Counter-current Chromatography Research Articles

HSCCC Straightforward Fast Preparative Method for Isolation of Two Major Cytotoxic Withanolides from Athenaea fasciculata (Vell.) I.M.C. Rodrigues & Stehmann

Athenaea fasciculata belongs to the Solanaceae family and is a promising source of cytotoxic withanolides known as aurelianolides A and B. In the last years, the pharmacological studies of these aurelianolides on different leukemia cell lines have stimulated new studies on their potential as alternative candidates for new lead anticancer drugs. However, the obtention of these two pure compounds by traditional preparative is a costly and long time-consuming process, which is performed in several steps. This study aimed to propose a straightforward approach for isolating aurelianolides A and B using high-speed countercurrent chromatography (HSCCC). In this study, among 10 different solvent systems, the system composed of n-hexane/ethyl acetate/methanol/water 3:6:2:1 (v/v/v/v) was chosen for optimization. This HEMWat system was optimized to 4:8:2:4 (v/v/v/v) and chosen for HSCCC separation in a tail-to-head elution mode. After the HSCCC scale-up procedure, a withanolides mixture (200.0 mg) was separated within 160 min in a single-step purification process. In total, 78.9 mg of aurelianolide A (up to 95.0% purity) and 54.3 mg of aurelianolide B (up to 88.5% purity) was obtained by this fast sequential liquid–liquid partition process. The isolated withanolides were identified by 1H and 13C NMR spectroscopy (this method has proven to be faster and more efficient than classical procedures (CC and Prep-TLC)).

Preparation of esculin acetates through transesterification reaction catalyzed by Novozyme 435® and their Purification followed by NMR characterization

In this study, biocatalytic transesterification reaction using Novozyme 435® (N435) lipase was employed to enhance the hydrophobicity of esculin, aiming to improve its solubility for commercial applications and enhance its bioactivity and oral viability. The acylation reaction of esculin with vinyl acetate was conducted at 60 °C and 200 rpm for 24 h. After chromatographic and spectroscopic analysis, two products were identified: the first one was monoacylated at the 6′-OH position of the glucosyl moiety of esculin (TR: 10.3 min and m/z 382.93 [M + H]+), and the second one was diacylated at the 6′-OH and 3′-OH positions (TR: 13.0 min and m/z 424.93 [M + H]+). The latter was the major product, with a conversion rate of 53.550 ± 0.368%, while the monoacetylated one showed 8.715 ± 0.064%. Both products were isolated by high-speed counter-current chromatography (HSCCC) using a two-phase system HEMWat 1:9:1:9 and characterized by NMR. In this way, these results improve the practical application of esculin, through the obtention of esculin mono and diacetates by fast and efficient biocatalysis reaction.

Plukenetia volubilis leaves as source of anti-Helicobacter pylori agents

IntroductionHelicobacter pylori infection is a major issue worldwide, with widespread prevalence, combined with its link to gastritis, peptic ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Meanwhile, effectiveness of current treatment protocols is limited by increasing antibiotic resistance and patient compliance issues due to long regimens and side effects. Plukenetia volubilis, or sacha inchi, is a valuable source of bioactive molecules. However, studies on its antimicrobial activity, especially against H. pylori, are lacking.MethodsIn this study, the anti-H. pylori activity of P. volubilis leaves water extract was explored using in vitro and in silico approaches. High-Performance Liquid Chromatography coupled to Electrospray Ionisation and Quadrupole Time-of-Flight Mass Spectrometry (HPLC-ESI- QTOF-MS-MS) analysis of the water extract from the leaves was used to characterise the chemical composition of the plant and allowed identification of some flavonoids, such as astragalin, and some phenolic compounds. Then, high-speed counter current chromatography (HSCCC) was used to fractionate the ethyl acetate partition obtained from the water extract from the leaves.Results and DiscussionThe presence of flavonoids derived from kaempferol was confirmed and astragalin was isolated for the first time in P. volubilis. The P. volubilis water infusion, ethyl acetate extract and the isolated astragalin exhibited anti-bacterial activity against H. pylori J99 and two clinical isolates (e.g., minimum inhibitory concentrations of 0.53, 0.51 and 0.49 μg/mL, respectively, for clarithromycin-resistant clinical isolate SSR366). Then, using molecular docking for potential protein targets for H. pylori, it was verified that astragalin could interact with these proteins by in silico analysis.ConclusionThese findings highlight that P. volubilis and astragalin produce a bacteriostatic activity against H. pylori and may have potential to be used in treatment against H. pylori, after further research.

