Quantitative Analysis of Caffeoylquinic Acids and Styrylpyrones in Sweetia panamensis Bark by UPLC

Six secondary metabolites from the methanolic extract of Sweetia panamensis (Fabaceae) bark were isolated and characterised. Along with the pyrones desmethylangonine β-d-O-glucopyranoside and desmethylangonine β-d-O-glucopyranosyl-(1→6)-O-β-d-glucopyranoside, already reported in this species, 5-O-caffeoylquinic acid (chlorogenic acid), 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid and the isoflavonoid 5-O-methylgenistein 7-O-β-d-glucopyranoside were isolated for the first time from S. panamensis. Additionally, an LC-ESI-MS qualitative analysis was performed and an ultra performance liquid chromatography (UPLC) method was developed and validated for the determination of these compounds. The UPLC method was applied to the quantitative analysis of plant samples. Pyrones and caffeoylquinic acids resulted to be the main compounds in the extract; in particular desmethylangonine β-d-O-glucopyranosyl-(1→6)-O-β-d-glucopyranoside was the most abundant compound.

     

Synthesis of N-glycyl-β-glycopyranosyl amines,derivatives of natural oligosaccharides — glucose analogs of Lex antigen

Treatment of the natural tri-, tetra-, and pentasaccharides, β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-Glcp, α-l-Fucp-(1→2)-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-Glcp, and α-l-Fucp-(1→2)-[α-d-GalNAcp-(1→3)]-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-Glcp, which are glucose analogs of Le^x, with ammonium carbamate in aqueous methanol gave the corresponding β-glycopyranosyl amines. After their N-acylation with N-Z-glycine N-hydroxysuccinimidyl ester (Z is benzyloxycarbonyl) with subsequent hydrogenolytic removal of Z-group, corresponding N-glycyl-β-glycopyranosyl amines were obtained in yields up to 70%.     

Asteropsiside A and other asterosaponins from the starfish Asteropsis carinifera

Three asterosaponins were isolated from the tropical starfish Asteropsis carinifera: a new one, asteropsiside A, and two known ones, regularoside A and thornasteroside A. The structure of the new compound was established using 2D NMR spectroscopy and ESI mass spectrometry as the sodium salt of 3-O-sulfonato-(20E)-6-O-{β-d-fucopyranosyl-(1→2)-β-d-galactopyranosyl-(1→4)-[β-d-quinovopyranosyl-(1→2)]-β-d-xylopyranosyl-(1→3)-β-d-quinovopyranosyl}-3β,6α-dihydroxy-5α-cholesta-9(11),20(22)-dien-23-one. Regularoside A and thornasteroside A were shown to display the ability to inhibit the growth of the T-47D and RPMI-7951 tumor cell colonies in vitro.     

Synthesis of β-d-glucopyranosyl- and β-d-galactopyranosylamines from 4-bromo-3-methylaniline and 2-amino-5-bromopyridine

The possibility of coupling of d-glucose and d-galactose with 4-bromo-3-methylaniline, 2,4,6-tribromoaniline, and 2-amino-5-bromopyridine was studied. The substituent in the aromatic ring was found to influence the conditions and possibility of the reaction. The yields of β-d-glucopyranosyl- and β-d-galactopyranosylamines from 4-bromo-3-methylaniline and 2-amino-5-bromopyridine were 50–65%; 2,4,6-tribromoaniline did not react at all.     

Carbohydrate recognition of symmetrical tripodal receptor type tris(2-aminoethyl)amine derivatives

Carbohydrate recognition of some bioactive symmetrical tripodal receptor type tris(2-aminoethyl)amine (TAEA) derivatives was investigated. In calorimetric experiments, the highest binding constant (Ka) of compound C (C₃₅H₄₉N₅O₄S) with methyl α-d-mannopyranoside was Ka = 858 M^?1 with 1:1 stoichiometry. Formation of hydrogen bonds in binding between symmetrical tripodal receptor type compound C and sugars was suggested by the large negative values of ?H° (=?34 to ?511 kJ mol^?1). In a comparison of each set of α- and β-anomers of some monosaccharides (methyl α/β-d-galactopyranoside, methyl α/β-d-glucopyranoside, and methyl α/β-l-fucopyranoside), compound C showed that the binding constant of β-anomer was larger than that of the corresponding α-anomer, indicating higher β-anomer selectivity. The calculated energy-minimized structure of the complex of compound C with guest methyl α-d-mannopyranoside is also presented. The experimental results obtained from this work indicated that symmetrical tripodal receptor type TAEA derivative C has a lectin-like carbohydrate recognition property.     

