6-hydroxyflavonoids as alpha-glucosidase inhibitors from marjoram (Origanum majorana) leaves

A methanol extract of marjoram leaves strongly inhibited rat intestinal alpha-glucosidase. Five 6-hydroxyflavonoids, 6-hydroxyapigenin (scutellarein; IC50 for sucrose hydrolysis by rat intestinal alpha-glucosidase, 12 microM), 6-hydroxyapigenin-7-O-beta-D-glucopyranoside (> 500 microM), 6-hydroxyluteolin-7-O-beta-D-glucopyranoside (300 microM), 6-hydroxyapigenin-7-O-(6-O-feruloyl)-beta-D-glucopyranoside (>500 microM), and 6-hydroxyluteolin-7-O-(6-O-feruloyl)-beta-D-glucopyranoside (> 500 microM), were isolated as active principles and related compounds. The two feruloylglucosides are novel compounds.     

Acute and subacute toxicity studies of Aegle marmelos Corr., an Indian medicinal plant.

This study was designed to elucidate the toxicity of the widely used plant Aegle marmelos in rats. We have taken total alcoholic, total aqueous, whole aqueous and methanolic extracts isolated from the leaves of A. marmelos and studied their toxic effects. Acute, subacute and LD(50) values were determined in experimental rats. The dead animals were obtained from primary screening studies, LD(50) value determination experiments and acute studies subjected to postmortem studies. The external appearance of the dead animals, the appearance of the viscera, heart, lungs, stomach, intestine, liver, kidney, spleen and brain were carefully noted and any apparent and significant features or differences from the norm were recorded. Following the chronic administration of A. marmelos for 14 days, the vital organs such as heart, liver, kidney, testis, spleen and brain were carefully evaluated by histopathological studies and any apparent and significant changes or differences from the norm were studied. From the acute administration of A. marmelos, the LD(50) values were determined using graphical method. The hearts stopped in systolic stand-still in the acute experiments. There were no remarkable changes noticed in the histopathological studies after 50 mg/kg body wt of the extracts of A. marmelos when administered intraperitoneally for 14 days successively. Pathologically, neither gross abnormalities nor histopathological changes were observed. After calculation of LD(50) values using graphical methods, we found a broad therapeutic window and a high therapeutic index value for A. marmelos extracts. Intraperitoneal administration of the extracts of the leaves of A. marmelos at doses of 50, 70, 90 and 100 mg/kg body wt for 14 consecutive days to male and female Wistar rats did not induce any short-term toxicity. Collectively, these data demonstrate that the extracts of the leaves of A. marmelos have a high margin of drug safety.     

Yeast and mammalian alpha-glucosidase inhibitory constituents from Himalayan rhubarb Rheum emodi Wall.ex Meisson

The methanolic extract of rhizome of Himalayan rhubarb Rheum emodi displayed mild yeast as well as mammalian intestinal alpha-glucosidase inhibitory activity. However, further fractionation of active extract led to the isolation of several potent molecules in excellent yields, displaying varying degrees of inhibition on two test models of alpha-glucosidase. Rhapontigenin, desoxyrhapontigenin, chrysophanol-8-O-beta-d-glucopyranoside, torachrysone-8-O-beta-d-glucopyranoside displayed potent yeast alpha-glucosidase inhibition. However chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside displayed potent to moderate mammalian alpha-glucosidase inhibitory activity. Other compounds displayed mild activity on both the tests. Except desoxyrhapontigenin and rhapontigenin that increased Vmax, other compounds including crude extract decreased the Vmax significantly (p<0.02) in yeast alpha-glucosidase test. Further kinetic analysis on mammalian alpha-glucosidase inhibition showed that chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside may be classified as mixed-noncompetitive inhibitors. However, desoxyrhapontigenin and rhapontigenin may be classified as modulators of enzyme activity. Presence and position of glycoside moiety in compounds appear important for better inhibition of mammalian alpha-glucosidase. This is the first report assigning particularly, mammalian intestinal alpha-glucosidase inhibitory activity to these compounds. Chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin, desoxyrhapontigenin and rhapontigenin have been isolated in substantial yields from R. emodi for the first time. Therefore, these compounds may have value in the treatment and prevention of hyperglycemia associated diabetes mellitus.     

Synthesis and biological evaluation of novel 8-aminomethylated oroxylin A analogues as alpha-glucosidase inhibitors

A series of 8-aminomethylated derivatives (1a-1j) were prepared by Mannich reaction of oroxylin A (1) with appropriate primary or secondary amines and para-formaldehyde. All the compounds were tested for their alpha-glucosidase inhibition activity against both yeast and rat intestinal alpha-glucosidase. Some of the compounds demonstrated significantly better alpha-glucosidase inhibitory activity than the parent compound (oroxylin A).     

