Preparative separation of ten flavonoids from Scutellaria baicalensis Georgi roots using two-dimensional countercurrent chromatography with an online-storage, dilution, and mixing interface.

The process of counter-current chromatography (CCC) separation for natural products typically necessitates the use of multiple solvent systems to accommodate constituents with a wide range of polarities. However, the incompatibility between these different solvent systems often results in unsuccessful online 2D successive separations. In this study, a 2D CCC system was developed, featuring an interface for online-storage, dilution, and mixing. It facilitated the implementation of online 2D CCC using different solvent systems. The method was subsequently applied for the preparative isolation of flavonoids from Scutellaria baicalensis Georgi roots. For 1D CCC, n-heptane-ethyl acetate-methanol-water (HepEMWat, 5:5:4:6, v/v) was utilized, while for 2D CCC, ethyl acetate-n-butanol-water (EBuWat, 0:5:5, v/v) was employed. The eluent with low resolution in 1D CCC was stored online, diluted three times using the lower phase of EBuWat (0:5:5, v/v), and subsequently transferred into 2D CCC for further isolation utilizing the same EBuWat (0:5:5, v/v) system. As results, six lipophilic compounds were isolated in 1D CCC in a normal mode, while two major hydrophilic constituents were isolated in a pH-peak-focusing mode in 2D CCC. Additionally, two additional compounds were purified through subsequent semi-preparative HPLC separation in order to resolve co-elution in 2D CCC. The developed 2D CCC system with a multifunctional interface demonstrated to be an exceptionally efficient and promising approach for the high-throughput purification of complex natural products.

Separation and purification of benzylester glucosides and derivatives from tubers of Gymnadenia conopsea (L.) R. Br. by linear gradient counter-current chromatography combined with elution-extrusion mode.

Tubers of Gymnadenia conopsea (L.) R. Br. (Orchidaceae), a traditional medicine and food homologous plant, has a broad application and development prospect in the food and drug industries. Benzylester glucosides, the main effective active components in this plant, are difficult to separate due to their similar structures and high polarity. In this study, linear gradient counter-current chromatography was used to separate benzylester glucosides and derivatives, combined with elution-extrusion mode. The main separation parameters were optimized, including the ratio of mobile phase and sample loading. Finally, seven compounds were successfully separated, including 4-hydroxybenzyl alcohol (1), 4-hydroxybenzaldehyde (2), dactylorhin B (3), loroglossin (4), dactylorhin A (5), 4-(ethoxymethyl) phenol (6), and militarine (7). The structures were analyzed by mass spectrometry and nuclear magnetic resonance spectrometry. According to our findings, the established method was an efficient approach to separate benzylester glucosides and derivatives from tubers of G. conopsea. The established strategy could be applied to purify other similar high-polarity compounds from complex natural products.

Development of temperature-controlled batch and 3-column counter-current protein A system for improved therapeutic purification.

Maximizing product quality attributes by optimizing process parameters and performance attributes is a crucial aspect of bioprocess chromatography process design. Process parameters include but are not limited to bed height, eluate cut points, and elution pH. An under-characterized chromatography process parameter for protein A chromatography is process temperature. Here, we present a mechanistic understanding of the effects of temperature on the protein A purification of a monoclonal antibody (mAb) using a commercial chromatography resin for batch and continuous counter-current systems. A self-designed 3D-printed heating jacket controlled the 1 mL chromatography process temperature during the loading, wash, elution, and cleaning-in-place (CIP) steps. Batch loading experiments at 10, 20, and 30 °C demonstrated increased dynamic binding capacity (DBC) with temperature. The experimental data were fit to mechanistic and correlation-based models that predicted the optimal operating conditions over a range of temperatures. These model-based predictions optimized the development of a 3-column temperature-controlled periodic counter-current chromatography (TCPCC) and were validated experimentally. Operating a 3-column TCPCC at 30 °C led to a 47% increase in DBC relative to 20 °C batch chromatography. The DBC increase resulted in a two-fold increase in productivity relative to 20 °C batch. Increasing the number of columns to the TCPCC to optimize for increasing feed concentration resulted in further improvements to productivity. The feed-optimized TCPCC showed a respective two, three, and four-fold increase in productivity at feed concentrations of 1, 5, and 15 mg/mL mAb, respectively. The derived and experimentally validated temperature-dependent models offer a valuable tool for optimizing both batch and continuous chromatography systems under various operating conditions.