In vitro and in silico inhibition of angiotensin-converting enzyme by carbohydrates and cyclitols

Fifteen carbohydrates (d-mannose, d-glucose, d-galactose, methyl-α-d-glucose, l-rhamnose, d-xylose, d-fructose, d-arabinose, dulcitol, mannitol, β-maltose, α-lactose, melibiose, sucrose, and raffinose) and four cyclitols [l-(+)-bornesitol, myo-inositol, per-O-acetyl-1-l-(+)-bornesitol, and quinic acid] were assayed for in vitro ACE inhibition. Of these molecules, per-O-Acetyl-1-l-(+)-bornesitol, quinic acid, methyl-α-d-glucose, d-rhamnose, raffinose, and the disaccharides were determined to be either inactive or weak ACE inhibitors, whereas l-(+)-bornesitol, d-galactose, d-glucose, and myo-inositol exhibited significant ACE inhibition. Molecular docking studies were performed to investigate interactions between active compounds and human ACE (Protein Data Bank, PDB 1O83). The results of various calculations showed that all active sugars bind to the same enzyme region, which is a tunnel directed towards the active site. With the exception of myo-inositol (K _i = 13.95 μM, IC₅₀ = 449.2 μM), the active compounds presented similar K _i and IC₅₀ values. d-Galactose (K _i = 19.6 μM, IC₅₀ = 35.7 μM) and l-(+)-bornesitol (K _i = 25.3 μM, IC₅₀ = 41.4 μM) were the most active compounds, followed by d-glucose (K _i = 32.9 μM, IC₅₀ = 85.7 μM). Our docking calculations are in agreement with the experimental data and show a new binding region for sugar-like molecules, which may be explored for the development of new ACE inhibitors.     

Preparative Isolation and Purification of Flavonoids and Protocatechuic Acid from Sea Buckthorn Juice Concentrate (Hippophaë rhamnoides L. ssp. rhamnoides) by High-Speed Counter-Current Chromatography

High-speed counter-current chromatography (HSCCC)—a support free all liquid–liquid chromatography technique—has been successfully used for the preparative isolation of isorhamnetin 3-O-β-d-glucoside, isorhamnetin 3-O-β-rutinoside, quercetin 3-O-β-d-glucoside, syringetin 3-O-β-d-glucoside and protocatechuic acid from sea buckthorn juice concentrate (Hippophaë rhamnoides L. ssp. rhamnoides, Elaeagnaceae). The preparative HSCCC instrument was a multilayer coil planet centrifuge equipped with three preparative coils. Separation was performed with a two phase solvent system (n-hexane–n-butanol–water, 1:1:2 v/v/v) in ‘head-to-tail’ mode. Each injection of 4.1 g crude ethyl acetate extract yielded isorhamnetin 3-O-β-d-glucoside (95 mg), isorhamnetin 3-O-β-rutinoside (10 mg), quercetin 3-O-β-d-glucoside (5 mg), and protocatechuic acid (34 mg) with purities >98%. The flavonoid syringetin 3-O-β-d-glucoside (2 mg) was a novel compound for H. rhamnoides. Chemical structures of all compounds were determined by HPLC–ESI–MS–MS, 1D-NMR (¹H, ¹³C, DEPT 135) spectroscopy and for elucidation of glycosidic linkages 2D-NMR (HMBC) spectroscopy was used.     

Concise synthesis of two natural steroidal glycosides isolated from <Emphasis Type="Italic">Allium schoenoprasum</Emphasis>

Two natural steroidal glycosides, diosgenin 3-O-α-l-rhamnopyranosyl-(1→2)-[β-d-glucopyranosyl-(1→4)]-β-d-glucopyranoside (1) and laxogenin 3-O-α-l-rhamnopyranosyl-(1→2)-[β-d-glucopyranosyl-(1→4)]-β-d-glucopyranoside (2) with important cytotoxic activity against the HCT 116 and HT-29 human colon cancer cell lines have been efficiently synthesized via straightforward sequential glycosylation reaction with the combined use of N-phenyltrifluoroacetimidates and trichloroacetimidates donors at room temperature. All structures of the synthesized new compounds were identified by ¹H NMR, ¹³C NMR and HRMS spectra.     

A new steroidal glycoside from Corypha taliera Roxb., a globally endangered species

The reversed-phased HPLC analysis of the methanol extract of the pericarp of C. taliera Roxb. (Talipalm), a rare species of Arecaceae family, afforded a new steroidal glycoside, β-sitosterol-3-O-α-l-rhamnopyranosyl-(1→4)-β-d-xylopyranosyl-(1→4)-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside (1). The structure of the compound was elucidated unequivocally by UV, IR, HR-ESI-MS, ¹H and ¹³C NMR spectroscopic studies.     

