Rapid screening, isolation, and activity evaluation of potential xanthine oxidase inhibitors in Polyporus umbellatus and mechanism of action in the treatment of gout.

INTRODUCTION: Studies show that Polyporus umbellatus has some pharmacological effects in enhancing immunity and against gout. OBJECTIVES: We aimed to establish new techniques for extraction, biological activity screening, and preparation of xanthine oxidase inhibitors (XODIs) from P. umbellatus. METHODS: First, the extraction of P. umbellatus was investigated using the back propagation (BP) neural network genetic algorithm mathematical regression model, and the extraction variables were optimised to maximise P. umbellatus yield. Second, XODIs were rapidly screened using ultrafiltration, and the change of XOD activity was tested by enzymatic reaction kinetics experiment to reflect the inhibitory effect of active compounds on XOD. Meanwhile, the potential anti-gout effects of the obtained active substances were verified using molecular docking, molecular dynamics simulations, and network pharmacology analysis. Finally, with activity screening as guide, a high-speed countercurrent chromatography (HSCCC) method combined with consecutive injection and two-phase solvent system preparation using the UNIFAC mathematical model was successfully developed for separation and purification of XODIs, and the XODIs were identified using MS and NMR. RESULTS: The results verified that polyporusterone A, polyporusterone B, ergosta-4,6,8(14),22-tetraen-3-one, and ergosta-7,22-dien-3-one of P. umbellatus exhibited high biological affinity towards XOD. Their structures have been further identified by NMR, indicating that the method is effective and applicable for rapid screening and identification of XODIs. CONCLUSION: This study provides new ideas for the search for natural XODIs active ingredients, and the study provide valuable support for the further development of functional foods with potential therapeutic benefits.

Target-Guided Isolation and Purification of Antioxidants from Urtica laetevirens Maxim. by HSCCC Combined with Online DPPH-HPLC Analysis.

Urtica laetevirens Maxim. is used extensively in traditional Chinese medicine (TCM) for its potent antioxidative properties. In this study, three antioxidants were purified from U. laetevirens. using HSCCC guided by online DPPH-HPLC analysis. Firstly, the online DPPH-HPLC analysis was performed to profile out the antioxidant active molecules in U. laetevirens. The ultrasonic-assisted extraction conditions were optimized by response surface methodology and the results showed the targeted antioxidant active molecules could be well enriched under the optimized extraction conditions. Then, the antioxidant active molecules were separated by high-speed countercurrent chromatography ethyl acetate/n-butanol/water (2:3:5, v/v/v) as the solvent system. Finally, the three targets including 16.8 mg of Isovitexin, 9.8 mg of Isoorientin, and 26.7 mg of Apigenin-6,8-di-C-ß-d-glucopyranoside were obtained from 100 mg of sample. Their structures were identified by 1H NMR spectroscopy.

Separation of Flavonoids and Purification of Chlorogenic Acid from Bamboo Leaves Extraction Residues by Combination of Macroporous Resin and High-Speed Counter-Current Chromatography.

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

Bioactivity-Guided Separation of Anti-Cholinesterase Alkaloids from Uncaria rhynchophlly (Miq.) Miq. Ex Havil Based on HSCCC Coupled with Molecular Docking.

As an important source of cholinesterase inhibitors, alkaloids in natural products have high potential value in terms of exerting pharmacological activities. In this study, a strategy for targeted preparation of cholinesterase inhibitors in Uncaria rhynchophlly (Miq.) Miq. ex Havil (UR) by high-speed counter-current chromatography was provided. In the method, a two-phase polar solvent system composed of ethyl acetate/n-butanol/water (1:4:5, v/v/v) was used, which isolated five alkaloids from the UR extract for the first time. All alkaloids were identified by HR-ESI-MS and NMR as 7-epi-javaniside (1), vincosamide (2), strictosamide (3), cadambine (4), and 3α-dihydrocadambine (5). The poorly resolved compounds 2 and 3 were separated by preparative HPLC (prep-HPLC). Among them, compounds 1, 4, and 5 were firstly obtained from UR. The purity of these plant isolates was 98.8%, 98.7%, 99.2%, 95.7%, and 98.5%, respectively. Compounds 1-5 exhibited an inhibitory effect on acetyl-cholinesterase and butyryl-cholinesterase with an IC50 from 1.47 to 23.24 µg/mL and 1.01 to 18.24 µg/mL. Molecular docking and inhibitory activities indicated that compound 1 showed stronger inhibitory activity on acetyl-cholinesterase and butyryl-cholinesterase.