Rapid purification and scale-up of tilianin using counter-current chromatography with rectangular horizontal tubing.

In counter-current chromatography (CCC), linear scale-up is an ideal amplification strategy. However, when transferring from analytical to predictable preparative processes with high throughput, linear scale-up would be challenging due to limitations imposed by differences in instrument parameters, such as gravitational forces, tubing cross-section area, tubing length, column volume and flow rate. Some effective scale-up strategies have been studied for different instrument parameters, but so far, these scale-up works have only been tested on standard circular (SC) tubing. The previous research of our group found that rectangular horizontal (RH) tubing can double the separation efficiency compared with conventional SC tubing, and has industrial production potential. This paper used the separation of tilianin from Dracocephalum moldavica L. as an example to demonstrate how to scale up the optimized process from analytical SC tubing to preparative RH tubing. After systematic optimization of solvent systems, sample concentration and flow rate on the analytical CCC, the optimized parameters obtained were successfully transferred to the preparative CCC. The results showed that a crude sample of 2.07 g was successfully separated using a solvent system of n-hexane - ethyl acetate - ethanol - water (1:4:1:5, v/v/v/v) in reversed phase mode, and the three consecutive separations produced a total of 380 mg tilianin in 75 min with high purities of 98.3%, as analyzed by HPLC. The total throughput achieved from the analytical to semi-preparative scale was improved by 138 times (from 12 mg/h to 1.66 g/h), while the column volume was increased by only 46.5 times (from 15.5 mL to 720 mL). This is the successful application of CCC for the separation and purification of tilianin. Given that SC tubing is the traditional configuration for CCC columns, this study is a necessary step to prove the applicability of RH tubing columns for routine use and potential large-scale industrial applications.

"Sweet space" strategy for solvent system selection in countercurrent chromatography: A case study on separation of polyunsaturated fatty acids from borage oil.

This study presents a comprehensive strategy for the selection and optimization of solvent systems in countercurrent chromatography (CCC) for the effective separation of compounds. With a focus on traditional organic solvent systems, the research introduces a "sweet space" strategy that merges intuitive understanding with mathematical accuracy, addressing the significant challenges in solvent system selection, a critical bottleneck in the widespread application of CCC. By employing a combination of volume ratios and graphical representations, including both regular and trirectangular tetrahedron models, the proposed approach facilitates a more inclusive and user-friendly strategy for solvent system selection. This study demonstrates the potential of the proposed strategy through the successful separation of gamma-linolenic acid, oleic acid, and linoleic acid from borage oil, highlighting the strategy's effectiveness and practical applicability in CCC separations.

Elution behavior of drugs in high-speed counter-current chromatography using on-column complexation with metal ions.

In this study, determination of (nitrogen containing) drugs by on-column complexation with metal ions in high-speed counter-current chromatography (HSCCC) was investigated. Bromazepam (BMP) was strongly retained in the organic upper stationary phase (UP) of the two-phase solvent system composed of tert-butyl methyl ether-acetonitrile-water (2:2:3, v/v/v) by eluting the aqueous lower mobile phase (LP) at a flow rate of 2 mL min-1. On the other hand, BMP (200 µg mL-1) was eluted faster without retention to the organic UP with the two-phase system containing 100 µg mL-1 of copper ions (CuCl2) because a very polar BMP-Cu2+ complex was immediately formed in the aqueous LP. The dramatic change in the retention behavior of BMP resulted from on-column complexation. The on-column complexation in HSCCC was further investigated for five (nitrogen containing) drugs and seven metal ions. In the result, tizanidine and phentolamine formed complexes with Al3+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+, ambroxol formed complexes with Al3+, Fe2+, and Cu2+, but voriconazole formed no complexes with all metal ions tested.

