Reversed-phase Medium-pressure Liquid Research Articles

Three new nonenes from culture broth of marine-derived fungus Albifimbria verrucaria and their cytotoxic and anti-viral activities.

Three new nonenes, verrucanonenes A‒C (1‒3), were isolated from culture broth of marine-derived fungus Albifimbria verrucaria. These compounds were isolated using silica gel column chromatography, reversed-phase medium pressure liquid chromatography, Sephadex LH-20 column chromatography, and preparative HPLC. Their structures were determined using a spectroscopic method. Cytotoxicities of these isolated compounds to A549, DU145, HCT116, and HT1080 cancer cell lines were assessed. Compounds 1‒3 exhibited cytotoxicities to DU145 cancer cell line, with IC50 values of 23.4, 28.6, and 20.1 µM, respectively. Compound 2 decreased H1N1-induced cytopathic effects on MDCK cells in a dose-dependent manner.

P-Terphenyl glucosides from the culture broth of Phlebiopsis castanea.

A new p-terphenyl derivative, hydroxystrepantibin D (1), was isolated along with two known p-terphenyls (2 and 3) from the culture broth of Phlebiopsis castanea. These compounds were isolated using silica gel column chromatography, reversed-phase medium-pressure liquid chromatography, Sephadex LH-20 column chromatography, and preparative HPLC. Their structures were determined based on spectroscopic methods. These compounds exhibited free radical scavenging activities with IC50 values in the range from 22.2 to 158.4 μM against 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical and in the range from 161.1 to 356.1 μM against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical.

Cinnamic acid derivatives from welsh onion (Allium fistulosum) and their antibacterial and cytotoxic activities.

Cinnamic acids are a class of compounds based on phenyl propanoid backbone (C6-C3) isolated from plants and microorganisms, exhibiting interesting biological activities. To characterise cinnamic acids through the phytochemical study of welsh onion, Allium fistulosum, and to evaluate their antibacterial and cytotoxic properties. The phytochemical study of A. fistulosum was performed through chromatographic techniques, including reversed phase medium-pressure liquid chromatography (MPLC) and high-pressure liquid chromatography (HPLC). Preliminary analysis of crude chromatographic fractions from the organic extracts was carried out by proton nuclear magnetic resonance (1 H-NMR) in order to prioritise the study of those having phenyl propanoid skeleton. The structural identification of the isolated compounds was performed through analysis of spectroscopic data, mainly one-dimensional (1D) and two-dimensional (2D) NMR. The antibacterial activity was assessed against gram negative (Escherichia coli) and gram positive (Staphylococcus aureus) bacteria while the cytotoxic property was evaluated on breast cancer cell line (MCF-7). The 1 H-NMR study of crude fractions and application of a straightforward method to purify the phenyl propanoid compounds by reversed phase MPLC and HPLC, allowed the effortless isolation of several cinnamic acids, including two new rare phenolic imidates (1 and 2). The use of an entirely NMR approach for structural elucidation of the isolated metabolites allowed the isolated material to be kept for further pharmacological tests. These results corroborate the importance of the use of 1D and 2D NMR to the identification of new phenyl propanoids, potential lead compounds against bacteria and cancer cells.

Simultaneous separation of three isomeric sennosides from senna leaf (Cassia acutifolia) using counter‐current chromatography

Senna leaf is widely consumed as tea to treat constipation or to aid in weight loss. Sennoside A, A1 , and B are dirheinanthrone glucosides that are abundant and the bioactive constituents in the plant. They are isomers that refer to the (R*R*), (S*S*), and (R*S*) forms of protons on C-10 and C-10' centers and it is difficult to refine them individually due to their structural similarities. The new separation method using counter-current chromatography successfully purified sennoside A, A1 , and B from senna leaf (Cassia acutifolia) while reversed-phase medium-pressure liquid chromatography yielded sennoside A only. n-Butanol/isopropanol/water (5:1:6, v/v/v) was selected as the solvent system for counter-current chromatography operation, and the partition coefficients were carefully determined by adding different concentrations of formic acid. High-resolution mass spectrometry and NMR spectroscopy were performed to verify the chemical properties of the compounds.

