"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.

Uncovering the Power of HSCCC: Separation of Diastereomeric and Regioisomeric Styrylpyrones from the Stem Bark of Goniothalamus leiocarpus.

The plant Goniothalamus leiocarpus of the Annonaceae family is used as an alternative medicine in tropical regions. Applying high-speed counter current chromatography (HSCCC), eight new bioactive styrylpyrone isomers, including 6R,7S,8R,2'S-goniolactone B (1), 6S,7S,8S,2'S-goniolactone B (2), 6R,7R,8R,2'S-goniolactone B (3), 6R,7S,8S,2'S-goniolactone C (4), 6R,7S,8R,2'S-goniolactone C (5), 6S,7R,8S,2'S-goniolactone C (6), and two positional isomers, 6R,7R,8R,2'S-goniolactone G (7) and 6S,7R,8R,2'S-goniolactone G (8), were isolated from a chloroform fraction (2.1 g) of G. leiocarpus, which had a prominent spot by TLC analysis. The structures of the new compounds were elucidated by MS, NMR, IR, and UV spectra, and their absolute configurations were determined by Mosher's method, ECD, and X-ray diffraction analysis. The isolates are characteristic components found in plants of the genus Goniothalamus and consist of two structural moieties: a styrylpyrone and a dihydroflavone unit. The isolation of the eight new compounds demonstrates the effectiveness of HSCCC in separating the isomers of natural styrylpyrone. In a bioactivity assessment, compounds 1 and 6 exhibited cytotoxic effects against the human colon carcinoma cell lines LS513 and SW620 with IC50 values ranging from 1.6 to 3.9 µM. Compounds 1, 2, 7, and 8 showed significant synergistic activity against antibiotic-resistant Staphylococcus aureus strains.

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.

A novel strategy by combining foam fractionation with high-speed countercurrent chromatography for the rapid and efficient isolation of antioxidants and cytostatics from Camellia oleifera cake.

Camellia oleifera cake is a by-product, which is rich in functional chemical components. However, it is typically used as animal feed with no commercial value. The purpose of this study was to isolate and identify compounds from Camellia oleifera cake using a combination of foam fractionation and high-speed countercurrent chromatography (HSCCC) and to investigate their biological activities. Foam fractionation with enhanced drainage through a hollow regular decahedron (HRD) was first established for simultaneously enriching flavonoid glycosides and saponins for further separation of target compounds. Under suitable operating conditions, the introduction of HRD resulted in a threefold increase in enrichment ratio with no negative effect on recovery. A novel elution-extrusion countercurrent chromatography (EECCC) coupled with the consecutive injection mode was established for the successful simultaneous isolation of flavonoid glycosides and saponins. As a result, 38.7 mg of kaemferol-3-O-[2-O-D-glucopyranosyl-6-O-α-L-rhamnopyranosyl]-ß-D-glucopyranoside (purity of 98.17%, FI), 70.8 mg of kaemferol-3-O-[2-O-ß-D-xylopyranosyl-6-O-α-L-rhamnopyranosyl]-ß-D-glucopyranoside (purity of 97.52%, FII), and 560 mg of an oleanane-type saponin (purity of 92.32%, FIII) were separated from the sample (900 mg). The present study clearly showed that FI and II were natural antioxidants (IC50 < 35 µg/mL) without hemolytic effect. FIII displayed the effect of inhibiting Hela cell proliferation (IC50 < 30 µg/mL). Further erythrocyte experiments showed that this correlated with the extremely strong hemolytic effect of FIII. Overall, this study offers a potential strategy for efficient and green isolation of natural products, and is beneficial to further expanding the application of by-products (Camellia oleifera cake) in food, cosmetics, and pharmacy.

Efficient and fast screening and separation based on computer-aided screening and complex chromatography methods for lipoxygenase inhibitors from Ganoderma lucidum.