An efficient method for the preparative isolation and purification of alkaloids from Gelsemium by using high speed counter-current chromatography and preparative HPLC.

We established an efficient method using high-speed countercurrent chromatography (HSCCC) combined with preparative high-performance liquid chromatography (prep-HPLC) for isolating and purifying Gelsemium elegans (G. elegans) alkaloids. First, the two-phase solvent system composed of 1% triethylamine aqueous solution/n-hexane/ethyl acetate/ethanol (volume ratio 4:2:3:2) was employed to separate the crude extract (350 mg) using HSCCC. Subsequently, the mixture that resulted from HSCCC was further separated by Prep-HPLC, resulting in seven pure compounds including: 14-hydroxygelsenicine (1, 12.1 mg), sempervirine (2, 20.8 mg), 19-(R)-hydroxydihydrogelelsevirine (3, 10.1 mg), koumine (4, 50.5 mg), gelsemine (5, 32.2 mg), gelselvirine (6, 50.5 mg), and 11-hydroxyhumanmantenine (7, 12.5 mg). The purity of these seven compounds were 97.4, 98.9, 98.5, 99, 99.5, 96.8, and 85.5%, as determined by HPLC. The chemical structures of the seven compounds were analyzed and confirmed by electrospray ionization mass spectrometry (ESI-MS), 1H-nuclear magnetic resonance (1H NMR), and 13 C-nuclear magnetic resonance (13 C NMR) spectra. The results indicate that the HSCCC-prep-HPLC method can effectively separate the major alkaloids from the purified G. elegans, holding promising prospects for potential applications in the separation and identification of other traditional Chinese medicines.

Integrated analysis and separation of 5-lipoxygenase inhibitors from triterpenes of Poria cocos using bioaffinity ultrafiltration UPLC-Q-Exactive, molecular docking and target-based multiple complex networks.

Poria cocos (Schw.) Wolf (P. cocos) has been widely used as medical plant in East Asia with remarkable anti-Alzheimer's disease (anti-AD) activity. However, the underlying mechanisms are still confused. In this study, based on the ß-Amyloid deposition hypothesis of AD, an integrated analysis was conducted to screen and separation 5-lipoxygenase (5-LOX) inhibitors from triterpenoids of P. cocos and investigate the anti-AD mechanisms, containing bioaffinity ultrafiltration UPLC-Q-Exactive, molecular docking, and multiple complex networks. Five triterpenoids were identified as potential 5-LOX inhibitors, including Tumulosic acid, Polyporenic acid C, 3-Epi-dehydrotumulosic acid, Pachymic acid and Dehydrotrametenolic acid. Five potential 5-LOX inhibitors were screened by ultrafiltration affinity assay in P. cocos. The molecular docking simulation results are consistent with the ultrafiltration experimental results, which further verifies the accuracy of the experiment. The commercial 5-LOX inhibitor that Zileuton was used as a positive control to evaluate the inhibitory effect of active ingredients on 5-LOX. Subsequently, the established separation method allowed the five active ingredients (Pachymic acid, 3-Epi-dehydrotumulosic acid, Dehydrotrametenolic acid, Tumulosic acid and Polyporenic acid C) with high purity to be isolated. Targeting network pharmacology analysis showed that five active ingredients correspond to a total of 286 targets. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis found that target cells were mainly enriched in Pathways in cancer, Lipid and atherosclerosis. Our results indicate that P. cocos extract has the potential to be used in the prevention and treatment of neurodegenerative diseases. This will help elucidate the mechanisms of action of various medicinal plants at the molecular level and provide more opportunities for the discovery and development of new potential treatments from health food resources.