Ultrafiltration LC-PDA-ESI/MS combined with reverse phase-medium pressure liquid chromatography for screening and isolation potential α-glucosidase inhibitors from Scutellaria baicalensis Georgi

Diabetes mellitus (DM) has become a worldwide health problem. α-Glucosidase inhibitors are widely used in the treatment of type 2 diabetes, and natural products are considered as the promising sources of α-glucosidase inhibitors. In this research, ultrafiltration liquid chromatography with photodiode array detection coupled to electrospray ionization tandem mass spectrometry (Ultrafiltration LC-PDA-ESI/MS) were applied to screen and identify α-glucosidase inhibitors from Scutellaria baicalensis. Following this, reverse-phase medium-pressure liquid chromatography (RP-MPLC) was applied to separate and isolate the active constituents. As a result, eleven major compounds in S. baicalensis were screened and identified as α-glucosidase inhibitors by Ultrafiltration LC-PDA-ESI/MS and six ligands, baicalin, wogonoside, 5,7,3,2′,6′-pentahydroxy flavanone, chrysin-6-C-arabinosyl-8-C-glucoside, chrysin-6-C-glucosyl-8-C-arabinoside and wogonin, were isolated by RP-MPLC. The purities of the six compounds were 89.7%, 83.5%, 76.2%, 93.7%, 75.1% and 96.2%, respectively, as determined by high performance liquid chromatography. The results demonstrate that the method for systematic isolation of bioactive components in S. baicalensis guided by Ultrafiltration LC-PDA-ESI/MS is a feasible and efficient technique and could be expected to extend for the separation of other enzyme inhibitors.

Preparation of an aspartic acid-containing protected peptide

Treatment of the peptide-resin Ac-Asn-Ser(Bzl)-Gly-Asp(OFm)-pMeBHA with HF/anisole cleaves the peptide-resin bond and removes the benzyl group, as expected, but also yields an impurity (ca. 25%) in which the benzyl group is linked to the fluorenylmethyl protecting group. Separation of the desired peptide from the impurity can be accomplished by reversed-phase medium-pressure liquid chromatography. The side reaction does not take place when the gamma-carboxyl of aspartic acid is protected with the 2-(4-acetyl-2-nitrophenyl)ethyl group.

An efficient method for the purification and characterization of nematicidal azadirachtins A, B, and H, using MPLC and ESIMS.

Azadirachtin A enriched concentrate containing 60% active ingredient (a.i.) was prepared from the methanolic extract of the de-fatted neem (Azadirachta indica A. Juss) seed kernels. Azadirachtins A, B, and H, the three major bioactive constituents of neem seed kernel, were purified from this methanolic concentrate by employing reverse phase medium-pressure liquid chromatography (MPLC), using methanol-water solvent system as an eluant. The three pure azadirachtin congeners thus obtained were characterized by their unique mass spectral fragmentation, using electrospray probe in positive ion mode (ESI). All three azadirachtins exhibited nematicidal and antifungal activities. Azadirachtin B was the most effective against the reniform nematode Rotylenchulus reniformis (EC(50) 96.6 ppm), followed by Azadirachtin A (119.1 ppm) and H (141.2 ppm). At 200-ppm concentration, the test compounds caused 50-65% mortality of Caenorhabditis elegans nematode. Azadirachtin H showed the highest activity against the phytophagous fungi Rhizoctonia solani (EC(50) 63.7 ppm) and Sclerotium rolfsii (EC(50) 43.9 ppm), followed by B and A. The isolation of pure azadirachtins A, B, and H directly by MPLC purification from its concentrate and their characterization by ESIMS are unique and less time-consuming.

Different phagostimulants in potato foliage for Manduca sexta and Leptinotarsa decemlineata

