High-speed Counter-current Chromatography Fractions Research Articles

Combining high-speed countercurrent chromatography three-phase solvent system with electrospray ionization-mass spectrometry and nuclear magnetic resonance to profile the unconventional food plant Syzygium malaccense

Syzygium malaccense (L.) Merr. & L.M. Perry is a native tree to Malaysia, but also occurs in other tropical regions of the world, including Brazil. The increasing interest in the consumption of its leaves motivated the investigation of compounds of the plant. Metabolite profiling of S. malaccense leaves was achieved by high-speed countercurrent chromatography (HSCCC) fractionation coupled off-line to electrospray mass-spectrometry (ESI-MS) detection and nuclear magnetic resonance (NMR) analysis. The ethanolic leaf extract was submitted to HSCCC using a three-phase solvent system (TPSS) composed by n-hexane – ethyl acetate – acetonitrile – H2O (2:1:1:1, v/v). The stepwise gradient elution was employed due to the extract's chemical complexity. HSCCC fractions were further analyzed by ESI-MS/MS using a flow injection experiment and by NMR acquiring 1H, HSQC and HMBC spectra. MS based dereplication was achieved by comparing acquired data to those available in public and commercial databases. Results were also correlated to previously isolated compounds described for the Syzygium genus. This process led to the annotation of 90 compounds. The NMR data provided structural confirmation and substitution patterns for some of them. Extract chemical composition is characterized by having flavonoids, benzoic acids, hydroxycinnamic acids, quinic acids, hydrolizable tannins, fatty acids, anacardic acids and others primary metabolites. Most of these compounds were described for the first time in the plant. This approach greatly facilitates phytochemical analysis and could be applied to improve metabolite discovery in other studies.

Extraction process and method validation for bioactive compounds from Citrus reticulata cv. Chachiensis: Application of response surface methodology and HPLC–DAD

AbstractCitrus reticulata cv. Chachiensis, a traditional Chinese herb, has extensive medicinal and edible effects. 3′,4′,5,6,7,8-Hexamethoxyflavone (HM) and 5,6,7,8,4′-pentamethoxyflavone (PM) are main bioactive compounds in Chachiensis, which have been reported to possess various biological properties. In this study, supercritical CO2 extraction (SCE) and high-speed countercurrent chromatography (HSCCC) were utilized to prepare HM and PM from Chachiensis. The contents of target compounds were determined by a high-performance liquid chromatography method with diode-array detection (HPLC-DAD), which was validated using the following parameters: linearity, sensitivity, repeatability, stability, precision and accuracy. The SCE conditions were optimized using response surface methodology with central composite design. Obtained optimum conditions were temperature of 37.9 °C, pressure of 26.3 MPa, and modifier volume of 81.0 mL. Under above conditions, the recoveries of target compounds were 92.52 ± 0.83 and 96.36 ± 0.43%, respectively. The most appropriate solvent system for HSCCC was selected as n-hexane/ethyl acetate/methanol/water (1:0.8:1:1.2, v/v). The HSCCC fractions were detected by HPLC-DAD, liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (1H NMR and 13C NMR). The results indicated that this method was successfully applied to obtain HM and PM with high purities and high recoveries from Chachiensis.

Extraction and separation of flavonoids from Malus hupehensis using high-speed countercurrent chromatography based on deep eutectic solvent