INTRODUCTION: Accurate screening and targeted preparative isolation of active substances from natural medicines have long been technical challenges in natural medicine research. OBJECTIVES: This study outlines a new approach for improving the efficiency of natural product preparation, focusing on the rapid and accurate screening of potential active ingredients in Ganoderma lucidum and efficient preparation of lipoxidase inhibitors, with the aim of providing new ideas for the treatment of Alzheimer's disease with G. lucidum. METHODS: The medicinal plant G. lucidum was selected through ultrafiltration coupled with liquid chromatography and mass spectrometry (UF-LC-MS) and computer-assisted screening for lipoxygenase (LOX) inhibitors. In addition, the inhibitory effect of the active compounds on LOX was studied using enzymatic reaction kinetics, and the underlying mechanism is discussed. Finally, based on the earlier activity screening guidelines, the identified ligands were isolated and purified through complex chromatography (high-speed countercurrent chromatography and semi-preparative high-performance liquid chromatography). RESULTS: Five active ingredients, ganoderic acids A, B, C2, D2, and F, were identified and isolated from G. lucidum. We improved the efficiency and purity of active compound preparation using virtual computer screening and enzyme inhibition assays combined with complex chromatography. CONCLUSION: The innovative methods of UF-LC-MS, computer-aided screening, and complex chromatography provide powerful tools for screening and separating LOX inhibitors from complex matrices and provide a favourable platform for the large-scale production of bioactive substances and nutrients.

[Off-line comprehensive two-dimensional countercurrent chromatography-liquid chromatography separation of Curcuma volatile oil].

The chemical constituents of volatile oils used in traditional Chinese medicine are highly complex. Thus, achieving the complete separation of volatile oils by one-dimensional chromatography is difficult owing to the low peak capacity of the technique. Although comprehensive two-dimensional gas chromatography provides an efficient means for separating volatile oils, it cannot be used to screen bioactive components because of its limitations. Therefore, developing a new method to separate volatile oils based on liquid chromatography is of great significance in efforts to obtain new approaches to screen bioactive components in volatile oil. The objectives of the present study are to establish an efficient method for separating the chemical constituents of Curcuma volatile oil using off-line comprehensive two-dimensional countercurrent chromatography-liquid chromatography (CCC-LC) and to investigate the two-dimensional peak capacity, orthogonality, and spatial coverage of this method. Both CCC and LC conditions were optimized. A biphasic n-hexane-methanol-water solvent system was selected via the colorimetric method, and the lower phase was used as the mobile phase in gradient-elution mode: 0-55 min, n-hexane-methanol-water (5∶2∶3 v/v/v); 55-170 min, n-hexane-methanol-water (5∶3∶2, v/v/v); 170-290 min, n-hexane-methanol-water (5∶4∶1, v/v/v). After gradient elution, elution-extrusion elution mode was applied within 290-375 min. Good resolution was achieved by the CCC separation process. The HPLC separation process was carried out with gradient elution using a mobile phase composed of acetonitrile (A)-water (B): 0-10 min, 50%A-65%A; 10-14 min, 65%A; 14-21 min, 65%A-85%A; 21-25 min, 85%A-95%A; 25-30 min, 95%A-55%A; 30-40 min, 55%A. Curcuma volatile oil was separated under the above optimized two-dimensional separation conditions, and the data obtained were drawn into a two-dimensional contour map using Matlab software. The calculated total peak capacity exceeded 954, which was 10 times more than that of one-dimensional chromatography. High orthogonality (r=0.17) and spatial coverage factor (68.1%) were also obtained. Our research provides a new methodology for separating volatile oils used in traditional Chinese medicine as well as an approach for evaluating the quality of traditional Chinese medicinal herbs using two-dimensional chromatographic fingerprints.

Enantioseparation of two antifungal azole drugs by analytical countercurrent chromatography using sulfobutyl ether-ß-cyclodextrin as chiral selector.

