Search Results (182)

The chemical composition of natural products is complex and the investigation of bioactivities of compounds of interest demands their isolation. S. terebinthifolia Raddi is a tree belonging to the Anacardiaceae family and is used in Brazilian folk medicine; its fruit (pink peppers) are used in cooking and its bark in phytomedicine. Extracts of other parts of this plant contain a plethora of components and merit further studies. Countercurrent chromatography (CCC) is frequently employed with natural products due to the high sample recovery rate. The objective of this work was to determine the best solvent system (SS) to fraction the ethanol extracts of leaves, flowers and fruit of Schinus terebinthifolia by CCC and isolate compounds of interest and elucidate their structures through nuclear magnetic resonance (NMR) and mass spectrometry (MS). In addition, antiproliferative, potential cell regeneration and antioxidant activities of the fractions of interest were evaluated. In the present work, three compounds were isolated; two were identified as anacardic acids [(6-(8′, 11′-heptadecadienyl)-salicylic acid and 6-(8′-heptadecenyl)-salicylic acid], as well as (Z)-masticadienoic acid. These compounds showed antiproliferative and potential cell regeneration activities as well as varying degrees of antioxidant capacity. Although these compounds present potential therapeutic activity, more studies are necessary to confirm their safety. 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

Pasuchaca (Geranium dielsianum Knuth), a traditional Peruvian medicinal plant from the Geraniaceae family used for diabetes management, was investigated for its antiglycative properties. This study aimed to screen, isolate, and identify the active antiglycative compounds from its aerial parts. By coupling a methylglyoxal (MGO)-HPLC screening assay with high-speed counter-current chromatography (HSCCC), seven dihydroflavonol derivatives were separated and identified from the 80% methanol extract. The compounds were identified as 2,3-dihydromyricetin 3-O-α-rhamnopyranoside (1), (+)-taxifolin 3-O-β-D-xylopyranoside (2), astilbin (6), isoastilbin (8), 3″-acetyl astilbin (9), and 2″-acetyl astilbin (11). Astilbin was identified as the major constituent, with remarkably high contents of 252.41 mg/g in the 80% methanol extract and 541.04 mg/g in the partitioned upper layer fraction. Astilbin demonstrated potent antiglycation activity across all stages of protein glycation (early, middle, late, and whole stages), significantly surpassing the positive control aminoguanidine. Furthermore, the formation of MGO-astilbin adducts was confirmed by LC-ESI-MS, validating its role as an effective MGO scavenger. This report is the first to isolate these phytochemicals from Pasuchaca. The findings establish astilbin as the key antiglycative component of Pasuchaca, substantiating its traditional use and highlighting its potential as a source of functional food ingredients or natural therapeutics for mitigating glycative stress. Full article

Lepidium meyenii Walpers (syn. Lepidium peruvianum Chacon) has been cultivated for centuries in the Peruvian Andes as both a vegetable and a traditional medicine resource. Maca is classified as a superfood and is widely used as a dietary supplement, particularly noted for its potential to enhance endurance, fertility, and endocrine balance. In recent years, there has been a growing interest in the cytotoxic effects of lepidilines and their derivatives; however, these compounds have been less extensively studied due to challenges associated with their isolation. This study aims to establish optimal extraction conditions to enrich lepidiline content in the extracts and to propose an efficient isolation method for four lepidilines using a green purification technique known as Centrifugal Partition Chromatography (CPC). The isolated compounds will be evaluated for their anticancer potential utilizing the MTT assay on SK-N-SH (ATCC^® HTB-11™) and SK-N-AS (ATCC^® CRL-2137™) neuroblastoma cell lines. The findings indicate that Soxhlet extraction with dichloromethane resulted in the highest recovery of lepidilines, with a content of 10.24% expressed as lepidiline A. The optimal biphasic solvent mixture suitable for CPC chromatographic applications was identified as a combination of chloroform, methanol, and water (4:3:2 v/v/v) containing 60 mM HCl. When utilized in conjunction with semi-preparative high-performance liquid chromatography (HPLC), this method successfully isolated lepidilines A–D, achieving a purity exceeding 95%. Notably, lepidiline B exhibited the highest cytotoxic potential, with an IC50 value of 14.85 µg/mL in SK-N-AS cells. Full article

The downstream process of monoclonal antibodies (mAbs) is expensive and significantly contributes to overall manufacturing costs. One primary reason is the extensive consumption of water and chemicals required for preparing large volumes of various buffers, essential for multiple chromatography and filtration steps. Reducing the water consumption in biopharmaceutical processes is critical to drive down costs and improve sustainability, which can be achieved through the introduction of buffer recycling. In this study, we implemented buffer recycling in an integrated two-step mAb downstream process consisting of a Protein A capture step in a periodic counter-current (PCC) set-up, followed by a mixed-mode polishing step in flowthrough mode. Buffer recycling was implemented during the cleaning-in-place (CIP) phases of the integrated steps, where the CIP buffer from the polishing column was recovered and reused counter-currently in the CIP phase of the capture column. Compared to the reference process without buffer recycling, this approach resulted in 29% savings in CIP buffer, while maintaining product purity within 0.66% and yield within 1.68% of the reference process. These minor differences confirm that buffer recycling can be implemented without compromising product quality. Through buffer recycling, we see significant potential to improve process sustainability in biomanufacturing by conserving water and reducing chemical waste. Full article