In the present experiment, a green and highly efficient extraction method for flavonoids established on deep eutectic solvents (DESs) was investigated by using the response surface methodology. The DES-based high-speed countercurrent chromatography (HSCCC) solvent systems were developed for the separation of high purity compounds from the DES extract of Malus hupehensis for the first time. Under the optimal conditions (liquid-to-solid ratio of 26.3 mL/g, water content of 25.5%, and extraction temperature of 77.5°C), the yield of flavonoids was 15.3 ± 0.1%, which was superior to that of the methanol extraction method. In accordance with the physical property of DES-based HSCCC solvent systems and K values of target compounds, DES-based HSCCC solvent systems composed of choline chloride/glucose-water-ethyl acetate (ChCl/Glu-H2O-EAC, 1:1:2, v/v) was selected for the HSCCC separation. Thus, five flavonoids (two novel compounds 1-2, 6´´-O-coumaroyl-2´-O-glucopyranosylphloretin and 3´´´-methoxy-6´´-O-feruloy-2´-O-glucopyranosylphloretin; three know compounds 3-5, namely, avicularin, phloridzin, and sieboldin) were efficiently separated from Malus hupehensis. DESs are the environment friendly and highly efficient solvents as the components of extraction solvent and HSCCC solvent system, and can be re-utilized many times. However, ethyl acetate can be soluble with a few hydrogen bond donors, such as urea, carboxylic acid and polyol, through the shake flask test. It is the great difficulty for the efficient and rapid separation of target compounds from the DESs extract because of the DESs residual in the HSCCC fractions. ChCl and Glu are the great choices of DESs without this problem. In addition, K values increased with the increase of the molar ratio of ChCl/Glu and the content of water, which could effectively guide us to choose the suitable DES-based HSCCC solvent system. The twice HSCCC separation results indicated that DES was the valuable and green solvent for the HSCCC separation of pure compounds from the extract for the first time, and showed the recycle superiority of DES-based HSCCC solvent system.

Separation of betacyanins from Iresine herbstii Hook. ex Lindl. leaves by high-speed countercurrent chromatography in a polar solvent system.

Betacyanins, natural plant pigments, from Iresine herbstii Hook. ex Lindl. leaf extract were separated for the first time by high-speed countercurrent chromatography (HSCCC) in a highly polar solvent system composed of PrOH-ACN-(NH4)2SO4satd.soln-H2O (1.0:0.5:1.2:1.0; v/v/v/v) in the tail-to-head mode. The flow rate of the mobile phase was 2.0 ml/min and the column rotation speed was 860 rpm. The retention of the stationary phase was 81.0%. For the identification of separated betacyanins in the crude extract and in the HSCCC fractions, as well as for the molecular formulas and multi-step fragmentation pattern elucidation, liquid chromatography with tandem mass spectrometry and high-resolution ion-trap time-of-flight mass spectrometry were performed. The innovative application of the HSCCC system for fractionation of the betacyanins present in I. herbstii leaves enabled effective separation as well as preconcentration of the pigments for further low- and high-resolution LC-MS/MS analysis. HSCCC separation enabled identification of 22 betacyanins, of which 18 had not been detected previously in the leaves of I. herbstii, and four of these betacyanins (sinapoyl-gomphrenin and coumaroyl-gomphrenin as well as their epimers) were identified for the first time in the Iresine genus.

Preparative Isolation of Piceatannol Derivatives from Passion Fruit (Passiflora edulis) Seeds by High-Speed Countercurrent Chromatography Combined with High-Performance Liquid Chromatography and Screening for α-Glucosidase Inhibitory Activities.

Passiflora edulis Sims (passion fruit) seeds are often discarded as byproducts during juice processing. In fact, the seeds are of considerable commercial value in the food and cosmetics industry because of their rich polyphenols, especially piceatannol. In this study, high-speed countercurrent chromatography (HSCCC) was applied for the separation of stilbene polyphenols from passion fruit seeds. The n-hexane-ethyl acetate-methanol-water (1:2:1:2.8, v/v) was found to be the optimum two-phase solvent for the preparation of two major stilbenes, scirpusin B (8) and piceatannol (9) with purities of 90.2% and 94.8%, respectively. In addition, a continuous semipreparative HPLC was applied to further purify the HSCCC fractions containing minor stilbenes and obtain four new piceatannol derivatives (1-4) along with three known ones (5-7). The structures of these new compounds were determined using spectroscopic methods, including NMR, high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), and circular dichroism (CD). The isolated compounds were evaluated for α-glucosidase inhibitory activities in vitro. The result suggested that all of them exhibited more significant activity than acarbose, and passiflorinol B (2) had the strongest activity, with a IC50 value of 1.7 μM.

