Centrifugal Partition Chromatography Research Articles

Separation of ochratoxins by centrifugal partition chromatography

The present research work was dedicated to developing an efficient method based on liquid-liquid chromatography (centrifugal partition chromatography, CPC) applicable to routine purifications of ochratoxins (OT) from the liquid culture of the strain A. albertensis SZMC 2107. The crude extract contained numerous components in addition to OTA (90.1 %,) and OTB (1.1 %,) according to HPLC examinations. For the separation of OTs by CPC, several tertiary systems based on acetonitrile, acetone, and short-chain alcohols were examined to find the most applicable biphasic system. The hexane/i-propanol/water 35:15:50 system supplemented with 0.1 % acetic acid was found to be the most efficient for use in CPC separation. Using liquid–liquid instrumental separation, the two OTs, namely OTA (2.23 mg) and OTB (0.031 mg), were successfully isolated with 96.3 % and–72.8 % purity, respectively, from 1 L ferment broth. The identities and purities of the purified components were confirmed and the performance parameters of each separation step and the whole procedure were determined. The developed method could be used effectively to purify OTs for analytical or toxicological applications.

Optimizing Chromatographic Separation with Redosing: Effects on Separation Efficiency of a Model System in Centrifugal Partition Chromatography

This study investigates and optimizes chromatographic separation in a Centrifugal Partition Chromatograph. Therefore, a model system is separated in a single-disc rotor. The occurring loss of the stationary phase lowers the separation efficiency over time. We introduced a new mode of operation, called the redosing of the stationary phase, to counteract this hydrodynamic phenomenon. Experiments with redosing at an optimized operating point demonstrate almost constant separation performance over 12 h, reducing solvent consumption by 45% and increasing chromatographic resolution by 37%. The improvement in retention by 69% contributes to this enhancement. Accordingly, reference experiments without redosing were conducted as a benchmark, highlighting the automated mode’s benefits, as mentioned.

Comparative Assessment of Lignan Profiling and Biological Activities of Schisandra henryi Leaf and In Vitro PlantForm Bioreactor-Grown Culture Extracts.

This research's scope encompassed biotechnological, phytochemical, and biological studies of Schisandra henryi, including investigations into its in vitro microshoot culture grown in PlantForm bioreactors (temporary immersion systems, TISs), as well as extracts from leaves of the parent plant, focusing on anti-inflammatory, antioxidant, anticancer, and antimicrobial activities. The phytochemical analysis included the isolation and quantification of 17 compounds from dibenzocyclooctadiene, aryltetralin lignans, and neolignans using centrifugal partition chromatography (CPC), HPLC-DAD, and UHPLC-MS/MS tandem mass spectrometry with triple quadrupole mass filter methods. Higher contents of compounds were found in microshoots extracts (max. 543.99 mg/100 g DW). The major compound was schisantherin B both in the extracts from microshoots and the leaves (390.16 and 361.24 mg/100 g DW, respectively). The results of the anti-inflammatory activity in terms of the inhibition of COX-1, COX-2, sPLA2, and LOX-15 enzymes indicated that PlantForm microshoot extracts showed strong activity against COX-1 and COX-2 (for 177 mg/mL the inhibition percentage was 76% and 66%, respectively). The antioxidant potential assessed using FRAP, CUPRAC, and DPPH assays showed that extracts from microshoot cultures had 5.6, 3.8, and 3.3 times higher power compared to extracts from the leaves of the parent plant, respectively. The total polyphenol content (TPC) was 4.1 times higher in extracts from the in vitro culture compared to the leaves. The antiproliferative activity against T-cell lymphoblast line Jurkat, breast adenocarcinoma cultures (MCF-7), colon adenocarcinoma (HT-29), and cervical adenocarcinoma (HeLa), showed that both extracts have considerable effects on the tested cell lines. The antimicrobial activity tested against strains of Gram-positive and Gram-negative bacteria and fungi showed the highest activity towards H. pylori (MIC and MBC 0.625 mg/mL).