In the past decades, several studies have focused on the identification of feeding stimulants for specialists that feed on solanaceous plants, especially potato (Solanum tuberosum). In the 1950's, a phagostimulant was isolated from potato and tomato for Manduca sexta and characterised as a glycoside. It was suggested that the phagostimulant for M. sexta and Leptinotarsa decemlineata is identical. We tested whether these insects indeed share the same recognition factor. Previous bioassays for L. decemlineata larvae and adults were found to be unsatisfactory, so a new assay system was developed, using starch and wheat flour-based wafers as a neutral substrate. An aqueous extract of potato foliage that was highly active as a stimulant was fractionated by reverse phase medium pressure liquid chromatography (MPLC). Both insects were stimulated by a MPLC fraction that eluted with 25% methanol in water. Further separation of this and the following fraction (35% methanol, stimulatory for the beetle only) by semi-preparative high performance liquid chromatography (HPLC), using gradients of water and acetonitrile, resulted in the isolation of two distinct fractions that stimulated feeding by L. decemlineata. None of the HPLC fractions was active for M. sexta. However, fractions of the flash chromatography with less polarity (45–75% methanol) had a stimulatory effect only on M. sexta. Thus, the two insects do not use a common feeding stimulant, and for both at least two compounds of different polarity are active.

Purification of stearidonic acid (18:4(n-3)) and hexadecatetraenoic acid (16:4(n-3)) from algal fatty acid with lipase and medium pressure liquid chromatography.

Stearidonic acid (18:4(n-3)) and hexadecatetraenoic acid (16:4(n-3)) are included in some edible marine algae such as Undaria pinnatifida and Ulva pertusa with relatively high compositions (up to 40%) of total fatty acids. In order to prepare 16:4(n-3) and 18:4(n-3) enriched fatty acid concentrates, we screened for a suitable lipase which concentrates these acids by the removal of other fatty acids in the selective esterification reaction reported by Shimada et al. (Shimada et al. (1997), J. Am. Oil Chem. Soc., 74, 1465-1470). In combination with the lipase reaction and reversed-phase medium pressure liquid chromatography, we purified 18:4(n-3) and 16:4(n-3) to more than 95% purity.

Palmitic acid is the major fatty acid responsible for significant anti- N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) activity in yogurt

We describe here the isolation and identification of palmitic acid as being responsible for significant anti- N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) activity in yogurt. The Ames test ( Salmonella typhimurium TA 100) was used to direct fractionation of activity. Yogurt was freeze-dried and extracted with acetone to yield a crude extract. The crude extract was purified by normal phase silica gel, Sephadex LH-20, and reversed phase medium pressure liquid chromatographies. The major compound in the active medium pressure liquid chromatographic fractions was determined to be palmitic acid on the basis of gas chromatography-mass spectrometry (GC/MS), MS/MS, by co-elution with authentic palmitic acid on GC and high pressure liquid chromatography (HPLC) systems, and by nuclear magnetic resonance (NMR) analysis. Other saturated straight chain and methyl branched fatty acids were detected by GC/MS and were later shown to possess anti-MNNG activity. Of the straight chain fatty acids, palmitic acid had the highest anti-MNNG activity. All ω-1 methyl branched fatty acids tested were more active than their straight chain counterparts. A trace amount of isopalmitic acid (14-methyl pentadecanoic acid), a minor milk lipid, was detected in one of the active fractions, and was later shown to be five times more active than palmitic acid. Isopalmitic acid also inhibited mutagenesis induced by 4-nitroquinoline- N-oxide (4NQO), and 7,12-dimethyl benz[ a]anthracene (DMBA), and was found to inhibit the metabolic activation of DMBA.

Plant growth regulators G 1, G 2, G 3: Synthesis, extraction and determination of leaf content in Eucalyptus grandis

Endogenous concentrations of plant growth regulators G 1, G 2, G 3 from leaves of French-planted Eucalyptus grandis were determined. The leaves were extracted with methanol and the resulting extracts were purified by means of silica gel column chromatography and reversed-phase medium-pressure liquid chromatography. Improved synthesis, purification and physico-chemical characterization of the G factors, for use as external standards, were developed.

Application of reversed-phase medium-pressure liquid chromatography to the isolation, separation and amino acid analysis of two closely related peptide toxins of the cyanobacterium Microcystis aeruginosa strain PCC 7806

A mixture of two compounds, related to cyanoginosin-LR, was isolated from the cyanobacterium Microcystis aeruginosa strain PCC 7806. Using an optimized reversed-phase fast protein liquid chromatography (RP-FPLC) method, up to 1.5 mg of this toxin mixture could be separated into two distinct compounds by a single preparative scale run. To perform an amino acid analysis of the two components, the hydrolysates derivatized with phenylisothiocyanate were analysed separately by RP-FPLC. A method was developed which could be used for the automatic sequential analysis of up to fourteen samples. The amino acid analysis by RP-FPLC was linear in the range 1–5 nmol. On the basis of this analysis, the two compounds differ by only a methylated versus a demethylated aspartate. Only six of the seven amino acids known for cyanoginosin- LR could be reliably detected.