Isolation of high-purity peptide Val-Val-Tyr-Pro from Globin Peptide using MCI gel column combined with high-speed counter-current chromatography.

Peptides have gained increased interest over the past several decades because of their therapeutics. In this research, a strategy combining MCI gel column chromatography and high-speed countercurrent chromatography was developed for the separation of high-purity peptide Val-Val-Tyr-Pro from Globin Peptide. First, the fraction of Val-Val-Tyr-Pro mixtures with a purity of 15.8% was obtained by using MCI gel column with a mixture of ethanol/water (20:80, v/v/v). Then, the high-purity Val-Val-Tyr-Pro was separated by high-speed countercurrent chromatography with a aqueous two phase systems of ethanol/acetonitrile/iso-propyl alcohol/(NH4 )2 SO4 Saturated solution /H2 O (0.5:0.5:0.25:1.5:0.7,v/v). The ammonium sulfate from high-speed countercurrent chromatography fractions was removed from target compound by MCI gel column chromatography using ethanol/water in stepwise elution mode. A 78mg of Val-Val-Tyr-Pro was successfully purified with the purities of 98.80% from 30g crude Globin Peptide. The amino acid sequence of the Val-Val-Tyr-Pro was determined by electrospray ionization high resolution tandem mass spectrometry. The method presents a practical strategy for the large-scale separation of pure peptide Val-Val-Tyr-Pro from Globin Peptide, and provides a reference method for obtaining high-purity peptide from other polypeptide mixtures.

Studies on polar high-speed counter-current chromatographic systems in separation of amaranthine-type betacyanins from Celosia species

Betacyanins, natural plant pigments exhibiting antioxidant and chemopreventive properties, were extracted from Celosia spicata (Thouars) Spreng. inflorescences and separated by high-speed counter-current chromatography (HSCCC) in two polar solvent systems composed of: TBME – 1-BuOH – ACN – H2O (0.7% HFBA, 2:2:1:5, v/v/v/v) (system I) and EtOH – ACN – 1-PrOH – (NH4)2SO4satd.soln – H2O (0.5:0.5:0.5:1.2:1, v/v/v/v/v) (system II). The systems were used in the head-to-tail (system I) and tail-to-head (system II) mode. The flow rate of the mobile phase was 2.0 ml/min and the column rotation speed was 860 rpm. The retention of the stationary phase was 73.5% (system I) and 80.0% (system II). For the identification of separated betacyanins in the crude extract as well as in the HSCCC fractions, LC-DAD-ESI–MS/MS analyses were performed. Depending on the target compounds, each of the systems exhibit meaningfully different selectivity and applicability. For the pairs of amaranthines (1/1′) and betanins (2/2′), the best choice is the system II, but the acylated amaranthine pairs (3/3′ and 4/4′) can be resolved only in the ion-pair system I. For the indication of the most suitable solvent system for Celosia plumosa hort., Celosia cristata L. and Celosia spicata (Thouars) Spreng. species, the profiles of betacyanins in different plant parts were studied.

Identifying Natural syNergist from Pongamia pinnata Using High-Speed Counter-Current Chromatography Combined with Isobolographic Analysis.