Counteracting Bleeding in Centrifugal Partition Chromatography: Redosing of the Stationary Phase

Centrifugal Partition Chromatography is a type of Liquid-Liquid Chromatography that offers several advantages compared to Liquid Chromatography. Two immiscible liquids are utilized, and one phase has to be immobilized to implement chromatographic separation. This is performed with the help of centrifugal force. As this immobilization is not ideal, the stationary phase continuously leaks out of the apparatus (so-called bleeding). We measured the stationary phase’s loss precisely and implemented a controller to compensate for it during operation. This innovative mode of operation is called redosing of the stationary phase and prolongs the experimental runtime significantly. In a first step, we implemented an open-loop controller, which was capable of counteracting bleeding but could not dial to a given setpoint precisely. Therefore, a closed-loop controller with a moving frame shifting factor was programmed. This controller reached and maintained setpoints with high accuracy. The last experimental step was to check for boundaries of this new degree of freedom. In addition, we highlighted the accompanying hydrodynamics during redosing with the help of Computational Fluid Dynamics. We were able to show the influence of different volumes of the redosed stationary phase on the flow regime.

Optimized Centrifugal Partition Chromatography (CPC) Protocol for Isolation of Urease Inhibitors: Magnoflorine and Berberine from Berberis vulgaris Extracts

In recent years, an increasing interest in phytotherapy has been observed. Parallel to the research on the total extracts of plant material, numerous studies on the activity of single molecules derived from plants are being conducted to address their mechanisms of action and determine active doses and eventual interactions. Despite this phenomenon, the isolation of individual compounds is a bottleneck due to its difficulty and cost. This work presents the results of a careful optimization of magnoflorine and berberine (isoquinoline alkaloids) recovery from a commonly distributed shrub, Berberis vulgaris, growing in Poland and Georgia, using CPC. Both compounds are known for their numerous medicinal properties, which makes the isolation methodology an important area of research. Additionally, CPC has the ability to isolate high-quality compounds in large quantities, which makes it an effective and easy-to-commercialize method. For a successful separation, the biphasic solvent system composed of hexane, butanol, ethanol, and water in a ratio (3:12:4:16 v/v/v/v) was used in the ascending mode, together with the flow rate of 8 mL/min and rotation speed of 1600 rpm. The method was selective for both compounds, and it delivered good results for both root and stem extracts from the plant. The qualitative composition of alkaloids in the studied extracts determined by HPLC-ESI-QTOF-MS/MS confirmed the presence of berberine, magnoflorine, jatrorhizine, and palmatine alkaloids from the group of isoquinolines. The isolates, magnoflorine and berberine, were subjected to the Helicobacter pylori growth inhibition assay and urease inhibition test to assess whether, next to the previously proved anticancer properties, these compounds are characterized by H. pylori inhibition. MGN was found to exhibit inhibitory potential against urease (IC50 = 25 mg/L).

Isolation of Hydrolyzable Tannins from Castanea sativa Using Centrifugal Partition Chromatography.

Castanea sativa wood is a rich source of hydrolyzable tannins, known for their diverse bioactivities. To investigate these bioactive properties further, it is crucial to isolate and characterize hydrophilic compounds effectively. To address this issue, we developed a centrifugal partition chromatography (CPC) method and applied it to an aqueous C. sativa wood extract. We determined the partition coefficients (KD) of the six major compounds using four butanol-/water-based biphasic solvent systems. Initially, we utilized the n-butanol/propanol/water (3:1:4, v/v/v) systems for the first fractionation step. Subsequently, we employed the water/methyl tert-butyl ether/butanol/acetone (8:5:3:4, v/v/v/v) system to fractionate moderately and highly hydrophilic fractions. We calculated the KD values for major compounds of the most hydrophilic fractions using the butanol/ethanol/water (4:1:5, v/v/v) and butanol/isopropanol/water (2:1:3, v/v/v) systems. In total, we isolated 23 compounds through a combination of CPC, size exclusion chromatography, and preparative HPLC. Among these compounds, six have never been previously described. We characterized them by 1D and 2D NMR experiments and high-resolution mass spectroscopy acquisitions.

