Isolation and purification of honokiol and magnolol from cortex Magnoliae officinalis by high-speed counter-current chromatography

High-speed counter-current chromatography was used to isolate and purify honokiol and magnolol from cortex Magnoliae Officinalis (Magnolia officinalis Rehd. et Wils.), a plant used in the traditional Chinese medicine. A crude sample, 150 mg, was successfully separated with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:0.4:1:0.4, v/v), and the fractions were analyzed by high-performance liquid chromatography. The separation produced 80 and 45 mg of honokiol and magnolol with purities of 99.2 and 98.2%, respectively, in 2.5 h.     

Preparative purification of anti-tumor derivatives of honokiol by high-speed counter-current chromatography

In our program to synthesize a series of novel derivatives as potential analogs of honokiol for anti-tumor treatment, we have found that at least three of the derivatives of honokiol showed more potency to inhibit the proliferation of K562 leukemia cells and SPC-A1 adenocarcinoma cells. As a critical step to our further series synthesis of derivatives of honokiol, three derivatives of honokiol composed of two isomers and one compound with two formyl groups, which were hardly separated by common purification methods, needed to be rapidly separated and purified. The present work describes analytical and preparative high-speed counter-current chromatography (HSCCC) for the isolation and purification of these three C-formylation derivatives of honokiol, named 3'-formylhonokiol, 5-formylhonokiol and 3',5-diformylhonokiol, respectively. The solvent system for HSCCC separation was composed of hexane-ethyl acetate-methanol-water with the ratio of 1:0.4:1:0.4 (v/v). The one-step purification produced 157.8 mg, 121.6 mg and 21.2 mg of 3'-formylhonokiol, 5-formylhonokiol, 3',5-diformylhonokiol from crude sample of 400mg with purities of 98.6%, 99.2% and 99.6%, respectively, in an elution time of 2.5 h. The purities and structural identification were determined by HPLC, (1)H NMR, (13)C NMR and mass spectroscopy. Their anti-proliferation effects on K562, A549 and SPC-A1 cell lines were evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay.     

Separation of honokiol and magnolol by intermittent counter-current extraction

Recently, intermittent counter-current extraction (ICcE) has been developed and shown its advantage in improving resolution between targeted compounds. However, how to choose suitable parameters to increase the throughput has not been systematically studied yet. In present work, we first calculated theoretically the conditions to carry out ICcE elution mode. Then, honokiol and magnolol were separated as model compounds using ICcE elution mode to confirm our conclusion. After parameters like sample concentration and sample feed were optimized in analytical high-performance counter-current chromatography (HPCCC), the separation process was scaled up to preparative HPCCC successfully. 12.8 g honokiol and 16.1 g magnolol were separated from 30 g mixture with purities of 98.6% and 93.7%. And the throughput of target isolation of ICcE elution mode was at least 3.75× higher than isocratic elution mode with the same HPCCC instruments. Our results confirmed our theory calculation and demonstrated the enormous potential of ICcE on preparative separation of binary mixture.     

高速逆流色谱在分离纯化木蝴蝶活性成分中的线性放大

利用高速逆流色谱分离纯化中草药木蝴蝶乙酸乙酯粗提物中的黄酮类活性成分,并将分离规模从分析型线性放大到制备型,以获得大量的活性成分,为进一步的药物筛选提供物质基础。实验在分析型高速逆流色谱上对分离参数进行了系统优化,并将优化条件放大到制备型高速逆流色谱上对911.6 mg木蝴蝶乙酸乙酯粗提物进行分离,得到5种化合物,经高效液相色谱、电喷雾电离质谱和核磁共振氢谱、碳谱分析鉴定,分别为白杨素(160.9 mg,纯度为97.3%)、黄芩素(130.4 mg,纯度为97.6%)、黄芩素-7-O-葡萄糖苷(314.0 mg,纯度为98.3%)、黄芩素-7-O-双葡萄糖苷(179.1 mg,纯度为99.2%)和一种新的白杨素双葡萄糖苷(21.7 mg,纯度为98.8%)。该放大过程不仅将处理量提高了53倍,还保持了在分析型设备上的分离度和分离时间。该工作为天然产物的研究提供了一个高效的分离纯化方法。     

How to realize the linear scale-up process for rapid purification using high-performance counter-current chromatography

