Search Results (26)

β-carotene, a high-value carotenoid widely used in the food, pharmaceutical, and cosmetics industries, is naturally synthesized by the microalga Dunaliella sp. However, the efficient extraction and purification of β-carotene from microalgae biomass remain a technical challenge. This study presents the development of a scalable and efficient isolation method employing high-performance countercurrent chromatography (HPCCC) to recover β-carotene from Dunaliella sp. The separation process was optimized by integrating two elution strategies (reverse phase and extrusion) using a biphasic solvent system of n-heptane and methanol (1:1, v/v). The upper phase served as the stationary phase, while the lower phase was used as the mobile phase. Two consecutive injections of 800 mg of microalgal extract each resulted in the isolation of 225.4 mg of β-carotene with a purity of 97% and a recovery of 98%. The developed HPCCC approach represents an efficient method for β-carotene purification and serves as a promising model for future scale-up in microalgae-based production platforms. Full article

Electrospray mass spectrometry off-line profiling monitored the recovery of targeted indole alkaloids from a fortified crude extract of Catharanthus roseus (790 mg) using semi-preparative high-performance countercurrent chromatography (HPCCC) fractionation. Visualization of selected single-ion traces projected the HPCCC molecular weight elution profile. Experimental partition-ratio values K_D and peak widths for detected metabolites were determined. Structural characterization of metabolites and co-elution effects were monitored in the scan range m/z 100–2000. In this study, the biphasic solvent system containing n-hexane–n-butanol–water with 0.5% ion-pair reagent trifluoro-acetic acid [1:1:2, v/v/v] was used based on partition ratio K_D-value liquid chromatography–electrospray ionization–mass spectrometry (LC-ESI-MS) analysis prediction. The monitoring of target ions resulted in the isolation of six major concentrated indole alkaloids (akuammicine, catharanthine, perivine, vindoline, vindorosine, and 19R-vindolinine), which were fully elucidated by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. Full article

Endemic in 21 countries, Chagas disease, also known as American Trypanosomiasis, is a neglected tropical disease (NTD) caused by the protozoan parasite Trypanosoma cruzi. The available drugs for the treatment of this disease, benznidazole and nifurtimox, are outdated and display severe side effects. Thus, the discovery of new drugs is crucial. Based on our continuous studies aiming towards the discovery of natural products with anti-T. cruzi potential, the MeOH extract from aerial parts of Baccharis sphenophylla Dusén ex. Malme (Asteraceae) displayed activity against this parasite and was subjected to high-performance countercurrent chromatography (HPCCC), to obtain one unreported syn-labdane diterpene — sphenophyllol (1) — as well as the known compounds gaudichaudol C (2), ent-kaurenoic acid (3), hispidulin (4), eupafolin (5), and one mixture of di-O-caffeoylquinic acids (6–8). Compounds 1–8 were characterized by analysis of nuclear magnetic resonance (NMR) and mass spectrometry (MS) data. When tested against trypomastigote forms, isolated labdane diterpenes 1 and 2 displayed potent activity, with EC₅₀ values of 20.1 μM and 2.9 μM, respectively. The mixture of chlorogenic acids 6–8, as well as the isolated flavones 4 and 5, showed significant activity against the clinically relevant amastigotes, with EC₅₀ values of 24.9, 12.8, and 2.7 μM, respectively. Nonetheless, tested compounds 1–8 displayed no cytotoxicity against mammalian cells (CC₅₀ > 200 μM). These results demonstrate the application of HPCCC as an important tool to isolate bioactive compounds from natural sources, including the antitrypanosomal extract from B. sphenophylla, allowing for the development of novel strategic molecular prototypes against tropical neglected diseases. Full article

Grapevine canes are an important source of bioactive compounds, such as stilbenoids. This study aimed to evaluate an in silico method, based on the Conductor-like Screening Model for Real Solvents (COSMO-RS) to isolate stilbenoids from a grapevine cane extract by offline heart-cut high-performance countercurrent chromatography (HPCCC). For the following extraction of resveratrol and ε-viniferin from grapevine canes, natural deep eutectic solvents (NADES) were used as an environmentally friendly alternative to the traditionally used organic solvents. In order to evaluate a variety of combinations of hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs) for the targeted extraction of stilbenoids, COSMO-RS was applied. In particular, ultrasonic-assisted extraction using a solvent mixture of choline chloride/1,2-propanediol leads to higher extraction yields of resveratrol and ε-viniferin. COSMO-RS calculations for NADES extraction combined with HPCCC biphasic solvent system calculations are a powerful combination for the sustainable extraction, recovery, and isolation of natural products. This in silico-supported workflow enables the reduction of preliminary experimental tests required for the extraction and isolation of natural compounds. Full article

