Search Results (72)

Peptide purification by preparative reversed-phase liquid chromatography remains one of the most resource-intensive stages in synthetic peptide manufacturing. Production processes commonly rely on acetonitrile/trifluoroacetic acid (ACN/TFA) mobile phases mainly because of their high chromatographic resolution. However, both components raise significant environmental and safety concerns related to toxicity, flammability, waste generation, and, in the case of TFA, environmental persistence as a per- and polyfluoroalkyl substance (PFAS, known as a “forever chemical”). Green alternatives based on ethanol, dimethyl carbonate, and sustainable acidic additives such as acetic acid have been proposed, but their industrial adoption remains limited due to reduced chromatographic performance, often resulting in lower yield and productivity under conventional batch operation. In this work, multi-column counter-current solvent gradient purification (MCSGP) was investigated as a strategy to integrate the use of green eluent systems without compromising process performance. Two therapeutic peptides, Tirzepatide and Tetracosactide, were selected as representative case studies with different structural complexity. Using ethanol/acetic acid for Tirzepatide and dimethyl carbonate/acetic acid for Tetracosactide, the MCSGP process achieved purity levels equivalent to those obtained with conventional ACN/TFA batch chromatography, with 88.1% yield at 89.0% purity for Tirzepatide and 93.8% yield at 94.0% purity for Tetracosactide. Productivity for Tirzepatide was improved, reaching 6.3 g/L_resin/h. These results demonstrate that MCSGP can compensate for the reduced separation efficiency typically associated with green eluent systems, enabling sustainable peptide purification without compromising process performance. By combining green solvents with the MCSGP process, this work paves the way for more sustainable peptide purification processes while maintaining high yield and productivity. Full article

Development of novel methods for the separation, characterization, and analysis of microplastics is an urgent task. Countercurrent chromatography (CCC) has been proven to be an efficient method for the separation and preconcentration of microplastics from aqueous samples using two-phase water–oil systems. However, the efficiency of separation of microplastics from natural seawater by CCC has not been studied so far. Here we demonstrate the high efficiency of separation of microplastics from Black Sea water samples by CCC. The separation efficiency of PE, PP, PS, PVC, PET microparticles of different size (<63, 63–100, 100–250 μm) from spiked seawater samples is about 100%. The method enables the separation of microplastics with size at least down to 1 μm to be performed. The combination of CCC and pyro-GC-MS was applied to the quantification of microplastics in Black Sea water samples. Seven microplastics (μPE, μPP, μSBR, μPVC, μPET) were determined in the seawater samples under study. The total concentration of determined microplastics was about 6.5 μg/L. It was shown that the combination of CCC and pyro-GC-MS enabled robust analytical data to be obtained and hence can be applied to an accurate quantification of microplastics in seawater. Full article

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

An antiviral effect of extracts prepared from aerial parts of nine species and from leaves of two species of the genus Spiraea L. was investigated for potential antiviral activity toward influenza A (H1N1) virus. The toxicity of dry extracts was analyzed, and the most selective extract was identified in vitro. The study’s material was collected in the Asian part of Russia. The plant extracts were prepared via three-stage countercurrent repercolation involving a complete cycle. All 40%-ethanolic extracts from Spiraea manifested antiviral activity against influenza A (H1N1) virus, with a selectivity index (SI) ranging from 1 to 10. IC₅₀ values indicated that the S. salicifolia L. S15 leaf extract (5.9 µg/mL) has the most pronounced antiviral effect and the lowest toxicity (CC₅₀ = 57.6 µg/mL) among the studied samples. The SI of this extract was 10, which exceeded that of the antiviral agent rimantadine (SI = 6). Biologically active compounds in the extract with the highest antiviral activity were identified using UV spectrometry and high-performance liquid chromatography. The S. salicifolia leaf extract was found to contain phenolic acids (chlorogenic, gentisic, caffeic, ferulic, and cinnamic acids), flavonols (quercetin, quercetin-3-glucuronoside, hyperoside, isoquercitrin, rutin, spiraeoside, avicularin, quercitrin, kaempferol, nicotiflorin, astragalin, and isorhamnetin-3-rutinoside), flavones (orientin, luteolin-7-glucoside, and vitexin), and coumarin. Predominant biologically active compounds in the S. salicifolia S15 leaf extract were such flavonols as rutin (19.3 mg/g), isoquercitrin (16.6 mg/g), isorhamnetin-3-rutinoside (10.6 mg/g), and astragalin (9.5 mg/g). Extraction of S. salicifolia leaves by repercolation is a more suitable method for extracting active ingredients with an antiviral effect. Full article

