Medium-pressure Chromatography Research Articles

Preparative isolation of maltol glycoside from Dianthus superbus and its anti-inflammatory activity in vitro.

Dianthus superbus is a traditional Chinese medicine that is commonly utilized as a treatment for inflammation, pain, and immunological conditions. In this study, an anti-inflammatory maltol glycoside derived from Dianthus superbus was isolated for the first time via medium and high-pressure liquid chromatography, and at the same time, the in vitro anti-inflammatory activity of this maltol glycoside was preliminarily explored. Initially, crude samples of Dianthus superbus were preprocessed via MCI GEL® CHP20P and Spherical C18 medium-pressure chromatography, under the guidance of evaluation of in vitro anti-inflammatory activity. Fr44 was found to be the target fraction, and it was further isolated via two-dimensional reversed-phase/hydrophilic interaction liquid chromatography, yielding > 95% pure and was identified as tunicoside B. MTT assay, nitric oxide and nitric oxide synthase were used to evaluate the effects of tunicoside B on murine macrophage Raw264.7 by nitric oxide synthase assay kit, molecular docking, and western blotting. The results showed that tunicoside B did not affect the viability of cells and exhibited significant anti-inflammatory activity. As far as we know, this is the first report of tunicoside B from Dianthus superbus and the first study on the anti-inflammatory activity of tunicoside B. More importantly, the approach established in this study is expected to provide a theoretical basis for the separation and pharmacological activity study of maltol glycosides from other natural products.

Large-scale preparative isolation of bergenin standard substance from Saxifraga atrata using polyamide coupled with MCI GEL® CHP20P as stationary phases in medium pressure chromatography

In this study, polyamide and MCI GEL® CHP20P were employed as stationary phases in medium pressure chromatography (MPC) for the efficient preparative separation of bergenin from Saxifraga atrata. Ethanol–water, methanol-water, and acetonitrile-water mobile phases all showed good enrichment capacity for bergenin fraction when polyamide was used as a stationary phase. After 5 cycles of polyamide MPC using acetonitrile/water, 1.2 g of bergenin fraction was isolated from 180 g Saxifraga atrata herb. Further purification of this fraction was conducted using MCI GEL® CHP20P styrene-divinylbenzene beads. The bergenin fraction was separated into two fractions, and after three runs of MPC, 714.2 mg of bergenin with purity above 99% was obtained. The results demonstrate that the combination of polyamide and styrene-divinylbenzene MPC can be utilized for preparative isolation of compounds from natural products with high yield and purity.

Metabolite profile of Nectandra oppositifolia Nees & Mart. and assessment of antitrypanosomal activity of bioactive compounds through efficiency analyses.

EtOH extracts from the leaves and twigs of Nectandra oppositifolia Nees & Mart. shown activity against amastigote forms of Trypanosoma cruzi. These extracts were subjected to successive liquid-liquid partitioning to afford bioactive CH2Cl2 fractions. UHPLC-TOF-HRMS/MS and molecular networking were used to obtain an overview of the phytochemical composition of these active fractions. Aiming to isolate the active compounds, both CH2Cl2 fractions were subjected to fractionation using medium pressure chromatography combined with semi-preparative HPLC-UV. Using this approach, twelve compounds (1-12) were isolated and identified by NMR and HRMS analysis. Several isolated compounds displayed activity against the amastigote forms of T. cruzi, especially ethyl protocatechuate (7) with EC50 value of 18.1 μM, similar to positive control benznidazole (18.7 μM). Considering the potential of compound 7, protocatechuic acid and its respective methyl (7a), n-propyl (7b), n-butyl (7c), n-pentyl (7d), and n-hexyl (7e) esters were tested. Regarding antitrypanosomal activity, protocatechuic acid and compound 7a were inactive, while 7b-7e exhibited EC50 values from 20.4 to 11.7 μM, without cytotoxicity to mammalian cells. These results suggest that lipophilicity and molecular complexity play an important role in the activity while efficiency analysis indicates that the natural compound 7 is a promising prototype for further modifications to obtain compounds effective against the intracellular forms of T. cruzi.

Preparative isolation of 1,1-diphenyl-2-picrylhydrazyl inhibitors from Ribes himalense using medium-pressure and two-dimensional reversed-phase/reversed-phase liquid chromatography guided by an online HPLC-1, 1-diphenyl-2-picrylhydrazyl assay.

