Preparative isolation of osthol and xanthotoxol from Common Cnidium Fruit (Chinese traditional herb) using stepwise elution by high-speed counter-current chromatography

Preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of osthol and xanthotoxol from Cnidium monnieri (L.) Cusson (Common Cnidium Fruit) using stepwise elution with a pair of two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water at (1:1:1:1, v/v), and (5:5:6:4, v/v), which had been selected by analytical high-speed counter-current chromatography. Using a preparative unit of the HSCCC centrifuge, about a 308 mg amount of the crude extract was separated, yielding 88.3 mg of osthol and 19.4 mg of xanthotoxol at a high purity of over 98%.     

Simultaneous separation and purification of five bioactive coumarins from the Chinese medicinal plant Cnidium monnieri by high-speed counter-current chromatography

Cnidium monnieri (L.) Cusson is a well-known Chinese medicinal plant, which has been used for the treatment of impotence, frigidity, and skin-related diseases, and exhibits strong antipruritic, antiallergic, antidermatophytic, antibacterial, antifungal, and antiosteoporotic activities. A high-speed counter-current chromatography method was developed for the separation and purification of five bioactive coumarins from this plant. The crude coumarins were obtained by ethanol extraction from the dried fruits of Cnidium monnieri (L.) Cusson under sonication. High-speed counter-current chromatography with the two-phase solvent systems n-hexane-ethyl acetate-ethanol-water (5:5:4:6, v/v) and n-hexane-ethyl acetate-ethanol-water (5:5:6:4, v/v) was successfully performed with stepwise elution. The five relatively pure coumarins were obtained from 500 mg of the crude extract in a single run. Their purities were 90.6-98.9%, and the recoveries were 85.7-94.2%.     

Preparative high-speed counter-current chromatography for purification of shikonin from the Chinese medicinal plant Lithospermum erythrorhizon

The bioactive compound shikonin was successfully isolated and purified from the crude extract of the traditional Chinese medicinal plant Lithospermum erythrorhizon Sieb. et Zucc. by preparative high-speed counter-current chromatography (HSCCC). The preparative HSCCC was performed using a two-phase solvent system composed of n-hexane-ethylacetate-ethanol-water (16:14:14:5 (v/v)). A total amount of 19.6 mg of shikonin at 98.9% purity was obtained from 52 mg of the crude extract (containing 38.9% shikonin) with 96.9% recovery. The preparative isolation and purification of shikonin by HSCCC was completed in 200 min in a one-step separation.     

蛇床子香豆素的薄层分离-直接进样-质谱鉴定

用丙酮提取,石油醚冷冻结晶,从蛇床子中得到混合香豆素。经薄层分离 ,直接进样和质谱分析 ,鉴定出8种香豆素化合物:Ⅰ.蛇床子素(osthol) ,Ⅱ.佛手内酯(bergapten) ,Ⅲ.欧芹属素乙(imperatorin) ,Ⅳ.花椒毒素(xanthotoxin) ,Ⅴ.异回芹内酯(isopimpinellin) ,Ⅵ.别异英波托林(alloisoimperatorin) ,Ⅶ.6 -甲氧基 -8 -甲基香豆精(6_methoxy_8_methylcoumarin) ,Ⅷ.花椒毒酚(xanthotoxol)。其中Ⅵ、Ⅶ首次于蛇床子中发现。     

Application of complexation high-speed counter-current chromatography in the separation of 5-hydroxyisoflavone isomers from Belamcanda chinensis (L.) DC

A novel separation technique of complexation high-speed counter-current chromatography (HSCCC) using copper ion as a complexation agent was first developed to isolate 5-hydroxyisoflavone isomers from Belamcanda chinensis (L.) DC. According to the partition coefficient and separation factor, the two-phase solvent system composed of light petroleum-ethyl acetate-methanol-water (3:5:3:5, v/v) and copper nitrate (0.10mol/L in the lower phase) was selected. 9.2mg isoirigenin (1), 46.4mg irigenin (2) and 1.2mg 5,7,4'-trihydroxy-6,3',5'-trimethoxyisoflavone (3) were simultaneously purified from 100mg crude extract by HSCCC with the purity of 95.06%, 96.98% and 93.69%, respectively. As evidenced by the results of UV-Vis spectroscopy, the stoichiometries of the copper ion with the three 5-hydroxyisoflavones were all 1:1 and their chelating power was 3>2>1. Those explained the complexation HSCCC behavior. It is the first report that includes the practical application of complexation HSCCC and explanation of its chromatographic behavior.     