Spatially and Temporally Resolved Analysis of Bleeding in a Centrifugal Partition Chromatography Rotor

Centrifugal Partition Chromatography (CPC) is a separation technique that utilizes immiscible liquid phases to purify compounds. The selection of solvents in Liquid–Liquid Chromatography offers flexibility and optimization possibilities for specific separation tasks. Understanding the hydrodynamics inside the apparatus is crucial for optimizing a CPC process. The phase retention ratio (Sf) determines the apparatus’s operating point and separation efficiency. However, stationary phase leakage, known as bleeding, complicates the immobilization of this phase. We used a partly transparent single-disc rotor to investigate the time and space dependency of bleeding inside a CPC apparatus, enabling real-time and localized determination of the phase retention ratio. By tracking the retention values over time, we observed the bleeding phenomenon and its progression from the inlet to the rotor outlet. Depending on the phase system used, the CPC was utilizable for a separation task for only 173–500 dimensionless residence times. Systems with a higher stability parameter (as described in the literature) showed a lower bleeding rate and increased stability over time. Accordingly, our results demonstrate the importance of maintaining an optimal ratio of mobile to stationary phase for efficient separation.

Prediction of Bleeding via Simulation of Hydrodynamics in Centrifugal Partition Chromatography

Centrifugal Partition Chromatography (CPC) utilizes a two-phase liquid–liquid system as mobile and stationary phases. During operation, the latter continuously drains out of the rotor, despite it being in fact stationary, leading to decreasing separation efficiency over time, a phenomenon still poorly understood today because neither simulations nor extensive experimental investigations have addressed this so-called bleeding. With the model presented in this study, the underlying hydrodynamics are discussed in detail. This model can simulate bleeding over 60 s and is verified experimentally for different operating points (volumetric flow rates of 5, 12, and 20 mL⋅min−1) of the Centrifugal Partition Chromatograph utilizing an aqueous–organic phase system. We simulated two interconnected chambers at the rotor inlet and analyzed the loss of the stationary phase over time. The results of the simulated second chamber are closely aligned with the experimental validation results. Thus, the prediction of bleeding utilizing the simulation of hydrodynamics was successful. Moreover, we highlighted the benefits of the two-chamber setup modeled in this study compared to single-chamber models.

Isolation of Polyphenols from Aqueous Extract of the Halophyte Salicornia ramosissima.

Polyphenols from residual non-food grade Salicornia ramosissima have health-promoting effects in feed, food, or nutraceutical applications. Therefore, the isolation of polyphenols is of interest from a series of environmentally friendly isolation methods with recyclable solvents. The isolation of polyphenols from non-food grade S. ramosissima was investigated using sequential membrane filtration with and without acid pretreatment, liquid-liquid extraction, resin adsorption, and centrifugal partition chromatography (CPC); analyzed by the Folin-Ciocalteu assay for total polyphenols; and finally analyzed using UPLC-TQMS in negative ion-spray mode for detection of 14 polyphenols. Sequential membrane filtration and acid hydrolysis indicated the polyphenols forming complexes with other compounds, retaining the polyphenols in the retentate fraction of large molecular weight cut-off membrane sizes. Conventional liquid-liquid extraction using sequential ethyl acetate and n-butanol showed most polyphenols were extracted, apart from chlorogenic acids, indicating a low isolation efficiency of higher polarity polyphenols. Analysis of the extract after resin adsorption by Amberlite XAD-4 resin showed high efficiency for separation, with 100% of polyphenols adsorbed to the resin after 13 bed volumes and 96.7% eluted from the resin using ethanol. CPC fractionations were performed to fractionate the concentrated extract after resin adsorption. CPC fractionations of the 14 polyphenols were performed using an organic or aqueous phase as a mobile phase. Depending on the mobile phase, different compounds were isolated in a high concentration. Using these easily scalable methods, it was possible to comprehensively study the polyphenols of interest from S. ramosissima and their isolation mechanics. This study will potentially lead the way for the large-scale isolation of polyphenols from S. ramosissima and other complex halophytes. The compounds of the highest concentration after CPC fractionation were isoquercitrin and hyperoside (155.27 mg/g), chlorogenic acid (85.54 mg/g), cryptochlorogenic acid (101.50 mg/g), and protocatechuic acid (398.67 mg/g), and further isolation using CPC could potentially yield novel polyphenol nutraceuticals.

Large-scale separation of alkaloids from Corydalis decumbens by pH-zone-refining centrifugal partition chromatography and their anticomplement activity.