Efficient discovery of active isolates from Dioscorea spongiosa by the combination of bioassay-guided macroporous resin column chromatography and high-speed counter-current chromatography.

In the present study, twelve compounds from Dioscorea spongiosa were successfully purified by an efficient technique combined bioassay-guided fractionation macroporous resin column chromatography (MRCC) pretreatment and high-speed counter-current chromatography (HSCCC) separation for the first time. Then, D101 MRCC was used to fractionate the crude extract into five parts, which further applied the bioassay-guided fractionation strategy to screen the active fractions of 2 and 4. As for the separation, 200 mg Fr.2 was purified by HSCCC using EtOAc/n-BuOH/H2 O (2:2:3, v/v), leading to annulatomarin (1), dioscoresides C (2), diosniponol C (3), methyl protodioscin (4), pseudoprotodioscin (5), protogracillin (6), as well as 200 mg Fr.4 yielding montroumarin (7), dioscorone A (8), diosniponol D (9), protodioscin (10), gracillin (11), and dioscin (12) using CH2 Cl2 /MeOH/H2 O (3:3:2, v/v) with the purities over 95.0%. Finally, the isolates were assayed for their anti-inflammatory, urico-lowering, and anti-diabetic activities in vitro, which indicated that the steroidal saponins of 5, 6, and 11 showed all these three activities.

Comparison of two different multiple dual-mode counter-current chromatograph for separation of ketoconazole enantiomers.

In this work, two different multiple dual-mode (MDM) counter-current chromatography methods, conventional MDM and modified MDM elution modes, were compared for the chiral separation of the ketoconazole enantiomers. The biphasic solvent system which consisted of n-hexane: isobutyl acetate: 0.1 mol/L phosphate buffer (2:4:6, v/v) (pH = 8.5) was employed as stationary phase and mobile phase. And the hydroxypropyl-ß-cyclodextrin (HP-ß-CD) with a concentration of 100 mmol/L was dissolved in the phosphate buffer, as the chiral selector. Under two different methods, dual-mode (DM) elution was performed to determine the time of the transformed phase roles and multiple cycles were performed to isolate ketoconazole, respectively. The result indicated that the modified MDM elution had a significant improvement on the separation, increasing the resolution from 0.51 to 1.19, while the resolution was increased from 0.40 to 0.79 by the conventional MDM elution. Ultimately, baseline separation of ketoconazole enantiomers was essentially achieved by high-speed counter-current chromatography under optimized modified MDM separation conditions. The final recoveries of the two enantiomers, R-(K) and S-(K), were 92.5 % and 83.3 %, respectively, corresponding to enantiomeric excess values of 99.0 % and 97.0 %, as determined by HPLC.

A new combined technology: Macroporous adsorption resin and high speed counter current chromatography for hydroxytyrosol separation from olive leaf enzymatic hydrolysate.

Due to three free hydroxyl groups, hydroxytyrosol (HT) presents strong bioactivity and has broad food and drug application prospects. However, there is no good separation and purification technology. In this study, separation and purification technology of HT from the ethyl acetate extraction of enzymatic hydrolysate from olive leaf (EEEH) was investigated with macroporous adsorption resin (MAR) and high-speed counter-current chromatography (HSCCC) and the separation factors were optimized. First, the adsorption properties of eight MARs (AB-8, S-8, D-101, X-5, XAD-1, XAD-5, NKA-Ⅱ, H-103) for HT enrichment were studied. The results showed that H-103 macroporous resin was adsorbent, sample concentration was 1.5 mg/mL, eluent was 30 % ethanol solution, sample loading rate was 3.0 BV/h, elution velocity was 2.0 BV/h, and HT purity of EEEH was increased from 10.23 % to 40.78 %. Then, solvent systems were examined according to partition coefficients of target component and petroleum ether: ethyl acetate: methanol: water (4:6:4:6, v/v) system was chosen. The critical experimental parameters of HSCCC were optimized as following: revolution speed was 1200 rpm and flow rate was 3 mL/min. The HT purity of macroporous resin purified EEEH was increased from 40.78 % to 85.7 %. Therefore, MAR-HSCCC combined technology could be a very effective approach to separate and purify HT from EEEH.

