Search Results (98)

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

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

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

Hericium erinaceus has long been favored for its remarkable nutritional and health-promoting benefits, and erinacine A is the key component responsible for the neuroprotective properties of H. erinaceus. Establishing an efficient method for separating erinacine A from H. erinaceus and screening the erinacine A-enriched strains is crucial to maximizing its benefits. Herein, we first reported that high-speed counter current chromatography (HSCCC) is an effective method for separating high-purity erinacine A. Using a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (4.5:5:4.5:5, v/v/v/v), erinacine A with a purity of over 95% was separated. Then, we evaluated the content and yield of erinacine A in the liquid-fermented mycelia of Hericium germplasms. Both the content and yield of erinacine A varied greatly among the surveyed strains. The significant effect of the strain on the erinacine A content and yield was revealed by an analysis of variance. The highest erinacine A content and yield were observed in the mycelia of a wild strain HeG, reaching 42.16 mg/g and 358.78 mg/L, which is superior to the current highest outcomes achieved using submerged cultivation. The isolation method established and the strains screened in this study can be beneficial for the scaling up of erinacine A extraction and nutraceutical development to industrial levels. Full article

Essential oils (EOs) are vital secondary metabolites in plants. They have garnered substantial attention owing to their distinct flavors and desirable attributes, including potent antioxidant, antibacterial, and antitumor properties. Nevertheless, the active constituents of EOs exhibit intricate chemical structures, and conventional separation techniques are inadequate for purifying the individual chemical components from EOs. High-speed countercurrent chromatography, based on the principles of a hydrodynamic equilibrium system, has emerged as a liquid–liquid chromatographic separation method renowned for its ability to handle substantial single injection volumes and the absence of irreversible adsorption. Consequently, in recent years, this technique has been widely employed in the isolation and refinement of natural products. In this review, a comprehensive analysis is conducted, contrasting the merits and demerits of high-speed countercurrent chromatography with conventional separation methods. The solvent systems, elution modes, commonly employed detectors, and practical applications are reviewed in the context of high-speed countercurrent chromatography for essential oil separation and purification. Furthermore, this review offers a glimpse into the potential prospects of applying this technique, with the intention of serving as a valuable reference for the use of high-speed countercurrent chromatography in the purification of EOs. Full article

Myrica rubra (Lour.) Siebold & Zucc bark is a traditional natural medicine used by the people of the Dong minority in western Hunan in China. In this study, the main compounds in Myrica rubra bark including epigallocatechin gallate, myricetrin, myricetin, taraxerol, myricanol, and 11-O-acetylmyricanol were separated using both silica gel column chromatography and high-speed countercurrent chromatography (HSCCC). Notably, it is the first report of discovering 11-O-acetylmyricanol from Myrica rubra bark. The results of the bioactivity studies suggested that epigallocatechin gallate showed the highest α-glucosidase inhibitory activity, while myricetin exhibited the highest reactive oxygen species (ROS) scavenging ability in zebrafish embryos. Intriguingly, myricanol exhibited strong apoptosis-inducing activity on HepG2 cells, and further studies revealed that myricanol was capable of promoting the cleavage of caspase 3, 8, and 9, then resulting in the apoptosis in HepG2 cells. The findings of the present study have important implications for the separation of the main compounds in Myrica rubra and will provide credence to the ethnomedicinal application of the isolated compounds against cardiovascular disease and cancer. 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

Poria cocos is traditionally used as both food and medicine. Triterpenoids in Poria cocos have a wide range of pharmacological activities, such as diuretic, sedative and tonic properties. In this study, the anti-tumor activities of poricoic acid A (PAA) and poricoic acid B (PAB), purified by high-speed counter-current chromatography, as well as their mechanisms and signaling pathways, were investigated using a HepG2 cell model. After treatment with PAA and PAB on HepG2 cells, the apoptosis was obviously increased (p < 0.05), and the cell cycle arrested in the G2/M phase. Studies showed that PAA and PAB can also inhibit the occurrence and development of tumor cells by stimulating the generation of ROS in tumor cells and inhibiting tumor migration and invasion. Combined Polymerase Chain Reaction and computer simulation of molecular docking were employed to explore the mechanism of tumor proliferation inhibition by PAA and PAB. By interfering with phosphatidylinositol-3-kinase/protein kinase B, Mitogen-activated protein kinases and p53 signaling pathways; and further affecting the expression of downstream caspases; matrix metalloproteinase family, cyclin-dependent kinase -cyclin, Intercellular adhesion molecules-1, Vascular Cell Adhesion Molecule-1 and Cyclooxygenase -2, may be responsible for their anti-tumor activity. Overall, the results suggested that PAA and PAB induced apoptosis, halted the cell cycle, and inhibited tumor migration and invasion through multi-pathway interactions, which may serve as a potential therapeutic agent against cancer. 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