For identifying the synergistic compounds from Pongamia pinnata, an approach based on high-speed counter-current chromatography (HSCCC) combined with isobolographic analysis was designed to detect the synergistic effects in the complex mixture. In the approach, the complex mixture was considered as the combination of two individual samples for isobolographic analysis: the target compound and the mixture with complete removal of the target compound (subtracted residue). The two samples were prepared by HSCCC, and were used for the calculation of the expected effect of their combination. Using this approach, three compounds representing the major peaks in the HPLC of the brine shrimp toxic extract from P. pinnata (brine shrimp lethality test (BST) LC50 36.5 μg/mL), pinnatin (1), 3,7-Dimethoxy-3′,4′-methylenedioxy flavone (2), and karanjin (3), were prepared from the extract, and were tested for their synergistic potency by BST. The two-phase solvent system containing n-hexane-ethyl acetate–MeOH–water (14:7:10:10, v/v/v/v) was selected for the one-step HSCCC separation according to the partition coefficient values (K). The extract was separated into seven fractions (Fr1–7) by HSCCC with a total mass recovery of 96.3%. Fr2, 4, and 6 were the peak fractions corresponding to compounds 3, 2, and 1, respectively. The purities and recoveries of the target compounds after the chromatographic analysis were 95.9%–97.5% and 92.2%–96.1%, respectively. The subtracted residue of each target compound was performed by recombining all HSCCC fractions except the fraction containing the target compound. Isobolographic analysis disclosed a significant synergistic effect between karanjin and its subtracted residue (potency ratio of 0.47), which gave clear evidence that the toxicity of the extract results from synergistic interactions, and karanjin was one of the synergists participating in the interaction. The other two compounds were excluded from the synergism because these two compounds showed additive effects with their subtracted residues. Karanjin was the first synergistic compound identified from P. pinnata.

High-speed counter-current chromatography in separation of betacyanins from flowers of red Gomphrena globosa L. cultivars.

Antioxidant and possible chemopreventive properties of betacyanins, natural plant pigments, contribute to a growing interest in their chemistry and separation. Mixtures of betacyanins from fresh red Gomphrena globosa L. cultivar flowers were separated in three highly polar solvent systems by high-speed counter-current chromatography (HSCCC) for a direct comparison of their separation effectiveness. Three samples of crude extract (600mg) were run on semi-preparative scale in solvent system (NH4)2SO4soln - EtOH (2.0:1.0, v/v) (system I) and the modified systems: EtOH - ACN - 1-PrOH - (NH4)2SO4satd.soln - H2O (0.5:0.5:0.5:1.2:1.0, v/v/v/v/v) (system II) and EtOH - ACN - (NH4)2SO4satd.soln - H2O (1.0:0.5:1.2:1.0, v/v/v/v) (system III). The systems were used in the head-to-tail (system I) or tail-to-head (systems II and III) mode. The flow rate of the mobile phase was 2.0ml/min and the column rotation speed was 860rpm. The retention of the stationary phase was 52.0% (system I), 80.2% (systems II) and 82.0% (system III). The betacyanins in the crude extract as well as HSCCC fractions were analyzed by LC-MS/MS. System I was applied for the first time in HSCCC for the separation of betacyanins and was quite effective in separation of amaranthine and 17-decarboxy-amaranthine (αI=1.19) and very effective for 17-decarboxy-amaranthine and betanin (αI=2.20). Modification of system I with acetonitrile (system III) as well as acetonitrile and propanol (system II) increased their separation effectiveness. Systems II-III enable complete separation of 17-decarboxy-amaranthine (KD(II)=2.94,KD(III)=2.42) and betanin (KD(II)=2.46,KD(III)=1.10) as well as betanin and gomphrenin I (KD(II)=1.62, KD(III)=0.74). In addition, separation of amaranthine and 17-decarboxy-amaranthine is the most effective in system II, therefore, this system proved to be the most suitable for the separation of all polar betacyanins.

Enantioseparation of (RS)-ibuprofen by closed recycling high-speed counter-current chromatography using hydroxypropyl-β-cyclodextrin as chiral selector

High-speed counter-current chromatography combined with closed recycling elution mode was developed to enantioseparate (RS)-ibuprofen by using hydroxypropyl-β-cyclodextrin (HP-β-CD) as the chiral selector. Key parameters for high-speed counter-current chromatography resolution including the concentration of HP-β-CD, the two-phase solvent system composition, equilibrium temperature, and the pH of aqueous phase were extensively investigated. Under the optimized conditions, the enantiomers of ibuprofen were successfully separated by preparative recycling high-speed counter-current chromatography and the resulting enantiopurity of each enantiomer was over 97.5% as determined by HPLC. Moreover, the recovery for (R,S)-ibuprofen from high-speed counter-current chromatography fractions was achieved in the range of 82–89%.