Peanut hulls (Arachis hypogaea, Leguminosae), which are a side stream of global peanut processing, are rich in bioactive flavonoids such as luteolin, eriodictyol, and 5,7-dihydroxychromone. This study aimed to isolate these flavonoid derivatives by liquid-liquid chromatography with as few steps as possible. To this end, luteolin, eriodictyol and 5,7-dihydroxychromone were isolated from peanut hulls using two different techniques, high-performance countercurrent chromatography (HPCCC) and fast-centrifugal partition chromatography (FCPC). The suitability of the biphasic solvent system composed of n-hexane/ethyl acetate/methanol/water (1.0/1.0/1.0/1.5; v/v/v/v) was determined by the Conductor like Screening Model for Real Solvents (COSMO-RS), which allowed the partition ratio K_D-values of the three main flavonoids to be calculated. After a one-step HPCCC separation of ~1000 mg of an ethanolic peanut hull extract, 15 mg of luteolin and 8 mg of eriodictyol were isolated with purities over 96%. Furthermore, 3 mg of 5,7-dihydroxychromone could be isolated after purification by semi-preparative reversed-phase liquid chromatography (semi-prep. HPLC) in purity of over 99%. The compounds were identified by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy (NMR). Full article

Marine organisms have gained considerable biotechnological interest in recent years due to their wide variety of bioactive compounds with potential applications. Mycosporine-like amino acids (MAAs) are UV-absorbing secondary metabolites with antioxidant and photoprotective capacity, mainly found in organisms living under stress conditions (e.g., cyanobacteria, red algae, or lichens). In this work, five MAAs were isolated from two red macroalgae (Pyropia columbina and Gelidium corneum) and one marine lichen (Lichina pygmaea) by high-performance countercurrent chromatography (HPCCC). The selected biphasic solvent system consisted of ethanol, acetonitrile, saturated ammonium sulphate solution, and water (1:1:0.5:1; v:v:v:v). The HPCCC process for P. columbina and G. corneum consisted of eight separation cycles (1 g and 200 mg of extract per cycle, respectively), whereas three cycles were performed for of L. pygmaea (1.2 g extract per cycle). The separation process resulted in fractions enriched with palythine (2.3 mg), asterina-330 (3.3 mg), shinorine (14.8 mg), porphyra-334 (203.5 mg) and mycosporine-serinol (46.6 mg), which were subsequently desalted by using precipitation with methanol and permeation on a Sephadex G-10 column. Target molecules were identified by HPLC, MS, and NMR. Full article

High-performance counter-current chromatography (HPCCC) was used as a tool for the isolation and fractionation of phenolic compounds (PCs) in extracts from wine lees (WL) and grape pomace (GP). The biphasic solvent systems applied for HPCCC separation were n-butanol:methyl tert-butyl ether:acetonitrile:water (3:1:1:5) with 0.1% trifluoroacetic acid (TFA) and n-hexane:ethyl acetate:methanol:water (1:5:1:5). After refining the ethanol:water extracts of GP and WL by-products by ethyl acetate extraction, the latter system yielded an enriched fraction of the minor family of flavonols. Recoveries of 112.9 and 105.9 mg of purified flavonols (myricetin, quercetin, isorhamnetin, and kaempferol) in GP and WL, respectively, from 500 mg of ethyl acetate extract (equivalent to 10 g of by-product) were obtained. The HPCCC fractionation and concentration capabilities were also exploited for the characterization and tentative identification of constitutive PCs by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). In addition to the isolation of the enriched flavonol fraction, a total of 57 PCs in both matrixes were identified, 12 of which were reported for the first time in WL and/or GP. The application of HPCCC to GP and WL extracts may be a powerful approach to isolate large amounts of minor PCs. The composition of the isolated fraction demonstrated quantitative differences in the individual compound composition of GP and WL, supporting the potential exploitation of these matrixes as sources of specific flavonols for technological applications. Full article

