Unusual stilbenoids and a stilbenolignan from seeds of Syagrus romanzoffiana

Stilbenoids, syagrusins A-B (1-2), and a stilbenolignan, 5-hydroxyaiphanol (3), along with three known phenylpropanoids (4-6), were isolated from seeds of Syagrus romanzoffiana. Compounds 1 and 2 possess unusual 1,4,4a,9a-tetrahydrofluoren-9-one and bicyclo[3.3.0]octanedione skeletons, respectively, whereas compound 3 is a stilbenolignan belonging to a very rare structural class of plant secondary metabolites. Their structures were elucidated by spectroscopic analyses. Compounds 1-3 exhibited moderate inhibitory activity against α-glucosidase with IC₅₀ values of 16.9 μM (1), 23.7 μM (2) and 12.8 μM (3), respectively.     

alpha-Glucosidase inhibitory pentacyclic triterpenes from the stem bark of Fagara tessmannii (Rutaceae)

In addition to fatty acids, a mixture of sterols (beta-sitosterol, stigmasterol, campesterol and stigmastanol), lupeol, arctigenin methylether, sesamin, vanillic acid (1), 2,6-dimethoxy-1,4-benzoquinone (2), betulinic acid and two pentacyclic triterpene acetates were isolated from Fagara tessmannii Engl. They were identified as 3beta-acetoxy-16beta-hydroxybetulinic acid (3a) and 3beta,16beta-diacetoxybetulinic acid (3b), and their structures were established using 1 and 2D NMR spectra and by comparison with published data. Two derivatives of the compounds were prepared. Some isolated compounds were evaluated for their antifungal and antibacterial activities. Compounds 1 and 3a showed significant inhibition of alpha-glucosidase.     

alpha-Glucosidase inhibitory constituents from stem bark of Terminalia superba (Combretaceae)

The CH(2)Cl(2)/CH(3)OH (1/1) extract of the dried stem bark of Terminalia superba afforded two compounds, (7S,8R,7'R,8'S)-4'-hydroxy-4-methoxy-7,7'-epoxylignan and meso-(rel 7S,8R,7'R,8'S)-4,4'-dimethoxy-7,7'-epoxylignan along with 11 known compounds. The structures of the compounds were established by analysing the spectroscopic data and also comparing it with the data of previously known analogues. All the isolated compounds were evaluated for their glycosidase inhibition activities. Gallic acid and methyl gallate showed significant alpha-glucosidase inhibition activity.     

Isolation of alpha-glusosidase inhibitors from hyssop (Hyssopus officinalis)

alpha-Glucosidase inhibitory activity was found in aqueous methanol extracts of dried hyssop (Hyssopus officinalis) leaves. Active principles against alpha-glucosidase, prepared from rat small intestine acetone powders, were isolated and characterized. The structures of these isolated compounds were determined to be (7S, 8S)-syringoylglycerol-9-O-(6'-O-cinnamoyl)-beta-D-glucopyranoside and (7S, 8S)-syringoylglycerol 9-O-beta-D-glucopyranoside by analysis of physical and spectroscopic data (FDMS, 1H NMR, 13C NMR, HMQC, and HMBC experiments) together with chemical syntheses.     

Acylated flavonol monorhamnosides, alpha-glucosidase inhibitors, from Machilus philippinensis

Bioassay-guided fractionation and isolation of the active constituents from the leaf extract of Machilusphilippinense Merr. yielded two active compounds, kaempferol-3-O-alpha-l-rhamnopyranoside 3',4'-di-E-p-coumaroic acid ester (1) and 3'-E,4'-Z-di-p-coumaroic acid ester (2) when tested against a Bacillus stearothermophilus, a alpha-glucosidase type IV. The IC(50) values of 1 and 2 were 6.10 and 1.00muM, respectively. Further application of the HPLC-SPE-NMR hyphenated technique in the on-line characterization of other active ingredients present in the CH(2)Cl(2) - soluble fraction led to identification of luteolin (3) and seven additional 3-O-(coumaroyl-rhamnopyranosyl)-flavonols (4-10). Their structures were determined mainly by (1)H NMR spectroscopic analyses. Among the compounds identified, compounds 2, 4, 5, and 7 were hitherto unknown natural products.     