Assignment of the NMR Parameters of the Branch-Point Trisaccharide of Ahylopectin Using 2-D NMR Spectroscopy at 500 MHz

Abstract

The proton and carbon nuclear magnetic resonance spectroscopic data for methyl 4-O-α-d-glucopyranosyl-[6-O-a-u-glucopyranosyl]-β-d-glucopyranoside (1), a model for the branch-point trisacch-aride of amylopectin, have been analysed using 2-D-heteronuclear correlated spectroscopy. Similar data are presented for the related disaccharide structures methyl β-d-maltopyranoside and β-d-isomal topyranoside.     

Synthesis ofS-(Carboxymethyl)-d-cysteine by 3-chloro-d-alanine chloride-lyase ofPseudomonas putida CR 1-1

S-(Carboxymethyl)-d-cysteine, which is an important component of semisynthetic cephalosporin, MT-141, was enzymatically synthesized.S-(Ethoxy-carbonyl-methyl)-d-cystein was synthesized from 3-chloro-d-alanine and ethyl thioglycolate by the β-replacement reaction of 3-chloro-d-alanine chloride-lyase fromPseudomonas putida CR 1-1 and subsequently hydrolyzed by alkali. The synthesizedS-(carboxymethyl)-d-cysteine was isolated from a large scale reaction mixture and identified physicochemically. The reaction conditions for the synthesis ofS-(ethoxycarbonylmethyl)-d-cysteine were optimized using resting cells ofP. putida CR 1-1.     

Synthesis of (1→5)-β-d-galactofuranan and cyclo[(1→5)-β-d-galactofurano]oligosaccharides

Synthesis of a linear (1→5)-β-d-galactofuranan was accomplished by trityl-cyanoethylidene polycondensation. On 10-fold reduction in the monomer concentration, the condensation products are cyclic oligosaccharides; the formation of 1,5-anhydro-α-d-galactofuranose was also demonstrated.     

Characterization of a d-Stereoselective Aminopeptidase (DamA) Exhibiting Aminolytic Activity and Halophilicity from Aspergillus oryzae

β-Aminopeptidases exhibit both hydrolytic and aminolytic (peptide bond formation) activities and have only been reported in bacteria. We identified a gene encoding the β-aminopeptidase homolog from a genome database of the filamentous fungus Aspergillus oryzae. The gene was overexpressed in A. oryzae, and the resulting recombinant enzyme was purified. Apart from bacterial homologs [β-Ala-para-nitroanilide (pNA)], the enzyme preferred d-Leu-pNA and d-Phe-pNA as substrates. Therefore, we designated this gene as d-stereoselective aminopeptidase A (damA). The purified recombinant DamA was estimated to be a hexamer and was composed of two subunits with molecular masses of 29.5 and 11.5 kDa, respectively. Optimal hydrolytic activity of DamA toward d-Leu-pNA was observed at 50 °C and pH 8.0. The enzyme was stable up to 60 °C and from pH 4.0–11.0. DamA also exhibited aminolytic activity, producing d-Leu-d-Leu-NH₂ from d-Leu-NH₂ as a substrate. In the presence of 3.0 M NaCl, the amount of pNA liberated from d-Leu-pNA by DamA was 3.1-fold higher than that in the absence of NaCl. Thus, DamA is a halophilic enzyme. The enzyme was utilized to synthesize several hetero-dipeptides containing a d-amino acid at the N-terminus as well as physiologically active peptides.     

Enzymatic Oxidation and Separation of Various Saccharides with Immobilized Glucose Oxidase

Glucose oxidase from Aspergillus niger, the specific enzyme for β-d-glucose oxidation, can also oxidize other related saccharides at very slow or negligible rates. The present study aimed to compare the kinetics of d-glucose oxidation using immobilized glucose oxidase on bead cellulose for the oxidation of related saccharides using the same biocatalyst. The significant differences were observed between the reaction rates for d-glucose and other saccharides examined. As a result, k _cat/K _M ratio for d-glucose was determined to be 42 times higher than d-mannose, 61.6 times higher than d-galactose, 279 times higher than d-xylose, and 254 times higher than for d-fructose and d-cellobiose. On the basis of these differences, the ability of immobilized glucose oxidase to remove d-glucose from d-cellobiose, d-glucose from d-xylose, and d-xylose from d-lyxose was examined. Immobilized catalase on Eupergit and mixed with immobilized glucose oxidase on bead cellulose or co-immobilized with glucose oxidase on bead cellulose was used for elimination of hydrogen peroxide from the reaction mixture. The accelerated elimination of d-glucose and d-xylose in the presence of co-immobilized catalase was observed. The co-immobilized glucose oxidase and catalase were able to decrease d-glucose or d-xylose content to 0–0.005% of their initial concentrations, while a minimum decrease of low oxidized saccharides d-xylose, d-cellobiose, and d-lyxose, respectively, was observed.     