Combined Ultrahigh Pressure Extraction and High-Speed Counter-Current Chromatography for Separation and Purification of Three Glycoside Compounds from Dendrobium officinale Protocorm.

As an alternative to Dendrobium candidum, protocorm-like bodies (PLBs) of Dendrobium candidum are of great value due to their high yield and low cost. In this work, three glycoside compounds, ß-D-glucopyranose 1-[(E)-3-(4-hydroxyphenyl)-2-propenoat] (I), ß-D-glucopyranose 1-[(E)-3-(3, 4-dihydroxyphenyl)-2-propenoat] (II), and 1-O-sinapoyl glucopyranoside (III), were extracted and isolated by ultrahigh pressure extraction (UPE) coupled with high-speed counter-current chromatography (HSCCC) from PLBs of D. officinale. First, the target compounds were optimized and prepared with 50% ethanol solution at a 1:30 (g/mL) solid/liquid ratio in 2 min under 300 MPa by UPE. Then, the crude extract was chromatographed with a silica gel column, and primary separation products were obtained. In addition, the products (150 mg) were separated by HSCCC under the solvent system of MTBE-n-butyl alcohol-acetonitrile-water (5:1:2:6, v/v/v/v), yielding 31.43 mg of compound I, 10.21 mg of compound II, and 24.75 mg of compound III. Their structures were further identified by ESI-MS, 1H NMR, and 13C NMR. The antioxidant results showed that the three compounds expressed moderate effects on the DPPH· scavenging effect. Compound II had the best antioxidant capacity and its IC50 value was 0.0497 mg/mL.

Efficient Preparation of Bafilomycin A1 from Marine Streptomyces lohii Fermentation Using Three-Phase Extraction and High-Speed Counter-Current Chromatography.

An efficient strategy was developed for the rapid separation and enrichment of bafilomycin A1 (baf A1) from a crude extract of the marine microorganism Streptomyces lohii fermentation. This strategy comprises liquid-liquid extraction (LLE) with a three-phase solvent system (n-hexane-ethyl acetate-acetonitrile-water = 7:3:5:5, v/v/v/v) followed by separation using high-speed counter-current chromatography (HSCCC). The results showed that a 480.2-mg fraction of baf A1-enriched extract in the middle phase of the three-phase solvent system was prepared from 4.9 g of crude extract after two consecutive one-step operations. Over 99% of soybean oil, the main hydrophobic waste in the crude extract, and the majority of hydrophilic impurities were distributed in the upper and lower phase, respectively. HSCCC was used with a two-phase solvent system composed of n-hexane-acetonitrile-water (15:8:12, v/v/v) to isolate and purify baf A1 from the middle phase fraction, which yielded 77.4 mg of baf A1 with > 95% purity within 90 min. The overall recovery of baf A1 in the process was determined to be 95.7%. The use of a three-phase solvent system represents a novel strategy for the simultaneous removal of hydrophobic oil and hydrophilic impurities from a microbial fermentation extract.

Quality Control of Psoralea corylifolia L. Based on High-Speed Countercurrent Chromatographic Fingerprinting.