Isolation and partial characterisation of a novel lectin from Aegle marmelos fruit and its effect on adherence and invasion of Shigellae to HT29 cells

Lectins are a class of ubiquitous proteins/glycoproteins that are abundantly found in nature. Lectins have unique carbohydrate binding property and hence have been exploited as drugs against various infectious diseases. We have isolated one such novel lectin from the fruit pulp of Aegle marmelos. The isolated lectin was partially characterised and its effect against Shigella dysenteriae infection was evaluated. The isolated lectin was found to be a dimeric protein with N-acetylgalactosamine, mannose and sialic acid binding specificity. The effect of Aegle marmelos fruit lectin on the adherence of Shigella dysenteriae to human colonic epithelial cells (HT29 cells) was evaluated by Enzyme Linked Immune Sorbent Assay and invasion was analysed. The protective nature of the Aegle marmelos fruit lectin was assessed by analyzing apoptosis through dual staining method. Aegle marmelos fruit lectin significantly inhibited hemagglutination activity of Shigella and its minimum inhibitory concentration is 0.625 μg/well. Further, at this concentration lectin inhibited Shigella dysenteriae adherence and invasion of HT29 cells and protects the HT29 cells from Shigella dysenteriae induced apoptosis. To conclude, isolated lectin dimeric protein with N-acetylgalactosamine, Mannose and sialic acid binding specificity and inhibits adherence and invasion of Shigellae to HT29 cells thus, protects the host.     

Cloning a cDNA for the lysosomal alpha-glucosidase

Messenger RNA was isolated from monkey testes and size-fractionated on sucrose gradients. In vitro translation of these mRNA fractions resulted in nascent, labeled alpha-glucosidase that could be precipitated with anti human alpha-glucosidase antiserum. A cDNA library was constructed from the most enriched fraction. The library was screened with cDNA made from mRNA obtained from immunoselected polysomes. Five cross-hybridizing clones were isolated and identified by their selection of alpha-glucosidase mRNA, as shown by hybrid released translation and further by their ability to hybridize with DNA from human chromosome 17, on which the gene coding for acid alpha-glucosidase is located.     

In vitro glucose uptake activity of Aegles marmelos and Syzygium cumini by activation of Glut-4, PI3 kinase and PPARγ in L6 myotubes

The purpose of the present study is to investigate the effect of methanolic extracts of Aegles marmelos and Syzygium cumini on a battery of targets glucose transporter (Glut-4), peroxisome proliferator activator receptor gamma (PPARgamma) and phosphatidylinositol 3' kinase (PI3 kinase) involved in glucose transport. A. marmelos and S. cumini are anti-diabetic medicinal plants being used in Indian traditional medicine. Different solvent extracts extracted sequentially were analysed for glucose uptake activity at each step and methanol extracts were found to be significantly active at 100ng/ml dose comparable with insulin and rosiglitazone. Elevation of Glut-4, PPARgamma and PI3 kinase by A. marmelos and S. cumini in association with glucose transport supported the up-regulation of glucose uptake. The inhibitory effect of cycloheximide on A. marmelos- and S. cumini-mediated glucose uptake suggested that new protein synthesis is required for the elevated glucose transport. Current observation concludes that methanolic extracts of A. marmelos and S. cumini activate glucose transport in a PI3 kinase-dependent fashion.     

2-Aminoresorcinol is a potent alpha-glucosidase inhibitor

A series of aminoresorcinols and related compounds were tested for rat intestinal alpha-glucosidase inhibition and these results suggested that the 2-aminoresorcinol moiety of 6-amino-5,7-dihydroxyflavone (2) is important to exert the intestinal alpha-glucosidase inhibitory activity and 2-aminoresorcinol (4), itself, is a potent alpha-glucosidase inhibitor and inhibited sucrose-hydrolyzing activity of rat intestinal alpha-glucosidase uncompetitively.     