Target-guided isolation and purification of cyclooxygenase-2 inhibitors from Meconopsis integrifolia (Maxim.) Franch. by high-speed counter-current chromatography combined with ultrafiltration liquid chromatography.

Meconopsis integrifolia (Maxim.) Franch. is used extensively in traditional Tibetan medicine for its potent anti-inflammatory properties. In this study, six cyclooxygenase-2 (COX-2) inhibitors were purified from M. integrifolia using high-speed counter-current chromatography guided by ultrafiltration liquid chromatography (ultrafiltration-LC). First, ultrafiltration-LC was performed to profile the COX-2 inhibitors in M. integrifolia. The reflux extraction conditions were further optimized using response surface methodology, and the results showed that the targeted COX-2 inhibitors could be well enriched under the optimized extraction conditions. Then the six target COX-2 inhibitors were separated by high-speed countercurrent chromatography with a solvent system composed of ethyl acetate/n-butanol/water (4:1:4, v/v/v. Finally, the six COX-2 inhibitors, including 21.2 mg of 8-hydroxyluteolin 7-sophoroside, 29.6 mg of 8-hydroxyluteolin 7-[6'''-acetylallosyl-(1→2)-glucoside], 42.5 mg of Sinocrassoside D3, 54.1 mg of Hypolaetin 7-[6'''-acetylallosyll-(l→2)-3''-acetylglucoside, 30.6 mg of Hypolaetin 7-[6'''-acetylallosyll-(l→2)-6''-acetylglucoside and 17.8 mg of Hypolaetin were obtained from 500 mg of sample. Their structures were elucidated by 1 H-NMR spectroscopy. This study reveals that ultrafiltration-LC combined with high-speed counter-current chromatography is a robust and efficient strategy for target-guided isolation and purification of bioactive molecules. It also enhances the scientific understanding of the anti-inflammatory properties of M. integrifolia but also paves the way for its further medicinal applications.

Evaluation of dynamic control of continuous capture with periodic counter-current chromatography under feedstock variations.

Multi-column periodic counter-current chromatography is a promising technology for continuous antibody capture. However, dynamic changes due to disturbances and drifts pose some potential risks for continuous processes during long-term operation. In this study, a model-based approach was used to describe the changes in breakthrough curves with feedstock variations in target proteins and impurities. The performances of continuous capture of three-column periodic counter-current chromatography under ΔUV dynamic control were systematically evaluated with modeling to assess the risks under different feedstock variations. As the concentration of target protein decreased rapidly, the protein might not breakthrough from the first column, resulting in the failure of ΔUV control. Small reductions in the concentrations of target proteins or impurities would cause protein losses, which could be predicted by the modeling. The combination of target protein and impurity variations showed complicated effects on the process performance of continuous capture. A contour map was proposed to describe the comprehensive impacts under different situations, and nonoperation areas could be identified due to control failure or protein loss. With the model-based approach, after the model parameters are estimated from the breakthrough curves, it can rapidly predict the process stability under dynamic control and assess the risks under feedstock variations or UV signal drifts. In conclusion, the model-based approach is a powerful tool for continuous process evaluation under dynamic changes and would be useful for establishing a new real-time dynamic control strategy.

Modeling of analytical, preparative and industrial scale counter-current chromatography separations.