Polysaccharides of algae 65. Unusual polysaccharide composition of the pacific brown alga Punctaria plantaginea

Quantitative determination revealed the presence of storage glucan (6.0%), fucoidan (19.2%) and alginate (12.7%) in the biomass of the brown alga Punctaria plantaginea collected from the Sea of Japan. The polysaccharides were isolated from the alga by fractional extraction followed by additional purification procedures. Unlike the well-known laminarans the storage polysaccharide from P. plantaginea was shown to be a linear (1→6)-β-d-glucopyranan, which is new for brown alga. The content of guluronic acid (G) residues in the alginate molecules exceeded the content of mannuronic acid residues (M), M/G = 0.5. Poly-G and poly-MG blocks were isolated from the products of partial hydrolysis of alginic acid; however, a heterogeneous mixture of polysaccharide fragments was obtained instead of the expected poly-M fraction. Preliminary data suggests that fucoidan from this alga is a new for brown algae type of sulfated polysaccharide (xylofucan) with a main backbone built of α-l-fucopyranose residues. This chain contains multiple sulfate groups and single non-sulfated β-d-xylopyranose residues as substituents.     

β-d-Xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-Directed Mutagenesis, Alternate Substrates, and Active-Site Inhibitor

β-d-Xylosidase/α-l-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme known for catalyzing hydrolysis of 1,4-β-d-xylooligosaccharides to d-xylose. Catalysis and inhibitor binding by the GH43 β-xylosidase are governed by the protonation states of catalytic base (D14, pK _a 5.0) and catalytic acid (E186, pK _a 7.2). Biphasic inhibition by triethanolamine of E186A preparations reveals minor contamination by wild-type-like enzyme, the contaminant likely originating from translational misreading. Titration of E186A preparations with triethanolamine allows resolution of binding and kinetic parameters of the E186A mutant from those of the contaminant. The E186A mutation abolishes the pK _a assigned to E186; mutant enzyme binds only the neutral aminoalcohol $ \left( {{\text{pH}} - {\text{independent}}\;K_{\text{i}}^{\text{triethanolamine}} = 19\,{\text{mM}}} \right) $ , whereas wild-type enzyme binds only the cationic aminoalcohol $ \left( {{\text{pH}} - {\text{independent}}\;K_{\text{i}}^{\text{triethanolamine}} = 0.065\,{\text{mM}}} \right) $ . At pH 7.0 and 25°C, relative kinetic parameter, $ k_{\text{cat}}^{\text{4NPX}}/k_{\text{cat}}^{\text{4NPA}} $ , for substrates 4-nitrophenyl-β-d-xylopyranoside (4NPX) and 4-nitrophenyl-α-l-arabinofuranoside (4NPA) of E186A is 100-fold that of wild-type enzyme, consistent with the view that, on the enzyme, protonation is of greater importance to the transition state of 4NPA whereas ring deformation dominates the transition state of 4NPX.     

LC-MS Determination and Pharmacokinetic Study of Luteolin-7-O-β-d-glucoside in Rat Plasma after Administration of the Traditional Chinese Medicinal Preparation Kudiezi Injection

A rapid and sensitive LC-MS method has been developed for the determination of luteolin-7-O-β-d-glucoside in rat plasma after solvent extraction. Separation was on an Elite Hypersil ODS2 column (250 mm × 4.6 mm i.d., 5 μm) with a mobile phase of acetonitrile-0.3% acetic acid (26:74, v/v). The samples were analyzed by using positive electrospray ionization MS in selected ion monitoring mode. The selected ions for luteolin-7-O-β-d-glucoside and the internal standard, isoquercitrin, were m/z 448.95 and m/z 464.95. Good linearity was observed over the range of 20–2,000 ng mL^?1 with a lower limit of quantification of 20 ng mL^?1. No interference peaks or matrix effects were observed. The validated method was applied to the pharmacokinetic study of luteolin-7-O-β-d-glucoside in rat plasma after intravenous administration of Kudiezi Injection.     

