Hexane Ethyl Acetate Methanol Water Research Articles

Solvent strength of organic phase for two biphasic solvent systems in high speed countercurrent chromatography

In this work, relationships between solvent strength of organic phase (ψ) for two biphasic solvent systems in high speed countercurrent chromatography, hexane-ethyl-acetate-methanol-water (HEMWat) and ethyl acetate-n-butanol-water (EBuWat), and partition coefficient (K) were investigated using four retention models, including Jandera's model (ABM), Neue-Kuss model (NK), linear-solvent-strength model (LSS) and quadratic-solvent-strength model (QSS). Experimental results showed that ABM model had the best fitting results for HEMWat system while NK model and QSS model had good fitting results in EBuWat system. Thus, a mathematical relationship between partition coefficient (K) and solvent strength of organic phase (ψ) could be obtained by measurement of partition coefficients of the target compounds with three different volume ratios of organic phase. At the same time, a functional map was proposed to construct to get a maneuverable region so that an optimal two-phase solvent system for separation of a target compound could be selected easily, which saved a lot of manpower for high speed countercurrent chromatographic separation. The application of this new method was declared by successful separation of two components, apigenin-6-C-β-D-xylopyranosyl-8-C-α-L-arabinopyranoside and vicenin-3, from dried leaves of Dendrobium officinale Kimura et Migo using high speed countercurrent chromatography.

Aporphine Alkaloids from Triclisia dictyophylla Diels by pH-Zone Refining Countercurrent Chromatography

A new dimeric aporphine alkaloid, biscocsarmine, 2, together with lirioferine, 1, and cocsarmine, 3, were isolated from the ethanol extract of the stems of Triclisia dictyophylla Diels (Menispermaceae) using a preparative pH-zone refining CCC in a single step. The first solvent system family tested for this fractionation was hexane–ethyl acetate–methanol–water (HEMWat) at a series of solvent ratios, and hydrochloric acid and triethylamine at 5 mM as eluter/retainer. None of these HEMWat ratios, however, gave suitable K for the target alkaloids. Ethyl acetate–n-butanol–water (EBuWat) was tested instead and the selected solvent system was composed of ethyl acetate–n-butanol–water 5:5:10 (v/v), where triethylamine (60 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. The chemical structures of the isolated compounds were elucidated based on comprehensive spectroscopic and spectrometric techniques. This is the first report of aporphine alkaloids in this plant species.

Efficient separation of five flavonoids from Oxytropis falcata bunge by high-speed counter-current chromatography and their anticancer activity

Oxytropis falcata Bunge, known as the “king of herbs” in Tibetan medicine, is used for treatment of hyperpyrexia, pain, wounds, inflammation, and anthrax. However, it is difficult to efficiently isolate compounds with high purity from O. falcata because of the complexity of traditional Tibetan medicines. In this study, the 80% ethanol elution fraction from extract by AB-8 macroporous resin column chromatography was demonstrated to have anticancer activity on human hepatoma SMMC-7721 cells in vitro. Then, a high-speed counter-current chromatography (HSCCC) method was successfully established for separation of compounds by using hexane–ethyl acetate–methanol–water (10:4:10:10, v/v/v/v) as the solvent system. Five flavonoids (7-hydroxyflavonone [1], 5,7-dihydroxy-4′-methoxy flavonol [2], 5,7-dihydroxyflavanone [3], 2′,4′-dihydroxychalcone [4], and 2′,4′-dihydroxydihydrochalcone [5]) were obtained in one-step separation with purities of 97.7%, 98.1%, 98.3%, 99.0%, and 98.3%, respectively. Finally, anticancer activities against the growth of SMMC-7721 cells of 5 flavonoids were confirmed. The IC50 values of the separated compounds were 213.45 μg/mL, 197.74 μg/mL, 375.16 μg/mL, 17.44 μg/mL, and 136.83 μg/mL in 24 h, respectively. The present study provided a basis for further development and utilization of this medicinal herb as a source of a new potential anticancer agent.