Analytical, preparative and industrial scale counter-current chromatography (CCC) processes differ in the volumes of the loaded solution of components to be separated and in the design of the equipment. Preliminary mathematical modeling is necessary for selection of the optimal design and operation mode of these CCC separations. This study aims to compare simulations of CCC separations at different scales, using an exact description based on the model of equilibrium cells and a much simpler approximate solution based on the Gaussian distribution. Equations for modeling CCC separations of different scales and examples of simulation these separations are presented. It is shown that the discrepancy between the two simulations increases with an increase in the volume of the loaded solution of the components and a decrease in the number of equilibrium cells of a CCC device. In analytical and preparative separations, which are based on complex centrifugal devices, and relatively small sample volumes are injected, approximate equations can be used to simulate various options of CCC separation. In industrial-scale CCC separations, large volumes of the solution of components may be loaded, and as we have proposed previously, these separations can be based on a cascade of mixer-settler extractors. In this case, a more accurate mathematical description based on the cell model equations should be used for modeling.

UV-based solvent system screening for high-speed counter-current chromatography fractionation of compounds with similar UV absorption from complex samples followed by preparative HPLC purification: Flavonoids from barley seedlings as sample.

This article proposes a solvent system screening strategy for compounds with similar UV absorption in complex samples by UV spectrophotometer. There is no need to calculate the partition coefficient value of each compound, only the partition coefficient of the whole sample. The partition coefficient value should be close to 1 in order to obtain as many high-speed counter-current chromatography fractions as possible. Then, preparative HPLC was used to purify the high-speed counter-current chromatography fractions. Based on the above strategy, seven c-glycosyl flavonoids and an amino acid were successfully obtained from barley seedlings through high-speed counter-current chromatography fractionation with ethyl acetate/n-butanol/water (8:2:10, v:v:v) system followed by preparative HPLC purification. The research shows that high-speed counter-current chromatography could be well developed as a tool for fractionation before purification, and greatly improves the separation efficiency.

Application of one-step inner-recycling counter-current chromatography for the preparative separation and purification of chemical constituents from the rhizome of Bergenia ciliate (haw.) Sternb.

Bergenia ciliata (haw.) Sternb, the renowned pharmaceutical plant in Jammu and Kashmir of Pakistan, is widely applied in treating different illnesses including diabetes, diarrhea, and vomiting. This work employed an efficient one-step inner-recycling counter-current chromatography for preparative separating and purifying compounds with similar partition coefficients from the rhizome of Bergenia ciliate (haw.). Five compounds, including quercetin rhamnodiglucoside (1), quercetin-3-O-rutinoside (2), bergenine (3), kaempferol (4), and palmatic acid (5), were successfully separated using the optimized biphasic solvent system that contained ter-butylmetylether/n-butanol/acetonitrile/water (2:2:1:5, v/v) with the purities over 98%. Mass spectrometry and nuclear magnetic resonance were conducted for structural identification. As a result, our proposed strategy might be applied in separating compounds with similar partition coefficients, which was advantageous with regard to the less solvent and time consumption, and the increased number of theoretical plates.

Integrated continuous downstream process of monoclonal antibody developed by converting the batch platform process based on the process characterization.

A continuous downstream process of monoclonal antibody was developed based on the process characterization. Periodic-counter current chromatography (PCCC) with two protein A columns was used for the capture step. For low pH virus inactivation (VI), a batch reactor was employed, which can work as a surge (buffer) tank. Flow-through chromatography (FTC) with two connected columns of different separation modes (anion-mixed mode and cation exchange) was designed as a polish step. After 24 h PCCC run, the collected pool was processed for VI. After adjusting pH and electric conductivity, the solution was fed to the two connected FTC columns for 24 h. Virus filter was also connected to the exit of the connected-column. PCCC and FTC were run in parallel. Six runs of different feed rates (0.5-10 L/day) and feed concentrations (1-3.2 g/L) were performed with protein A columns of 1-5 mL and FTC columns of 3-10 mL. The largest run (feed rate 10 L/day, feed concentration 2 g/L) was carried out at a GMP facility with 15 mL protein A columns and 100 mL FTC columns. Good recovery and purity values were obtained for all runs. The process was found to be flexible and stable for feed fluctuations. Only three surge or pool tanks were needed in addition to the final product pool tank.

Separation and purification of alkaloids and phenolic acids from Phellodendron chinense by pH-zone refining and online-storage inner-recycling counter-current chromatography.