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 achyrobichalcone from Achyrocline satureioides by high- speed countercurrent chromatography.

Achyrobichalcone is a biflavonoid recently found in Achyrocline satureioides. This substance has unprecedented chemical structure and occurrence, but resembles other bioactive bichalcones, which have important pharmacological properties, such as anticancer activity. The major challenge for evaluation of the physicochemical and biological properties of this new molecule is the isolation step, which affects the purity and yield of the isolated product. Thus, the objective of this work was to develop a semi-preparative method for achyrobichalcone isolation from Achyrocline satureioides by high-speed countercurrent chromatography. The high-speed countercurrent chromatography separation was achieved in two steps. In the first step, an enriched fraction of achyrobichalcone from the freeze-dried extract was obtained, using the solvent system hexane-ethyl acetate-methanol-water 0.8:1:0.8:1, v/v. The purification of achyrobichalcone from the enriched fractions was achieved by further high-speed countercurrent chromatography fractionation with hexane-ethyl acetate-methanol-water 0.9:0.9:0.8:1, v/v. The final isolated product was obtained using preparative thin layer chromatography and crystallization procedure. A yellow semi-crystalline solid with purity close to 90% was obtained as the final product. The mass recovery of achyrobichalcone isolation was near 67%. The structural identification from spectroscopic and chromatographic techniques confirmed the achyrobichalcone structure. This is the first report of achyrobichalcone isolation on a semi-preparative scale by high-speed countercurrent chromatography. This method afforded achyrobichalcone in good yield and purity for further biopharmaceutical studies.

Separation of Four Phenylpropanoid Glycosides from a Chinese Herb by HSCCC.

Four phenylpropanoid glycosides (PPGs), such as echinacoside, wiedemannioside C, forsythoside B and verbascoside, were isolated and purified from the Tibetan medicinal herb Pedicularis longiflora Rudolph. var. tubiformis (Klotz) Tsoong by high-speed counter-current chromatography (HSCCC) combined with macroporous resin (MR) column separation for the first time. In the present study, the two-phase solvent system composed of ethyl acetate-n-butanol-water (10 : 6:15, v/v/v) was used for HSCCC separation. A total of 3.5 mg of echinacoside, 12.6 mg of wiedemannioside C, 22.7 mg of forsythoside B and 48.7 mg of verbascoside with the purity of 93.6, 97.9, 97.8 and 98.1%, respectively, were obtained from 120 mg of crude sample. The HSCCC fractions were analyzed by HPLC, and the chemical structures were identified by (1)H NMR and (13)C NMR. The results demonstrate that MR coupled with HSCCC is a powerful technique for separation of PPGs from natural products.

ISOLATION AND PURIFICATION OF SEVEN COMPOUNDS FROM EXTRACT OF RHEUM PALMATUM L. BY HIGH SPEED COUNTER CURRENT CHROMATOGRAPHY AND RAPID PREPARATIVE CHROMATOGRAPHY