A specific acid alpha-glucosidase in lamellar bodies of the human lung

In the present investigation, we have demonstrated that three lysosomal-type hydrolases, alpha-glucosidase, alpha-mannosidase and a phosphatase, are present in lamellar bodies isolated from adult human lung. The hydrolase activities that were studied, all showed an acidic pH optimum, which is characteristic for lysosomal enzymes. The properties of acid alpha-glucosidase in the lamellar body fraction and that in the lysosome-enriched fraction were compared. Using specific antibodies against lysosomal alpha-glucosidase from human placenta, two alpha-glucosidases could be distinguished in the lamellar body fraction: one with a high affinity to the antibodies as found in the lysosome-enriched fraction and another with a much lower affinity. Both forms showed an acidic pH optimum. The same heterogeneity of alpha-glucosidase in the lamellar body fraction could be observed using immobilized concanavalin A. The lectin was able to precipitate nearly all alpha-glucosidase activity of the lysosome-enriched fraction. In contrast, 30% of the alpha-glucosidase activity in the lamellar body fraction was not precipitable. Furthermore, the lamellar body alpha-glucosidase with the low antibody affinity could not be bound to concanavalin A. The results suggest that lamellar bodies contain at least two acid alpha-glucosidases: one similar to the lung lysosomal alpha-glucosidase, and another lamellar body-specific isoenzyme with a different immunoreactivity and lectin affinity. The lamellar body-specific alpha-glucosidase should prove useful as a lamellar body-specific marker enzyme.     

Antioxidant and hepatoprotective potential of Aegle marmelos Correa. against CCl4-induced oxidative stress and early tumor events

The antioxidant properties and inhibitory effect on early tumor promoter markers of A. marmelos (25 and 50 mg/Kg b. wt. orally) have been evaluated. Male Wistar rats were pre-treated for seven consecutive days with A. marmelos prior to CCl4 (1 mL Kg(- 1) body weight p. o., in corn oil [1:1 v/v]) treatment. Pre-treatment with A. marmelos suppressed lipid peroxidation (LPO), xanthine oxidase (XO) and release of serum toxicity marker enzymes viz, SGOT, LDH, SGPT dose-dependently and significantly (p < 0.001). Hepatic antioxidant status viz, reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), quinone reductase (QR), catalase (CAT) were concomitantly restored in A. marmelos-treated groups (p < 0.001). In addition, A. marmelos pretreatment also prevented the CCl4-enhanced ornithine decarboxylase (ODC) and hepatic DNA synthesis significantly (p < 0.001). In conclusion, carbon tetrachloride-induced liver toxicity was strikingly attenuated by A. marmelos treatment and the study gives some insight into the mechanisms involved in diminution of free radical generating toxicants and enhancement of the antioxidant armory, hence preventing further tissue damage, injury and hyperproliferation. Thus, these findings indicate that A. marmelos attenuates CCl4-mediated hepatic oxidative stress, toxicity, tumor promotion and subsequent cell proliferation response in Wistar rats.     

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.     

Xanthones from Cudrania tricuspidata displaying potent alpha-glucosidase inhibition

We have proven that xanthones 1-8 isolated from the root of C. tricuspidata possess highly potent alphaalpha-glucosidase inhibition properties. Compound 1 was identified as a new isoprenylated tetrahydroxy xanthone, 1,3,6,7-tetrahydroxy-2-(3-methylbut-2-enyl)-8-(2-methylbut-3-en-2-yl)-9H-xanthen-9-one (1). These are the first natural xanthones documented to exhibit such inhibition. The IC(50) values of compounds 1-8 inhibiting alpha-glucosidase activity were determined to be up to 16.2microM. Mechanistic analysis showed the xanthones 1-8 exhibited full mixed inhibition.     

Regional distribution and substrate specificity of digestive enzymes involved in terminal digestion in Musca domestica hind-midguts.

One membrane-bound alpha-glucosidase and two soluble alpha-glucosidases were isolated from homogenates of the hind-midgut, the main digestive region in Musca domestica larvae. The membrane-bound alpha-glucosidase and the low-Mr soluble alpha-glucosidase hydrolyze maltopentaose better than maltose, maltotriose, and maltotetraose, the reverse being true for the high-Mr soluble alpha-glucosidase. A membrane-bound glucoamylase previously described in Musca domestica midgut was shown by gradient centrifugation and dialysis against EDTA to result from the combined action of an amylase and an alpha-glucosidase. The determination of amylase, alpha-glucosidases, soluble and membrane-bound carboxypeptidase A, membrane-bound aminopeptidase and dipeptidase along the tissue and luminal contents of the hind-midgut is described. The data support a proposal concerned with how starch and protein are digested in Musca domestica larval hind-midguts and where and how midgut glycosidases and peptidases are secreted.     