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

Flavonoids are major active small-molecule compounds in bamboo leaves, which can be easily obtained from the bamboo leaves extraction residues (BLER) after the polysaccharides extraction. Six macroporous resins with different properties were screened to prepare and enrich isoorientin (IOR), orientin (OR), vitexin (VI), and isovitexin (IVI) from BLER, and the XAD-7HP resin with the best adsorption and desorption performance was selected for further evaluation. Based on the static adsorption experiments, the experimental results showed that the adsorption isotherm fitted well with the Langmuir isotherm model, and the adsorption process was better explained by the pseudo-second-order kinetic model. After the dynamic trial of resin column chromatography, 20 bed volume (BV) of upload sample and 60% ethanol as eluting solvent was used in a lab scale-up separation, and the results demonstrated that the content of four flavonoids could be increased by 4.5-fold, with recoveries between 72.86 and 88.21%. In addition, chlorogenic acid (CA) with purity of 95.1% was obtained in water-eluted parts during dynamic resin separation and further purified by high-speed countercurrent chromatography (HSCCC). In conclusion, this rapid and efficient method can provide a reference to utilize BLER to produce high-value-added food and pharmaceutical products. Full article

The characteristics of high polarity and susceptibility to oxidation in phenolic glycosides increase the difficulty of their separation from natural products. In the present study, two new phenolic glycosides with similar structures were isolated from Castanopsis chinensis Hance using a combination of multistep CC and high-speed countercurrent chromatography. Preliminary separation of the target fractions was carried out by Sephadex LH-20 chromatography (100–0% EtOH in H₂O). High-speed countercurrent chromatography with an optimized solvent system of N-Hexane/Ethyl acetate/Methanol/Water (1:6:3:4, v/v/v/v) with a satisfactory stationary phase retention and separation factor was used for further separation and purification of the phenolic glycosides. Consequently, two new phenolic glycoside compounds were obtained with purities of 93.0% and 95.7%. 1D-NMR and 2D-NMR spectroscopy, mass spectrometry, and optical rotation were employed to identify their structures, which were assigned as chinensin D and chinensin E. The antioxidant and α-glucosidase inhibitory activities of these two compounds were evaluated using a DPPH antioxidant assay and a α-glucosidase inhibitory assay. Both compounds showed good antioxidant activity with IC₅₀ values of 54.5 ± 0.82 µg/mL and 52.5 ± 0.47 µg/mL. The α-glucosidase inhibitory activity of the compounds was poor. The successful isolation and structure identification of the two new compounds provides materials not only for a systematic isolation method of phenolic glycosides with similar structures, but also for the screening of antioxidants and enzyme inhibitors. Full article

Poria cocos (P. cocos) is a traditional Chinese medicinal product with the same origin as medicine and food. It has diuretic, anti-inflammatory and liver protection properties, and has been widely used in a Chinese medicine in the treatment of Alzheimer’s disease (AD). This study was conducted to explore the activity screening, isolation of acetylcholinesterase inhibitors (AChEIs), and in vitro inhibiting effect of P. cocos. The aim was to develop a new extraction process optimization method based on the Matlab genetic algorithm combined with a traditional orthogonal experiment. Moreover, bio−affinity ultrafiltration combined with molecular docking was used to screen and evaluate the activity of the AChEIs, which were subsequently isolated and purified using high-speed counter−current chromatography (HSCCC) and semi−preparative high-performance liquid chromatography (semi−preparative HPLC). The change in acetylcholinesterase (AChE) activity was tested using an enzymatic reaction kinetics experiment to reflect the inhibitory effect of active compounds on AChE and explore its mechanism of action. Five potential AChEIs were screened via bio−affinity ultrafiltration. Molecular docking results showed that they had good binding affinity for the active site of AChE. Meanwhile, the five active compounds had reversible inhibitory effects on AChE: Polyporenic acid C and Tumulosic acid were non-competitive inhibitors; 3−Epidehydrotumulosic acid was a mixed inhibitor; and Pachymic acid and Dehydrotrametenolic acid were competitive inhibitors. This study provided a basis for the comprehensive utilization of P. cocos and drug development for the treatment of AD. Full article

Obtaining an ideal solvent system for target compounds is still an obstacle to the wide application of high-speed counter-current chromatography (HSCCC). The partition coefficient and retention of the stationary phase are two key parameters for solvent system selection. The retention of the stationary phase of the solvent system is roughly judged by settling time using a test tube, which is subjective and inaccurate. In this study, we demonstrated that high-resolution separation of HSCCC is tightly connected with the retention of the stationary phase. Notably, unlike the in vitro test of settling time, we investigated the retention of the stationary phase of classical biphasic solvent systems by a TBE300C HSCCC apparatus. Our results revealed that settling time is not always inversely proportional to the retention of the stationary phase. The n-hexane–ethylacetate–methanol–water solvent systems showed the highest correlation coefficient of settling time and retention of the stationary phase (r = −0.91, n = 16). N-heptane–n-butanol–acetonitrile–water solvent system showed the lowest correlation coefficient (r = −0.26, n = 7). These results may be helpful for HSCCC solvent system selection and accelerate the application of this technique. Full article