high-performance liquid chromatography (HPLC) analysis. Their structures were identified by MS, 1H NMR and 13C NMR.     

Isolation of a novel flavanone 6-glucoside from the flowers of Carthamus tinctorium (Honghua) by high-speed counter-current chromatography

A novel flavanone glycoside, (2S)-4',5,6,7-tetrahydroxyflavavone 6-O-beta-D-glucopyranoside was isolated from the ethyl acetate extract of the flowers of Carthamus tinctorium by high-speed counter-current chromatography (HSCCC). Using an optimized two-phase solvent system composed of ethyl acetate-methanol-water (5:1:5, v/v), target compound (52 mg) with purity of 98.0% was obtained from 2.0 g of sample by HSCCC in seven times run. The structure of the target compound was elucidated by means of spectroscopic methods including IR, MS, 1D and 2D NMR techniques.     

One step purification of flavanone glycosides from Poncirus trifoliata by centrifugal partition chromatography

Flavanone glycosides were successfully separated from the crude extract of Poncirus trifoliata by preparative centrifugal partition chromatography with a two-phase solvent system composed of ethyl acetate-acetonitrile-water (3:2:5, v/v/v). Naringin (50.0 mg), neoponcirin (16.8 mg), and poncirin (71.9 mg) were purified from the 524 mg crude extract in only one step. The purities of the isolated compounds were determined to be over 90% by HPLC analysis and their structures were elucidated by (1)H NMR, (13)C NMR, and ESI-MS.     

Preparative separation of isovitexin and isoorientin from Patrinia villosa Juss by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) with a solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v) was used to isolate and separate two C-glycosylflavones from Patrinia villosa Juss, a traditional Chinese medicine. The separation produced 42.9 mg isovitexin and 20.1 mg isoorientin with purities of 99.3% and 98.5%, respectively as determined by high-performance liquid chromatography (HPLC) in one step elution from 250 mg crude extract, and identification was performed by MS, 1H NMR and 13C NMR. It is the first report of discovering isovitexin and isoorientin from the plant of Patrinia genus.     

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.     

Separation of salidroside from Rhodiola crenulata by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was used to purify salidroside from an extract of Rhodiola crenulata with two steps using a two-phase solvent system composed of ethyl acetate-n-butanol-water (1:4:5, v/v) in the first run and chloroform-methanol-isopropanol-water (5:6:1:4) in the second run. The method yielded 21.9 mg of salidroside from 1.216 g of the crude sample at 98% purity determined by HPLC analyses. Identification was performed by 1H NMR, 13C NMR, and MS.     

Preparative isolation and purification of antioxidative diarylheptanoid derivatives from Alnus japonica by high-speed counter-current chromatography

This study employed the online HPLC-2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)(+) bioassay to rapidly determine the antioxidant compounds occurring in the crude extract of Alnus japonica. The negative peaks of the ABTS(+) radical scavenging detection system, which indicated the presence of antioxidant activity, were monitored by measuring the decrease in absorbance at 734 nm. The ABTS(+)-based antioxidant activity profile showed that three negative peaks exhibited antioxidant activity. High-speed counter-current chromatography (HSCCC) was used for preparative scale separation of the three active peaks from the extract. The purity of the isolated compounds was analyzed by HPLC and their structures were identified by (1)H- and (13)C-nuclear magnetic resonance spectrometry (NMR), heteronuclear multiple bond correlation (HMBC), and heteronuclear single quantum correlation (HSQC). Two solvent systems composed of n-hexane/ethylacetate/methanol/water (4:6:4:6, v/v) and of ethyl acetate/methanol/water (1:0.1:1, v/v) were performed in high-speed counter-current chromatography. Consequently, a total of 527 mg of hirsutanonol 5-O-β-D-glucopyranoside, 80.04 mg of 3-deoxohirsutenonol 5-O-β-D-glucopyranoside, and 91.0 mg of hirsutenone were obtained with purity of 94.7, 90.5, and 98.6%, respectively.     