The naphthazarins shikonin and alkannan are strongly chromogenic, dark red enantiomers, each of which has biological activity, that are found primarily in the plant family Boraginaceae. These compounds and their many chemical metabolites, derivatives, oligomers, and analogs (“shikonoids”) are an important group of phytochemicals, utilized since antiquity as components of dyes, traditional medicines, and food and cosmetics. They are now recognized for their potent anti-inflammatory and regulatory activity on a variety of molecular signaling pathways in humans. Since many Boraginaceae species are overly exploited or endangered, we developed a pilot-scale in vitro shikonoid production system using Plagiobothrys arizonicus (Gray) Greene ex A.Gray, the Arizona popcorn flower, native to the southwestern USA and the Sonoran floristic province in the Madrean region of Mexico. Aseptic root cultures were initiated from fresh leaf tissue and stimulated to continuously produce shikonoids in liquid shake cultures layered under paraffin oil from which the shikonoids were extracted and concentrated. The crude, red extracellular product from these rapidly expanding root masses was also fractionated by Centrifugal Counter-Current Chromatography (CCC) into its component shikonin derivatives. A number of these shikonoids profoundly up-regulated detoxification and antioxidant proteins (phase 2 enzymes) and inhibited inflammation in mammalian cell bioassay systems. This prototype shikonoid production methodology can be readily scaled to either batch or chemostat culture. 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

Alzheimer’s disease (AD) is a progressive neurodegenerative condition and one of the most prevalent types of dementia in older adults. Currently, the primary drugs used to treat AD are acetylcholinesterase (AChE) inhibitors. The development of natural substances has become a research hotspot due to the high number of adverse effects of synthetic drugs. In this study, a new assay based on ultrafiltration–liquid chromatography–high-speed counter-current chromatography (UF-HPLC-HSCCC) was developed for the rapid screening and identification of AChE inhibitors from Olea europaea L. fruit. In this research, we screened and isolated two AChE inhibitors from O. europaea fruit extracts, identified by EI-MS and NMR as secologanoside and oleuroside-11-methyl ester. These compounds were identified for the first time from O. europaea and found to possess AChE inhibitory activity using an in vitro AChE inhibition assay and molecular docking. The IC₅₀ values of the two compounds were 0.76 ± 0.04 mM and 1.08 ± 0.05 mM. The results demonstrated that secologanoside showed better AChE inhibition activity than oleuroside-11-methyl ester, suggesting that this compound is a promising AChE inhibitor. At the same time, the results showed that the combination of UF-HPLC- HSCCC provides a powerful tool for screening and isolating AChE inhibitors in complex samples. Full article

Gynura divaricata (L.) DC is a long-used medicinal and edible plant in China folk. Its hyperglycemic effects have garnered increasing public attention in recent years. This study revealed that the ethyl acetate (EtOAc) and butanol (BuOH) partition fractions of G. divaricata crude extract exhibited significantly higher α-glucosidase inhibition activity and enhanced glucose uptake ability compared to other fractions. Guided by the hypoglycemic bioassay, these two fractions were subjected to isolation of active compounds using high-speed countercurrent chromatography (HSCCC). A two-phase solvent system composed of hexane-methyl tert-butyl ether (MtBE)-methanol-0.1% TFA water was employed for the separation of the EtOAc fraction by conventional HSCCC through a gradient elution strategy. Five major compounds were obtained and identified as chlorogenic acid (1), 3,4-dicaffeoylquinic acid (2), 3,5-dicaffeoylquinic acid (3), 4,5-dicaffeoylquinic acid (4), and kaempferol-3-O-β-D-glucopyranoside (5) by ESI-MS, ¹HNMR, and ¹³CNMR. The chlorogenic acid and the three dicaffeoylquinic acids were found to display higher inhibitory activities against α-glucosidase compared to the flavonoid. Considering their acidic nature, pH-zone-refining CCC (PHZCCC) was then applied for further scale-up separation using a solvent system MtBE: n-butanol: acetonitrile: water with trifluoroacetic acid (TFA) as a retainer and ammonium hydroxide (NH₄OH) as an eluter. A significantly higher yield of chlorogenic acid was obtained from the BuOH fraction by PZRCCC. Molecular docking between the caffeoylquinic acids and α-glucosidase confirmed their hypoglycemic activities. This study demonstrates that CCC is a powerful tool for preparative separation of active constituents in natural products. This research presents a novel and effective method for the preparative isolation of hypoglycemic compounds from Gynura divaricata. Full article