Traditional Chinese medicine (TCM)has played an important role in promoting the health of Chinese people. The TCM Psoralea corylifolia L. has been used in the treatment of various kinds of diseases including enuresis, vitiligo, and calvities. However, therapeutic effects of P. corylifolia L. have often influenced by the quality of plants. So, it is very important to control the quality of P. corylifolia L. In this study, analytical high-speed countercurrent chromatography (HSCCC) was successfully used to fingerprint P. corylifolia L. Samples of P. corylifolia L. were extracted by ultrasonic extraction. n-hexane-ethyl acetate-methanol-water at a ratio of 5:5.5:6.5:5 (v/v) was selected as a two-phase solvent system and the condition of HSCCC were optimized in order to good separation. And the method of HSCCC was verified (reproducibility, precision, and stability). HSCCC chromatograms exhibited six common peaks, which were selected as indicator compounds for the quality control of P. corylifolia L. Within 20 types of medicinal materials, chemical components are similar, but the levels of components are quite different in HSCCC fingerprint. The present results demonstrate that the HSCCC method provides a reliable basis for the quality control of P. corylifolia L. and can also be applied to confirm the authenticity of plant materials.

Isolation and Identification of Anthocyanin Component in the Fruits of Acanthopanax Sessiliflorus (Rupr. & Maxim.) Seem. by Means of High Speed Counter Current Chromatography and Evaluation of Its Antioxidant Activity.

Acanthopanax sessiliflorus (Rupr. & Maxim.) Seem. (Araliaceae) is one of the most abundant species of genus Acanthopanax. The fruits of A. sessiliflorus are used in traditional medical protocols as an analgesic, tonic, antidiabetic, antihypertensive, anti-inflammatory, antitumor, and immune-stimulating agent. In this work, we carried out a comprehensive investigation into the anthocyanin components in the fruits of A. sessiliflorus. The anthocyanin content in the fresh fruits of A. sessiliflorus was determined by high performance liquid chromatography-diode array detection (HPLC/DAD), and the anthocyanin component was isolated from these using high-speed counter-current chromatography (HSCCC) and elucidated by electro-spray ionization-mass spectrometry (ESI/MS), 1H- and 13C-NMR. Its antioxidant activity was evaluated by ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). We found that A. sessiliflorus contained a gross anthocyanin content of 121.35 mg/100 g. HSCCC was successfully used for separation and purification of the primary anthocyanin component, cyanidin 3-xylosyl-galactoside. The antioxidant and radical scavenging tests indicated that cyanidin 3-xylosyl-galactoside is a potent antioxidant.

Pharmacological Evaluation of Artemisia cina Crude CO2 Subcritical Extract after the Removal of Santonin by Means of High Speed Countercurrent Chromatography.

Artemisia species are highly important due to their economic significance as medicines, fodder and food. Artemisia cina is an endemic species to Kazakhstan. In folk medicine, water extract of A. cina was used in the treatment of bronchial asthma while the alcohol extract has larvicidal and antituberculosis activity. The most common and most extensively studied compound from this species is the terpenoid santonin. The toxicity of this compound occurs at the doses of 60 mg for children and 200 mg for adults causing among other issues xanthopsia, leading to blindness. Having this in mind, the main idea of this work was to remove santonin from the crude extract and to check if the santonin-free extract would still be of any pharmacological importance. A CO2 subcritical extract was chromatographed using high-speed countercurrent chromatography (HSCCC) for the removal of santonin. The santonin-free CO2 subcritical extract (SFCO2E) as well as the isolated compound pectolinarigenin, a flavonoid, were assessed for their pharmacological actions. From the results obtained we can safely suggest that HSCCC is an efficient methodology to completely remove santonin from the CO2 subcritical extract. It was also possible to observe promising antinociceptive and anti-inflammatory activities for both SFCO2E and pectolinarigenin at concentrations that can justify the production of a phytomedicine with this endemic plant from Kazakhstan.

Phytochemical Profile and Antidepressant Effect of Ormosia henryi Prain Leaf Ethanol Extract.