Potent alpha-glucosidase inhibitors purified from the red alga Grateloupia elliptica

Diabetes mellitus is a most serious and chronic disease whose incidence rates are increasing with incidences of obesity and aging of the general population over the world. One therapeutic approach for decreasing postprandial hyperglycemia is to retard absorption of glucose by inhibition of α-glucosidase. Two bromophenols, 2,4,6-tribromophenol and 2,4-dibromophenol, were purified from the red alga Grateloupia elliptica. IC₅₀ values of 2,4,6-tribromophenol and 2,4-dibromophenol were 60.3 and 110.4 μM against Saccharomyces cerevisiae α-glucosidase, and 130.3 and 230.3 μM against Bacillus stearothermophilus α-glucosidase, respectively. In addition, both mildly inhibited rat-intestinal sucrase (IC₅₀ of 4.2 and 3.6 mM) and rat-intestinal maltase (IC₅₀ of 5.0 and 4.8 mM). Therefore, bromophenols of G. elliptica have potential as natural nutraceuticals to prevent diabetes mellitus because of their high α-glucosidase inhibitory activity.     

椰子粗腿豆象的检疫鉴定

南京禄口机场海关截获了一批旅客从巴西携带入境的金山葵种子,送南京海关实验室检出豆象。通过形态学和分子生物学方法,对该虫进行了物种鉴定。鉴定结果表明该虫为椰子粗腿豆象。该虫在我国尚无分布记录,为国内口岸首次截获。该虫一般随带果的棕榈寄主种苗进行远距离传播,一旦传入我国,将对我国的农林安全造成巨大威胁,各口岸须加强对进境种苗的检验检疫。     

Purification and characterization of an alpha-glucosidase from germinating millet seeds

An α-glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) was isolated from germinating millet (Panicum miliaceum L.) seeds by a procedure that included ammonium sulfate fractionation, chromatography on CM-cellulofine/Fractogel EMD SO₃, Sephacryl S-200 HR and TSK gel Phenyl-5 PW, and preparative isoelectric focusing. The enzyme was homogenous by SDS-PAGE. The molecular weight of the enzyme was estimated to be 86,000 based on its mobility in SDS-PAGE and 80,000 based on gel filtration with TSKgel super SW 3000, which showed that it was composed of a single unit. The isoelectric point of the enzyme was 8.3. The enzyme readily hydrolyzed maltose, malto-oligosaccharides, and α-1,4-glucan, but hydrolyzed polysaccharides more rapidly than maltose. The K_m value decreased with an increase in the molecular weight of the substrate. The value for maltoheptaose was about 4-fold lower than that for maltose. The enzyme preferably hydrolyzed amylopectin in starch, but also readily hydrolyzed nigerose, which has an α-1,3-glucosidic linkage and exists as an abnormal linkage in the structure of starch. In particular, the enzyme readily hydrolyzed millet starch from germinating seeds that had been degraded to some extent.     

Purification and biochemical characterisation of a membrane-bound alpha-glucosidase from the parasite Entamoeba histolytica