The separation of large polar constituents presents a substantial challenge in natural product research when employing column chromatography techniques, as the process is both complex and time-consuming. In this study, an acetonitrile/tetrahydrofuran/di-(2-ethylhexyl) phosphoric acid/aqueous saturated sodium chloride solvent system was developed and utilized for the countercurrent chromatography of polar constituents from Ginkgo biloba L. seeds. Five polar constituents were effectively isolated using an acetonitrile/tetrahydrofuran/di-(2-ethylhexyl) phosphoric acid/aqueous saturated sodium chloride (2:2:0.8:3, v/v) solvent system using a two-step countercurrent chromatography method. In the initial countercurrent chromatography process, three constituents were successfully purified from the methanol extract: compound 1, compound 4, and compound 5. Compounds 2 and 3, co-eluted from the column, were further subjected to three inner-recycling chromatographic procedures. At last, five constituents were purified and identified, including 4′-O-methylpyridoxine (1); two indole alkaloid N-glucosides, ginkgoside B (2) and ginkgoside A (3); 2-(4-hydroxybenzyl) malic acid (4); and coniferyl alcohol (5). The results demonstrated that the acetonitrile/tetrahydrofuran/di-(2-ethylhexyl) phosphoric acid/aqueous saturated sodium chloride solvent system serves as a feasible system for the efficient countercurrent chromatography separation of polar components. Full article

The exploration of natural antifungal substances from algal origins is significant due to the increasing resistance of pathogens to conventional antifungal agents and the growing consumer demand for natural products. This manuscript represents the inaugural investigation into the antifungal attributes of bioactive compounds extracted from Fucus vesiculosus via supercritical carbon dioxide (scCO₂) extraction utilizing contemporary countercurrent chromatography (CCC). In aligning with the prospective utilization of this extract within the agricultural sector, this study also serves as the preliminary report demonstrating the capability of Fucus vesiculosus scCO₂ extract to enhance the activity of plant resistance enzymes. The fractions obtained through CCC were subjected to evaluation for their efficacy in inhibiting the macrospores of Fusarium culmorum. The CCC methodology facilitated the successful separation of fatty acids (reaching up to 82.0 wt.% in a given fraction) and fucosterol (attaining up to 79.4 wt.% in another fraction). All CCC fractions at the concentration of 1.0% were found to inhibit 100% of Fusarium culmorum growth. Moreover, Fucus vesiculosus scCO₂ extract was able to activate plant resistance enzymes (Catalase, Ascorbic Peroxidase, Guaiacol Peroxidase, Phenylalanine Ammonia-Lyase, and Phenylalanine Ammonia-Lyase Activity). 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

Adonis tianschanica is a lesser-known plant species belonging to the genus Adonis that grows in Kazakhstan. The aim of this study was to characterize the composition of the ethanolic, water, and hydroethanolic extracts from the aerial parts of A. tianschanica by HPLC-ESI-QTOF-MS/MS to isolate the major compound isoquercitrin by HSCCC (High-Speed Counter-Current Chromatography) and to determine the cytotoxicity and anti-inflammatory potential of the extracts produced with this plant. Fingerprinting of the analyzed extracts showed the presence of a multitude of metabolites comprising polyphenols, organic acids, and coumarins, and only trace quantities of cardiac glycosides in the analyzed samples. Flavonoids were certainly the best-represented group, with kaempferol, quercetin, and their derivatives as the major components of the extracts. Key findings in this paper were that the ethanol: water (50:50 v/v) extract of A. tianschanica and its major compound isoquercitrin were able to reduce the production of NO induced by LPS, in addition to demonstrating anti-inflammatory effects by reducing cytokines such as IL-6, TNF-α, and IL-1β. Full article

Deep eutectic solvents (DESs) are mixtures of organic compounds displaying excellent solvent properties while keeping an ecofriendly character. In this study, DESs have been applied to the extraction of phenylpropanoid glycosides from Pedicularis oederi Vahl, successively separated by means of counter-current chromatography. Firstly, the ultrasonic-assisted extraction conditions were optimized by response surface methodology, and the results showed phenylpropanoid glycosides could be well extracted under the optimized extraction conditions with deep eutectic solvents. Then, the sample was separated by counter-current chromatography using ethyl acetate/aqueous solution of choline chloride and glycerol (6:6, v/v) as the solvent system. In about 360 min, four phenylpropanoid glycosides, including 31.6 mg of echinacoside, 65.3 mg of Jionoside A1, 28.9 mg of Forsythoside B, 74.1 mg of verbascoside, and 21.2 mg of kaempferol-3-O-rutinoside were obtained from about 900 mg of the sample. It revealed deep eutectic solvents could be well employed as a green solvent for the extraction and counter-current separation of natural products. Full article