The Ormosia henryi Prain leaf (OHPL) is a new bioactive resource with potential antidepressant activity, but few reports have confirmed its chemical composition or antidepressant effect. To investigate the phytochemical profile of OHPL ethanol extract (OHPLE), six flavone C-glycosides and two flavone O-glycosides were purified by high-speed counter-current chromatography combined with preparative high-performance liquid chromatography (HSCCC-prep-HPLC). The eight isolated compounds were identified by NMR and MS. Forty-six flavonoids, including flavones, flavone C-glycosides, flavone O-glycosides, isoflavones, isoflavone O-glycosides, prenylflavones and polymethoxyflavones were definitively or tentatively identified from OHPLE using ultra-performance liquid chromatography/ electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS/MS) on the basis of fragment ions that are characteristic of these isolated compounds. The results of the antidepressant assay suggest that OHPLE significantly improved depression-related behaviors of chronic unpredictable mild stress (CUMS) mice. The observed changes in these mice after OHPLE treatment were an increased sucrose preference index, reduced feeding latency, prolonged tail suspension time, and upregulated expression of brain-derived neurotrophic factor (BDNF). The details of the phytochemicals and the antidepressant effect of OHPLE are reported here for the first time. This study indicates that the OHPL, enriched in flavone C-glycosides, is a new resource that might be potentially applied in the field of nutraceuticals (or functional additives) with depression-regulating functions.

Separation of Five Flavonoids from Aerial Parts of Salvia Miltiorrhiza Bunge Using HSCCC and Their Antioxidant Activities.

The aerial parts of Salvia miltiorrhiza Bunge, as the non-medicinal parts, are always discarded during harvesting, resulting in a huge waste of resources and environmental pressure. Due to the high flavonoid content and their antioxidant activities characteristics, the aerial parts of S. miltiorrhiza can be developed into natural antioxidants and used in foods. A high-speed counter-current chromatography (HSCCC) method, using a two-phase solvent system composed of tert-butyl methyl ether/n-butanol/acetonitrile/water (3:1:1:20, v/v), was the first to successfully isolate five flavonoids from the aerial parts of S. miltiorrhiza in one attempt, and separately categorized as rutin (1), isoquercitrin (2), kaempferol-3-O-α-l-rhamnopyranosyl-(1→6)-ß-d-glucopyranoside (3), kaempferol-3-O-ß-d-glucopyranoside (4) and apigenin-7-O-ß-d-glucopyranoside (5) after identification. The purities of these plant isolates were 97.3%, 99.5%, 92.8%, 98.1% and 98.7%, respectively. All the flavonoids were identified by HR-ESI-MS, 1D and 2D NMR. Compounds 3 and 5 were firstly isolated from the plant of S. miltiorrhiza. Results from antioxidant assays showed that rutin (1) and isoquercitrin (2) had higher antioxidant capacities compared to L-ascorbic acid as the positive control.

Screening and Isolating Major Aldose Reductase Inhibitors from the Seeds of Evening Primrose (Oenothera biennis).

Aldose reductase (AR) is a drug target for therapies to treat complications caused by diabetes mellitus, and the development of effective AR inhibitors (ARIs) of natural origin is considered to be an attractive option for reducing these complications. In this research, the rat lens AR (RLAR) inhibitory activity of evening primrose (Oenothera biennis) seeds was investigated for the first time. In our results, the 50% (v/v) methanol extract of evening primrose seeds exhibits excellent RLAR inhibitory activity (IC50 value of 7.53 µg/mL). Moreover, after enrichment of its bioactive components, the ARIs are more likely to be present in the ethyl acetate fraction of 50% (v/v) methanol extract (EME) of evening primrose seeds, which exhibits superior RLAR inhibitory activity (IC50 value of 3.08 µg/mL). Finally, gallic acid (1), procyanidin B3 (2), catechin (3), and methyl gallate (4) were identified as the major ARIs from the EME by affinity-based ultrafiltration-high-performance liquid chromatography and were isolated by high speed countercurrent chromatography, with gallic acid (11.46 µmol/L) and catechin (14.78 µmol/L) being the more potent inhibitors of the four ARIs identified. The results demonstrated that evening primrose seeds may be a potent ingredient of ARIs.

Target-Guided Isolation of Three Main Antioxidants from Mahonia bealei (Fort.) Carr. Leaves Using HSCCC.