The lack of suitable chromatographic purification methods makes it a challenge to effectively isolate the chemical components of traditional Tibetan medicines. Ribes himalense is a rarely studied Tibetan medicine, reputed to have free radical-scavenging effects. In the present work, we utilized it as a model herb to highlight an approach for the separation of 1,1-diphenyl-2-picrylhydrazyl inhibitors via medium-pressure chromatography and two-dimensional reversed-phase/reversed-phase interaction liquid chromatography under the guidance of an online high-performance liquid chromatography-1,1-diphenyl-2-picrylhydrazyl assay. Finally, we obtained two free radical inhibitors (>95% purity) from the R. himalense extract. This is the first report of the rapid isolation of these free radical inhibitors from R. himalense. This method can be useful in quality standard assessment and further pharmacological activity research, and may be used as a reference for the composition research of various natural products.

A Review Article on Flash Chromatography

Flash chromatography is a purification technique that can be elucidated as a modified preparative column chromatography. Flash chromatography is a technique obtained by the blend of medium pressure and short column chromatography, which results in quick segregation of mixture of components. The efficiency and speed of classical column chromatography can be increased by the use of high pressurized gas to pump the solvent across the column. In contrast to the many other chromatographic techniques, large samples can be separated and isolated using flash chromatography. This technique in many cases is used as an antecedent step to the highly sophisticated methods such as HPLC, NMR and FT-IR so as to obtain purified samples. The main applications of flash chromatography include refinement of synthetic products, segregation of intended compounds from natural products, isolation of purified compounds for further sophisticated analysis and the segregation of complex blend of mixtures into simpler fractions for analysis. The present review focuses on the different aspects of flash chromatographic technique.

Synthesis and characterization of novel astragalin galactosides using β-galactosidase from Bacillus circulans.

Astragalin (kaempferol-3-O-β-d-glucopyranoside, Ast) is a kind of flavonoid known to have anti-oxidant, anti-HIV, anti-allergic, and anti-inflammatory effects. It has low solubility in water. In this study, novel astragalin galactosides (Ast-Gals) were synthesized using β-galactosidase from Bacillus circulans and reaction conditions were optimized to increase the conversion yield of astragallin. Purified Ast-Gal1 (11.6% of Ast used, w/w) and Ast-Gal2 (6.7% of Ast used, w/w) were obtained by medium pressure chromatography (MPLC) with silica C18 column and open column packed with Sephadex LH-20. The structures of Ast-Gal1 and Ast-Gal2 were identified by nuclear magnetic resonance (NMR) to be kaempferol-3-O-β-d-glucopyranosyl-(1→6)-β-d-galactopyranoside and kaempferol-3-O-β-d-glucopyranosyl-(1→6)-β-d-galactopyranosyl-(1→4)-β-d-galactopyranoside, respectively. The water solubility of Ast, Ast-Gal1, and Ast-Gal2 were 28.2±1.2mg/L, 38,300±3.5mg/L, and 38,800±2.8mg/L, respectively. The SC50 value (the concentration required to scavenge 50% of the ABTS+) of Ast, Ast-Gal1, and Ast-Gal2 were 5.1±1.6μM, 6.5±0.4μM, and 4.9±1.1μM, respectively. The IC50 values (the half maximal inhibitory concentration) of Ast, Ast-Gal1, and Ast-Gal2 against angiotensin converting enzyme (ACE) were 171.0±1.2μM, 186.0μM, and 139.0±0.2μM, respectively.

EXTRACTION AND ISOLATION OF NUCIFERINE FROM LOTUS LEAVES USING FLASH CHROMATOGRAPHY

Nelumbo nucifera Gaertn (Family: Nelumbonaceae), commonly known as lotus, is an aquatic perennial plant. The lotus leaves are used to treat fever, hematemesis, hyperlipidaemia and obesity. The leaves are rich in alkaloids such as nuciferine, roemerine, pronuciferine and N-nornuciferine, which are responsible for biological activity of leaf. In the present work, rapid and efficient flash chromatographic process for selective isolation of nuciferine from Nelumbo nucifera Gaertn was developed. The rate of extraction and purity of nuciferine depends upon the solvent used for extraction and optimized chromatographic conditions. Methanol was found to be best solvent for highest possible recovery of the nuciferine from Nelumbo nucifera Gaertn. Flash chromatography, also known as medium pressure chromatography, is used to speed up the flow of solvent, which decreases the time needed to purify the sample. The purified sample of nuciferine was then characterized by UV spectroscopy, HPTLC and HPLC-MS.