Preparative separation of high-purity cordycepin from Cordyceps militaris(L.) Link by high-speed countercurrent chromatography

A high-speed counter-current chromatography (HSCCC) technique in a preparative scale has been applied to separate and purify cordycepin from the extract of Cordyceps militaris(L.) Link by a one-step separation. A high efficiency of HSCCC separation was achieved on a two-phase solvent system of n-hexane-n-butanol-methanol-water (23:80:30:155, v/v/v/v) by eluting the lower mobile phase at a flow rate of 2 ml/min under a revolution speed of 850 rpm. HSCCC separation of 216.2 mg crude sample (contained cordycepin at 44.7% purity after 732 cation-exchange resin clean-up) yielded 64.8 mg cordycepin with purity of 98.9% and 91.7% recovery. Identification of the target compound was performed by UV, IR, MS, (1)H NMR and (13)C NMR.     

高速逆流色谱法分离制备鱼藤根中的2种鱼藤酮类化合物

建立了分离制备鱼藤根中2种鱼藤酮类化合物的高速逆流色谱法。以正己烷-乙酸乙酯-甲醇-水(体积比为7:0.25:5:3)为两相溶剂系统,上相为固定相,下相为流动相,在主机转速850 r/min、流速2.0 mL/min、检测波长254 nm条件下进行分离制备,从50 mg鱼藤根粗提物中得到了2种鱼藤酮类化合物,分别为6.4 mg纯度为96.60%的鱼藤酮和23.4 mg纯度为97.87%的鱼藤素。该方法为鱼藤酮类化合物的深入研究提供了物质基础。     

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.     

高速逆流色谱法分离纯化续随子种子中的七叶内酯

建立了高速逆流色谱(HSCCC)技术分离纯化续随子种子中七叶内酯的方法。将续随子种子的乙酸乙酯萃取物直接进行高速逆流色谱分离,考察了不同溶剂系统的分离效果。结果表明,最佳的溶剂系统为氯仿-甲醇-水(体积比为4:3:2),以其上相为固定相,下相为流动相。从200 mg续随子种子乙酸乙酯萃取物中分离得到80 mg七叶内酯,纯度为99.04%。HSCCC技术可高效分离纯化续随子种子中的七叶内酯,为得到高纯度的七叶内酯提供了制备技术。     

Isolation and purification of oridonin from the whole plant of Isodon rubescens by high-speed counter-current chromatography

Semi-preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of oridonin from Isodon rubescens by using a two-phase-solvent system composed of n-hexane-ethyl acetate-methanol-water (2.8:5:2.8:5, v/v/v/v). The targeted compound isolated, collected and purified by HSCCC was analyzed by high performance liquid chromatography (HPLC). A total of 40.6 mg of oridonin with the purity of 73.5% was obtained in less than 100 min from 100 mg of crude Isodon rubescens extract. The chemical structure of the compound was identified by IR, 1H-NMR and 13C-NMR.     

Isolation of bioactive components from Flaveria bidentis (L.) Kuntze using high-speed counter-current chromatography and time-controlled collection method

Semipreparative high-speed counter-current chromatography (HSCCC) by time-controlled collection method was successfully applied for isolation and purification of α-terthienyl, 5-(3-buten-1-ynyl)-2,2'-bithienyl, and 5-(3-penten-1-ynyl)-2,2'-bithienyl from Flaveria bidentis (L.) Kuntze for the first time. The two-phase solvent system composed of n-hexane and acetonitrile at the volume ratio of 1:1 (v/v) was used for the semipreparative HSCCC. The 5.2 mg α-terthienyl, 2.2 mg 5-(3-buten-1-ynyl)-2,2'-bithienyl, and 4.3 mg 5-(3-penten-1-ynyl)-2,2'-bithienyl with the purity of 99.9, 90.2, and 92.1% were produced from 265.6 mg crude extract, respectively, and 5-(3-penten-1-ynyl)-2,2'-bithienyl was first isolated from Flaveria bidentis (L.) Kuntze. The structures of the separated compounds were identified by electrospray-ionization mass spectrometry and proton and carbon nuclear magnetic resonance ((1)H- and (13)C-NMR).     

Separation of epigallocatechin and flavonoids from Hypericum perforatum L. by high-speed counter-current chromatography and preparative high-performance liquid chromatography

High-speed counter-current chromatography (HSCCC) and preparative high-performance liquid chromatography (prep-HPLC) were successively used for the separation of epigallocatechin and flavonoids from Hypericum perforatum L. The two-phase solvent system composed of ethyl acetate–methanol–water (10:1:10, v/v) was used for HSCCC. About 900 mg of the crude extract was separated by HSCCC, yielding 7.8 mg of quercitrin at a purity of over 97%, 12.6 mg of quercetin at a purity of over 93%, and 38.9 mg of a mixture of hyperoside, isoquercitrin and miquelianin constituting over 97% of the fraction. A mixture of epigallocatechin and avicularin pooled from three HSCCC runs, a total amount of 54.3 mg, was further separated by prep-HPLC yielding 23.4 mg of epigallocatechin and 15.3 mg of avicularin each at a purity of over 97%.     