Centrifugal partition chromatography in the pH-zone-refining mode was successfully applied to the separation of alkaloids from the crude extract of Corydalis decumbens. The experiment was performed with a two-phase solvent system composed of petroleum ether-ethyl acetate-ethanol-water (5:5:3:7, v/v/v/v) where triethylamine (10mM) was added to the stationary phase and hydrochloric acid (10mM) to the mobile phase. From 1.6g of the crude extract, 43mg protopine, 189mg (+)-egenine, and 158mg tetrahydropalmatine were obtained with a purity of 98.2%, 94.6%, and 96.7%, respectively. Tetrahydropalmatine showed an interesting anticomplement effect with CH50 0.11 and AP50 0.25mg/mL, respectively. In a mechanistic study, tetrahydropalmatine interacted with C1, C3, C4, and C5 components in the complement activation cascade.

A modern purification by accelerated solvent extraction and centrifugal partition chromatography and biological evaluation of capsaicin from Capsicum chinense

A special alkaloid compound known as capsaicin, which can only be found in the fruit of the Capsicum plant, was isolated and tested for its anti-inflammatory activity. The purpose of this work is to establish a simple and quick approach for capsaicin purification utilizing centrifugal partition chromatography (CPC) as well as an effective method - accelerated solvent extraction (ASE), for extracting capsaicin from Capsicum chinense. After purification, capsaicin was validated by HPLC-DAD at 281 nm to be > 90% purity. The in vivo anti-inflammatory activity of the isolated capsaicin was also investigated, and the IC50 value of the capsaicin was determined to be 57.61 µg/mL. The current work emphasizes how an ASE and CPC system may combine to extract high-purity capsaicin from Capsicum chinense, which have the anti-inflammatory activity, as we evaluated in the experiment.

Discovery of Anti-Coronavirus Cinnamoyl Triterpenoids Isolated from Hippophae rhamnoides during a Screening of Halophytes from the North Sea and Channel Coasts in Northern France

The limited availability of antiviral therapy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spurred the search for novel antiviral drugs. Here, we investigated the potential antiviral properties of plants adapted to high-salt environments collected in the north of France. Twenty-five crude methanolic extracts obtained from twenty-two plant species were evaluated for their cytotoxicity and antiviral effectiveness against coronaviruses HCoV-229E and SARS-CoV-2. Then, a bioguided fractionation approach was employed. The most active crude methanolic extracts were partitioned into three different sub-extracts. Notably, the dichloromethane sub-extract of the whole plant Hippophae rhamnoides L. demonstrated the highest antiviral activity against both viruses. Its chemical composition was evaluated by ultra-high performance liquid chromatography (UHPLC) coupled with mass spectrometry (MS) and then it was fractionated by centrifugal partition chromatography (CPC). Six cinnamoyl triterpenoid compounds were isolated from the three most active fractions by preparative high-performance liquid chromatography (HPLC) and identified by high resolution MS (HR-MS) and mono- and bi-dimensional nuclear magnetic resonance (NMR). Specifically, these compounds were identified as 2-O-trans-p-coumaroyl-maslinic acid, 3β-hydroxy-2α-trans-p-coumaryloxy-urs-12-en-28-oic acid, 3β-hydroxy-2α-cis-p-coumaryloxy-urs-12-en-28-oic acid, 3-O-trans-caffeoyl oleanolic acid, a mixture of 3-O-trans-caffeoyl oleanolic acid/3-O-cis-caffeoyl oleanolic acid (70/30), and 3-O-trans-p-coumaroyl oleanolic acid. Infection tests demonstrated a dose-dependent inhibition of these triterpenes against HCoV-229E and SARS-CoV-2. Notably, cinnamoyl oleanolic acids displayed activity against both SARS-CoV-2 and HCoV-229E. Our findings suggest that Hippophae rhamnoides could represent a source of potential antiviral agents against coronaviruses.

Phytochemical profiling and evaluation of the anti-aging and anti-inflammatory potential of cultivated Eclipta alba (L.)