Mahonia bealei (Fort.) Carr. is an economically important plant that is widely cultivated in Southwest China. Its leaves are commonly used for tea and contain an abundance of antioxidant compounds. However, methods of the systematic purification of antioxidants from M. bealei are lacking. In this study, antioxidants from this plant were effectively and rapidly enriched. First, antioxidants were screened using online 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH)-high performance liquid chromatography (HPLC), followed by separation using high-speed countercurrent chromatography with an optical solvent system composed of n-hexane/ethyl acetate/methanol/water (1:5:1:5, v/v/v/v). Three phenolics-chlorogenic acid (1, 8.3 mg), quercetin-3-O-ß-d-glucopyranoside (2, 20.5 mg), and isorhamnetin-3-O-ß-d-glucopyranoside (3, 28.4 mg)-were obtained from the ethyl acetate-soluble fraction (240 mg) by one-step separation. The chemical structures of the phenolics were characterized by MS and NMR techniques, and the purity of each compound was >92.0% as determined by HPLC. The isolated compounds were assessed by scavenging activities on DPPH and superoxide radicals as well as cytoprotective assays, all of which showed similar trends regarding the antioxidant capacities of the compounds. Moreover, compounds 1-3 significantly attenuated the lipid peroxidation and antioxidant enzyme activities in H2O2-treated RAW264.7 cells. Our study demonstrated the efficiency of a newly developed integrative system for the comprehensive characterization of pure compounds from M. bealei, which will allow their use as reference substances.

Fingerprint Analysis of Cnidium monnieri (L.) Cusson by High-Speed Counter-Current Chromatography.

Cnidium monnieri (L.) Cusson is a popular Traditional Chinese Medicine (TCM) with a variety of bioactivities. However, there are some problems that have affected the development of Cnidium monnieri (L.) Cusson. At present, many methods have been reported for the analysis of coumarins in Cnidium monnieri (L.) Cusson. However, the quality control of coumarins in Cnidium monnieri (L.) Cusson by high-speed counter-current chromatography (HSCCC) has not been reported. In this study, analytical high-speed counter-current chromatography (HSCCC) was successfully used for fingerprint of Cnidium monnieri (L.) Cusson with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water at 4:6:6.5:3.5 (v/v). The UV wavelength was set at 254 nm. Six coumarin compounds with high biological activity were selected as indicator compounds for the quality control. The HSCCC fingerprint of the Cnidium monnieri (L.) Cusson was successfully established and there were some differences according to the results of the fingerprint analysis. The present results demonstrate that HSCCC is an established and efficient technique for the fingerprint analysis of Cnidium monnieri (L.) Cusson and can be used to control the quality of Cnidium monnieri (L.) Cusson. In brief, HSCCC is a useful technology for the fingerprint analytical method for TCM.

Comprehensive optimization of precursor-directed production of BC194 by Streptomyces rochei MB037 derived from the marine sponge Dysidea arenaria.

BC194, a derivative of borrelidin (BN) that features a lower cytotoxicity than that of BN due to an altered starter unit, trans-1,2-cyclobutanedicarboxylic acid (trans-1,2-CBDA), is a potent inhibitor of angiogenesis. However, BC194 production has only been reported to occur via mutasynthesis, which requires tedious, multistep genetic manipulation. In this study, we surveyed several factors contributing to the precursor-directed biosynthesis of BC194 and provided an alternative method for the production of BC194 that is directly applicable to other BN-producing strains. First, the precursor-directed biosynthesis of BC194 by a BN-producing strain, Streptomyces rochei MB037 derived from sponge Dysidea arenaria, was carried out in modified Radix astragali (RA) medium with 5 mM trans-1,2-CBDA. Next, possible inhibitors of BN starter unit trans-1,2-cyclopentanedicarboxylic acid (trans-1,2-CPDA) biosynthesis were investigated. It was found that potassium ferricyanide was a possible inhibitor of 3,4-dihydroxyphenylacetate 2,3-dioxygenase (DHPAO) and capable of suppressing the yield of BN and increasing the BC194 yield by 112.5% (from 5.2 ± 0.76 to 11.9 ± 0.59 mg/L). BC194 yield was further enhanced in the presence of 50 mM trans-1,2-CBDA, reaching 20.2 ± 0.62 mg/L. Furthermore, 3% macroporous adsorbent DA-201 resin was added to the fermentation broth, enabling a further 36.6% increase in BC194 production and reaching 27.59 ± 1.15 mg/L. Moreover, an efficient separation of BC194 with approximately 95% purity was developed by employing high-speed counter-current chromatography (HSCCC), achieving an improved recovery (approximately 93%).