Isolation of quinolone alkaloids from Tetradium ruticarpum via preparative high-speed counter-current chromatography and evaluation of their binding affinities for bovine serum albumin

By application of preparative high-speed counter-current chromatography (HSCCC) to the crude quinolone alkaloids (1.1 g) from the fruit of Tetradium ruticarpum, 1-methyl-2((6Z,9Z)-pentadecadienyl)-4(1H)-quinolone (1, 8.4 mg), dihydroevocarpine (2, 27.0 mg), and 1-methyl-2-pentadecyl-4(1H)-quinolone (3, 18.8 mg) were isolated in one step with sufficient purity using the solvent system composed of hexane–ethyl acetate–methanol–water (Hex–EtOAc–MeOH–H2O, 5:2:5:3). Further purification of the subfraction was performed by amending the solvent composition and achieved another three quinolone alkaloids, i.e., 1-methyl-2-undecylquinolin-4(1H)-one (4, 13.7 mg), (Z)-1-methyl-2-(tridec-5-en-1-yl) quinolin-4(1H)-one (5, 14.0 mg) from subfraction FR3-A3-85 using Hex–EtOAc–MeOH–H2O (5:3.5:8.75:8.25), and 1-methyl-2-nonylquinolin-4(1H)-one (6, 15.1 mg) from subfraction FR3-A3-36 using Hex–EtOAc–MeOH–H2O (5:3.8:5:4.8). The relationship between the structure of the six alkaloids and their affinities for bovine serum albumin (BSA) was investigated using fluorescence titration analysis. The length and the presence of double bond of the side chain affected their binding process with BSA. The binding behavior might influence their other biological activities.

Preparative Separation and Purification of Four Glycosides from Gentianae radix by High-Speed Counter-Current Chromatography and Comparison of Their Anti-NO Production Effects.

Secoiridoid and iridoid glycosides are the main active components of Gentianae radix. In this work, one iridoid and three secoiridoid glycosides from Gentianae radix have been purified by high-speed counter-current chromatography in two runs using different solvent systems. Ethyl acetate–n-butanol–water (2:1:3, v/v/v) was the optimum solvent system to purify ca. 4.36 mg of loganic acid, 3.05 mg of swertiamarin, and 35.66 mg of gentiopicroside with 98.1%, 97.2% and 98.6% purities, respectively, while 31.15 mg of trifloroside with 98.9% purity was separated using hexane–ethyl acetate–methanol–water (1:3:1:3, v/v/v/v). The structures of the glycosides were identified by mass spectrometry and NMR. After separation, the anti-nitric oxide production effects of the compounds on lipopolysaccharide-induced BV-2 murine microglial cells were also evaluated. All of the compounds inhibited the production of nitric oxide in lipopolysaccharide-induced BV-2 cells with high cell viabilities in a concentration-dependent manner, which demonstrated that were able to be used as a nitric oxide inhibitor.

An approach based upon the consecutive separation and the economical two-phase solvent system preparation using UNIFAC mathematical model for increasing the yield of high-speed counter-current chromatography

Abstract A HSCCC method combined with the consecutive injection and the economical two-phase solvent system preparation using UNIFAC mathematical model was successfully developed for increasing the yield of target compound. HSCCC was performed with a two-phase solvent system composed of n -hexane–ethyl acetate–methanol–water (3:5:3:5, v/v) at a flow rate of 6 mL/min. The components of the upper and lower phases were calculated according to UNIFAC mathematical model to prepare the stationary and mobile phases, separately. Only 300 mL stationary phase was filled into the column, and the crude sample (200 mg, ten times) was consecutive loaded into the column at the given interval time and eluted using the individually prepared mobile phase without reestablishing hydrodynamic equilibrium. After recrystallized, 168 mg tiliroside with the purity of 95.8% was obtained from 2 g crude sample of Pinus massoniana L. The structure was identified by 1 H NMR and 13 C NMR. The present method not only avoided the time consuming of exchanging the new solvent system and reestablishing hydrodynamic equilibrium, but also coped with a large amount of organic solvent waste.

Isolation of a Tetrameric A-type Proanthocyanidin Containing Fraction from Fresh Tea (Camellia Sinensis) Leaves Using High-Speed Counter-Current Chromatography

A proanthocyanidin (PA) fraction containing tetrameric A- and B-type PAs was obtained after Sephadex LH-20 chromatography and high-speed counter-current chromatography (HSCCC) of a PA extract from fresh tea (Camellia sinensis) leaves. The biphasic solvent system hexane–ethyl acetate–methanol–water system (1:5:1:5), with the aqueous phase as the mobile phase, was selected on the basis of partition coefficient (KD1.02), settling time (18 s), and stationary phase retention (60%). Electrospray ionization mass spectrometry (ESI-MS) indicated the presence of A-type linkages in the tetrameric PAs in one of the HSCCC fractions.