Natural products are increasingly studied for pharmaceutical and cosmetic applications. In our continuing search for new natural active ingredients for cosmetic uses, we investigated the phytochemistry and studied the bioactivity of Eclipta alba (L.) cultivated in center-Val de Loire Region (CVLR) in France to develop a new anti-aging active ingredient. This plant is known as the false daisy and is traditionally used in Ayurveda for the treatment of digestive, respiratory, hepatic, and cutaneous disorders and for its activity as a hair growth promoter.To highlight the compounds involved in different bioactivities, a metabolomic study of the plant was undertaken. The extract of the aerial parts, obtained by ultrasound-assisted extraction in ethanol, was tested for antioxidant activity and for its inhibitory effect on various enzymes involved in skin aging. The raw extract was fractionated using liquid/liquid extraction (LLE) and Centrifugal Partition Chromatography (CPC), then fraction activity was evaluated to target the main active compounds. HRMS/MS and NMR were used to identify the compounds contained in the active fractions.Some fractions had showed strong collagenase, cyclooxygenase-2 (COX-2) and xanthine oxidase (XO) inhibition. The two most active fractions contained mostly coumestans (demethylwedelolactone (DWL) and wedelolactone (WL)), and other phenolic compounds present in lower amount, which were identified as carriers of the anti-collagenase, anti-cyclooxygenase 2 and anti-xanthine oxidase activity.Therefore, this study suggested that Eclipta alba grown in CVLR has a phytochemistry in line with the description reported in literature and is highly composed of coumestans, phenolic compounds and saponins. The ethanolic extract has shown anti-inflammatory and antioxidant activity and for the first time an anti-collagenase activity has been demonstrated. These activities have been correlated with the presence of wedelolactone (WL) and demethylwedelolactone (DWL), the two majors’ compounds, and other phenolics compounds. Thus, Eclipta alba (L.) cultivated in CVLR could be used as an interesting antioxidant and anti-aging agent for cosmetical applications.

Emodin-8-O-Glucoside-Isolation and the Screening of the Anticancer Potential against the Nervous System Tumors.

Emodin-8-O-glucoside (E-8-O-G) is a glycosylated derivative of emodin that exhibits numerous biological activities, including immunomodulatory, anti-inflammatory, antioxidant, hepatoprotective, or anticancer activities. However, there are no reports on the activity of E-8-O-G against cancers of the nervous system. Therefore, the aim of the study was to investigate the antiproliferative and cytotoxic effect of E-8-O-G in the SK-N-AS neuroblastoma, T98G human glioblastoma, and C6 mouse glioblastoma cancer cells. As a source of E-8-O-G the methanolic extract from the aerial parts of Reynoutria japonica Houtt. (Polygonaceae) was used. Thanks to the application of centrifugal partition chromatography (CPC) operated in the descending mode using a mixture of petroleum ether:ethyl acetate:methanol:water (4:5:4:5 v/v/v/v) and a subsequent purification with preparative HPLC, E-8-O-G was obtained in high purity in a sufficient quantity for the bioactivity tests. Assessment of the cancer cell viability and proliferation were performed with the MTT (3-(bromide 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium), CTG (CellTiter-Glo®) and BrdU (5-bromo-2'-deoxyuridine) assays, respectively. E-8-O-G inhibits the viability and proliferation of SK-N-AS neuroblastoma, T98G human glioblastoma multiforme, and C6 mouse glioblastoma cells dose-dependently. E-8-O-G seems to be a promising natural antitumor compound in the therapy of nervous system tumors.

Chemical profiling of botanical extracts obtained in NADES systems using centrifugal partition chromatography combined with 13 C NMR dereplication-Hypericum perforatum as a case study.

Natural deep eutectic solvents (NADES) have emerged as interesting extractants to develop botanical ingredients. They are nontoxic and biodegradable, nonflammable, easy to prepare, and able to solubilize a wide range of molecules. However, NADES extracts remain difficult to analyze because the metabolites of interest stay highly diluted in the nonvolatile viscous NADES matrix. This study presents a robust analytical workflow for the chemical profiling of NADES extracts. It is applied to Hypericum perforatum aerial parts extracted with the neutral mixture fructose/glycerol/water (3/1/1, w/w/w), and compared to the chemical profiling of a classical dry methanol extract. Exploiting polarity differences between metabolites, the H. perforatum NADES extract was partitioned in a liquid-liquid solvent system to trap the hydrophilic NADES constituents in the lower phase. The upper phase, containing a diversity of secondary metabolites from H. perforatum, was fractionated by centrifugal partition chromatography. All fractions were chemically investigated using a 13 C NMR dereplication method which involves hierarchical clustering analysis of the whole NMR dataset, a natural metabolite database for metabolite identification, and 2D NMR analyses for validation. Liquid chromatography-mass spectrometry (LC-MS) analyses were also performed to complete the identification process. A range of 21 metabolites were unambiguously identified, including glycosylated flavonols, lactones, catechins, phenolic acids, lipids, and simple sugars, and 15 additional minor extract constituents were annotated by LC-MS based on exact mass measurements. The proposed identification process is rapid and nondestructive and provides good prospects to deeply characterize botanical extracts obtained in nonvolatile and viscous NADES systems.