A New Megastigmane Nor-sesquiterpene Isolated from Pourouma Guianensis by High-Performance Countercurrent Chromatography

Introduction: Pourouma guianensis (Urticaceae) is widespread throughout Brazil and is commonly known as “mapati”. Terpenes are common in Urticaceae species. However, few studies have reported the isolation and characterisation of these compounds in Pourouma genus. Method: The aim of this study was to separate substances from stem barks of P. guianensis using a high-performance countercurrent chromatography gradient elution method. The dichloromethane extract of the P. guianensis stem bark was submitted to silica gel column chromatography, yielding three fractions. Fraction 3 was submitted to a gradient elution system in high-performance countercurrent chromatography using two solvent systems, hexane/ethyl acetate/methanol/water (1:2:x:1, x=1.0 and 1.5). Six compounds were isolated. Results: A new megastigmane nor-sesquiterpene named 2-acetyl-4,7-epoxy-3-oxy-5,5,9- trimethylhexahydrocoumaran (1) was obtained together with five known terpenes: vomifoliol (2), 2α,3β,19α,23-tetrahydroxyurs-12-en-28-oic acid (3), 3-β-D-arabinopyranoside-19-hydroxyurs-12-en- 28-oic acid (4), arjunolic acid (5), and tormentic acid (6). Fractions 1 and 2 were purified by binary gradient elution from hexane to ethyl acetate. Conclusion: This process isolated three known compounds: friedelin (7) and 3β-friedelanol (8) from fraction 1, and β-sitosterol (9) from fraction 2. Gradient elution in countercurrent chromatography was a suitable alternative, exploiting variations in the partition coefficient between substances with small structural differences.

Separation and Characterization of Wenjin Tongluo San Essential Oil with a Comprehensive Chromatographic Separation

The essential oil components of traditional Chinese medicine in-hospital preparation were complex, and one-dimensional chromatographic separation was difficult to completely separate them due to the limited peak capacity. This study was carried out to establish a comprehensive two-dimensional chromatographic separation and analysis method based on countercurrent chromatography (CCC) and gas chromatography (GC). In this paper, we focused on the separation of the essential oil of the traditional Chinese medicine in-hospital preparation Wenjing Tongluo San by CCC × GC, and explored the orthogonality between the two chromatographic techniques to provide the new technical support for the screening of the active ingredients. A solvent system composed of n-hexane-ethyl acetate-methanol-water (9.5:0.5:8.5:1.5, v/v) was chosen for the first-dimensional CCC separation. All the fractions collected from CCC were transferred to GC for plotting two-dimensional contours map. The calculated capacity of the two-dimensional separation system exceeded 3000, which was 8 times more than that of the one-dimensional separation system. High orthogonality (r = 0.42) and spatial coverage factor (70.42%) were obtained. Meanwhile, all the fractions were identified by GC-MS. Our research provided a new methodology for separating essential oils in traditional Chinese medicine as well as an approach for evaluating the quality of traditional Chinese medicinal in-hospital preparation based on two-dimensional chromatographic fingerprints.