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.     

An effective high-speed countercurrent chromatographic method for preparative isolation and purification of mollugin directly from the ethanol extract of the Chinese medicinal plant Rubia cordifolia

The medicinal plant Rubia cordifolia has been used widely in traditional Chinese medicine (TCM) for its antibacterial, antioxidant and anti-inflammatory activities. In this study, a preparative high-speed countercurrent chromatography (HSCCC) method for isolation and purification of the bioactive component mollugin directly from the ethanol extract of R. cordifolia was successfully established by using light petroleum (bp 60-90 degrees C)/ethanol/diethyl ether/water as the two-phase solvent system. The upper phase of light petroleum/ethanol/diethyl ether/water (5:4:3:1 v/v) was used as the stationary phase of HSCCC. Under the optimum conditions, 46 mg of mollugin at 98.5% purity, as determined by HPLC, could be yielded from 500 mg of the crude extract in a single HSCCC separation. The peak fraction of HSCCC was identified by 1H NMR and 13C NMR.     

高速逆流色谱分离纯化蔓荆子中的活性成分

应用高速逆流色谱法(HSCCC)分离纯化蔓荆子中的活性成分。以石油醚-乙酸乙酯-甲醇-水(体积比为3:6:3.6:3)为两相溶剂体系,在转速为800 r/min、流速为1.5 mL/min、检测波长为254 nm的条件下进行分离,所得馏分经高效液相色谱法(HPLC)检测,并经电喷雾电离(ESI)质谱和核磁共振谱(NMR)鉴定化合物的结构。从250 mg蔓荆子粗提物中一次性分离得到4个化合物,分别为23 mg对羟基苯甲酸、15 mg 3,6,7-三甲基槲皮万寿菊素、24 mg蔓荆子黄素和5 mg蒿黄素,其纯度约为93.1%、 97.3%、 98.7%和98.5%。该法具有简便、快速、重复性好的优点,为分离蔓荆子中的活性成分提供了新的方法。     

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 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.     

Preparative isolation and purification of phillyrin from the medicinal plant Forsythia suspensa by high-speed counter-current chromatography

Forsythia suspensa (Thunb.) Vahl. has been used widely in traditional medicines to treat gonorrhea, erysipelas, inflammation, pyrexia and ulcer. It has also shown antioxidant activity, as well as antibacterial, antiviral, choleretic and antiemetic effects. A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of the bioactive molecule phillyrin from F. suspensa (Thunb.) Vahl. The crude phillyrin was obtained by extraction with 50% ethanol from the dried fruits of F. suspensa (Thunb.) Vahl. under sonication. Preparative HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (1:9:1:9, v/v/v/v) was successfully performed, and the components purified and collected were analyzed by high-performance liquid chromatography. The method yielded 5.6 mg phillyrin at 98.6% purity from 500 mg of the crude extract (1.2% phillyrin) with the recovery of 92% in a one-step separation.     

Preparative isolation and purification of psoralen and isopsoralen from Psoralea corylifolia by high-speed counter-current chromatography

Psoralen and isopsoralen were separated from Psoralea corylifolia by high-speed counter-current chromatography (HSCCC). A two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:5:4.5:5.5, v/v) was used for HSCCC separation, and yielded, from 100 mg of crude extract, 39.6 mg of psoralen and 50.8 mg of isopsoralen each at over 99% purity as determined by high performance liquid chromatography (HPLC). The identification of psoralen and isopsoralen were performed with 1H NMR and 13C NMR.     

正交设计试验优化高速逆流色谱分离制备玛咖中芥子油苷的条件

基于高速逆流色谱（HSCCC）技术从玛咖中分离制备出两种芥子油苷,苄基芥子油苷（glucotropaeolin, GTL）和甲氧基苄基芥子油苷（glucolimnanthin, GLI）。使用正交设计试验对分离条件进行优化,采用高分辨质谱对制备的组分进行鉴定,采用高效液相色谱法（HPLC）对组分进行定量分析。确定了两个组分GTL与GLI的HSCCC最佳分离条件：溶剂系统为正丁醇-乙腈-200 g/L硫酸铵溶液（1:0.5:2.4, v/v/v）,上相为固定相,下相为流动相,流动相流速2 mL/min,主机转速900 r/min,从玛咖根粗提物中一次性分离得到157.72 mg/kg纯度为97.9%的苄基芥子油苷和31.93 mg/kg的甲氧基苄基芥子油苷,固定相保留率达57.6%。该方法成本低,简便易行,样品损失量小,可大量循环进样制备。