Investigation of alternative two-phase solvent systems for purification of natural products by centrifugal partition chromatography.

Centrifugal partition chromatography (CPC) is a liquid-liquid chromatography characterised by its solvent flexibility. The compounds undergoing separation are subjected to a continuous partition process between two immiscible phases in a column space free of solid support. In the context of green chemistry, it is important to substitute halogenated and petroleum-based solvents commonly used in purification processes. The main goal of the current study was to replace classical solvents used in CPC (e.g., hexane and methanol) by green and renewable alternatives. Solvents were first selected based on literature. Their commercial availability, price, recyclability, toxicity and ability to form two phases were particularly sought after. The new two-phase solvent systems were evaluated for the purification of two compounds of interest: piperine and cannabidiol. Using these alternative two-phase solvent systems allows us to isolate natural products with a high purity level (> 95%). Substituting petroleum-based solvents with bio-sourced, renewable alternatives reduces the environmental impact of CPC. Herein, new biphasic solvent systems were built using hexamethyldisiloxane, ethyl isobutyrate and 2-methyl tetrahydrofuran in combination with ethanol and water. Furthermore, this research provides a scientific basis for developing new and sustainable solvent systems in CPC.

Exploration of the main active metabolites from Tinospora crispa (L.) Hook. f. & Thomson stem as insulin sensitizer in L6.C11 skeletal muscle cell by integrating in vitro, metabolomics, and molecular docking

Ethnopharmacological relevanceTinospora crispa (L.) Hook. f. & Thomson stem (TCS) has long been used as folk medicine for the treatment of diabetes mellitus. Previous study revealed that TCS possesses multi-ingredients and multi-targets characteristic potential as insulin sensitizer activity. However, its mechanisms of action and molecular targets are still obscure. Aim of the studyIn the present study, we investigated the effects of TCS against insulin resistance in muscle cells through integrating in vitro experiment and identifying its active biomarker using metabolomics and in molecular docking validation. Materials and methodsWe used centrifugal partition chromatography (CPC) to isolate 33 fractions from methanolic extract of TCS, and then used UHPLC-Orbitrap-HRMS to identify the detectable metabolites in each fraction. We assessed the insulin sensitization activity of each fraction using enzyme-linked immunosorbent assay (ELISA), and then used confocal immunocytochemistry microscopy to measure the translocation of glucose transporter 4 (GLUT4) to the cell membrane. The identified active metabolites were further simulated for its molecular docking interaction using Autodock Tools. ResultsThe polar fractions of TCS significantly increased insulin sensitivity, as measured by the inhibition of phosphorylated insulin receptor substrate-1 (pIRS1) at serine-312 residue (ser312) also the increasing number of translocated GLUT4 and glycogen content. We identified 58 metabolites of TCS, including glycosides, flavonoids, alkaloids, coumarins, and nucleotides groups. The metabolomics and molecular docking simulations showed the presence of minor metabolites consisting of tinoscorside D, higenamine, and tinoscorside A as the active compounds. ConclusionsOur findings suggest that TCS is a promising new treatment for insulin resistance and the identification of the active metabolites in TCS could lead to the development of new drugs therapies for diabetes that target these pathways.

Evaluation of ethanol-based aqueous two-phase systems performance for application in centrifugal partition chromatography (CPC)