An alpha-glucosidase was solubilised from a mixed membrane fraction of Entamoeba histolytica and purified to homogeneity by a two-step procedure consisting of ion exchange chromatography in a Mono Q column and affinity chromatography in concanavalin A-sepharose. Although the enzyme failed to bind the lectin, this step rendered a homogenous and more stable enzyme preparation that resolved into a single polypeptide of 55 kDa after SDS-PAGE. As measured with 4-methylumbelliferyl-alpha-D-glucopyranoside (MUalphaGlc) as substrate, glycosidase activity was optimum at pH 6.5 with different buffers and at 45 degrees C. Although the enzyme preferentially hydrolysed nigerose (alpha1,3-linked), it also cleaved kojibiose (alpha1,2-linked), which was the second preferred substrate, and to a lesser extent maltose (alpha1,4), trehalose (alpha1,1) and isomaltose (alpha1,6). Activity on alpha1,3- and alpha1,2-linked disaccharides was strongly inhibited by the glycoprotein processing inhibitors 1-deoxynojirimycin and castanospermine but was unaffected by australine. Glucose and particularly 3-deoxy-D-glucose and 6-deoxy-D-glucose were strong inhibitors of activity, whereas 2-deoxy-D-glucose and other monosaccharides had no effect. Enzyme activity on MUalphaGlc was very sensitive to inhibition by diethylpyrocarbonate suggesting a critical role of histidine residues in enzyme catalysis. Other amino acid modifying reagents such as N-ethylmaleimide and N-(3-dimethylaminopropyl)-N'ethylcarbodiimide showed a moderate effect or none at all, respectively. Results are discussed in terms of the possible involvement of this glycosidase in N-glycan processing.     

Isolation and primary structures of seven serine proteinase inhibitors from Cyclanthera pedata seeds

Seven new trypsin inhibitors, CyPTI I–VII, were purified from ripe seeds of Cyclanthera pedata by affinity chromatography on immobilized chymotrypsin in the presence of 5 M NaCl followed by preparative native PAGE at pH 8.9. The CyPTIs (Cyclanthera pedata trypsin inhibitors) belong to a well-known squash inhibitor family. They contain 28–30 amino acids and have molecular weights from 3031 to 3367 Da. All the isolated inhibitors strongly inhibit bovine β-trypsin (K_a > 10¹¹ M^− 1) and, more weakly, bovine α-chymotrypsin (K_a ≈ 10⁴–10⁶ M^− 1). In the presence of 3 M NaCl the association constants of CyPTIs with α-chymotrypsin increased a few hundred fold. Taking advantage of this phenomenon, a high concentration of NaCl was used to isolate the inhibitors by affinity chromatography on immobilized chymotrypsin. It was found that although one of them, CyPTI IV, had split the Asn25–Gly26 peptide bond, its inhibitory activity remained unchanged. The hydrolyzed bond is located downstream of the reactive site. Presumably, the inhibitor is a naturally occurring, double-chain protein arising during posttranslational modifications.     

Ganoderol B: A potent α-glucosidase inhibitor isolated from the fruiting body of Ganoderma lucidum

α-Glucosidase inhibitor has considerable potential as a diabetes mellitus type 2 drug because it prevents the digestion of carbohydrates. The search for the constituents reducing α-glucosidase activity led to the finding of active compounds in the fruiting body of Ganoderma lucidum. The CHCl₃ extract of the fruiting body of G. lucidum was found to show inhibitory activity on α-glucosidase in vitro. The neutral fraction, with an IC₅₀ of 88.7 μg/ml, had stronger inhibition than a positive control, acarbose, with an IC₅₀ of 336.7 μg/ml (521.5 μM). The neutral fraction was subjected to silica gel column chromatography and repeated p-HPLC to provide an active compound, (3β,24E)-lanosta-7,9(11),24-trien-3,26-diol (ganoderol B). It was found to have high α-glucosidase inhibition, with an IC₅₀ of 48.5 μg/ml (119.8 μM).     

Glaucacyclopeptide A from the seeds of Annona glauca

The seeds of Annona glauca furnished two cyclopeptides one of which is novel. The structure was elucidated on the basis on mass spectrometry, 2D NMR methods and amino acids analysis.     