Isolation of Methylxantines from Cacao Beans (Theobroma cacao) by Counter-Current Chromatography (CCC)

Counter-current chromatography (CCC) was applied to the preparative separation of the methylxantines (theophylline, theobromine, and caffeine) from the methanol extract of Theobroma cacao seeds. The solvent system used was composed of hexane-ethyl acetate–methanol–water (2:3:1:1 v/v/v/v) and the extraction was monitored by high-performance liquid chromatography employing conditions for a rapid determination of the methylxantines. The mixture of methylxantines obtained were analyzed using Microsorb-MV 100-5, 3 µm (C18) column (250 mm × 4, 6 mm), and mobile phase water:methanol (75:25) in the isocratic mode at a flow-rate of 5 mL/min and detection was carried out at 275 nm.

A novel 9 × 9 map-based solvent selection strategy for targeted counter-current chromatography isolation of natural products

Counter-current chromatography (CCC) is an efficient liquid–liquid chromatography technique for separation and purification of complex mixtures like natural products extracts and synthetic chemicals. However, CCC is still a challenging process requiring some special technical knowledge especially in the selection of appropriated solvent systems. In this work, we introduced a new 9×9 map-based solvent selection strategy for CCC isolation of targets, which permit more than 60 hexane–ethyl acetate–methanol–water (HEMWat) solvent systems as the start candidates for the selection of solvent systems. Among these solvent systems, there are clear linear correlations between partition coefficient (K) and the system numbers. Thus, an appropriate CCC solvent system (i.e., sweet spot for K=1) may be hit by measurement of k values of the target only in two random solvent systems. Besides this, surprisingly, we found that through two sweet spots, we could get a line (“Sweet line”) where there are infinite sweet solvent systems being suitable for CCC separation. In these sweet solvent systems, the target has the same partition coefficient (K) but different solubilities. Thus, the better sweet solvent system with higher sample solubility can be obtained for high capacity CCC preparation. Furthermore, we found that there is a zone (“Sweet zone”) where all solvent systems have their own sweet partition coefficients values for the target in range of 0.4<K<2.5 or extended range of 0.25<K<16. All results were validated by using 14 pure GUESSmix mimic natural products as standards and further confirmed by isolation of several targets including honokiol and magnolol from the extracts of Magnolia officinalis Rehd. Et Wils and tanshinone IIA from Salvia miltiorrhiza Bunge. In practice, it is much easier to get a suitable solvent system only by making a simple screening two to four HEMWat two-phase solvent systems to obtain the sweet line or sweet zone without special knowledge or comprehensive standards as references. This is an important advancement for solvent system selection and also will be very useful for isolation of current natural products including Traditional Chinese Medicines.

Separation and Purification of Gardecin from Gardenia jasminoides Ellis by High-Speed Countercurrent Chromatography

In the present study, a high-speed counter-current chromatography (HSCCC) was investigated for separation and purification of gardecin on a preparative scale. Hexane–ethyl acetate–methanol–water (0.3:1:0.3:1, v/v) was selected as the optimum solvent system to purify gardecin from a fraction obtained from HPD100 column chromatography fractionation of gardenia. After HSCCC isolation, 20.1 mg of gardecin with purity of 97.4% was obtained from 322 g of dry gardenia fruits. Chemical structure identification of this pigment was carried out by MS, 1H NMR, and 13C NMR. Additionally, antioxidant activity of gardecin in comparison with crocin-1 was investigated. The present results demonstrated that gardecin could be efficiently obtained using HSCCC from this herb and this compound features strong antioxidant activity.