In this work, eight compounds from Phellodendron chinense were separated and purified by pH-zone refining counter-current chromatography and traditional counter-current chromatography coupled with online-storage inner-recycling counter-current chromatography (IRCCC). The pH-zone-refining mode was adopted for separating 2.0 g of crude extract with the solvent system of chloroform-methanol-water (4:3:3, v/v), in which 10 mM hydrochloric acid and 10 mM triethylamine were added in the stationary and mobile phases, respectively. Meanwhile, traditional counter-current chromatography coupled with online-storage IRCCC separation was performed by the solvent system of n-hexane-ethyl acetate-methanol-water (5:5:2:8, v/v). Finally, eight compounds, including six alkaloids as 6-methylpiperidin-2-one(1), isoplatydesmine(4), berlambine(5), epiberberine(6), palmatine(7), berberine(8) and two phenolic acids as ferulic acid(2), isoferulic acid(3), were successfully obtained using these three different CCC modes with the purities over 95.0%.

A novel and high-efficient method for the preparation of heat-stable antifungal factor from Lysobacter enzymogenes by high-speed counter-current chromatography.

Heat-stable antifungal factor (HSAF) produced by the biocontrol bacterium Lysobacter enzymogenes shows considerable antifungal activity and has broad application potential in the agricultural and medical fields. There is a great demand for pure HSAF compounds in academic or industrial studies. However, an efficient preparation method that produces a high yield and high purity of HSAF is lacking, limiting the development of HSAF as a new drug. In the present study, high-speed counter-current chromatography (HSCCC) combined with column chromatography was successfully developed for the separation and preparation of HSAF from the crude extract of L. enzymogenes OH11. The crude extract was obtained by macroporous resin adsorption and desorption, and the main impurities were partly removed by ultraviolet light (254 nm) and gel filtration (Sephadex LH-20). In the HSCCC procedure, the selected suitable two-phase solvent system (n-hexane/ethyl acetate/methanol/water = 3:5:4:5, v/v, the lower phase added with 0.1% TFA) with a flow rate of 2.0 mL/min and a sample loading size of 100 mg was optimized for the separation. As a result, a total of 42 mg HSAF with a purity of 97.6% and recovery of 91.7% was yielded in one separation. The structure elucidation based on HR-TOF-MS, 1H and 13C NMR, and antifungal activities revealed that the isolated compound was unambiguously identified as HSAF. These results are helpful for separating and producing HSAF at an industrial scale, and they further demonstrate that HSCCC is a useful tool for isolating bioactive constituents from beneficial microorganisms.

Isolation and purification of high polar glycosides from aerial parts of Gynostemma pentaphyllum (Thunb.) Makino by linear gradient counter-current chromatography coupled with inner-recycling mode.

Gynostemma pentaphyllum (Thunb.) Makino represents the popular health food and supplemental product with broad pharmacological activities. The highly polar glycosides, including flavonoids and saponins, are major effective active components that contain diverse sugar positions and quantities, which result in diverse chemical polarities, making it challenging to separate and isolate these components. The present work described the rapid and efficient linear gradient counter-current chromatography to preparatively separate glycosides from aboveground parts of G. pentaphyllum. Besides, the ethyl acetate and n-butanol binary mobile phases were achieved through adjusting associated proportions. Six glycosides, including quercetin-3-O-neohesperidoside (1), kaempferol-3-O-robinobioside (2), kaempferol-3-O-neohesperidoside (3), gypenoside LVI (4), ginsenoside Rb3 (5), and gypenoside XLVI (6), were isolated at the purities greater than 98%. Moreover, electrospray ionization mass spectrometry and nuclear magnetic resonance tandem mass spectrometry were conducted for structural identification. According to our findings, the established linear gradient counter-current chromatography was an efficient approach to separate the highly polar glycosides from aboveground parts of G. pentaphyllum. Our proposed strategy can be used to separate active compounds from other complex natural products.