Zwitterionic Azaspirocyclic Hydantoins

Background: Azaspirocycles are important frameworks in biologically active natural products and are an emerging pharmacophore in drug design. We prepared ten novel zwitterionic azaspirocyclic hydantoins along with their ring-opened and nitro-group reduced analogues. Methods: Zwitterionic azaspirocyclic hydantoins were synthesized in high yield from 2,4- dinitrobenzoic acid and a range of carbodiimides in dichloromethane as solvent. These novel molecules were purified by reverse-phase medium pressure (flash) chromatography and fully characterized. Some molecules were evaluated in the NIH NCI-60 anti-cancer screen. Results: Intense red zwitterionic azaspirocyclic hydantoins were fashioned from an electron-deficient benzoic acid, 2,4-dinitrobenzoic acid, in a dearomatizing intramolecular ipso-spiroannulation using 1,3- disubstituted carbodiimides. Our proposed reaction mechanism invokes a rearrangement to an N-acylurea intermediate that spontaneously ipso-spirocyclizes under our reaction conditions in a 100% atom efficient manner. A broad survey of available carbodiimides revealed that only molecules with sterically undemanding N-alkyl groups on this N-acylurea precursor were able to form the most stable products. A diagnostic analytical pattern was observed in tandem MS in ESI- mode that revealed an unusual degradation by way of R-NCO neutral fragment loss. Catalytic hydrogenation gave quantitative conversion to the ring-opened 2,4-diaminoaromatic N-acylurea. Stable structures were tested for anticancer activity, yet without success. Conclusion: We have presented an efficient and atom-economical synthetic approach to zwitterionic azaspirocyclic hydantoins using the in situ intramolecular dearomatization of electrophilic N-acylureas. Keywords: Azaspirocycle, carbodiimide, dearomatization, SNAr mechanism, spirocyclic, zwitterion.

Identification of Pyrogallol in the Ethyl Acetate-Soluble Part of Coffee as the Main Contributor to Its Xanthine Oxidase Inhibitory Activity.

In this study, ethyl acetate-soluble parts of hot-water extracts from roasted coffee beans were found to demonstrate potent xanthine oxidase (XO) inhibition. The XO inhibitory activities and chlorogenic lactone contents (chlorogenic lactones have previously been identified as XO inhibitors in roast coffee) were measured for ethyl acetate-soluble parts prepared from coffee beans roasted to three different degrees. Although chlorogenic lactone contents decreased with higher degrees of roasting, the XO inhibitory activity did not decrease. These data led us to investigate new potent inhibitors present in these ethyl acetate-soluble extracts. Repeated assay-guided purifications afforded a highly potent XO inhibitor, which was eluted before chlorogenic lactones via medium-pressure chromatography using an octadecylsilica gel column. The obtained inhibitor was identified as pyrogallol (1,2,3-trihydroxybenzene), which had an IC50 of 0.73 μmol L-1, much stronger than that of other related polyphenolic compounds. Quantitative analysis of pyrogallol and chlorogenic lactones revealed that pyrogallol (at concentrations of 33.9 ± 4.2 nmol mL-1 in light roast coffee and 39.4 ± 3.9 nmol mL-1 in dark roast coffee) was the main XO inhibitor in hot-water extracts of roasted coffee beans (i.e., drinking coffee).

Acetylcholinesterase inhibitors from Tetrapterys mucronata

In our search for acetylcholinesterase (AChE) inhibitors, the investigation of Tetrapterys mucronata Cav. (Malpighiaceae), a plant used in Brazil for its psychotropic properties, was pursued. The methanolic extract of the bark of T. mucronata presented an interesting AChE inhibition in a TLC bioautographic assay. In order to isolate the active compounds, bioassay-guided isolation was undertaken using HPLC-microfractionation in 96 well plates and agar-overlay bioautography to localize the active compounds. The analytical HPLC-PAD conditions were geometrically transferred to a preparative medium pressure chromatography (MPLC-UV) by chromatographic calculations for the efficient isolation of the active compounds at the milligram scale in one step. Using this approach, 22 compounds were isolated, six of them being new natural products, such as 5-hydroxy-2-(2,3-dihydroxy-1-(1 H-indol-3-yl)propyl)-1 H-indole-3-carboxamide and 3-(2-(dimethylamino)ethyl)-2-(2,3-dihydroxy-1-(1 H-indol-3-yl)propyl)-1 H-indol-5-ol. The structure of the isolated compounds was elucidated by classical spectroscopic methods including UV, 2D NMR and HR-MS and chemical derivatisation. Some of the isolated compounds presented a strong AChE inhibition on the bioautographic assay; however those compounds showed weak activity when it was measured in solution.