High speed counter current chromatography (HSCCC) combined with rapid preparative chromatography (RPC) was developed for the separation and purification of seven compounds from Rheum palmatum L. in this paper. Compounds dimmer-catechin, catechin, trans-3,5,4′-trihydroxystilbene-4′-O-β-D-(6′-O-gallayl)-glucoside, 1-O-Galloyl-6-O-cinnamoyl-glucose, 1-O-Galloyl-2-O-cinnamoyl-glucose, rhein and emodin were isolated and purified by HSCCC using an optimized two-phase solvent system composed of n-hexane–ethyl acetate–n-butanol–water (1:2:1:4, v/v/v/v) and RPC. The purities of compounds 1, 3, 4 and 6 from HSCCC separation were over 98.70%, 96.75%, 98.20% and 98.07%, and those of compounds 1, 2, 4, 5 and 7 from RPC separation were 95.33%, 89.40%, 93.7%, 92.89% and 94.76%, determined by HPLC. The HSCCC and RPC fractions were analyzed by HPLC and electrospray ion source mass spectroscopy (ESI-MSn) in negative ion mode. The identification of the seven compounds from the extract of R. palmatum L. was based on matching their retention time, the detection of the molecular ions and the fragment ions of the molecular ion obtained in the MSn experiments with those of the data reported in the literature. The results demonstrated that HSCCC and RPC are feasible and efficient techniques for systematic isolation of bioactive components from R. palmatum L.

Preparative mass-spectrometry profiling of bioactive metabolites in Saudi-Arabian propolis fractionated by high-speed countercurrent chromatography and off-line atmospheric pressure chemical ionization mass-spectrometry injection

Propolis is a glue material collected by honeybees which is used to seal cracks in beehives and to protect the bee population from infections. Propolis resins have a long history in medicinal use as a natural remedy. The multiple biological properties are related to variations in their chemical compositions. Geographical settings and availability of plant sources are important factors for the occurrence of specific natural products in propolis. A propolis ethylacetate extract (800mg) from Saudi Arabia (Al-Baha region) was separated by preparative scale high-speed countercurrent chromatography (HSCCC) using a non-aqueous solvent system n-hexane–ACN (1:1, v/v). For multiple metabolite detection, the resulting HSCCC-fractions were sequentially injected off-line into an atmospheric pressure chemical ionization mass-spectrometry (APCI-MS/MS) device, and a reconstituted mass spectrometry profile of the preparative run was visualized by selected ion traces. Best ion-intensities for detected compounds were obtained in the negative APCI mode and monitored occurring co-elution effects. HSCCC and successive purification steps resulted in the isolation and characterization of various bioactive natural products such as (12E)- and (12Z)-communic acid, sandaracopimaric acid, (+)-ferruginol, (+)-totarol, and 3β-acetoxy-19(29)-taraxasten-20a-ol using EI-, APCI-MS and 1D/2D-NMR. Cycloartenol-derivatives and triterpene acetates were isolated in mixtures and elucidated by EI-MS and 1D-NMR. Free fatty acids, and two labdane fatty acid esters were identified by APCI-MS/MS. In total 19 metabolites have been identified. The novel combination of HSCCC fractionation, and APCI-MS-target-guided molecular mass profiling improve efficiency of lead-structure identification.

Analysis and determination of oestrogen-active compounds in fructus amomi by the combination of high-speed counter-current chromatography and high performance liquid chromatography

Amomum longiligulare or Amomum villosum showed oestrogenic activity. In the present study, oestrogen-active components in fructus amomi, the seeds of A. longiligulare were separated by high-speed countercurrent chromatography (HSCCC) using stepwise elution of eight mobile phases with gradient polarity and advanced separation by high performance liquid chromatography (HPLC). The results yielded 17 compounds with the amount of 8-138mg and a purity of 94.3-99.8% from a 3g ethanolic extract of fructus amomi. The chemical structures of the compounds were identified by ESI-MS and NMR spectra, in which eight diarylheptanoids were demonstrated as the main oestrogen-active compounds in the fructus amomi. Determination of the diarylheptanoids in fructus amomi from various origins showed that fructus amomi contains more than 0.5% total diarylheptanoids. The results showed that fructus amomi is a diarylheptanoids-rich food resource possessing oestrogen-activity. The combination method of HSCCC and HPLC can be applied for the analysis of bioactive compounds by detecting the corresponding bioactivity in the HSCCC fractions and separating the target compounds with HPLC.