Solvent screening for the purification of monoterpenoids by countercurrent and centrifugal partition chromatography

AbstractBACKGROUNDCountercurrent chromatography (CCC) and centrifugal partition chromatography (CPC) are efficient techniques to purify terpenoids from essential oils. These methods require suitable solvent systems for the partition between the two immiscible liquid phases. In this study, using the analytical shake‐flask method, we measured the partition coefficients of three model monoterpenoids, namely carvone, eucalyptol and thymol, in 11 biphasic solvent systems, at 298.2 K. Moreover, the predictive COSMO‐RS model was applied to represent the partition coefficients of data measured in this work and retrieved from the literature, the liquid–liquid equilibrium (LLE) data of three solvent families suitable for CCC/CPC separations (i.e., Arizona, Modified Arizona and Green Arizona) and the solute's partition coefficients in the three solvent families.RESULTSThe partition coefficients obtained for thymol and eucalyptol are the first partition data available in the literature. The obtained root mean square deviations (RMSDs) between the experimental and predicted partition coefficients (log basis) varied between 0.28 and 0.49. For LLE, RMSDs of 0.040 and 0.048 were achieved for the Arizona and Green Arizona families, respectively.CONCLUSIONThe predictive COSMO‐RS model describes the monoterpenoid partition coefficients and the LLE data of the solvent families well. The most favorable solvent systems to perform the separations were identified, showing that systems with low and intermediate‐to‐low polarities are the most promising options for separating the selected monoterpenoids from their natural matrices by CCC/CPC techniques. © 2024 Society of Chemical Industry (SCI).

High-Speed Countercurrent Chromatography Isolation of Active Components from Evodia Rutaecarpa and Affinity Ultrafiltration Screening for Their Acetylcholinesterase Inhibitor Activity.

Acetylcholinesterase inhibitors from Evodia rutaecarpa were screened, prepared, and evaluated. To screen the lipophilic alkaloid active constituents in E. rutaecarpa, we improved and optimized an ultrafiltration system. Three acetylcholinesterase (AChE) inhibitors, dehydroevodiamine, evodiamine, and rutecarpine, were screened. Addressing the limitations of the traditional response surface methodology (RSM) for multiobjective screening, we integrated RSM with the Non-dominated Sorting Genetic Algorithm III to achieve the optimal extraction of these active ingredients. High-speed countercurrent chromatography was used to isolate the active components using a two-phase solvent system: n-hexane/ethyl acetate/methanol/water (3.0:2.5:3.5:2.0, v/v/v/v) and ethyl acetate/methanol/water (3.0:1.0:4.0, v/v/v). The nuclear magnetic resonance spectroscopy confirmed the structures of the compounds, and molecular docking and dynamics simulations assessed the inhibitory effects of the chemical components on AChE, which were consistent with the findings of the ultrafiltration experiments. We also confirmed the neuroprotective properties of these compounds against glutamate-induced apoptosis in PC12 cells. Overall, we achieved the systematic optimization of multitarget compound extraction and lipophilic alkaloid ultrafiltration screening, as well as preparation and activity validation, laying the groundwork for the development of AChE inhibitors from lipophilic alkaloids.

A modeling method based on incomplete mixed cells in counter-current chromatography: interphase mass transfer ratio

A modeling method based on incomplete mixed cells in counter-current chromatography: interphase mass transfer ratio

A new consecutive high-performance counter-current chromatography approach based on co-current and overlap mode for isolating active compounds from olive leaves

Conventional consecutive separation methods based on overlapping injection modes produce interactions between samples as the separation time increases. This will lead to a reduction in the purity of the target due to the gradual saturation of the stationary phase distribution. To enhance the purity of the sample preparation and separation efficiency, we devised a consecutive separation method under high-performance counter-current chromatography (HPCCC) utilizing co-current elution and overlapping injection. The method was used for the separation and preparation of olive leaves for their main bioactive polyphenolic constituents. Six HPCCC separations were conducted in reversed-phase mode using a two-phase solvent system consisting of petroleum ether/ethyl acetate/water (1:40:40, v/v). Finally, 302.7 mg of luteolin-4’-O-β-D-glucoside and oleuropein were isolated from 720 mg of crude sample with purities of 93.3 % and 96.2 %, respectively. The optimization strategy outlined in this study can be used to enhance the extraction of bioactive components from olive leaves.