This study reports a successful enantioseparation of two antifungal drugs, Ketoconazole and Voriconazole, using countercurrent chromatography (CCC) with synthesized sulfobutyl ether-ß-cyclodextrin (SBE-ß-CD) as chiral selector. Two biphasic solvent systems composed of dichloromethane: 0.1 mol L-1 of phosphate buffer solution (pH 3.0) (1:1, v/v) and n-hexane: ethyl acetate: 0.1 mol L-1 phosphate buffer solution (pH 3.0) (1.5:0.5:2, v/v/v) were selected. Influence factors were investigated, including degree of substitution of SBE-ß-CD, concentration of SBE-ß-CD, equilibrium temperature, and pH of aqueous phase. Under optimized separation conditions, a large enantioseparation factor of α ≥ 3.26 and a high peak resolution Rs= 1.82, was achieved for enantioseparation of Voriconazole by countercurrent chromatography, and purity of two azole stereoisomers collected from CCC separation reached 98.5%, as determined by HPLC. Molecular docking was employed to investigate the formation of inclusion complex.

Development of a general separation strategy by countercurrent chromatography using sanshools from Zanthoxylum bungeanum oleoresin as a case study.

Three kinds of sanshools were separated from Zanthoxylum bungeanum oleoresin by high-speed countercurrent chromatography. Sanshools are a series of amide compounds extracted from the Zanthoxylum bungeanum. Due to similar structures, polarities, and dissociation constants, it was challenging to select an appropriate solvent system for their complete separation by countercurrent chromatography. To address this challenge, a solvent-system-selection strategy was proposed to identify a relatively suitable solvent system. Additionally, a separation procedure incorporating multi-elution modes selection was established to separate similar compounds in a logical order. Ultimately, a solvent system comprising n-hexane:ethyl acetate:methanol:water in a ratio of 19:1:1:5.67 was selected. Three amide compounds with high purity were obtained through the use of recycling elution mode to improve separation resolution: hydroxy-ε-sanshool (8.4 mg; purity: 90.64%), hydroxy-α-sanshool (326.4 mg; purity: 98.96%), and hydroxy-ß-sanshool (71.8 mg; purity: 98.26%) were obtained from 600 mg sanshool crude extract. The summarized solvent-system-selection strategy and separation procedure incorporating multi-elution modes may instruct countercurrent chromatography users, particularly novices, seeking to separate compounds with highly similar chemical properties.

Continuous protein refolding and purification by two-stage periodic counter-current chromatography.

Matrix-assisted refolding (MAR) has been used as an alternative to conventional dilution-based refolding to improve recovery and reduce specific buffer consumption. Size exclusion chromatography (SEC) has been extensively used for MAR because of its ability to load and refold proteins at high concentrations. However, the SEC-based batch MAR processes have the disadvantages of requiring longer columns for better separation and product dilution due to a high column-to-sample volume ratio. In this work, a modified operational scheme is developed for continuous MAR of L-asparaginase inclusion bodies (IBs) using SEC-based periodic counter-current chromatography (PCC). The volumetric productivity of the modified SEC-PCC process is 6.8-fold higher than the batch SEC process. In addition, the specific buffer consumption decreased by 5-fold compared to the batch process. However, the specific activity of the refolded protein (110-130 IU/mg) was less due to the presence of impurities and additives in the refolding buffer. To address this challenge, a 2-stage process was developed for continuous refolding and purification of IBs using different matrices in sequential PCCs. The performance of the 2-stage process is compared with literature reports on single-stage IMAC-PCC and conventional pulse dilution processes for refolding L-asparaginase IBs. The 2-stage process resulted in a refolded protein with enhanced specific activity (175-190 IU/mg) and a high recovery of 84%. The specific buffer consumption (6.2 mL/mg) was lower than the pulse dilution process and comparable to the single-stage IMAC-PCC. A seamless integration of the two stages would considerably increase the throughput without compromising other parameters. High recovery, throughput, and increased operational flexibility make the 2-stage process an attractive option for protein refolding.

Campomanesia lineatifolia Ruiz & Pavón (Myrtaceae): Isolation of major and minor compounds of phenolic-rich extract by high-speed countercurrent chromatography and anti-inflammatory evaluation.