Separation and identification of polyphenols in apple pomace by high-speed counter-current chromatography and high-performance liquid chromatography coupled with mass spectrometry

Apple pomace, a by-product in the processing of apple juice, was investigated as a potential source of polyphenols. Two methods of separation and purification of polyphenols from apple pomace extract were established by combination of gel chromatography with high-speed counter-current chromatography (HSCCC) and solvent extraction with HSCCC, respectively. The optimal separation was performed on a Sephadex LH-20 column using gradient aqueous ethanol as eluting solvent from 0% to 100% in increments of 10%. HPLC analysis indicated that main polyphenols existed in fractions eluted between 40% and 50% aqueous ethanol. The fractions of interest from column were separated by HSCCC with the solvent system hexane–ethyl acetate–1% aqueous acetic acid (0.5:9.5:10, v/v/v). Ethyl acetate fractionation of the apple pomace extract followed by direct HSCCC separation by the same solvent system in the volume ratio of 1:9:10 also produced a good separation of the main polyphenols of interest. Six high-purity polyphenols were achieved tentatively and identified by HPLC/MS: chlorogenic acid (1, m/z 354), quercetin-3-glucoside/quercetin-3-glacaside (2, m/z 464), quercetin-3-xyloside (3, m/z 434), phloridzin (4, m/z 436), quercetin-3-arabinoside (5, m/z 434), and quercetin-3-rhamnoside (6, m/z 448). These results provided a preliminary foundation for further development and exploration of apple pomace.     

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.     

Preparative isolation and purification of syringin and edgeworoside C from Edgeworthia chrysantha Lindl by high-speed counter-current chromatography

The bioactive compound syringin along with edgeworoside C were separated from the n-butanol extract of the stems and barks of Edgeworthia chrysantha Lindl (E. papyrifera) by high-speed counter-current chromatography (HSCCC) while it was difficult to purify each compound by silica gel column chromatography. Syringin was isolated from this plant for the first time. The two-phase solvent system used was composed of ethyl acetate-ethanol-water at an optimized volume ratio of 15:1:15 (v/v/v). Preparative HSCCC yielded, from 110mg of the partially purified extract, 28mg of syringin and 45 mg edgeworoside C each at over 96% purity by high-performance liquid chromatography analysis. Their structures were identified by electron impact ionization MS, 1H NMR and 13C NMR.     

High‐performance countercurrent chromatography separation of Peucedanum cervaria fruit extract for the isolation of rare coumarin derivatives

For the first time, rare major and minor compounds from fruits of Peucedanum cervaria were isolated. High‐performance countercurrent chromatography with two different solvent systems, heptane/ethyl acetate/methanol/water (3:2:3:2 and 2:1:2:1, v/v), was successfully used in the reversed‐phase mode. A scale‐up process from analytical to semipreparative in a very short time was developed. The structures of isolated compounds were evaluated by high‐performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry, gas chromatography with mass spectrometry, and one‐ and two‐dimensional NMR spectroscopy. (8S,9R)‐9‐(3‐Methylbutenoyloxy)‐O‐acetyl‐8,9‐dihydrooroselol (compound B), (8S,9R)‐9‐(2‐methyl‐Z‐butenoyloxy)‐O‐acetyl‐8,9‐dihydrooroselol (edultin, compound C), and (8S,9R)‐9‐acetoxy‐O‐(2α‐methylbutyryl)‐8,9‐dihydrooroselol (compound D) were obtained using heptane/ethyl acetate/methanol/water (2:1:2:1, v/v) in <40 min. The method yielded 4.6 mg of a mixture of compounds B and C (11:89) and 3.7 mg of compound D. These amounts were obtained from the crude extract (0.5 g) in a single run. Although the compounds are known, their isolation by countercurrent chromatography and the analysis of their relative stereochemistry by two‐dimensional NMR spectroscopy have been performed for the first time. Additionally, heptane/ethyl acetate/methanol/water (3:2:3:2, v/v) led to the isolation of oxypeucedanin (1.2 mg; compound A). This is the first time that angular dihydrofuranocoumarin was isolated from plant extract by countercurrent chromatography.     

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 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.     

Accelerated solvent extraction and pH‐zone‐refining counter‐current chromatographic purification of yunaconitine and 8‐deacetylyunaconitine from Aconitum vilmorinianum Kom

This study aimed to seek an efficient method to extract and purify yunaconitine and 8‐deacetylyunaconitine from Aconitum vilmorinianum Kom. by accelerated solvent extraction combined with pH‐zone‐refining counter‐current chromatography. The major extraction parameters for accelerated solvent extraction were optimized by an orthogonal test design L₉ (3)⁴. Then a separation and purification method was established using pH‐zone‐refining counter‐current chromatography with a two‐phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (5:5:2:8, v/v) with 10 mM triethylamine in the upper phase and 10 mM HCl in the lower phase. From 2 g crude extract, 224 mg of 8‐deacetylyunaconitine (I) and 841 mg of yunaconitine (II) were obtained with a purity of over 98.0%. The chemical structures were identified by ESI‐MS and ¹H and ¹³C NMR spectroscopy.