EFFECTIVE ON-LINE COMBINATION OF HIGH SHEAR TECHNIQUE AND CONTINUOUS SAMPLE INJECTION IN HIGH-PERFORMANCE COUNTER-CURRENT CHROMATOGRAPHY FOR ISOLATION AND PURIFICATION OF POLYPHENOLS IN GREEN TEA

A new sample preparation method of high shear dispersing emulsifier (HSDE) coupled with continuous sample injection in high-performance counter-current chromatography (HPCCC) was successfully used for the extraction and online isolation of caffeine and two polyphenolic compounds from green tea. The lower phase of hexane-ethyl acetate–methanol–water (1:8:1:8, v/v/v/v) was used as both the HSDE solvent and HPCCC mobile phase. In one time sample loading, 1.5 mg of (-)-epicatechin-3-gallate, 5.0 mg of (-)-epigallocatechin-3-gallate, and 3.1 mg of caffeine with the purity over 95.0% determined by high performance liquid chromatography (HPLC), could be obtained from about 0.1 g of raw material of green tea. Ten time sample injections were realized in HSDE extraction−HPCCC separation process, and the quantity could be magnified about decuple, 13.8 mg of (-)-epicatechin-3-gallate, 46.3 mg of (-)-epigallocatechin-3-gallate, and 28.9 mg of caffeine could obtain from 1.0 g of raw material of green tea. The compounds were analyzed by HPLC and identified by their chromatograms and their retention time compared with that of authentic reference compounds. It was the first time combining HSDE with continuous sample injection in HPCCC for effective isolation and purification of active compounds from natural products. The mechanism of HSDE extraction was illustrated.

Large-scale separation of alkaloids from Gelsemium elegans by pH-zone-refining counter-current chromatography with a new solvent system screening method

A rapid and simple solvent system screening method was developed for pH-zone-refining counter-current chromatography (CCC) separation, which was much easier to find a suitable solvent system than the traditional method. Using this method, an optimal solvent system composed of hexane–ethyl acetate–methanol–water (3:7:1:9, v/v) was selected for pH-zone-refining CCC separation of alkaloids from the stems of Gelsemium elegans, where 10mM triethylamine (TEA) was added to the upper organic stationary phase as a retainer and 10mM hydrochloric acid (HCl) to the aqueous mobile phase as an eluter. As a result, six oxindole alkaloids, 420mg 19-xo-gelsenicine, 456mg gelsemine, 723mg koumine, 379mg 11-methoxygelsemamide, 342mg gelsenicine and 318mg humantenine were successfully purified in one step from 4.5g crude extract with the purity of over 95%, respectively. The structures of the oxindole alkaloids were identified by ESI-MS, 1H NMR and 13C NMR.

Dynamic pH junction high-speed counter-current chromatography coupled with microwave-assisted extraction for online separation and purification of alkaloids from Stephania cepharantha

A simple and efficient dynamic pH junction high-speed counter-current chromatography method was developed and further applied to the online extraction, separation and purification of alkaloids from Stephania cepharantha by coupling with microwave-assisted extraction. Mineral acid and organic base were added into the mobile phase and the sample solution, respectively, leading to the formation of a dynamic pH junction in the column and causing focus of alkaloids. Selective focus of analytes can be achieved on the basis of velocity changes of the pH junction through appropriate selection of solvent systems and optimization of additive concentrations. The extract can be directly introduced into the HSCCC for the online extraction, separation and purification of alkaloids from S. cepharantha. Continuous separation can be easily achieved with the same solvent system. Under the optimum conditions, 6.0g original sample was extracted with 60mL of the upper phase of hexane–ethyl acetate–methanol–water (1:1:1:1, v/v/v/v) containing 10% triethylamine under 50°C and 400W irradiation power for 10min, the extracts were directly separated and purified by high-speed counter-current chromatography. A total of 5.7mg sinomenine, 8.3mg 6,7-di-O-acetylsinococuline, 17.9mg berbamine, 12.7mg isotetrandrine and 14.6mg cepharanthine were obtained with purities of 96.7%, 93.7%, 98.7%, 97.3% and 99.3%, respectively. The online method provides good selectivity to ionizable compounds and improves the separation and purification efficiency of the high-speed counter-current chromatography technique. It has good potential for separation and purification of effective compounds from natural products.