The development of Dai medicine is relatively slow, and Zanthoxylum has great economic and medicinal value. It is still difficult to obtain medicinal components from the low-polarity parts of Zanthoxylum belonging to Dai medicine. In this study, we introduced one simple and quick strategy of separating target compounds from the barks of Z. acanthopodium var. timbor by high-performance countercurrent chromatography (HPCCC) with an off-line anti-inflammatory activity screening mode. The development of this strategy was based on the TLC-based generally useful estimation of solvent systems (GUESS) method and HPCCC in combination. This paper presented a rapid method for obtaining target anti-inflammatory compounds. Three lignins were enriched by HPCCC with an off-line inhibition mode of nitric oxide production in lipopolysaccharide-stimulated RAW264.7 macrophage cells, using petroleum ether–ethyl acetate–methanol–water (3:2:3:2) as the solvent system. The results showed that this method was simple and practical and could be applied to trace the anti-inflammatory components of the low-polarity part in Dai medicine. Full article

Paeoniflorin is a glycoside compound found in Paeonia lactiflora Pall that is used in traditional herbal medicine and shows various protective effects on the cardio-cerebral vascular system. It has been reported that the pharmacological effects of paeoniflorin might be generated by its metabolites. However, the bioavailability of paeoniflorin by oral administration is low, which greatly limits its clinical application. In this paper, a paeoniflorin-converting enzyme gene (G6046, GenBank accession numbers: OP856858) from Cunninghamella blakesleeana (AS 3.970) was identified by comparative analysis between MS analysis and transcriptomics. The expression, purification, enzyme activity, and structure of the conversion products produced by this paeoniflorin-converting enzyme were studied. The optimal conditions for the enzymatic activity were found to be pH 9, 45 °C, resulting in a specific enzyme activity of 14.56 U/mg. The products were separated and purified by high-performance counter-current chromatography (HPCCC). Two main components were isolated and identified, 2-amino-2-p-hydroxymethyl-methyl alcohol-benzoate (tirs-benzoate) and 1-benzoyloxy-2,3-propanediol (1-benzoyloxypropane-2,3-diol), via UPLC-Q-TOF-MS and NMR. Additionally, paeoniflorin demonstrated the ability to metabolize into benzoic acid via G6046 enzyme, which might exert antidepressant effects through the blood–brain barrier into the brain. Full article

High-performance countercurrent chromatography (HPCCC) was used for the target-guided isolation of precursors of 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN) from Riesling wine. In separated HPCCC fractions of an Amberlite^® XAD^®-2 extract obtained from a German Riesling, TDN-generating fractions were identified by the acid-catalyzed hydrolysis of the progenitors at pH 3.0 and subsequent HS-GC-MS/MS analysis. The presence of multiple TDN-generating precursors in Riesling wine could be confirmed. From polar HPCCC fractions (11–13 and 14–16), 3,4-dihydroxy-7,8-dihydro-β-ionone 3-O-rutinoside and 3,4-dihydroxy-7,8-dihydro-β-ionone 3-O-β-d-glucopyranoside were isolated as major TDN-precursors at a sufficient amount for structure elucidation by NMR spectroscopic studies. In the medium polar HPCCC factions (27–35), enzymatic hydrolysis liberated the aglycones 3-hydroxy-β-ionone and 3-hydroxy-TDN in minor amounts. In further less polar TDN-generation fractions (36–44 and 45–50), glycosidic progenitors were absent; instead, a minor TDN formation most likely from non-conjugated constituents was observed. Full article

Organoboron compounds are well known for their use as synthetic building blocks in several significant reactions, e.g., palladium-catalyzed Suzuki-Miyaura cross-coupling. As an element, boron is fascinating; as part of a molecule it structurally resembles a three-valent atom, but if there is a lone pair of electrons nearby, the boron atom’s empty p-orbital may capture the lone pair and form a covalent bond. This is the main aspect that is challenging chemistry during the synthesis of boron containing molecules and may lead into unexpected reactions and products. To study this, we synthesized and studied novel aromatic azaborines for better understanding of their structures and reactions. Here, we report a one-pot method for the synthesis of substituted aromatic azaborines and computational studies of their structure to explain their observed chemical properties. Full article