Isolation and characterization of alpha-glucosidase inhibitor from the fungus Ganoderma lucidum

An alpha-glucosidase inhibitor, SKG-3, was isolated from the fruiting bodies of Ganoderma lucidum and its physico-chemical properties were characterized. It was a highly specific and effective reversible inhibitor of alpha-glucosidase. It showed very potent inhibitory activity against alpha-glucosidase with an IC50 value of 4.6 micro g/ml, but no activity for any other glycosidases tested. Enzyme activity could be recovered upon dialysis, thus providing evidence for the reversibility of the inhibition. A Lineweaver-Burk plot indicated that the SKG-3 inhibition of alpha-glucosidase was competitive.     

Synthesis and bioevaluation of glycosyl ureas as alpha-glucosidase inhibitors and their effect on mycobacterium

Glycosyl amino esters (2-13) on reaction with different isocyanates resulted in quantitative conversion to glycosyl ureas (14--32). Few of the selected ureas (15-20, 22-28, 30 and 32) on cyclative amidation with DBU/TBAB/4 A MS gave respective dihydropyrimidinones in fair to good yields (33-47). The compounds were screened for alpha-glucosidase inhibitory activity and two (19 and 23) of them showed strong inhibition against rat intestinal alpha-glucosidase. The compounds were also screened against Mycobacterium aurum, however, only one (19) of them exhibited marginal antitubercular activity.     

Synthesis of andrographolide derivatives: a new family of alpha-glucosidase inhibitors

Andrographolide (1), the cytotoxic agent of the plant Andrographis paniculata, was subjected to semi-synthetic studies leading to a series of new derivatives, a novel family of glucosidase inhibitors. Nicotination of 3,19-hydroxyls in 15-alkylidene andrographolide derivatives (9) was favorable to alpha-glucosidase inhibition activity. Among them, 15-p-chlorobenzylidene-14-deoxy-11,12-didehydro-3,19-dinicotinateandrographolide (11c) was a very potent inhibitor against alpha-glucosidase with an IC50 value of 6 microM. However, all compounds concerned for beta-glucosidase showed no inhibition. All compounds synthesized were characterized by the analysis of NMR, IR, HRMS spectra and the stereochemistry of 2 was confirmed by X-ray analysis.     

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.     

Human lysosomal alpha-glucosidase. Characterization of the catalytic site.

The substrate analogue conduritol B epoxide (CBE) is demonstrated to be an active site-directed inhibitor of human lysosomal alpha-glucosidase. A competitive mode of inhibition is obtained with glycogen as natural and 4-methylumbelliferyl-alpha-D-glucopyranoside as artificial substrate. The inactivation of the enzyme is time and concentration dependent and results in the covalent binding of CBE. Catalytic activity is required for binding to occur. CBE-labeled peptides containing the catalytic residue of lysosomal alpha-glucosidase were isolated and identified by microsequencing and amino acid analysis. The peptides appeared to originate from a protein domain which is highly conserved among alpha-amylases, maltase, glucoamylases, and transglucanosylases. Based on the sequence similarity and the mechanism of CBE binding, Asp-518 is predicted to be the essential carboxylate in the active site of lysosomal alpha-glucosidase. The functional importance of Asp-518 and other residues around the catalytic site was studied by expression of in vitro mutagenized alpha-glucosidase cDNA in transiently transfected COS cells. Substitution of Asp-513 by Glu-513 is shown to interfere with the posttranslational modification and the intracellular transport of the alpha-glucosidase precursor. The residues Trp-516 and Asp-518 are demonstrated to be critical for catalytic function.     

In vitro antiproliferative effects on human tumor cell lines of extracts from the Bangladeshi medicinal plant Aegle marmelos Correa.

In the present paper we show that extracts from Aegle marmelos Correa are able to inhibit the in vitro proliferation of human tumor cell lines, including the leukemic K562, T-lymphoid Jurkat, B-lymphoid Raji, erythroleukemic HEL, melanoma Colo38, and breast cancer MCF7 and MDA-MB-231 cell lines. Molecules present within the studied Aegle marmelos C. extracts were identified by gas-chromatography/mass-spectrometry analysis; three derivatives (butyl p-tolyl sulfide, 6-methyl-4-chromanone and butylated hydroxyanisole) were found to exhibit strong activity in inhibiting in vitro cell growth of human K562 cells. The antiproliferative activity of these compounds was found to be comparable to that of known antitumor agents, including cisplatin, chromomycin, cytosine arabinoside and 5-fluorouracil. In addition, the antiproliferative activity of butyl-p-tolyl sulfide, 6-methyl-4-chromanone and 5-methoxypsolaren was associated to activation of the differentiation pattern of K562 cells.     

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.