Conical coils counter-current chromatography for preparative isolation and purification of tanshinones from Salvia miltiorrhiza Bunge

Modern counter-current chromatography (CCC) originated from the helical coil planet centrifuge. Recently, spiral coils were found to possess higher separation efficiency in both the retention of stationary phase and solutes resolution than other CCC coils like the helical and toroidal coils used on type-J CCC and cross-axis CCC. In this work, we built a novel conical coil CCC for the preparative isolation and purification of tanshinones from Salvia miltiorrhiza Bunge. The conical coils were wound on three identical upright tapered holders in head-to-tail and left-handed direction and connected in series. Compared with helical and spiral coil CCC, conical coil CCC not only placed CCC column in a two-dimensional centrifugal field, but also provided a potential centrifugal force gradient both in axial and radial directions. The extra centrifugal gradient made mobile phase move faster and enabled CCC much higher retention of stationary phase and better resolution. As a result, higher efficiency has been obtained with the solvent system of hexane–ethyl acetate–methanol–water (HEMWat) with the volume ratio of 5:5:7:3 by using conical coil CCC apparatus. Four tanshinones, including cryptotanshinone (1), tanshinone I (2), 1,2-dihydrotanshinquinone (3) and tanshinone IIA (4), were well resolved from 500mg to 1g crude samples with high purity. Furthermore, the conical coil CCC can make a much higher solid phase retention, which makes it to be a powerful separation tool with high throughput. This is the first report about conical coil CCC for separation of tanshinones and it may also be an important advancement for natural products isolation.

Preparation of two novel monobrominated 2-(2′,4′-dihydroxybenzoyl)-3,4,5,6-tetrachlorobenzoic acids and their separation from crude synthetic mixtures using vortex counter-current chromatography

The present work describes the preparation of two compounds considered to be likely precursors of an impurity present in samples of the color additives D&C Red No. 27 (Color Index 45410:1) and D&C Red No. 28 (Color Index 45410, phloxine B) submitted to the U.S. Food and Drug Administration for batch certification. The two compounds, 2-(2′,4′-dihydroxy-3′-bromobenzoyl)-3,4,5,6-tetrachlorobenzoic acid (3BrHBBA) and its 5′-brominated positional isomer (5BrHBBA), both not reported previously, were separated from synthetic mixtures by vortex counter-current chromatography (VCCC). 3BrHBBA was prepared by chemoselective ortho-bromination of the dihydroxybenzoyl moiety. Two portions of the obtained synthetic mixture, 200mg and 210mg, respectively, were separated by VCCC using two two-phase solvent systems that consisted of hexane–ethyl acetate–methanol–aqueous 0.2% trifluoroacetic acid (TFA) in the volume ratios of 8:2:5:5 and 7:3:5:5, respectively. These separations produced 35mg and 78mg of 3BrHBBA, respectively, each product of over 98% purity by HPLC at 254nm. 5BrHBBA was prepared by monobromination of the dihydroxybenzoyl moiety in the presence of glacial acetic acid. To separate the obtained synthetic mixture, VCCC was performed in the pH-zone-refining mode with a solvent system consisting of hexane–ethyl acetate–methanol–water (6:4:5:5, v/v) and with TFA used as the retainer acid and aqueous ammonia as the eluent base. Separation of a 1-g mixture under these conditions resulted in 142mg of 5BrHBBA of ∼99% purity by HPLC at 254nm. The isolated compounds were characterized by high-resolution mass spectrometry and proton nuclear magnetic resonance spectroscopy.

Development and evaluation of a spiral tube column for counter-current chromatography

An improved type-J counter-current chromatography (CCC) planet centrifuge with two spiral tube columns (volume 2×15 mL, β value 0.3-0.7, tubing 0.8 mm id) was developed and evaluated for its retention ability of four typical different solvent systems including heptane-methanol (1:1, v/v) (A), hexane-ethyl acetate-methanol-water (1:1:1:1, v/v) (B), n-butanol-acetic acid-water (4:1:5, v/v) (C), PEG1000-K(2)HPO(4)-water (12.5:12.5:75, w/w) (D) under eight different operation modes. The results indicated that the spiral tube column could significantly increase the retention of four typical solvent systems compared with a traditional multilayer coil column with similar parameters (volume 35 mL, β value 0.3-0.7, tubing 0.8 mm id). The retention of stationary phase (S(f)) for the less polar system (A) and moderately polar solvent system (B) can be increased by about 10%, and for the polar system (C) and aqueous two-phase system (ATPS) (D) by 30-40%. The preliminary applications of this spiral tube column to the separation of small molecular compounds such as moderately polar theaflavins, polar anthocyanins and dipeptides were successful. Acceptable resolution can be obtained between cytochrome c and myoglobin, lysozyme and myoglobin when it was applied on protein separation; however, it still needs to be improved with regard to its column efficiency.