Preparative separation of ten flavonoids from Scutellaria baicalensis Georgi roots using two-dimensional countercurrent chromatography with an online-storage, dilution, and mixing interface

The process of counter-current chromatography (CCC) separation for natural products typically necessitates the use of multiple solvent systems to accommodate constituents with a wide range of polarities. However, the incompatibility between these different solvent systems often results in unsuccessful online 2D successive separations. In this study, a 2D CCC system was developed, featuring an interface for online-storage, dilution, and mixing. It facilitated the implementation of online 2D CCC using different solvent systems. The method was subsequently applied for the preparative isolation of flavonoids from Scutellaria baicalensis Georgi roots. For 1D CCC, n-heptane-ethyl acetate–methanol-water (HepEMWat, 5:5:4:6, v/v) was utilized, while for 2D CCC, ethyl acetate-n-butanol-water (EBuWat, 0:5:5, v/v) was employed. The eluent with low resolution in 1D CCC was stored online, diluted three times using the lower phase of EBuWat (0:5:5, v/v), and subsequently transferred into 2D CCC for further isolation utilizing the same EBuWat (0:5:5, v/v) system. As results, six lipophilic compounds were isolated in 1D CCC in a normal mode, while two major hydrophilic constituents were isolated in a pH-peak-focusing mode in 2D CCC. Additionally, two additional compounds were purified through subsequent semi-preparative HPLC separation in order to resolve co-elution in 2D CCC. The developed 2D CCC system with a multifunctional interface demonstrated to be an exceptionally efficient and promising approach for the high-throughput purification of complex natural products.

Prediction model for retention volumes of the three-phase solvent system in high-speed countercurrent chromatography.

Owing to its ability to separate substances with a broad scope of polarities, exploring the three-phase solvent systems (TPSSs) with high-speed countercurrent chromatography is a topic of interest in separation science, and their retention volumes should be more concerned. This study primarily investigates the behavior of retention volumes while examining the isolation abilities of the TPSS in the technique above. We took standard compounds, including sophoricoside, Sudan red 7B, and rotenone, which have a broad range of polarity, for investigation in this study and separated them using different four-liquid TPSSs made up of water, acetonitrile, methyl acetate, and n-hexane (WAMH). Our findings show that the retention volumes gradually alter in response to changes in phase polarity within the proposed solvent systems. With TPSSs, we preliminarily studied compound isolation and the promising formula of their retention volumes. The proposed solvent systems WAMH in different ratios showed high correlations and adjusted correlation coefficients above 0.9978 and 0.9913 for the actual and calculated retention volumes. This study will be particularly beneficial for researchers focusing on countercurrent chromatography with TPSSs, as it offers valuable time-saving insights.

Separation of astragaloside IV from Astragalus membranaceus based on high-speed countercurrent chromatography in continuous injection mode.

Astragaloside IV (AS-IV) is an index for the quality evaluation of the traditional Chinese medicine Astragalus and an important material basis for Astragalus to exert its medicinal effects, and it is difficult to obtain a single AS-IV by ordinary separation methods. To find a new isolation method that can prepare AS-IV quickly and efficiently. AS-IV was isolated from Astragalus membranaceus extract by high-speed countercurrent chromatography using a two-phase solvent system consisting of ethyl acetate/n-butanol/water (4.2:0.8:5, v/v) at a speed of 950 rpm at a flow rate of 2mL/min using one of the high-speed countercurrent chromatographic sequential injection models developed during the previous study. Compared with the common countercurrent chromatographic separation, this separation method increased the injection volume and yield by 4-fold and 4.47-fold, respectively, with only about 1.2-fold increase in solvent consumption and separation time, and the purity was basically not reduced, and 55.9mg of AS-IV, with a purity of 96.95%, was finally prepared from 400 mg of the crude extract in 240 min. The continuous injection mode of high-speed countercurrent chromatography was able to successfully prepare a large amount of AS-IV with high purity at one time.

