Reversed-phase Medium-pressure Liquid Chromatography 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.

Screening and isolating potential α-glucosidase inhibitors from Rhizoma Coptidis by ultrafiltration LC-PDA-ESI/MS combined with high-speed countercurrent chromatography and reverse-phase medium-pressure liquid chromatography

Rhizoma Coptidis is an alkaloid-rich herbal drug used in Chinese herbal medicine. Currently, conventional methods for screening and isolating alkaloids are labor-intensive and time-consuming. In the present study, ultrafiltration liquid chromatography with photodiode array detection coupled to electrospray ionization tandem mass spectrometry (ultrafiltration liquid chromatography-photodiode array detector-electrospary ionization mass spectrometry (LC-PDA-ESI/MS)) were applied to screen and identify α-glucosidase inhibitors in R. Coptidis. High-speed countercurrent chromatography and reverse-phase medium-pressure liquid chromatography were applied to separate and isolate the active constituents. As a result, five major compounds in R. Coptidis were screened and identified as α-glucosidase inhibitors by ultrafiltration LC-PDA-ESI/MS. Five ligands, jatrorrhizine, epiberberine, coptisine, palmatine, and berberine, were isolated by reverse-phase medium-pressure liquid chromatography and high-speed countercurrent chromatography. The purities of the five compounds as determined by high-performance liquid chromatography were 76.20, 75.13, 82.24, 93.78, and 92.01%, respectively. The results indicate that systematic isolation of bioactive components in R. Coptidis guided by ultrafiltration LC-PDA-ESI/MS is a feasible and efficient technique that could be extended to separation of other enzyme inhibitors.

Potential of Pseudoshikonin I Isolated from Lithospermi Radix as Inhibitors of MMPs in IL-1β-Induced SW1353 Cells.

Pseudoshikonin I, the new bioactive constituent of Lithospermi radix, was isolated from this methanol extract by employing reverse-phase medium-pressure liquid chromatography (MPLC) using acetonitrile/water solvent system as eluents. The chemical structure was determined based on spectroscopic techniques, including 1D NMR (1H, 13C, DEPT), 2D NMR (gCOSY, gHMBC, gHMQC), and QTOF/MS data. In this study, we demonstrated the effect of pseudoshikonin I on matrix-metalloproteinase (MMPs) activation and expression in interleukin (IL)-1β-induced SW1353 chondrosarcoma cells. MMPs are considered important for the maintenance of the extracellular matrix. Following treatment with PS, active MMP-1, -2, -3, -9, -13 and TIMP-2 were quantified in the SW1353 cell culture supernatants using a commercially available ELISA kit. The mRNA expression of MMPs in SW1353 cells was measured by RT-PCR. Pseudoshikonin I treatment effectively protected the activation on all tested MMPs in a dose-dependent manner. TIMP-2 mRNA expression was significantly upregulated by pseudoshikonin I treatment. Overall, we elucidated the inhibitory effect of pseudoshikonin on MMPs, and we suggest its use as a potential novel anti-osteoarthritis agent.

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.

PREPARATIVE SEPARATION OF HYDROXYL SAFFLOWER YELLOW A AND ANHYDROSAFFLOR YELLOW B IN PLANT EXTRACT OF CARTHAMUS TINCTORIUS L. BY REVERSE-PHASE MEDIUM-PRESSURE LIQUID CHROMATOGRAPHY

Two known flavonoids—hydroxyl safflower yellow A (HSYA) and anhydrosafflor yellow B (anhydro-SYB)—were isolated and purified from plant extract of Carthamus tinctorius L. for the first time by reverse-phase medium-pressure liquid chromatography (RP-MPLC) using a mobile phase system consisting of acetonitrile and water containing 0.5% acetic acid with a Biotage® SNAP Cartridge KP- C18-HS column. A total of 2.92 g of HSYA and 1.07 g of anhydro-SYB from 9.2 g plant extract of C. tinctorius L., each at 100% and 93% as determined by high-performance liquid chromatography (HPLC), respectively. The identification of the two purified compounds was achieved by congruent retention times, the data of the liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MSn) with those of the authentic standards and literature reports. The fragmentation pathway was proposed according the LC-ESI-MSn data.

PREPARATIVE SEPARATION OF CHROMONES IN PLANT EXTRACT OF SAPOSHNIKOVIA DIVARICATA BY REVERSE-PHASE MEDIUM-PRESSURE LIQUID CHROMATOGRAPHY AND HIGH PERFORMANCE COUNTER-CURRENT CHROMATOGRAPHY

Five known chromones were isolated and purified from Saposhnikovia divaricata for the first time by a combination of reverse-phase medium-pressure liquid chromatography (RP-MPLC) and high performance counter-current chromatography (HPCCC). The n-butanol extracts of S. divaricata were preliminarily chromatographed by RP-MPLC and further isolated by preparative HPCCC. A total of 582.8 mg of prim-O-glucosylcimifugin, 228.9 mg of 4′-O-β-D-glucosyl-5-O- methylvisamminol, 113 mg of cimifugin, 14.5 mg of sec-O-glucosylhamaudol, and 4.68 mg of 5-O-methylvisamminol were purified from 7.48 g of the n-butanol extract of S. divaricata, each at over 95% purity as determined by high performance liquid chromatography (HPLC). The identification of the five purified compounds was achieved by congruent retention times, the data of the liquid chromatography- electrospray ionization mass spectrometry (LC-ESI-MSn), and nuclear magnetic resonance spectroscopy (NMR) with those of the authentic standards and literature reports.

Preparative purification of peoniflorin and albiflorin from peony rhizome using macroporous resin and medium‐pressure liquid chromatography

Peoniflorin (PF) and albiflorin (AF) are two principal components of Paeonia species, which exhibit various biological activities such as improvement of blood circulation and immunoregulating function. To further utilization of waste parts of peony plants, an efficient method for preparative purification of these two ingredients from white peony rhizome was developed based on macroporous resin (MAR) and medium-pressure liquid chromatography (MPLC). The separation characteristics of nine typical MARs were investigated by static adsorption/desorption experiments, and LX38 was revealed as optimal one. Further static experiments with LX38 resin indicated that the adsorbents fitted well to the pseudo-second-order kinetics model and both Langmuir and Freundlich isotherm models. Based on the optimal process parameters, a large-scale preparation was successfully applied. After one run treatment with LX38, the contents of PF and AF were increased 15-fold to 24.5 and 16.8% in the refined extract, respectively. Both purified compounds were obtained from refined extract by reversed-phase MPLC at second-stage separation. The process developed is better because of its low cost, high efficiency, and procedural simplicity making it a potential approach for large-scale production of PF and AF for their further applications in functional foods and pharmaceuticals.

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.

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.

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.