Preparation of flavonoids and diarylheptanoid from Alpinia katsumadai hayata by microwave-assisted extraction and high-speed counter-current chromatography

In the present work, five flavonoids and diarylheptanoid, namely, alpinetin, pinocembrin chalcone, pinocembrin, cardamomin and 1,7-diphenyl-4,6-heptadien-3-one, were simply and rapidly prepared from Alpinia katsumadai hayata ( A. katsumadai) by microwave-assisted extraction (MAE) followed with high-speed counter-current chromatography (HSCCC). The critical conditions of MAE and HSCCC were optimized. 2.0 g crude sample was extracted with MAE, and then directly separated and purified by HSCCC with a two-phase solvent system composed of hexane–ethyl acetate–methanol–water (3:7:6:4, v/v/v/v), and 7.5 mg of alpinetin, 1.9 mg of pinocembrin chalcone, 2.6 mg of 1,7-diphenyl-4,6-heptadien-3-one with purities of 98.3%, 96.9% and 93.0% were obtained respectively in a single step within 4 h. 4.0 mg mixture of cardamomin and pinocembrin from HSCCC was further separated by prep-HPLC, 2.1 mg of 99.4% pinocembrin and 1.4 mg of 99.8% cardamomin were obtained respectively. The purities of compounds were determined by HPLC and their chemical structures were further identified by ESI-MS and 1H NMR. The results demonstrated that MAE coupled with HSCCC was a feasible, economical and efficient technique for rapid extraction, separation and purification of effective compounds in natural products.

Spiral counter-current chromatography of small molecules, peptides and proteins using the spiral tubing support rotor

An important advance in countercurrent chromatography (CCC) carried out in open flow-tubing coils, rotated in planetary centrifuges, is the new design to spread out the tubing in spirals. More spacing between the tubing was found to significantly increase the stationary phase retention, such that now all types of two-phase solvent systems can be used for liquid–liquid partition chromatography in the J-type planetary centrifuges. A spiral tubing support (STS) frame with circular channels was constructed by laser sintering technology into which FEP tubing was placed in 4 spiral loops per layer from the bottom to the top and a cover affixed allowing the tubing to connect to flow-tubing of the planetary centrifuge. The rotor was mounted and run in a P.C. Inc. type instrument. Examples of compounds of molecular weights ranging from <300 to approximately 15,000 were chromatographed in appropriate two-phase solvent systems to assess the capability for separation and purification. A mixture of small molecules including aspirin was completely separated in hexane–ethyl acetate–methanol–water. Synthetic peptides including a very hydrophobic peptide were each purified to a very high purity level in a sec-butanol solvent system. In the STS rotor high stationary phase retention was possible with the aqueous sec-butanol solvent system at a normal flow rate. Finally, the two-phase aqueous polyethylene glycol–potassium phosphate solvent system was applied to separate a protein from a lysate of an Escherichia coli expression system. These experiments demonstrate the versatility of spiral CCC using the STS rotor.

Evaluation of dual flow counter-current chromatography and intermittent counter-current extraction

The aim of this research is to compare two continuous extraction technologies, intermittent counter-current extraction (ICcE) and dual flow counter-current chromatography (DFCCC), in terms of loading and throughput using the GUESSmix, and show the advantages and disadvantages of the two methods. A model sample containing caffeine, vanillin, naringenin and carvone, with a total load of 11.2g, was employed with a hexane–ethyl acetate–methanol–water (2:3:2:3) phase system to evaluate an ICcE method on a preparative (912ml coil volume) DE-Midi instrument. While DFCCC was carried out on a specially designed preparative (561ml coil volume) bobbin installed in a similar Midi instrument case. While similar throughputs of 7.8g/h and 6.9g/h were achieved for the ICcE and DFCCC methods respectively, ICcE was demonstrated to have a number of advantages over DFCCC.

Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography

Counter-current chromatography (CCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane–methanol–acetonitrile (1.5:0.5:0.5, v/v) and n-heptane–methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane–ethyl acetate–methanol–water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol–ethyl acetate–water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC–MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, 1HNMR and 13CNMR.