Bioassay-guided Fractionation of Anti-Leishmania amazonensisExtracts from Ampelozizyphus amazonicus by High-speed Countercurrent Chromatography.

Ampelozizyphus amazonicus Ducke, known as "saracura-mirá" in the Amazon rainforest, is valued for its traditional use in malaria prevention and treatment. The plant's roots and bark are employed for these purposes, while the wood is often overlooked. Given the global importance of leishmaniasis, research focused on A. amazonicus anti-Leishmania amazonensis potential. The ethyl acetate extract from the bark (EAEB) exhibited the most effective inhibition of intracellular amastigote growth with IC50 7.0 µg.mL-1 but showed high toxicity (CC50 9.0 µg.mL-1). The wood ethanol (EW) and ethyl acetate (EAEW) extracts demonstrated promising results against L. amazonensis with IC50 15.8 µg.mL-1 and 10.0 µg.mL-1, CC50 50.5 µg.mL-1 and 22.7 µg.mL-1, respectively. High-speed countercurrent chromatography combined with mass spectrometry off-line detection was employed for the bioassay-guided fractionation of EAEB and EAEW using HEMWat as solvent system. These extracts showed to be rich in saponins and triterpenes, besides minor glycosylated flavonoids. HSCCC was effective in obtaining high purity fractions, leading to the identification of a di-glycosylated triterpene saponin from EAEB, and melaleucic acid from EAEW. These findings highlight A. amazonicus as a potential source for developing new therapeutic alternatives for leishmaniasis and HSCCC as a technique enabling better separation efficiency of complex saponin matrices.

Countercurrent chromatography isolation of green propolis biomarkers: Potential blockers of SARS-COV-2 RBD and ACE2 interaction

Propolis is a natural resinous mixture produced by honeybees with numerous biological activities. Considering the recently reported potential of propolis as an adjuvant in COVID-19 treatment, a methodology for the fractionation of the hexane extract of Brazilian green propolis (HEGP) was developed for the obtention of prenylated biomarkers by countercurrent chromatography. The inhibition of the interaction between the receptor binding domain (RBD) of spike and ACE2 receptor was evaluated by the Lumitᵀᴹ immunoassay. Fractionation of HEGP was performed by both normal (CCC1 and CCC2, with extended elution) and reversed (CCC3) phase elution-extrusion modes with the solvent system hexane–ethanol–water 4:3:1. The normal elution mode of CCC1 (471 mg HEGP in a 80 mL column volume, 1.6 mm id) was scaled-up (CCC5, 1211 mg HEGP in a 112 mL column volume, 2.1 mm id), leading to the isolation of 89.9 mg of artepillin C, 1; 52.7 mg of baccharin, 2; and 26.6 mg of chromene, with purities of 93 %, 83 % and 88 %, respectively, by HPLC-PDA. Among the isolated compounds, artepillin C, 1, and baccharin, 2, presented the best results in the Lumitᵀᴹ immunoassay, showing 67% and 51% inhibition, respectively, at the concentration of 10 μM. This technique proved to be of low operational cost and excellent reproducibility.

Impact of the degree of substitution on the enantioseparation of anionic β-cyclodextrin in liquid‒liquid extraction and countercurrent chromatography: Insights from molecular docking simulations

Five types of sulfobutylether-β-cyclodextrin (SBE-β-CD) and carboxymethyl-β-cyclodextrin (CM-β-CD) with different degrees of substitution were synthesized, and six and five racemates were respectively chosen to study the influence of the degree of substitution on the enantioseparation factor. The synthesized SBE-β-CD and CM-β-CD were characterized using 1H NMR spectroscopy and mass spectrometry. The results indicated that the influence of the degree of substitution on enantioseparation for distinct racemates exhibited significant variability. Increasing the degree of substitution of CM-β-CD led to an increasing enantioseparation factor, while SBE-β-CD with a specific degree of substitution provided the optimum enantioseparation factor for some racemates. The optimum enantioseparation factors of N-methyl duloxetine and duloxetine were obtained when a relatively low degree of substitution (DS = 3.5) was selected. And the optimum enantioseparation factor was obtained with a relatively high degree of substitution (DS = 7.5). SBE-β-CD with a low substitution degree of 3.5 was chosen to optimize the countercurrent chromatographic enantioseparation of N-methyl-duloxetine, which resulted in a significant improvement in peak resolution from 0.51 to 0.83. Molecular docking was used to construct three SBE-β-CDs with different degrees and distributions of substitution, and a good agreement was found between the docking results and the experimental results.