多糖誘導体系のキラル充填剤を使用した中圧クロマト用キラルカラムとキラルTLCの開発

多糖誘導体系のキラル充填剤を使用した中圧クロマト用キラルカラムとキラルTLCの開発

Antifungal compounds isolated from Diospyros bipindensis

Diospyros bipindensis (Gürke) stem barks are used in Cameroon by pygmies Baka for the treatment of pulmonary diseases. One hypothesis was that this type of diseases could be caused by fungi. Thus the antifungal activity of the traditional preparation as well as that of different extracts was tested against Candida albicans. Some extracts presented a significant antifungal activity. The bioguided isolation was undertaken using HPLC-microfractionation in 96 well plates combined with bioautography in order to localize the active compounds in the HPLC profiling of the extracts. In a second step, medium pressure chromatography was used to isolate the active compounds. Using this approach seventeen compounds were isolated, nine of them are new natural products. The structures of the isolated compounds were elucidated by classical spectroscopic methods including UV, NMR and HR-MS.

Limited contribution of isoflavones to hepatocellular phase II enzyme-inducing activity of soybean ( Glycine max) extracts

Soy flour was successively extracted with ethyl acetate, ethanol, and water and tested for quinone reductase (QR) induction in the Hepa 1c1c7 cell line. The ethanol extract was found to be the most potent, with a CD value (concentration required to double QR specific activity) of 460 μg/ml. Phase separation of the ethanol extract and a phospholipid removal procedure yielded a fraction that doubled QR at 80 μg/ml. Reverse phase medium pressure chromatography of the purified ethanol extract yielded 13 fractions. Constituents of selected fractions were identified using mass spectrometry and NMR analysis. Fractions 2, 8, and 10 contained 47% of the recovered mass and were the least active of the isolates. These three fractions contained isoflavone glycosides. The latest eluting fractions 11–13 were found to be the most potent, and contained the majority of recovered inducing units (IUs), a measure of potency.

An efficient, one-pot synthesis of various ceramide 1-phosphates from sphingosine 1-phosphate

An efficient, one-pot procedure for the synthesis of ceramide 1-phosphates with varying N-acyl substituents, to serve as tool compounds for analytical and biological investigations, was developed. Sphingosine 1-phosphate was silylated in situ to increase its solubility and to protect the 3-hydroxy functionality and then allowed to react with activated acid derivatives in the presence of diisopropylethylamine. Simultaneous cleavage of the silyl protecting groups and separation from reagents and by-products was achieved by medium pressure chromatography on reversed phase material. Thus, ceramide 1-phosphates with various fatty acid chains and with fluorescent and affinity labels attached to the sphingoid backbone were prepared in good yields.

Synthesis and characterization of ( S)-amino alcohol modified M41S as effective material for the enantioseparation of racemic compounds

A new chiral stationary phase (CSP) was synthesized based on ( S)-1-anilino-3-propyl-2-propanol covalently bonded to the mesoporous semi-crystalline material M41S. Direct semipreparative enantioseparation of mandelic acid could be achieved using medium pressure chromatography. Partly separated could also be the enantiomers of 2,2′-dihydroxy-1,1′-binaphthalene, cyanochromene oxide, diethyl tartrate and 2-phenyl propionic acid. The characterization of CSP was accomplished by microanalysis, cross polarized magic angle spinning (CP-MAS) 13C NMR, powder X-ray diffraction (XRD), FTIR, thermo-gravimetric analysis (TGA), N 2 adsorption–desorption isotherm, scanning electron microscopy (SEM) and solid reflectance UV–vis spectroscopy. Furthermore the stability of CSP was satisfactory as it could withstand three washing and reuse experiments of enantioseparation of mandelic acid without loss in its performance.

Characterization of the Bifunctional γ-Glutamate-cysteine Ligase/Glutathione Synthetase (GshF) of Pasteurella multocida

Glutamate-cysteine ligase (gamma-ECL) and glutathione synthetase (GS) are the two unrelated ligases that constitute the glutathione biosynthesis pathway in most eukaryotes, purple bacteria, and cyanobacteria. gamma-ECL is a member of the glutamine synthetase family, whereas GS enzymes group together with highly diverse carboxyl-to-amine/thiol ligases, all characterized by the so-called two-domain ATP-grasp fold. This generalized scheme toward the formation of glutathione, however, is incomplete, as functional steady-state levels of intracellular glutathione may also accumulate solely by import, as has been reported for the Pasteurellaceae member Haemophilus influenzae, as well as for certain Gram-positive enterococci and streptococci, or by the action of a bifunctional fusion protein (termed GshF), as has been reported recently for the Gram-positive firmicutes Streptococcus agalactiae and Listeria monocytogenes. Here, we show that yet another member of the Pasteurellaceae family, Pasteurella multocida, acquires glutathione both by import and GshF-driven biosynthesis. Domain architecture analysis shows that this P. multocida GshF bifunctional ligase contains an N-terminal gamma-proteobacterial gamma-ECL-like domain followed by a typical ATP-grasp domain, which most closely resembles that of cyanophycin synthetases, although it has no significant homology with known GS ligases. Recombinant P. multocida GshF overexpresses as an approximately 85-kDa protein, which, on the basis of gel-sizing chromatography, forms dimers in solution. The gamma-ECL activity of GshF is regulated by an allosteric type of glutathione feedback inhibition (K(i) = 13.6 mM). Furthermore, steady-state kinetics, on the basis of which we present a novel variant of half-of-the-sites reactivity, indicate intimate domain-domain interactions, which may explain the bifunctionality of GshF proteins.