High-speed countercurrent chromatographic recovery and off-line electrospray ionization mass spectrometry profiling of bisdesmodic saponins from Saponaria officinalis possessing synergistic toxicity enhancing properties on targeted antitumor toxins

Saponaria officinalis L. (Caryophyllaceae), also known as fuller's herb or soapwort is a medicinal plant, which grows from Europe to Central Asia. Medicinal properties attributed to this plant include its antitussive and galactogogue properties. Recently, bisdesmodic saponins with very specific structural features from S. officinalis have been shown to strongly enhance the efficacy of specific targeted toxins (anti-tumor antibodies connected to protein toxins) in-vitro and in-vivo in a synergistic manner. In the presently reported novel approach we used preparative all-liquid high-speed countercurrent chromatography (HSCCC) to recover a total of 22 fractions using biphasic solvent system tert-butylmethylether/n-butanol/acetonitrile/water 1:3:1:5 (v/v/v/v) from a complex precipitated crude saponin mixture. Out of these 22 fractions, 3 fractions had the enhancer effect on anti-tumor toxins out of which one fraction (F7) was further tested elaborately in different cell lines. The molecular weight distribution and compound profiles of separated saponins were monitored by off-line injections of the sequentially collected fractions to an electrospray ion-trap mass-spectrometry system (ESI-IT-MS). The functional saponin fractions were mainly bisdesmosidc and contained saponin m/z 1861 amongst other. Using the bio-assay guided monitoring, the highly active fractions containing 2 to 3 bisdesmodic saponins (5μg/mL) were screened for their effectiveness in enhancing the anti-tumor activity of targeted toxin Sap3-EGF, which was determined using the impedance based real-time cell cytotoxicity evaluation. This novel combination of HSCCC fractionation, MS-target-guided profiling procedure and bio-assay guided fractionation yielded 100mg of functional saponins from a 60g crude drug powder in a rapid and convenient manner.

Profiling the fatty acids from a strain of the microalgae Alexandrium tamarense by means of high-speed counter-current chromatography and gas chromatography coupled with mass spectrometry

Fatty acids of microalgae have been studied as potential chemotaxonomic markers, to reveal plausible lipid phycotoxins or in the context of mass production of algal biofuels. The planctonic microalgae Alexandrium tamarense (Dinophyceae) is a common harmful algal bloom species that often proliferates in eutrophic costal waters. Alexandrium blooms are the proximal source of toxins associated with paralytic shellfish poisoning (PSP), a neurological affliction that has caused human illness for centuries via consumption of contaminated shellfish. However, data on the fatty acid composition of A. tamarense is currently limited. For this reason, we cultivated a well-defined strain of A. tamarense (Alex2, group I, North American clade) in order to study both its major and minor fatty acids. The harvested microalgae were transesterified and the fatty acid methyl esters were fractionated by means of high-speed counter-current chromatography (HSCCC). The resulting 31 HSCCC fractions were analyzed by gas chromatography with mass spectrometry (GC/MS). Unknown substances were identified by transferring assorted HSCCC fractions into picolinyl or pyrrolidide derivatives. Twenty fatty acids (range 0.2–22.9% contribution to total fatty acids) were identified in the unfractionated sample with 14:0, 16:0, 18:1n-9, 18:4n-3, 18:5n-3 and 22:6n-3 representing>80% of the total fatty acids. HSCCC fractionation enabled the identification of further 22 trace fatty acids contributing between ∼0.01 and 0.2% to total fatty acids. The fatty acids included several branched-chain fatty acids as well as scarcely reported fatty acids like 11-methyl-18:1n-6tr or 18:2Δ4,9. In order to enable a better comparability and repeatability of HSCCC fractionations, we calculated for each HSCCC fraction the total volume of mobile phase, which had passed the HSCCC. From this volume we subtracted the volume of extruded stationary phase and divided the corrected volume by the total coil volume. These elution values were in good agreement with the partition ratios of randomly chosen fatty acid methyl esters obtained in shake flask tests, which allows the prediction of the elution from the HSCCC system when the partition ratio is known.