Establishing carbon dots assisted high speed countercurrent chromatography and its application for efficient separation of rare earth element ions

Rare earth elements with unique magnetic properties and optical properties, known as the ‘industrial vitamin’, has a very high commercial value. As a secondary resource of rare earth elements, low-concentration solution includes mixed rare earth ions, which need to realize efficient separation and recovery urgently. High speed countercurrent chromatography is suitable for the separation and purification of rare earth element ions due to its advantages of large loading, good tolerance to samples, and simple pretreatment. In this study, a carbon dots assisted high speed countercurrent chromatography method was designed and established, the carbon dots were applied to the mobile phase of high speed countercurrent chromatography for the first time. The low concentration of REEs solution was enriched, and the effective separation of La (III), Ce (III), Gd (III) and Er (III) was successfully achieved. The complete separation of La (III), Ce (III), Gd (III) and Er (III) was achieved with a solvent system of 0.05 mol L−1 P507 (PE), 0.05 mol L−1 HNO3, and 0.1 mol L−1 CDs2 carbon dots (1:1:0.01, v/v/v), the upper phase as stationary phase, the lower phase as mobile phase. Density functional theory result showed that the binding energy of REEs (III)-CDs2 was larger than that of REEs (III)-P507, so the affinity of CDs2 to REEs (III) was stronger than that of P507. Therefore, with the addition of CDs2, the ability of mobile phase to elute REEs from the stationary phase was enhanced, and the separation effect was improved.

Rapidly screening of pancreatic lipase inhibitors from Clematis tangutica using affinity ultrafiltration-HPLC-QTOFMS technique combined with targeted separation, in vitro validation, and molecular docking.

Screening of novel pancreatic lipase inhibitors from complex natural products is a meaningful task. Through accurately screening and separating pancreatic lipase inhibitors from Clematis tangutica (C. tangutica), to discover new leading compounds for slimming and accelerate the development and utilization of Tibetan medicine resources. An integrated strategy that combines affinity ultrafiltration and high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (AU-HPLC-QTOFMS), targeted separation, in vitro validation, and molecular docking was developed to screen pancreatic lipase inhibitors from C. tangutica. The AU-HPLC-QTOFMS technique was performed to fish for the potential active substances. Macroporous resin, preparative liquid chromatography, and high-speed countercurrent chromatography were implemented for the accurate and targeted separation of active compounds. The inhibitory activities of target compounds to pancreatic lipase were detected by the inhibition experiments in vitro. The binding affinities and binding sites were analyzed using molecular docking. A total of eleven kinds of pancreatic lipase inhibitory substances were screened from C. tangutica. Seven triterpenoid saponins were screened for the first time as lipase inhibitors and successfully prepared with purities higher than 97%. Tanguticoside B, clematangoticoside J, hederoside H1, and rutin showed stronger inhibitory effects with IC50 values of 1.539 ± 0.048, 1.661 ± 0.092, 1.793 ± 0.069, and 1.792 ± 0.094 mmol/l. Moreover, they have the lowest ΔG values of -10.84, -9.97, -10.87, and -9.39 kcal/mol to pancreatic lipase. The integrated strategy using AU-HPLC-QTOFMS, targeted separation, in vitro validation, and molecular docking was feasible for rapidly screening and directionally isolating pancreatic lipase inhibitors from C. tangutica.