Purified Phlorizin from DocynIa Indica (Wall.) Decne by HSCCC, Compared with Whole Extract, Phlorizin and Non-Phlorizin Fragment Ameliorate Obesity, Insulin Resistance, and Improves Intestinal Barrier Function in High-Fat-Diet-Fed Mice.

Natural products generally contain complex and multiple bioactive compounds that are responsible for the effects on health through complicated synergistic and/or suppressive actions. As an important raw material of local ethnic minority tea, ethnomedicines and food supplements in southwestern areas of China, Docynia indica (Wall.) Decne (DID) mainly consists of phlorizin (PHZ), which is the main active component. In this study, the holistic activities and the interactions of components of PHZ, non-phlorizin (NP) in the DID extract (DIDE) were evaluated. A rapid and effective high-speed counter-current chromatography (HSCCC) was performed to knock out PHZ from DIDE and the purity of PHZ was 96.01% determined by HPLC, with a recovery rate of 96.76%. After 13 weeks of treatment course in a high-fat diet (HFD)-induced obese mice model, the results revealed that the DIDE and PHZ significantly decreased weight gain, blood lipid levels, hyperplasia of adipocytes and alleviated inflammation (p < 0.05). Both DIDE and PHZ improves insulin resistance (p < 0.001). Meanwhile, the intestinal barrier function was improved compared to HFD group, through the determination of serum lipopolysaccharides (LPS), glucagon-likepeptide-2 (GLP-2) and hematoxylin-eosin staining of jejunum. Interestingly, after NP treatment, the metabolic syndrome of the HFD-induced obesity appeared to have a similar improvement. All the experiments showed that there is a synergistic weakening phenomenon when PHZ and NP interact with each other in the mixed state. In conclusion, for the PHZ and NP showing a good effect on anti-obesity, anti-inflammation, and intestinal barrier function, DIDE could be a good source of functional food to prevent obesity.

Isolation and Purification of Two Isoflavones from Hericium erinaceum Mycelium by High-Speed Counter-Current Chromatography.

High-speed counter-current chromatography (HSCCC) was used to separate and purify two isoflavones for the first time from Hericium erinaceum (H. erinaceum) mycelium using a two-phase solvent system composed of chloroform-dichloromethane-methanol-water (4:2:3:2, v/v/v/v). These two isoflavones were identified as genistein (4',5,7-trihydroxyisoflavone, C15H10O5) and daidzein (4',7-dihydroxyisoflavone, C15H10O4), using infrared spectroscopy (IR), electro-spary ionisation mass (ESI-MS), ¹H-nuclear magnetic resonance (NMR) and 13C-NMR spectra. About 23 mg genistein with 95.7% purity and 18 mg daidzein with 97.3% purity were isolated from 150 mg ethanolic extract of H. erinaceum mycelium. The results demonstrated that HSCCC was a feasible method to separate and purify genistein and daidzein from H. erinaceum mycelium.

Separation of Seven Polyphenols from the Rhizome of Smilax glabra by Offline Two Dimension Recycling HSCCC with Extrusion Mode.

An offline two-dimensional recycling high-speed countercurrent chromatography (2D R-HSCCC) strategy with extrusion mode was developed for isolating polyphenols from the rhizome of Smilax glabra. Firstly, the ethyl acetate extract was divided into two fractions, Fr.1 and Fr.2, by silica gel column chromatography. Then, HSCCC was applied to separate polyphenols from the two fractions using a solvent system consisting of petroleum ether-ethyl acetate-methanol-water (1:3:0.5:5, v/v). Fifty milligrams of Fr.1 was separated by conventional HSCCC, yielding 5-O-caffeoylshikimic acid (1, 15.8 mg) and taxifolin (2, 4.8 mg). Offline 2D R-HSCCC with extrusion mode was used to separate Fr.2, and astilbin (4, 37.3 mg), neoisoastilbin (5, 8.8 mg), engeletin (7, 7.9 mg), and a mixture of two polyphenols were obtained from 100 mg of Fr.2. The mixture of two polyphenols was further separated by pre-HPLC, yielding neoastilbin (3, 15.2 mg) and isoastilbin (6, 9.9 mg). The purities of these seven compounds were all over 96.0%. Their structures were identified by MS and NMR. The results demonstrated that the strategy based on offline 2D R-HSCCC with extrusion mode was a powerful tool to separate the main compounds from the rhizome of Smilax glabra and valued for the preparative separation compounds with broad K-values and similar structures.