This study used the isolation of six constituents from Selaginella tamariscina as an example to demonstrate how to achieve rapid and predictable linear scale-up processes in both normal- and reversed-phase high-performance counter-current chromatography. After systematic optimization of solvent systems, sample concentration, sample loading volume, rotation speed and flow rate on the analytical Mini-DE centrifuge, the optimized parameters obtained were directly transferred to the preparative Midi-DE centrifuge, with nearly the same purities, resolutions and elution times but with 50 times the throughput. Amentoflavone (446.7 mg, 97.8%), robustaflavone (21.6 mg, 89.4%), bilobetin (80.7 mg, 92.7%), hinokiflavone (15.1 mg, 85.5%), isocryptomerin (34.8 mg, 89.6%) and an apigenin-diglucoside (46.3mg, 96.4%) were obtained with amounts and purities shown in parentheses as analysed by HPLC. The process, therefore, offers an efficient and rapid method of obtaining sufficient quantities of target compounds with significantly increased throughput after a linear scale-up.     

Rapid separation of flavonoids by analytical high-speed counter-current chromatography

A commercial model of the analytical high-speed counter-current chromatography instrument was used for separation of flavonoids from a crude ethanol extract of dried fruits of sea buckthorn (Hippophae rhamnoides). Using a two-phase solvent system of chloroform-methanol-water (4:3:2), a five-fold increase in flow-rate of the mobile phase from 60 to 300 ml/h resulted in a rapid separation of five components in less than 15 min without significant loss in peak resolution. Major flavonoid component, isorhamnetin, was identified in its pure state by mass spectrometric analysis.     

Preparative purification of glycyrrhizin extracted from the root of liquorice using high-speed counter-current chromatography

Glycyrrhizin is one of the main bioactive components in liquorice (Glycyrrhiza uralensis Fisch) which has recently been found to be highly active in inhibiting replication of the severe acute respiratory syndrome (SARS)-associated virus. The separation and purification of glycyrrhizin from a methanol-water (70:30 (v/v)) extract of liquorice roots was achieved using high-speed counter-current chromatography. The separation was performed at a preparative scale in a one-step separation with a two-phase solvent system composed of ethyl acetate-methanol-water (5:2:5 (v/v)). The lower phase was used as the mobile phase in the head-to-tail elution mode. The present method yielded 42.2 mg glycyrrhizin at 96.8% purity from 130 mg of the crude exact with 95.2% recovery as determined by HPLC analysis.     

Preparative purification of gentamicin components using high-speed counter-current chromatography coupled with electrospray mass spectrometry

We developed a useful and preparative method based on high-speed counter-current chromatography with mass spectrometry (HSCCC/MS) to purify gentamicin C1a, C2/2a and C1 from standard powder. The analytes were purified on the HSCCC model CCC-1000 (multi-layer coil planet centrifuge) with a volatile two-phase solvent system composed of n-butanol/10% aqueous ammonia solution (50:50, v/v) and detected on an LCMS-2020EV quadrupole mass spectrometer fitted with an electrospray ionization (ESI) source system in positive ionization following scan mode (m/z 100-500). The HSCCC/ESI-MS peaks indicated that gentamicin C1a (m/z 450: [M+H](+)), C2/2a (m/z 464: [M+H](+)) and C1 (m/z 478: [M+H](+)) have the peak resolution values of 1.3 and 1.7 from 30 mg of loaded gentamicin powder. The HSCCC yielded 3.9 mg of gentamicin C1a, 12.6 mg of gentamicin C2/2a and 12.0 mg of gentamicin C1. These purified substances were analyzed by LC/MS with scan positive-mode. Based on the LC/MS chromatograms and spectra of the fractions, analytes were estimated to be over 95% pure. These gentamicin isomers of C1a, C2/2a and C1 were evaluated for their antibacterial activities. The overall results indicate that this approach of HSCCC/MS is a powerful technique for the purification of gentamicin components.     

Flow rate gradient high-speed counter-current chromatography separation of five diterpenoids from Triperygium wilfordii and scale-up

In this paper, high-speed counter-current chromatography (HSCCC) instruments with different gravitational forces were applied for the separation of bioactive compounds from Triperygium wilfordii Hook.f. The critical parameters including sample concentration, sample volume and flow rate were first optimized on an analytical Mini-DE HSCCC system, and then scaled up to a preparative TBE 300A HSCCC system. Although this scale-up process was performed using different CCC instruments with different centrifuges and gravitational forces, the same resolutions were obtained and the elution time could be predictable. Five diterpenoid compounds and one unknown compound were separated from Triperygium wilfordii Hook.f. by HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (HEMW) (3:2:3:2, v/v/v/v). This one-step flow gradient separation produced triptonide (25 mg), isoneotriptophenolide (77 mg), hypolide (83 mg), unknown compound (1 mg), triptophenolide (42 mg), triptonoterpene methyl ether VI (37 mg) from 320 mg crude extract with purities of 98.2%, 96.6%, 98.1%, 95.3%, 95.1%, and 96.5%, respectively. Their purities and structures were identified by high-performance liquid chromatography, mass spectrometry and NMR. This paper demonstrates that analytical CCC plays an important role in optimizing parameters and scale-up process when analytical CCC and preparative CCC are supplied by different manufacturers with different gravitational forces, and the scale-up process from analytical CCC to preparative CCC is still predictable.     