Investigation of flavonoids from Siparuna sarmentosa Perkins

Siparuna sarmentosa Perkins (Siparunaceae), known as 'Limão-bravo' is an herbaceous plant of an important medicinal genus from Brazilian Amazon. There are reports of medicinal uses of several species of Siparuna by indigenous and local communities. Literature reports the isolation of flavonoids, alkaloids and terpenoids from this genus but there are no reports on the chemistry of the species Siparuna sarmentosa (Ss). Previous work on S. guianensis by HPLC-DAD-ESI-MS/MS [Negri,2012] reports the presence of C-glycosylated flavonoids, never before reported for Siparuna. In this way, this study aims to evaluate the chemical profile of Ss by TLC, HPLC-DAD and HR-ESI-MS and to isolate the flavonoid derivatives by CCC in order to confirm the C-glycosyl pattern. Leaves of Ss, collected in the National Park Adolpho Ducke (Manaus, Brazil) were extracted with methanol and the crude extract obtained (SsEM) was subjected to clean-up by SPE. The clean-up performed by SPE was carried out in reversed phase (RP18) and the yielded fractions were analyzed by HR-ESI-MS. The error for each suggested molecular formula was calculated in ppm. The presence of lucenin-2 (1.5 ppm) and vicenin-2 (0.3 ppm), vitexin (4.6 ppm), quercitrin (2.9 ppm) and vanillic acid (0.4 ppm) [Negri,2012] is suggested. In order to obtain an enriched flavonoid fraction for further isolation of the proposed C-glycosyl flavonoids, high-speed countercurrent chromatography (HSCCC) of SsEM was performed with the biphasic solvent system hexane-ethyl acetate-methanol-water 3:6:6:5, in reversed phase mode, affording the flavonoid fraction SsFF. In this process a flavonoid aglycone and a terpenoid derivative were recovered from the organic stationary phase of the HSCCC fractionation. Their structure is under elucidation.

Bioassay-guided separation and purification of water-soluble antioxidants from Carthamus tinctorius L. by combination of chromatographic techniques

A combined chromatographic method using high-speed counter-current chromatography (HSCCC) and Sephadex LH-20 chromatography was established for bioassay-guided separation and purification of water-soluble antioxidants from Carthamus tinctorius L. florets. The crude sample II obtained by an initial cleanup step on the AB-8 macroporous resin exhibited a potential ABTS radical cation scavenging activity with the SC50 value of 49.28μg/mL. The HSCCC separation was performed with a two-phase solvent system composed of n-butanol-0.1mol/L HCl (1:1, v/v) at a flow rate of 1.2mL/min. After a single run, 3mg hydroxysafflor yellow A (HSYA) with 98% purity was separated as a major component from 20mg of crude sample II. In order to increase the yield and investigate the minor components, the sample size of HSCCC separation was enlarged to 1g. Then, HSCCC fractions were subjected to Sephadex LH-20 chromatography and eluted with distilled water at a flow rate of 1.5mL/min to remove the acid and further separate the components. This separation yielded 184.0mg of HSYA (1), 10.0mg of 5,4′-dihydroxyflavone-3,6-di-O-β-D-glucoside-7-O-β-D-glucuronide (2), 3.0mg of 3-O-caffeoylquinic acid (3), and 3.2mg of 4-O-β-D-glucosyl-trans-p-coumaric acid (4) from 1g crude sample II of C. tinctorius L. florets. The purity of the separated compounds were over 95% by HPLC analysis, and their chemical structures were confirmed by MS, 1HNMR and 13CNMR. Compounds 3 and 4 were found from this plant for the first time. Antioxidant activities assayed in vitro by ABTS radical cation scavenging showed that the SC50 values of the above four compounds were 44.39±1.62μg/mL, 78.13±1.00μg/mL, 21.85±1.96μg/mL, and 31.44±2.06μg/mL, respectively.