ETHNOPHARMACOLOGICAL RELEVANCE: Campomanesia lineatifolia Ruiz & Pavón (Myrtaceae), an edible species found in Brazilian Forest, possesses leaves that are traditionally used for the treatment of gastrointestinal disorders in Brazil. Extracts of C. lineatifolia are rich in phenolics and exhibit antioxidant, and gastric antiulcer properties. Furthermore, Campomanesia spp. have been described to possess anti-inflammatory properties, but studies related to chemical constituents of C. lineatifolia are scarce in the literature. AIM OF THE STUDY: This work aims to identify the chemical composition of the phenolic-rich ethanol extract (PEE) from C. lineatifolia leaves and evaluate the anti-inflammatory activity that could be related to its ethnopharmacological use. MATERIALS AND METHODS: The high-speed countercurrent chromatography (HSCCC), using an isocratic and a step gradient elution method, and NMR, HPLC-ESI-QTOF-MS/MS were used to isolate and identify the chemicals of PEE, respectively. Lipopolysaccharide-(LPS)-stimulated THP-1 cells were used to evaluate the anti-inflammatory activities from PEE and the two majority flavonoids isolated by measure TNF-α and NF-κB inhibition assays. RESULTS: Fourteen compounds were isolated from the PEE, further identified by NMR and HPLC-ESI-QTOF-MS/MS, twelve of them are new compounds, and two others are already known for the species. The PEE, quercitrin and myricitrin promoted a concentration-dependent inhibition of TNF-α, and PEE promoted an inhibition of NF-κB pathway. CONCLUSIONS: PEE from C. lineatifolia leaves demonstrated significant anti-inflammatory activity that may be related to the traditional use to treat gastrointestinal disorders.

Biphasic alcoholysis coupled with high-speed countercurrent chromatography for high performance on separating phorbol from Croton tiglium Linn extracts.

Phorbol is a tetracyclic diterpenoid found in Euphorbia tirucalli, Croton tiglium, and Rehmannia glutinosa, and is nuclear of various phorbol esters. The rapid obtaining of phorbol with high purity highly contributes to its application, such as synthesizing phorbol esters with designable side chains and particular therapeutic efficacy. This study introduced a biphasic alcoholysis method for obtaining phorbol from croton oil by using polarity imparity organic solvents in both phases and established a high-speed countercurrent chromatography method for simultaneous separation and purification of phorbol. The optimized operation conditions of biphasic alcoholysis were a reaction time of 91 min, a temperature of 14°C, and a croton oil-methanol ratio of 1:30 (g:ml). The phorbol during the biphasic alcoholysis was 3.2-fold higher in content than that obtained in conventional monophasic alcoholysis. The optimized high-speed countercurrent chromatography method was using the ethyl acetate/n-butyl alcohol/water at 4.7:0.3:5 (v:v:v) with Na2 SO4 at 0.36 g/10 ml as the solvent system, using the mobile phase flow rate of 2 ml/min, the revolution of 800 r/min, under which the retention of the stationary phase was achieved at 72.83%. The crystallized phorbol following high-speed countercurrent chromatography was obtained as high purity of 94%.

Nonlinear liquid-liquid chromatography: Beyond a constant distribution coefficient.

Liquid-liquid chromatography (LLC) is a technique in which the separation of mixture components is achieved due to their different distribution between the two phases of a pre-equilibrated biphasic solvent system. In this work, the LLC operation in the nonlinear range of the distribution isotherm was systematically examined for the first time. The influence of the feed concentration on the elution profiles of a model component (cannabidiol, CBD) was studied in three LLC units of different types and sizes ranging from ∼20 mL to ∼2 L. A series of pulse injections with CBD concentrations varying from 1 to 300 mg/mL was performed with n-hexane/methanol/water 5/4/1 (v/v/v) in descending mode (lower phase as the mobile phase). The elution profiles were simulated using the equilibrium-cell model and an anti-Langmuir-like equation for describing the CBD distribution equilibria. The distribution equilibria equation parameters were fitted to the CBD elution profiles using the peak fitting method. The model was validated and provided good predictions of the CBD elution profiles in the entire concentration range for all three LLC units.

Isolation of plastochromanol-8 from flaxseed oil by countercurrent separation methods.