To better control the quality of saponins, ensure their biological activity and clinical therapeutic effect, and expand the development and application of saponins, this paper systematically and comprehensively reviews the separation and analytical methods of saponins in the past decade. Since 2010, the electronic databases of PubMed, Google Scholar, ISI Web of Science, Science Direct, Wiley, Springer, CNKI (National Knowledge Infrastructure, CNKI), Wanfang Med online, and other databases have been searched systematically. As a result, it is found that ionic liquids and high-performance countercurrent chromatography are the most popular extraction and separation techniques for saponins, and the combined chromatography technique is the most widely used method for the analysis of saponins. Liquid chromatography can be used in combination with different detectors to achieve qualitative or quantitative analysis and quality control of saponin compounds in medicinal materials and their preparations. This paper provides the latest valuable insights and references for the analytical methods and continued development and application of saponins. Full article

Polyphenols show a spectrum of bioactive effects, including an influence on lipid metabolism. In this study, we performed activity-guided fractionations of black chokeberry (aronia), cranberry, and pomegranate extracts to identify the biologically active compounds. The extracts were prepared from fruit juice concentrates with the adsorbent resin Amberlite XAD-7 and were separated into a copigment and an anthocyanin fraction, followed by fractionation into a polymer and monomeric fraction by means of hexane precipitation. For further fractionation of the cranberry and pomegranate copigment fractions, high-performance countercurrent chromatography (HPCCC) was used. The compounds in each fraction were identified by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS), and the quantification was performed by ultra high-performance liquid chromatography-diode array detector (UHPLC-DAD) analyses. Each of the (sub-)fractions was tested in three in vitro assays: phosphodiesterase 3B (PDE) activity, lipid accumulation, and lipolysis in 3T3-L1 cells. The results showed that various fractions and subfractions can inhibit lipid accumulation and PDE activity as well as increase lipolysis, particularly copigments. Overall, our results indicate an influence of polyphenol-rich (sub-)fractions on the lipid metabolism. Full article

Phaeodactylum tricornutum is a rich source of fucoxanthin, a carotenoid with several health benefits. In the present study, high performance countercurrent chromatography (HPCCC) was used to isolate fucoxanthin from an extract of P. tricornutum. A multiple sequential injection HPCCC method was developed combining two elution modes (reverse phase and extrusion). The lower phase of a biphasic solvent system (n-heptane, ethyl acetate, ethanol and water, ratio 5/5/6/3, v/v/v/v) was used as the mobile phase, while the upper phase was the stationary phase. Ten consecutive sample injections (240 mg of extract each) were performed leading to the separation of 38 mg fucoxanthin with purity of 97% and a recovery of 98%. The process throughput was 0.189 g/h, while the efficiency per gram of fucoxanthin was 0.003 g/h. Environmental risk and general process evaluation factors were used for assessment of the developed separation method and compared with existing fucoxanthin liquid-liquid isolation methods. The isolated fucoxanthin retained its well-described ability to induce nuclear translocation of transcription factor FOXO3. Overall, the developed isolation method may represent a useful model to produce biologically active fucoxanthin from diatom biomass. Full article

The detailed metabolite profiling of Laguncularia racemosa was accomplished by high-performance countercurrent chromatography (HPCCC) using the three-phase system n-hexane–tert-butyl methyl ether–acetonitrile–water 2:3:3:2 (v/v/v/v) in step-gradient elution mode. The gradient elution was adjusted to the chemical complexity of the L. racemosa ethyl acetate partition and strongly improved the polarity range of chromatography. The three-phase solvent system was chosen for the gradient to avoid equilibrium problems when changing mobile phase compositions encountered between the gradient steps. The tentative recognition of metabolites including the identification of novel ones was possible due to the off-line injection of fractions to electrospray ionization mass spectrometry (ESI-MS/MS) in the sequence of recovery. The off-line hyphenation profiling experiment of HPCCC and ESI-MS projected the preparative elution by selected single ion traces in the negative ionization mode. Co-elution effects were monitored and MS/MS fragmentation data of more than 100 substances were used for structural characterization and identification. The metabolite profile in the L. racemosa extract comprised flavonoids, hydrolysable tannins, condensed tannins and low molecular weight polyphenols. Full article