Analysis of Three Flavonoids in Oxytropis kansuensis Bunge by RP-LC–DAD Coupled with Weighted Least-Squares Linear Regression

For the first time an RP-LC method with diode-array detection has been developed for simultaneous analysis of three flavonoids [rhamnocitrin-3-O-β-d-galactopyranoside-4′-O-β-d-glucospyranoside (RGG), rhamnocitrin-3-O-β-d-galactopyranoside (RG), and 10-methoxymedicarpin (MC)] in a methanol extract of Oxytropis kansuensis Bunge whole plant. Separation was achieved on an ODS column within 18 min. The effect of mobile phase pH on separation of the three flavonoids was investigated. Compared with relative errors obtained by use of least-squares linear regression and logarithmic regression for data processing, weighted least-squares linear regression was more accurate. Response was a linear function of concentration in the ranges 0.0091–3.4, 0.013–4.9, and 0.0085–3.2 mg mL^?1 for RGG, RG, and MC, respectively, with correlation coefficients >0.9997. The amounts of the three flavonoids in O. kansuensis Bunge were successfully analyzed with satisfactory repeatability and recovery.     

Molecular dynamics simulation study of xyloglucan adsorption on cellulose surfaces: effects of surface hydrophobicity and side-chain variation

The effect of surface hydrophobicity and side-chain variation on xyloglucan adsorption onto cellulose microfibrils (CMF) is investigated via molecular dynamics simulations. A molecular model of CMF with (100), (010), (1–10), (110) and (200) crystal faces was built. We considered xylogluco-oligosaccharides (XGO) with three repeating units, namely (XXXG)_3, (XXLG)₃, and (XXFG)₃ (where each (1,4)-β-d-glucosyl residue in the backbone is given a one-letter code according to its substituents: G = β-d-Glc; X = α-d-Xyl-(1,6)-β-d-Glc; L = β-d-Gal-(1,2)-α-d-Xyl-(1,6)-β-d-Glc; F = α-l-Fuc-(1,2)-β-d-Gal-(1,2)-α-d-Xyl-(1,6)-β-d-Glc). Our work shows that (XXXG)₃ binds more favorably to the CMF (100) and (200) hydrophobic surfaces than to the (110), (010) and (1–10) hydrophilic surfaces. The origin of this behavior is attributed to the topography of hydrophobic CMF surface, which stabilizes (XXXG)₃ in flat conformation. In contrast, on the rough hydrophilic CMF surface (XXXG)₃ adopts a less favorable random-coil conformation to facilitate more hydrogen bonds with the surface. Extending the xyloglucan side chains from (XXXG)₃ to (XXLG)₃ hinders their stacking on the CMF hydrophobic surface. For (XXFG)₃, the interaction with the hydrophobic surface is as strong as (XXXG)₃. All three XGOs have similar binding to the hydrophilic surface. Steered molecular dynamics simulation was performed on an adhesive model where (XXXG)₃ was sandwiched between two CMF hydrophobic surfaces. Our analysis suggests that this sandwich structure might help provide mechanical strength for plant cell walls. Our study relates to a recently revised model of primary cell walls in which extensibility is largely determined by xyloglucan located in limited regions of tight contact between CMFs.     

Molecular Cloning and Expression of a Novel β-Glucosidase Gene from Phialophora sp. G5

A novel β-glucosidase gene, bgl1G5, was cloned from Phialophora sp. G5 and successfully expressed in Pichia pastoris. Sequence analysis indicated that the gene consists of a 1,431-bp open reading frame encoding a protein of 476 amino acids. The deduced amino acid sequence of bgl1G5 showed a high identity of 85 % with a characterized β-glucosidase from Humicola grisea of glycoside hydrolase family 1. Compared with other fungal counterparts, Bgl1G5 showed similar optimal activity at pH 6.0 and 50 °C and was stable at pH 5.0–9.0. Moreover, Bgl1G5 exhibited good thermostability at 50 °C (6 h half-life) and higher specific activity (54.9 U mg^–1). The K _m and V _max values towards p-nitrophenyl β-d-glucopyranoside (pNPG) were 0.33 mM and 103.1 μmol?min^–1?mg^–1, respectively. The substrate specificity assay showed that Bgl1G5 was highly active against pNPG, weak on p-nitrophenyl β-d-cellobioside (pNPC) and p-nitrophenyl-β-d-galactopyranoside (ONPG), and had no activity on cellobiose. This result indicated Bgl1G5 was a typical aryl β-glucosidase.