A new lariciresinol-type butyrolactone lignan derived from hydroxymatairesinol and its identification in spruce wood.

When the natural lignan hydroxymatairesinol (1) was treated with an alkaline aqueous solution, it partially rearranged to isomeric forms of a lariciresinol-type butyrolactone lignan. The two major diastereomers formed (2 and 3) were isolated by column and medium-pressure chromatography, and their structures were elucidated by MS and NMR techniques. These previously unknown butyrolactone lignans were identified as naturally occurring in spruce knotwood by GC, GC-MS, and HPLC-ESI MS/MS analyses. The formation of isohydroxymatairesinol (2) and epi-isohydroxymatairesinol (3) from hydroxymatairesinol (1), and their detection in rat urine after administration of 1, is discussed.

Optically Active Telluronium Imides: Optical Resolution, Absolute Configuration, and Mechanism of Racemization.

Thermodynamically and kinetically stabilized asymmetric diaryltelluronium imides were synthesized and optically resolved into their enantiomers on an optically active column using medium-pressure column chromatography. The relationship between the absolute configuration and the optical properties of the chiral telluronium imides was clarified. The mechanism of the racemization, which involves the formation of telluroxides by the hydrolysis of the telluronium imides, was proposed. © 2003 Wiley Periodicals, Inc. Heteroatom Chem 14:523–529, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.10185

Preparation and diastereomeric separation of an ( S)- and ( R)-1-(methoxycarbonyl)tridec-10-yl glucoside derivative, a precursor for a monosaccharide constituent of resin glycosides

The 6- O-mesyl derivative of phenyl 1-thio-β- d-glucopyranoside was prepared from d-glucose as a synthetic equivalent of a 6-deoxy-hexosyl donor. Racemic methyl 11-hydroxytetradecanoate (methyl convolvulinolate) was synthesized by Grignard reaction of propylmagnesium bromide with 10-undecenal followed by hydroboration. Both intermediates were coupled by NIS–TfOH-promoted glycosidation to give a mixture of two diasteromeric glucopyranosides, which were separated on a preparative scale by medium pressure chromatography. One of the products was identified as having the natural ( S)-configuration by comparison of its 1H NMR spectrum with an authentic sample prepared from the corresponding chiral hydroxyfatty acid.

Identification of O-glycosylation sites and partial characterization of carbohydrate structure and disulfide linkages of human insulin-like growth factor binding protein 6.

The actions of insulin-like growth factors (IGFs) are modulated by a family of high-affinity binding proteins (IGFBPs), including IGFBP-6, which preferentially binds IGF-II and is O-glycosylated. Glycosylated and nonglycosylated recombinant human IGFBP-6, expressed in Chinese hamster ovary cells and Escherichia coli, respectively, were purified using IGF-II affinity chromatography and reverse-phase medium-pressure chromatography. Electrospray ionization mass spectrometry (ESMS) of glycosylated IGFBP-6 revealed considerable heterogeneity of carbohydrate composition. Major glycoforms contained 8-16 monosaccharides, including N-acetylhexosamine, hexose, and N-acetylneuraminic acid. Glycosylation sites of IGFBP-6 were identified as Thr126, Ser144, Thr145, Thr146, and Ser152 by using a combination of ESMS and Edman sequencing of tryptic fragments separated by reverse-phase high-pressure liquid chromatography. One oligosaccharide chain contained 5-6 monosaccharides, whereas the others contained 2-4 monosaccharides. Glycosylated IGFBP-6 exhibited greater resistance to proteolysis by chymotrypsin and trypsin than nonglycosylated IGFBP-6. Native disulfide bond positions in IGFBP-6 were localized by means of observed disulfide-linked tryptic fragments, revealing that there are two disulfide-linked subdomains within each of the N- and C-terminal regions and confirming a previous suggestion that the latter regions are not interconnected. A model of IGFBP-6 is developed in which these distinct domains are separated by a central region which is O-glycosylated.