Preparative isolation and purification of capsaicin and dihydrocapsaicin from Capsici Fructus using supercritical fluid extraction combined with high speed countercurrent chromatography.

BACKGROUND: Supercritical fluid extraction with CO2 (SFE-CO2 ) was utilized for extraction of capsaicin (CA) and dihydrocapsaicin (DHCA) from Capsici Fructus, and then a two-step enrichment method for separating capsaicinoids from SFE-CO2 extracts was developed. The process involved extraction with aqueous methanol and crystallization by alkali extraction and acid precipitation. Finally, a consecutive high-speed countercurrent chromatography (HSCCC) separation method was successfully applied in the purification of CA and DHCA from capsaicinoid crystal. RESULTS: The extraction pressure, extraction temperature and volume of co-solvent were optimized at 33 MPa, 41 °C and 75 mL, respectively, using response surface methodology; the extraction rates of CA and DHCA were about 93.18% and 93.49%, respectively. 407.43 mg capsaicinoid crystal was isolated from the SFE-CO2 extracts obtained from 100 g capsicum powder by the two-step enrichment method. About 506 mg and 184 mg CA and DHCA with purities up to 98.31% and 96.68%, respectively, were obtained from 1 g capsaicinoid crystal in one HSCCC of three consecutive sample loadings without exchanging any solvent system. CONCLUSIONS: This method comprising SFE-CO2 , a two-step enrichment and HSCCC was efficient, powerful and practical for the large-scale preparation of CA and DHCA from Capsici Fructus with high purity and high yield. © 2017 Society of Chemical Industry.

High-Speed Counter-Current Chromatography with an Online Storage Technique for the Preparative Isolation and Purification of Dihydroflavonoids from Sophora alopecuroides L.

INTRODUCTION: High-speed counter-current chromatography (HSCCC) is an efficient and non-absorption separation technique, but limitations still exist in simultaneous isolation of complex structures of natural products. Moreover, particular methods are various for different kinds of natural products. OBJECTIVE: A novel HSCCC strategy combined with an online storage recycling elution (OSR-CCC) technique was developed for the quick separation of naturally occurring dihydroflavonoids from the extract of the herb Sophora alopecuroides L. METHODOLOGY: In the separation procedure, a storage loop and two six-port valves were connected to a HSCCC system. Effluent A was subjected to an online storage loop and then to recycling separation three times after effluent B was collected in head-to-tail mode. After completion of the recycling separation of effluent A, the elution was switched to tail-to-head mode to collect effluent C. A biphasic solvent system of n-hexane/ethyl acetate/methanol/water (9:6:6:8, v/v/v/v) was used as the separation solvent during the whole elution. RESULTS: Six constituents were isolated simultaneously from the extract (200 mg) of S. alopecuroides by running HSCCC non-stop, and their purities were higher than 95.0%. Their structures were determined as the pterocarpan glycoside sophoratonkin (1) (10.0 mg) and five dihydroflavonoids, alopecurone F (2) (5.4 mg), lehmannin (3) (11.0 mg), alopecurone A (4) (35.0 mg), sophoraflavanone G (5) (21.0 mg), alopecurone B (6) (31.0 mg). CONCLUSION: This recycling HSCCC method combined with an online storage technique could be a rapid, effective and simple approach to isolate stilbene-dihydroflavonoids from herbs of the Sophora genus simultaneously. Copyright © 2017 John Wiley & Sons, Ltd.