The naturally occurring antioxidant plastochromanol-8 (PC-8) is a member of the tocochromanol (vitamin E) family which features eight unsaturated isoprene units in the side chain compared to three in the case of γ-tocotrienol. Due to the lack of a commercially available PC-8 standard, we developed a route to gain relevant amounts of highly pure PC-8. Specifically, ∼320 g flaxseed oil was saponified and the bulky PC-8 was enriched by gel permeation chromatography. It followed countercurrent chromatography using the solvent system n-hexane/benzotrifluoride/acetonitrile (20:7:13, v/v/v). The final purification was achieved by centrifugal partition chromatography using the novel solvent system hexamethyldisiloxane/acetonitrile (1:1, v/v). This step provided ∼26 mg PC-8 (>99.5 %, according to HPLC, GC and NMR analysis). Two further, hitherto unknown minor tocochromanols (<1 % of PC-8) were detected and could be identified to be plastochromanol-7 (PC-7) and plastochromanol-9 (PC-9), i.e. tocochromanols with seven and nine unsaturated isoprene units, respectively, in the side chain.

Comprehensive two-dimensional countercurrent chromatography × gas chromatography characterization of Artemisia argyi essential oil.

A comprehensive two-dimensional (2D) countercurrent chromatography (CCC) × gas chromatography (GC) was investigated for characterization of chemical constituents of Artemisia argyi essential oil, and orthogonality for the 2D chromatographic system was evaluated. A solvent system composed of n-hexane/acetonitrile/methanol (2:2:1, v/v/v) was selected for first dimensional separation of Artemisia argyi essential oil. Then all CCC fractions were analyzed by GC, which provided a wealth of information regarding the composition of the essential oil. Visualization of chemical compositions obtained from the comprehensive 2D CCC × GC separation was achieved by creation of a 2D contour plot map. Total peak capacity was evaluated and approximately 1392 peaks were obtained through a comprehensive 2D CCC × GC separation. A high spatial coverage and a low linear correlation coefficient were achieved. Meanwhile, all compounds were identified by GC-MS. The obtained 2D contour plot could be divided into six zones to show the characteristic chemical compositions. Six zones could be divided into different component groups, including monoterpenes, sesquiterpenes, monoterpene alcohols, phenols, aldehydes, ketones and esters, which could be used to identify compounds that have not been reported, and to predict the structure of unknown compounds in Artemisia argyi essential oil and comprehensively characterize fingerprint peak.

Large-scale preparation of five polar polyphenols including three isomers from Phyllanthus emblica Linn. by preparative high-speed counter-current chromatography.

The separation of polar compounds is challenging work due to poor retention and insufficient selectivity. In the present study, an efficient strategy for large-scale preparation of five polar polyphenols including three isomers from Phyllanthus emblica Linn has been established by preparative high-speed counter-current chromatography. Macroporous resin column chromatography was used for the enrichment of the polar polyphenols. However, sugar and other ultra-polar impurities were co-washed out with the targets. Liquid-liquid extraction with ethyl acetate/water (1/1, v/v) solvent system was developed to remove the ultra-polar impurities with a clearance rate of 95%. Finally, the targets were introduced to preparative high-speed counter-current chromatography for separation using ethyl acetate/n-butanol/acetic acid/water (2/7/1/10, v/v/v/v) solvent system. As a result, 191 mg of Mucic acid 1,4-lactone 5-O-gallate, 370 mg of ß-Glucogallin, 301 mg of Gallic acid, 195 mg of Mucic acid 1,4-lactone 3-O-gallate and 176 mg of Mucic acid 1,4-lactone 2-O-gallate with purity higher than 98% were obtained from 1.5 g of sample. Mucic acid 1,4-lactone 3-O-gallate, Mucic acid 1,4-lactone 3-O-gallate, and Mucic acid 1,4-lactone 2-O-gallate are isomers. The results showed that high-speed counter-current chromatography could be well developed for the separation of polar compounds from natural products.

Bioassay-guided separation and identification of the anticancer composition from Curcuma longa L. by the combination strategy of methanol gradient countercurrent chromatography and ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry.