Aqueous two-phase systems (ATPS) have recently been proposed for Centrifugal Partition Chromatography (CPC). This type of biphasic system has several advantages over conventional aqueous-organic liquid–liquid systems, namely, high biocompatibility owing to its large water content. The most commonly used ATPS are polymer-based systems. However, owing to their poor phase separation hydrodynamics compared to aqueous-organic systems, the use of short-length water-miscible alcohols has been evaluated as an alternative to polymers. In this study, an ATPS composed of ethanol and dipotassium hydrogen phosphate (K2HPO4) is proposed to overcome the common hydrodynamic drawbacks of polymer-based ATPS. The Ethanol/K2HPO4 system exhibited a very short settling time (ts = 30.85 ± 0.80 s), high interfacial tension (IFT = 6.38 mN/m), significant density differences between the phases (Δρ = 353.40 kg/m3), and low viscosity of the coexisting phases at 25 °C (maximum µ = 3.79 ± 0.11 mPa.s), making it an ideal choice for implementation in CPC apparatus. A comprehensive study of this biphasic system was conducted using CPC equipment by carefully adjusting the operating parameters, specifically, the mobile phase flow rate (F), rotational speed (ω), and mode of operation, that is, ascending (upper phase is mobile) or descending (lower phase is mobile). In this particular CPC equipment, the use of polymers as phase-forming compounds in ATPS resulted in higher CPC pump pressures compared to ethanol or phosphate salt aqueous solutions, thus confirming the superior performance of the ethanol/salt ATPS. The results obtained using this ATPS demonstrated that high F and ω values lead to lower stationary phase retention (SF) and higher backpressures, respectively. SF remained relatively constant and above 50% for F ≤ 6 mL/min but significantly decreased for F > 6 mL/min. This trend confirms that hydrostatically stable conditions can be achieved by employing the Ethanol/K2HPO4 system in the CPC mode by controlling F (i.e., the hydrodynamics of the mobile phase). The partition of the model solutes gallic acid and L-tyrosine was also evaluated using the ethanol/K2HPO4 system. The critical operating CPC parameters were compared, confirming the significant potential of the ethanol/salt ATPS for successful commercial application of this chromatographic technique in the separation of small value-added biomolecules.

Rapid Chemical Profiling of Filipendula ulmaria Using CPC Fractionation, 2-D Mapping of 13C NMR Data, and High-Resolution LC-MS.

Filipendula ulmaria, commonly known as meadowsweet, is a wild herbaceous flowering plant that is widely distributed in Europe. A range of salicylic acid derivatives and flavonol glycosides have been previously associated with the antirheumatic and diuretic properties of F. ulmaria. In the present work, a hydroalcoholic extract from F. ulmaria aerial parts was extensively profiled using an efficient NMR-based dereplication strategy. The approach involves the fractionation of the crude extract by centrifugal partition chromatography (CPC), 13C NMR analysis of the fractions, 2D-cluster mapping of the entire NMR dataset, and, finally, structure elucidation using a natural metabolite database, validated by 2D NMR data interpretation and liquid chromatography coupled with mass spectrometry. The chemodiversity of the aerial parts was extensive, with 28 compounds unambiguously identified, spanning various biosynthetic classes. The F. ulmaria extract and CPC fractions were screened for their potential to enhance skin epidermal barrier function and skin renewal properties using in vitro assays performed on Normal Human Epidermal Keratinocytes. Fractions containing quercetin, kaempferol glycosides, ursolic acid, pomolic acid, naringenin, β-sitosterol, and Tellimagrandins I and II were found to upregulate genes related to skin barrier function, epidermal renewal, and stress responses. This research is significant as it could provide a natural solution for improving hydration and skin renewal properties.

Isolation of Alkaloids from Sinomenium acutum by Centrifugal Partition Chromatography and Their Ameliorating Effects on Dexamethasone-Induced Atrophy in C2C12 Myotubes

Bioactivity-guided isolation was conducted using centrifugal partition chromatography (CPC) from an extract of Sinomenium acutum rhizome, which has shown promising preventive effects in a dexamethasone-induced C2C12 myotube atrophy model. CPC was operated with a solvent system of n-butanol–acetonitrile–water (10:2:8, v/v/v, containing 0.5% triethylamine) in dual mode (ascending to descending), which provided a high recovery rate (>99%) with a high resolution. Then, the preventive effects of the obtained CPC fractions were examined against dexamethasone-induced atrophy in C2C12 myotubes according to the weight ratios of the obtained fractions. The active fractions were further purified by semi-preparative HPLC that led to obtaining five alkaloids, one lignan glycoside, and one phenylpropanoid glycoside. Among these, at a concentration of 1 nM, sinomenine, magnoflorine, and acutumine could ameliorate dexamethasone-induced myotube atrophy in C2C12 myotubes by 9.3%, 13.8%, and 11.3%, respectively.