Separation of alkaloids from herbs using high-speed counter-current chromatography

Alkaloids represent a most widespread group of bioactive natural products. Because of their alkalinity and structural diversity, the fractionation and purification of the alkaloids from herbs can often present a number of practical difficulties using the conventional chromatographic techniques. High-speed counter-current chromatography (HSCCC) is a liquid-liquid partition chromatography with a support-free liquid stationary phase, and is gaining more and more popularity as a viable separation technique for bioactive compounds from natural resources. In the present review, focus is placed on the separation of alkaloids by both conventional HSCCC and pH-zone-refining counter-current chromatography (CCC) techniques from herbs. The review presents the separation of over 120 different alkaloid compounds from more than 30 plant species by the conventional HSCCC and pH-zone-refining CCC. Based on the data from the literature, the proper solvent systems for the separation of alkaloids by the conventional HSCCC and pH-zone-refining CCC are also summarized.     

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。     

Separation and purification of isoflavones from Pueraria lobata by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was applied to the semipreparative separation and purification of puerarin and related isoflavones from a crude extract of Pueraria lobata. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v). Using the above solvent system the preparative HSCCC was successfully performed yielding six relatively pure isoflavones including puerarin from 80 mg of the crude extract in one-step separation.     

Isolation and purification of coumarin compounds from Cortex fraxinus by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was successfully used for the isolation and purification of coumarin compounds from Cortex fraxinus, the Chinese herbal drug. n-Butanol-methanol-0.5% acetic acid (5:1.5:5, v/v) was used as the two-phase solvent system. 14.3 mg of fraxin, 26.5 mg of aesculin, 5.8 mg of fraxetin and 32.4 mg of aesculetin with the purity of 97.6, 99.5, 97.2 and 98.7%, respectively were obtained from 150 mg of crude extracts of C. fraxinus in a single run. The structures of the isolated compounds were identified by 1H NMR and 13C NMR.     

Separation and purification of glucosinolates from crude plant homogenates by high-speed counter-current chromatography

Glucosinolates are anionic, hydrophilic plant secondary metabolites which are of particular interest due to their role in the prevention of cancer and other chronic and degenerative diseases. The separation and purification of glucosinolates from a variety of plant sources (e.g. seeds of broccoli, arugula and the horseradish tree), was achieved using high-speed counter-current chromatography (HSCCC). A high-salt, highly polar system containing 1-propanol-acetonitrile-saturated aqueous ammonium sulfate-water (1:0.5:1.2:1), was run on a semi-preparative scale and then transferred directly to preparative scale. Up to 7 g of a concentrated methanolic syrup containing about 10% glucosinolates was loaded on an 850-ml HSCCC column, and good separation and recovery were demonstrated for 4-methylsulfinylbutyl, 3-methylsulfinylpropyl, 4-methylthiobutyl, 2-propenyl and 4-(rhamnopyranosyloxy)benzyl glucosinolates. Multiple injections (5 to 6 times) were performed with well-preserved liquid stationary phase under centrifugal force. Pooled sequential runs with broccoli seed extract yielded about 20 g of its predominant glucosinolate, glucoraphanin, which was produced at > 95% purity and reduced to powdered form.     

高良姜中二苯基庚烷类化合物的高速逆流色谱分离制备

应用高速逆流色谱法(HSCCC)分离纯化了高良姜中3种二苯基庚烷类化合物。以正己烷-乙酸乙酯-甲醇-水(2:3:1.75:1, v/v/v/v)为两相溶剂系统,下相为固定相,上相为流动相,在主机转速为858 r/min、流速1.5 mL/min的条件下,从122.20 mg高良姜石油醚萃取物中经一步HSCCC分离可制备得到5R-羟基-7-(4-羟基-3-甲氧基苯基)-1-苯基-3-庚酮(7.37 mg)、7-(4-羟基-3-甲氧基苯基)-1-苯基-4E-烯-3-庚酮(9.11 mg)和1,7-二苯基-4E-烯-3-庚酮(15.44 mg),经高效液相色谱分析,纯度均大于93%,各化合物的结构由质谱和核磁共振氢谱、碳谱鉴定确证。该方法简便、快速、高效,可用于高良姜中二苯基庚烷类化合物的快速分离制备。     