Separation of methylated and non-methylated cyanoginosin-LR homologues of the cyanobacterium Microcystis aeruginosa strain PCC 7806 by reversed-phase medium-pressure liquid chromatography

Cyanoginosins produced by Microcystis aeruginosa sp. are toxic cyclic heptapeptides with molecular weights of about 1000 dalton. Microcystis aeruginosa strain PCC 7806 proved to synthesize a methylated and non-methylated cyanoginosin-LR. A reversed-phase medium-pressure liquid cromatographic method was developed to separate the two cyanoginosin homologues on a preparative scale. The influence of chromatographic conditions on the separation such as solvent system, gradient parameters, flow-rate and temperature was investigated systematically. After optimization, up to 1.5 mg of the two compounds could be separated completely within 40 min.

Large fragments of nef‐protein and gp110 envelope glycoprotein from HIV‐1

Chemical synthesis of large peptide fragments (from 18 to 66 amino acid residues long) of the gp110 envelope glycoprotein and of nef-protein from HIV-1 was achieved by the solid phase method. Stepwise assembling of the peptide chains was carried out automatically on 4-(oxymethyl)-phenylacetamidomethyl resin using the N-alpha-butyloxycarbonyl amino acids with benzyl-based side chain protecting groups. Two elongation protocols were used depending on the peptide chain length: a standard cycle, mainly characterized by a single coupling step (Boc-amino acid symmetrical anhydride in dimethylformamide), and an optimized one for large peptides, based on a double coupling strategy (Boc-amino acid symmetrical anhydride first in dimethylformamide, then in dichloromethane). Final cleavage of the peptide from the solid support was carried out by anhydrous hydrogen fluoride and crude peptides were purified by C18 reverse phase medium pressure liquid chromatography after molecular filtration. Characterization of the purified peptides was done by analytical HPLC, amino acid content determination, and circular dichroism analysis both in polar (H2O) and in non-polar (TFE) environments. Immunoreactivity of anti-nef positive sera from HIV-1 infected patients by ELISA with the synthetic peptides was investigated. The results showed four major antigenic regions of nef-protein and mainly the immunodominance of the N- and C-termini of the molecule. Several of these peptides should prove to be useful for both diagnosis and vaccination purposes.

Inhibitors of sterol synthesis. Chromatography of acetate derivatives of oxygenated sterols.

The separation of the acetate derivatives of a number of oxygenated sterols was achieved by medium pressure liquid chromatography on silica gel columns and by normal and reversed phase high performance liquid chromatography. We have explored the application of these chromatographic systems for the analysis of oxygenated sterols of plasma samples from two normal human subjects. The addition of highly purified [14C]cholesterol to plasma permitted the detection and quantitation of oxygenated sterols formed by autoxidation of cholesterol during processing of the samples. Special attempts to suppress autoxidation of cholesterol included the use of an all-glass closed system for saponification and extraction under argon followed by rapid removal of cholesterol from the polar sterols by reversed phase medium pressure liquid chromatography. Chromatographic analyses of the [3H]acetate derivatives of the polar sterols provided a sensitive approach for the detection and quantitation of the individual oxygenated sterols. Oxygenated sterols detected in plasma included cholest-5-ene-3 beta,26-diol, (24S)-cholest-5-ene-3 beta,24-diol, and cholest-5-ene-3 beta,7 alpha-diol. After correction for their formation by autoxidation of cholesterol during processing of the samples, very little or none of the following sterols were observed: cholest-5-ene-3 beta,7 beta-diol, 5 alpha,6 alpha-epoxy-cholestan-3 beta-ol, 5 beta,6 beta-epoxy-cholestan-3 beta-ol, and cholestane- 3 beta, 5 alpha,6 beta-triol, and the 25-hydroxy, 22R-hydroxy, 21-hydroxy, 20 alpha-hydroxy, and 19-hydroxy derivatives of cholesterol.