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)). Full article

In recent years, discovering new drug candidates has become a top priority in research. Natural products have proven to be a promising source for such discoveries as many researchers have successfully isolated bioactive compounds with various activities that show potential as drug candidates. Among these compounds, phenolic compounds have been frequently isolated due to their many biological activities, including their role as antioxidants, making them candidates for treating diseases related to oxidative stress. The isolation method is essential, and researchers have sought to find effective procedures that maximize the purity and yield of bioactive compounds. This review aims to provide information on the isolation or separation methods for phenolic compounds with antioxidant activities using column chromatography, medium-pressure liquid chromatography, high-performance liquid chromatography, counter-current chromatography, hydrophilic interaction chromatography, supercritical fluid chromatography, molecularly imprinted technologies, and high-performance thin layer chromatography. For isolation or purification, the molecularly imprinted technologies represent a more accessible and more efficient procedure because they can be applied directly to the extract to reduce the complicated isolation process. However, it still requires further development and refinement. 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

The increasing use of rare-earth elements (REEs) in renewable technologies such as electric vehicles and wind turbines is driving a rapid rise in their economic importance. This work investigated the separation of REEs (Nd, Pr, Sm, and Dy) from NdFeB magnets using high-performance solid-phase extraction chromatography. More than 99% of the REEs were extracted from an Fe-rich sulfate-based leach liquor using a three counter-current stage solvent extraction of 34.7 vol.% and O/A 1.5. The REE-loaded H₂SO₄ strip solution was separated into a high-purity Nd and Pr fraction from separate Sm and Dy fractions in a single stage using a D2EHPA-impregnated column and H₂SO₄ gradient elution. 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

Urtica laetevirens Maxim. is used extensively in traditional Chinese medicine (TCM) for its potent antioxidative properties. In this study, three antioxidants were purified from U. laetevirens. using HSCCC guided by online DPPH-HPLC analysis. Firstly, the online DPPH-HPLC analysis was performed to profile out the antioxidant active molecules in U. laetevirens. The ultrasonic-assisted extraction conditions were optimized by response surface methodology and the results showed the targeted antioxidant active molecules could be well enriched under the optimized extraction conditions. Then, the antioxidant active molecules were separated by high-speed countercurrent chromatography ethyl acetate/n-butanol/water (2:3:5, v/v/v) as the solvent system. Finally, the three targets including 16.8 mg of Isovitexin, 9.8 mg of Isoorientin, and 26.7 mg of Apigenin-6,8-di-C-β-d-glucopyranoside were obtained from 100 mg of sample. Their structures were identified by ¹H NMR spectroscopy. Full article