In this study, a combined strategy with methanol gradient countercurrent chromatography and ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry was introduced for the fractionation and identification of active constituents from Curcuma longa L. The gradient countercurrent chromatography separation was performed using the heptane-ethyl acetate-methanol-water (5:5:2:8, v/v) solvent system, in which the lower phase and methanol were used as the mobile phases. Constituents of turmeric with large partition coefficients were well resolved. Subsequent cytotoxicity analysis showed that the fractions 10, 11, 12, and 15 expressed significantly higher cytotoxicity against B16 mouse melanoma than the other fractions. Four compounds with potent activity, curcumin, demethoxycurcumin, and bisdemethoxycurcumin from fraction 11 and galanal A from fraction 15, were purified, and the half-maximal inhibitory concentrations were 18.5 ± 1.3, 7.8 ± 0.4, 20.4 ± 1.3, and 14.1 ± 0.8 µM, respectively. Ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry was then applied for compound identification from fraction 10 with constituents in very low content. A total of 14 diarylheptanoids were identified, which are supposed to be cytotoxic constituents. It proved that the strategy based on the combination of bioassay-guided methanol gradient countercurrent chromatography separation and ultra-high-performance liquid chromatography coupled with tandem mass spectrometry-assisted peak identification could be an efficient method for natural product screening and new drug discovery.

Heparin purification by expanded bed anion exchange in a countercurrent chromatography column.

In this study, an expanded bed anion exchange in a countercurrent chromatography column (EB-CCC) technique for separation and purification of heparin, an anticoagulant and antithrombotic glycosaminoglycan, is reported for the first time. A comparative evaluation of the EB-CCC technique with the conventional fixed bed column chromatography (FBCC) revealed its effectiveness in improving adsorption at high flow rates and reducing separation time. A significantly higher maximum adsorption (91.66%) was exhibited by EB-CCC in comparison with FBCC (45.16%) at the eluent flow rate of 1 mL·min-1. Similarly, the experimental adsorption capacity of heparin was enhanced by 1.69, 2.06 and 2.58 times in the case of EB-CCC at the flow rates of 1, 2 and 5 mL·min-1, respectively. Moreover, the directly proportional amplification of double loaded resin and double column volume was demonstrated at an EB-CCC rotational speed of 300 rpm and a flow rate of 2 mL·min-1, and the experimental adsorption capacity was observed to increase from 66.42 to 136.48 mg·g-1 after amplification. Heparin purified by EB-CCC displayed higher potency (216.09 ± 11.89 IU·mg-1) as compared to FBCC (205.51 ± 7.90 IU·mg-1) and the initial crude heparin 134.17 ± 4.12 IU·mg-1. Furthermore, comparing to the purified heparin by FBCC, heparin purified by EB-CCC had low molecular weight, high FXa/FIIa, superior anticoagulation effect and enhanced suitability as an exogenous anticoagulant.

Large-scale preparation of two minor polar polyphenols from Phyllanthus emblica Linn. by macroporous resin column chromatography and high-temperature preparative high-speed counter-current chromatography.

In the present study, an efficient strategy for the large-scale preparation of two minor polar polyphenols from Phyllanthus emblica L. has been established by macroporous resin column chromatography and high-temperature preparative high-speed counter-current chromatography. Macroporous resin column chromatography was used for the enrichment of the target polyphenols. The target polyphenols could be well enriched in the 10% ethanol fraction and the peak area proportion increased from 2.2% in the crude extract to 85.3% in the 10% ethanol fraction. Then, the 10% ethanol fraction was introduced to preparative high-speed counter-current chromatography for separation using an n-butanol-ethyl acetate-water (17:3:20, v/v/v) solvent system. In order to improve the retention of the stationary phase, a high-temperature preparative high-speed counter-current chromatography had been developed by using a 316 L stainless steel pipe as a column. A 50°C of column temperature was used and the retention rate of the stationary phase could reach 75%. Finally, 871 mg of compound Ⅰ and 534 mg of compound Ⅱ with purities higher than 98% were obtained from 4 g of the sample. The chemical structures were identified as corilagin and 1, 6-di-O-galloyl-ß-D-glucoside by 1 H-NMR and 13 C-NMR.

[Preparation of cucurbitacin compounds in Siraitia grosvenorii roots by high speed countercurrent chromatography].