One step isolation and purification of liquiritigenin and isoliquiritigenin from Glycyrrhiza uralensis Risch. using high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) technique in semi-preparative scale has been successfully applied to the separation of bioactive flavonoid compounds, liquiritigenin and isoliquiritigenin in one step from the crude extract of Glycyrrhiza uralensis Risch. The HSCCC was performed using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-acetonitrile-water (2:2:1:0.6:2, v/v). Yields of liquiritigenin (98.9% purity) and isoliquiritigenin (98.3% purity) obtained were 0.52% and 0.32%. Chemical structures of the purified liquiritigenin and isoliquiritigenin were identified by electrospray ionization-MS (ESI-MS) and NMR analysis.     

Recent progress on the industrial scale-up of counter-current chromatography

The pharmaceutical industries are looking for rapid methods of purification and predictable scale-up for their drug development process that will cut their costs and enable them to reduce the time to market. In this paper, recent progress is reviewed in the development and demonstration of two types of industrial scale centrifugal liquid-liquid chromatography: hydrostatic and hydrodynamic. Industrial scale hydrostatic processes by Partus Technologies and Armen Instrument are just emerging. Results demonstrating scalability are presented for hydrodynamic processes by Dynamic Extractions. The review concludes that the time is now right, with this appropriate commercial support, for high performance counter-current chromatography to emerge as a major enabling technology for industry.     

Preparative isolation and purification of coumarins from Peucedanum praeruptorum Dunn by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of coumarins from Peucedanum praeruptorum Dunn (Baihuaqianhu in Chinese) was successfully established by using light petroleum-ethyl acetate-methanol-water as the two-phase solvent system in gradient elution mode. The upper phase of light petroleum-ethyl acetate-methanol-water (5:5:5:5, v/v) was used as the stationary phase of HSCCC. The mobile phase used in HSCCC was the lower phase of light petroleum-ethyl acetate-methanol-water (5:5:5:5, v/v) and light petroleum-ethyl acetate-methanol-water (5:5:6.5:3.5, v/v) that was changed in gradient. Four kinds of coumarins and another unknown compound were obtained and yielded 5.3 mg of qianhucoumarin D, 7.7 mg of Pd-Ib, 35.8 mg of (+)-praeruptorin A, 31.9 mg of (+)-praeruptorin B and 6.4 mg of unknown compound with the purity of 98.6%, 92.8%, 99.5%, 99.4% and 99.8% in one-step separation, respectively. The structures of the coumarins were identified by 1H NMR and 13C NMR.     

Separation and purification of isoflavones from a crude soybean extract by high-speed counter-current chromatography

A set of isoflavones with a broad range of polarity including daidzin, glycitin, genistin, acetyldaidzin, glycitein, acetylgenistin and daidzein was separated from a crude soybean extract by high-speed counter-current chromatography using a two-step operation. Three solvent systems were used: chloroform-methanol-water (4:3:2, v/v); chloroform-methanol-n-butanol-water (4:3:0.5:2, v/v); and methyl tert.-butyl ether-tetrahydrofuran-0.5% aqueous trifluoroacetic acid (2:2:0.15:4, v/v). The first solvent system was used for separating less polar isoflavones and the second for more polar isoflavones by eluting the lower organic phase. Genistin and glycitin, which were only partially resolved in the chloroform system, were separated by the third solvent system. Each isolated component showed 98-99% purity as determined by high-performance liquid chromatography analysis. Their structures were identified by LC-MS.     

Isolation and purification of canthaxanthin from the microalga Chlorella zofingiensis by high-speed counter-current chromatography

Certain microalgae are considered to be a potential source of canthaxanthin, which possesses strong antioxidant and anticancer activities. A high-speed counter-current chromatography (HSCCC) method was developed for the separation and purification of canthaxanthin from the microalga Chlorella zofingiensis. The crude canthaxanthin was obtained by extraction with organic solvents after the microalgal sample had been saponified. Preparative HSCCC, with a two-phase solvent system composed of n-hexane-ethanol-water (10:9:1 v/v), was successfully performed yielding canthaxanthin at 98.7% purity from 150 mg of the crude extract (2.1% canthaxanthin) in a one-step separation. The recovery of canthaxanthin was 92.3%. This was the first report that canthaxanthin was successfully separated and purified from microalgae.