Preparative separation of the constituents from Ginkgo biloba L. leaves by a combination of multi-stage solvent extraction and countercurrent chromatography.

In this study, we employed a combination approach for the preparative separation of constituents from Ginkgo biloba L. leaves. It involved multi-stage solvent extractions utilizing two-phase multi-solvent systems and countercurrent chromatography (CCC) separations using three different solvent systems. The n-heptane/ethyl acetate/water (1:1:2, v/v) and n-heptane/ethyl acetate/methanol/water (HepEMWat, 7:3:7:3, v/v) solvent systems were screened out as extraction systems. The polarities of the upper and lower phases in the multi-solvent systems were adjustable, enabling the effectively segmented separation of complex constituents in G. biloba L. The segmented products were subsequently directly utilized as samples and separated using CCC with the solvent systems acetate/n-butanol/water (4:1:5, v/v), HepEMWat (5:5:5:5, v/v), and HepEMWat (9:1:9:1, v/v), respectively. As a result, a total of 11 compounds were successfully isolated and identified from a 2g methanol extract of G. biloba L through two-stage extraction and three CCC separation processes; among them, nine compounds exhibited high-performance liquid chromatography purity exceeding 85%.

Separation of Non-alkaloid Toxin Lignans and New Flavonoid from Himalayan Mayapple (Podophyllum Hexandrum Royle) by High-Speed Counter-Current Chromatography and Their Anti-inflammatory Activity Evaluation

Podophyllum hexandrum Royle (Berberidaceae) is reported from the Himalayan region and China. It is also known as the Himalayan Mayapple and is reported for the treatment of constipation, fever, jaundice, liver disorders, etc. Herein, the isolation of chemical constituents using high-speed counter-current chromatography (HSCCC) from the EtOH extract of the rhizomes of Himalayan Mayapple is reported. As a result, kaempferol 3-glucoside (1), quercetin-3-O-β-D-glucopyranoside (2), quercetin 3-O-β-D-glucopyranosyl-(1→6)-3-O-ethyl-β-D-glucopyranoside (3), kaempferol 3- O-β-D-glucopyranoside (4), α-peltatin(5), podophyllotoxin (6), 4'-demethylpodophyllotoxin (7), 4',5'-didemethylpodophyllotoxin (8), and kaempferol (9)were separated. Compounds 6-9 were separated by the normal HSCCC while 1-5 were obtained by the offline-recycling HSCCC using HEMWat (1:9:4:6, v/v) solvent system. The pure components were tested in lipopolysaccharidesinduced mice macrophage cells. Compounds 6 and 7 showed significant inhibition. The nitric oxide production was inhibited by compounds 6 and 7, effectively, with IC50 values of 1.328 x 10-6 and 2.851 x 10-6 M, respectively. In this assay, kaempferol (9), a positive inhibitor expressively inhibited lipopolysaccharides-induced nitric oxide production.

Native Mexican black bean purified anthocyanins fractionated by high-performance counter-current chromatography modulate inflammatory pathways

In addition to their pigment properties, the potential health benefits of anthocyanins have made them a subject of interest in recent years. This study aimed to obtain purified anthocyanin fractions from native Mexican black bean cultivars using Amberlite XAD-7 resin column and HPCCC and evaluate their anti-inflammatory properties using RAW 264.7 cells. The major anthocyanins in the purified anthocyanin fractions were delphinidin 3-glucoside (61.8%), petunidin 3-glucoside (25.2%), and malvidin 3-glucoside (12.2%). Purified anthocyanin fractions at 12.5 μg/mL effectively prevented LPS-induced ERK1/ERK2 phosphorylation and reduced the protein expression of COX-2 and mRNA expression of iNOS. Results showed that purified anthocyanin fractions have the potential to modulate the inflammatory response by inhibiting the production of pro-inflammatory mediators through the ERK1/ERK2 and NF-κB pathways. This study suggests that anthocyanins from black beans could be used as a natural strategy to help modulate inflammation-associated diseases.