Euphorbia ebracteolata Hayata (Euphorbiaceae family) is a perennial plant that is widely distributed in Korea, Japan, and China. Its roots contain bioactive diterpenes that have anti-inflammatory properties. However, the anti-inflammatory mechanisms are not yet fully understood. This study aimed to identify the most active anti-inflammatory compound from the roots of E. ebracteolata Hayata, using bioassay-guided fractionation and a combinative method of high-speed countercurrent chromatography (HSCCC) and preparative high-performance liquid chromatography (HPLC). Then, we investigated its anti-inflammatory mechanism in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Ebractenoid F was identified as the most potent bioactive compound of E. ebracteolata Hayata. Ebractenoid F significantly decreased nitric oxide (NO) production and nuclear factor-κB (NF-κB) activation induced by LPS in RAW 264.7 macrophages. Moreover, ebractenoid F decreased the degradation of inhibitory κB-α, the nuclear translocation of the p65 and p50 subunits of NF-κB, and the expression of NF-κB downstream genes. Furthermore, ebractenoid F inhibited the phosphorylation of Akt and mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK) and c-Jun NH₂ terminal kinase (JNK), in LPS-stimulated RAW 264.7 cells. In conclusion, ebractenoid F exerts the most potent anti-inflammatory effect by suppressing NF-κB-mediated NO production in LPS-stimulated RAW 264.7 cells. Ebractenoid F may be a useful therapeutic compound for the prevention or treatment of inflammation-associated diseases. Full article

Alkylresorcinols (∑ARs) are bioactive lipid compounds predominantly found in cereals. These amphiphilic compounds exist in a high structural diversity and can be divided into two main groups, i.e., 5-alkylresorcinols (ARs) and 2-methyl-5-alkylresorcinols (mARs). The pseudocereal quinoa has a very unique AR profile, consisting not only of straight-chain alkyl chains but also iso- and anteiso-branched isomers. Here, we describe a method for the isolation of such methyl-branched ARs and mARs from quinoa. The enrichment of the ∑AR fraction from the lipid extracts by centrifugal partition chromatography (CPC) was followed by ∑AR profiling using countercurrent chromatography (CCC) and GC/MS analysis of CCC fractions. A total of 112 ∑ARs could be detected, 63 of which had not been previously described in quinoa. Due to this high number of ∑ARs, the direct isolation of individual ARs was not possible using conventional CCC. Instead, the more powerful heart-cut mode was applied to enrich the target compounds. A final purification step—the separation of CCC-co-eluting mARs from ARs —was performed via silver ion chromatography. Altogether, ten rare branched-chain ∑ARs (five iso-branched mARs and five anteiso-branched ARs, including mAR19:0-i and AR20:0-a) were isolated with purities up to 98% in the double-digit mg range. 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

The discovery of bioactive compounds from medicinal plants has played a crucial role in drug discovery. In this study, a simple and efficient method utilizing affinity-based ultrafiltration (UF) coupled with high-performance liquid chromatography (HPLC) was developed for the rapid screening and targeted separation of α-glucosidase inhibitors from Siraitia grosvenorii roots. First, an active fraction of S. grosvenorii roots (SGR2) was prepared, and 17 potential α-glucosidase inhibitors were identified based on UF-HPLC analysis. Second, guided by UF-HPLC, a combination of MCI gel CHP-20P column chromatography, high-speed counter-current countercurrent chromatography, and preparative HPLC were conducted to isolate the compounds producing active peaks. Sixteen compounds were successfully isolated from SGR2, including two lignans and fourteen cucurbitane-type triterpenoids. The structures of the novel compounds (4, 6, 7, 8, 9, and 11) were elucidated using spectroscopic methods, including one- and two-dimensional nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. Finally, the α-glucosidase inhibitory activities of the isolated compounds were verified via enzyme inhibition assays and molecular docking analysis, all of which were found to exhibit certain inhibitory activity. Compound 14 exhibited the strongest inhibitory activity, with an IC₅₀ value of 430.13 ± 13.33 μM, which was superior to that of acarbose (1332.50 ± 58.53 μM). The relationships between the structures of the compounds and their inhibitory activities were also investigated. Molecular docking showed that the highly active inhibitors interacted with α-glucosidase through hydrogen bonds and hydrophobic interactions. Our results demonstrate the beneficial effects of S. grosvenorii roots and their constituents on α-glucosidase inhibition. Full article