Three 1-thio-beta-D-glucopyranosides from the seeds of Afrostyrax lepidophyllus Mildbr

Three new 1-thioglycosides namely methylthiomethyl 1-thio-beta-D-glucopyranoside (Afrostyraxthioside A), methylsulfonylmethyl 1-thio-beta-D-glucopyranoside (Afrostyraxthioside B) and methylsulfonylmethylthiomethyl 1-thio-beta-D-glucopyranoside (Afrostyraxthioside C) were isolated from the seeds of Afrostyrax lepidophyllus Mildbr. Their structures were mainly elucidated by using one- and two-dimensional NMR and mass spectroscopies and also by an efficient one-step synthesis. Moreover, Afrostyraxthiosides A, B and C constitute a new subclass of 1-thioglycosides isolated from natural sources.     

An antifungal peptide from Fagopyrum tataricum seeds

A major trypsin inhibitor was isolated and characterized from the seeds of the tartary buckwheat (Fagopyrum tataricum) (FtTI) by ammonium sulfate precipitation, ion exchange chromatography and centrifugal ultrafiltration. SDS-PAGE analysis under reducing condition showed that FtTI is a single polypeptide chain with a molecular mass of approximately 14 kDa. The complete amino acid sequence of FtTI was established by automatic Edman degradation and mass spectrometry. It was found that the trypsin inhibitor molecule consists of 86 amino acid residues containing two disulfide bonds which connect Cys⁸ to Cys⁶⁵ and Cys⁴⁹ to Cys⁵⁸. The active site of the inhibitor was found to contain an Asp⁶⁶-Arg⁶⁷ bond. MALDI-TOF analysis showed that FtTI has two isoforms (Mr: 11.487 and 13.838 kDa). Dixon plots revealed a competitive inhibition of trypsin with inhibition constants (Ki) of 1.6 nM. Analysis of the amino acid sequence suggests that FtTI is a member of the protease inhibitor I family. What is more, FtTI exhibited strong inhibitory activity against phytopathogenic fungi.     

Characterization of the P5 subfamily of P-type transport ATPases in mice

In mammals, the most poorly understood P-type ATPases are those of the P(5) subfamily. To begin characterization of the mammalian P(5)-ATPases, BLAST searches of DNA sequence databases were performed. Five genes were identified in the mouse, human, dog, and rat genomes, and the coding sequences of the mouse genes, termed Atp13a1-Atp13a5, were determined. The intron/exon organization of Atp13a1 differs entirely from those of Atp13a2-5, which are closely related. Amino acid sequence comparisons between the five mouse and two yeast P(5)-ATPases suggest that Atp13a1 is orthologous to the yeast Cod1 gene and that Atp13a2-5 are orthologous to yeast Yor291w. Northern blot analysis showed that Atp13a1, Atp13a2, and Atp13a3 mRNAs were expressed in all mouse tissues, whereas Atp13a4 and Atp13a5 mRNAs were restricted to brain and stomach. While the substrate specificity of these transporters is unknown, their importance is underscored by the presence of homologs in fish, insects, worms, and other eukaryotes.     

Are the effects of α-glucosidase inhibitors on cardiovascular events related to elevated levels of hydrogen gas in the gastrointestinal tract?

The major side-effect of treatment with α-glucosidase inhibitors, flatulence, occurs when undigested carbohydrates are fermented by colonic bacteria, resulting in gas formation. We propose that the cardiovascular benefits of α-glucosidase inhibitors are partly attributable to their ability to neutralise oxidative stress via increased production of H₂ in the gastrointestinal tract. Acarbose, which is an α-glucosidase inhibitor, markedly increased H₂ production, with a weaker effect on methane production. Our hypothesis is based on our recent discovery that H₂ acts as a unique antioxidant, and that when inhaled or taken orally as H₂-dissolved water it ameliorates ischaemia-reperfusion injury and atherosclerosis development.     

Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: isolation, characterization and chemical synthesis

Five serine proteinase inhibitors (Mirabilis jalapa trypsin inhibitors, MJTI I and II and Spinacia oleracea trypsin inhibitors, SOTI I, II, and III) from the garden four-o'clock (M. jalapa) and spinach (S. oleracea) seeds were isolated. The purification procedures included affinity chromatography on immobilized methylchymotrypsin in the presence of 5M NaCl, ion exchange chromatography and/or preparative electrophoresis, and finally RP-HPLC on a C-18 column. The inhibitors, crosslinked by three disulfide bridges, are built of 35 to 37 amino-acid residues. Their primary structures differ from those of known trypsin inhibitors, but showed significant similarity to the antimicrobial peptides isolated from the seeds of M. jalapa (MJ-AMP1, MJ-AMP2), Mesembryanthemum crystallinum (AMP1), and Phytolacca americana (AMP-2 and PAFP-S) and from the hemolymph of Acrocinus longimanus (Alo-1, 2 and 3). The association equilibrium constants (K(a)) with bovine beta-trypsin for the inhibitors from M. jalapa (MJTI I and II) and S. oleracea (SOTI I-III) were found to be about 10(7)M(-1). Fully active MJTI I and SOTI I were obtained by solid-phase peptide synthesis. The disulfide bridge pattern in both inhibitors (Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) was established after their digestion with thermolysin and proteinase K followed by the MALDI-TOF analysis.     

Purification and characterization of a trypsin inhibitor from Putranjiva roxburghii seeds

A highly stable and potent trypsin inhibitor was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family by acid precipitation, cation-exchange and anion-exchange chromatography. SDS-PAGE analysis, under reducing condition, showed that protein consists of a single polypeptide chain with molecular mass of approximately 34 kDa. The purified inhibitor inhibited bovine trypsin in 1:1 molar ratio. Kinetic studies showed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 1.4x10(-11) M. The inhibitor retained the inhibitory activity over a broad range of pH (pH 2-12), temperature (20-80 degrees C) and in DTT (up to100 mM). The complete loss of inhibitory activity was observed above 90 degrees C. CD studies, at increasing temperatures, demonstrated the structural stability of inhibitor at high temperatures. The polypeptide backbone folding was retained up to 80 degrees C. The CD spectra of inhibitor at room temperature exhibited an alpha, beta pattern. N-terminal amino acid sequence of 10 residues did not show any similarities to known serine proteinase inhibitors, however, two peptides obtained by internal partial sequencing showed significant resemblance to Kunitz-type inhibitors.     

Germacranolides from seeds of the endangered Umbelliferae species Rouya polygama

From the etheral extract of Rouya polygama Coincy seeds (Umbelliferae) six new germacranolides 1-6 were isolated. The structures were elucidated by chemical methods and spectroscopic analysis. Compounds 1 and 4 exhibited activity against Artemia salina larvae.     

Furostanol glycoside 26-O-beta-glucosidase from the leaves of Solanum torvum

A beta-glucosidase (torvosidase) was purified to homogeneity from the young leaves of Solanum torvum. The enzyme was highly specific for cleavage of the glucose unit attached to the C-26 hydroxyl of furostanol glycosides from the same plant, namely torvosides A and H. Purified torvosidase is a monomeric glycoprotein, with a native molecular weight of 87 kDa by gel filtration and a pI of 8.8 by native agarose IEF. Optimum pH of the enzyme for p-nitrophenyl-beta-glucoside and torvoside H was 5.0. Kinetic studies showed that Km values for torvoside A (0.06 3mM) and torvoside H (0.068 mM) were much lower than those for synthetic substrates, pNP-beta-glucoside (1.03 mM) and 4-methylumbelliferyl-beta-glucoside (0.78 mM). The enzyme showed strict specificity for the beta-d-glucosyl bond when tested for glycone specificity. Torvosidase hydrolyses only torvosides and dalcochinin-8'-beta-glucoside, which is the natural substrate of Thai rosewood beta-glucosidase, but does not hydrolyse other natural substrates of the GH1 beta-glucosidases or of the GH3 beta-glucosidase families. Torvosidase also hydrolyses C5-C10 alkyl-beta-glucosides, with a rate of hydrolysis increasing with longer alkyl chain length. The internal peptide sequence of Solanum beta-glucosidase shows high similarity to the sequences of family GH3 glycosyl hydrolases.