Siraitia grosvenorii (Swingle) C. Jeffrey, belonging to the family Cucurbitaceae, is a natural sweetener. The roots of this plant are used in folk medicine for the treatment of rheumatoid arthritis. Cucurbitacins play an important role in the resistance of this plant to insects and adversity, and have anti-inflammatory, anti-tumor, and other biological activities. They usually exist as a variety of similar structures in Cucurbitaceae plants. Separation of a large amount of high-purity monomer compounds by the conventional separation method based on column chromatography is difficult, which limits the research and application of their activities. Therefore, we chose a new method for this separation. High-speed countercurrent chromatography (HSCCC) is a liquid-liquid chromatographic technique characterized by high recovery and reproducibility, and is considered a very effective method for the separation of natural compounds present in various plant extracts. An appropriate solvent system is the key for efficient separation, but its selection is tedious, which hampers the wider implementation of HSCCC in chemical research involving preparative separations. In this study, based on the general estimation strategy by using the TLC solvent system (GUESS), the corresponding relationship between the partition distribution coefficient (K value) and the TLC retention factor (Rf value) of the compounds was established by the partition experiment. The Rf value and separation coefficient α were calculated using the water-saturated organic phase as the expansion agent, which could minimize the number of countercurrent separation experiments required in solvent system selection. In this study, HSCCC was used to establish an efficient method for the extraction of cucurbitacins from the root extract of Siraitia grosvenorii. A fraction rich in cucurbitacins was obtained from the ethanol extract of Siraitia grosvenorii roots after separation by column chromatography on HPD-100, MCI, and C18 columns. Six types of solvent systems with different compositions were investigated using the GUESS method. The results showed that employing the solvent system of n-hexane-ethyl acetate-methanol-water (3∶7∶3∶7, v/v/v/v) to partition the cucurbitacin fraction could remove a large number of impurities. The components retained in the upper phase in the partition experiment were subsequently purified by HSCCC. The favorable solvent system for HSCCC was n-hexane-ethyl acetate-methanol-water (4∶6∶5∶5, v/v/v/v), while the upper and lower phases were selected as the stationary and mobile phases, respectively, with a flow rate of 2.0 mL/min, a rotation speed of 860 r/min, and an injected sample weight of 280 mg. Five cucurbitacin compounds were obtained by one-time separation. The weights of the five compounds were 14.73, 8.82, 30.74, 5.03, and 3.81 mg. The purities of these compounds were 97.0%, 95.4%, 96.3%, 91.6%, and 95.3%, respectively. Their structures were identified as cucurbitacin Q1, 23,24-dihydrocucurbitacin F-25-acetate, cucurbitacin B, 23,24-dihydrocucurbitacin B, and dihydroisocucurbitacin B-25-acetate by1H-NMR and 13C-NMR spectroscopies, along with comparison with the literature. This study demonstrated how GUESS guidance accelerates the selection of HSCCC solvent systems, simplifies the workflow, and it provides an efficient preparative method for the separation of chemical constituents from the Siraitia grosvenorii roots, which can also be used as a new method for the large-scale preparation of cucurbitacin compounds.

High-speed countercurrent chromatography as an efficient technique for large separation of plant polyphenols: A review.

Polyphenols, which are commonly found in fruits, vegetables, cereals, and legumes, are the most abundant dietary antioxidants. To date, numerous studies have demonstrated the ability of polyphenols to prevent the development of several diseases and established the corresponding structure-activity relationships. However, polyphenol standards are either not commercially available or very expensive, mainly because the structural complexity and diversity of polyphenols complicate their fractionation and isolation from plant extracts by conventional separation techniques. High-speed countercurrent chromatography (HSCCC) is based on continuous liquid-liquid partitioning, which enables one to eliminate irreversible adsorption on solid supports. This technique has been extensively used for natural product isolation and is well suited to the effective large-scale separation of polyphenols and their derivatives, achieving high purities and yields of up to several hundred milligrams per run within several hours. The present review briefly introduces briefly HSCCC technology and summarizes its applications in the separation and purification of plant polyphenols, including the recent achievements in the large-scale preparation of proanthocyanidins and anthocyanins by our laboratory.