Glucitol-core containing gallotannins inhibit the formation of advanced glycation end-products mediated by their antioxidant potential.

Glucitol-core containing gallotannins (GCGs) are polyphenols containing galloyl groups attached to a 1,5-anhydro-d-glucitol core, which is uncommon among naturally occurring plant gallotannins. GCGs have only been isolated from maple (Acer) species, including the red maple (Acer rubrum), a medicinal plant which along with the sugar maple (Acer saccharum), are the major sources of the natural sweetener, maple syrup. GCGs are reported to show antioxidant, α-glucosidase inhibitory, and antidiabetic effects, but their antiglycating potential is unknown. Herein, the inhibitory effects of five GCGs (containing 1-4 galloyls) on the formation of advanced glycation end-products (AGEs) were evaluated by MALDI-TOF mass spectroscopy, and BSA-fructose, and G.K. peptide-ribose assays. The GCGs showed superior activities compared to the synthetic antiglycating agent, aminoguanidine (IC50 15.8-151.3 vs. >300 µM) at the early, middle, and late stages of glycation. Circular dichroism data revealed that the GCGs were able to protect the secondary structure of BSA protein from glycation. The GCGs did not inhibit AGE formation by the trapping of reactive carbonyl species, namely, methylglyoxal, but showed free radical scavenging activities in the DPPH assay. The free radical quenching properties of the GCGs were further confirmed by electron paramagnetic resonance spectroscopy using ginnalin A (contains 2 galloyls) as a representative GCG. In addition, this GCG chelated ferrous iron, an oxidative catalyst of AGE formation, supported a potential antioxidant mechanism of antiglycating activity for these polyphenols. Therefore, GCGs should be further investigated for their antidiabetic potential given their antioxidant, α-glucosidase inhibitory, and antiglycating properties.

The hydrolyzable gallotannin, penta-O-galloyl-ß-D-glucopyranoside, inhibits the formation of advanced glycation endproducts by protecting protein structure.

Glycation is a spontaneous process initiated by a condensation reaction between reducing sugars and proteins that leads to the formation of advanced glycation endproducts (AGEs). The in vivo accumulation of AGEs is associated with several chronic human diseases and, thus, the search for AGE inhibitors is of great research interest. Hydrolyzable tannins (gallotannins and ellagitannins) are bioactive plant polyphenols which show promise as natural inhibitors of glycation and AGE formation. Notably, the gallotannin, 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (PGG), is a key intermediate involved in the biosynthesis of hydrolyzable tannins in plants. Herein, we investigated the effects of PGG on the individual stages of protein glycation and on protein structure (using bovine serum albumin; BSA). MALDI-TOF data demonstrated that PGG inhibited early glycation by 75% while the synthetic AGE inhibitor, aminoguanidine (AG), was not active (both at 50 µM). In addition, PGG reduced the formation of middle and late stage AGEs by 90.1 and 60.5%, respectively, which was superior to the positive control, AG. While glycation induced conformational changes in BSA from α-helix to ß-sheets (from circular dichroism and congo red binding studies), PGG (at 50 µM) reduced this transition by 50%. Moreover, BSA treated with PGG was more stable in its structure and retained its biophysical properties (based on zeta potential and electrophoretic mobility measurements). The interaction between PGG and BSA was further supported by molecular docking studies. Overall, the current study adds to the growing body of data supporting the anti-AGE effects of hydrolyzable tannins, a ubiquitous class of bioactive plant polyphenols.

Inhibitory effect of gold nanoparticles on the D-ribose glycation of bovine serum albumin.

Formation of advanced glycation end products (AGEs) by nonenzymatic glycation of proteins is a major contributory factor to the pathophysiology of diabetic conditions including senile dementia and atherosclerosis. This study describes the inhibitory effect of gold nanoparticles (GNPs) on the D-ribose glycation of bovine serum albumin (BSA). A combination of analytical methods including ultraviolet-visible spectrometry, high performance liquid chromatography, circular dichroism, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were used to determine the extent of BSA glycation in the presence of citrate reduced spherical GNPs of various sizes and concentrations. GNPs of particle diameters ranging from 2 nm to 20 nm inhibited BSA's AGE formation. The extent of inhibition correlated with the total surface area of the nanoparticles. GNPs of highest total surface area yielded the most inhibition whereas those with the lowest total surface area inhibited the formation of AGEs the least. Additionally, when GNPs' total surface areas were set the same, their antiglycation activities were similar. This inhibitory effect of GNPs on BSA's glycation by D-ribose suggests that colloidal particles may have a therapeutic application for the treatment of diabetes and conditions that promote hyperglycemia.

Pomegranate phenolics inhibit formation of advanced glycation endproducts by scavenging reactive carbonyl species.

Advanced Glycation Endproducts (AGEs) are a heterogeneous group of molecules produced from non-enzymatic glycation. Accumulation of AGEs in vivo plays an important role in the pathology of chronic human diseases including type-2 diabetes and Alzheimer's disease. Natural AGEs inhibitors such as the pomegranate (Punica granatum) fruit show great potential for the management of these diseases. Herein, we investigated the in vitro anti-glycation effects of a pomegranate fruit extract (PE), its phenolic constituents [punicalagin (PA), ellagic acid (EA) and gallic acid (GA)], and their in vivo derived colonic metabolites [urolithin A (UA) and urolithin B (UB)]. All of the samples showed anti-glycation activities and PE, PA, and EA were more potent inhibitors than the positive control, aminoguanidine. PE and the purified phenolics also exhibited carbonyl scavenger reactivity. Our study suggests that pomegranate may offer an attractive dietary strategy for the prevention and treatment of AGE-related diseases such as type-2 diabetes and Alzheimer's disease.

Transsulfuration Is a Significant Source of Sulfur for Glutathione Production in Human Mammary Epithelial Cells.

The transsulfuration pathway, through which homocysteine from the methionine cycle provides sulfur for cystathionine formation, which may subsequently be used for glutathione synthesis, has not heretofore been identified as active in mammary cells. Primary human mammary epithelial cells (HMEC's) were labeled with 35S-methionine for 24 hours following pretreatment with a vehicle control, the cysteine biosynthesis inhibitor propargylglycine or the gamma-glutamylcysteine synthesis inhibitor buthionine sulfoximine. Cell lysates were prepared and reacted with glutathione-S-transferase and the fluorescent labeling compound monochlorobimane to form a fluorescent glutathione-bimane conjugate. Comparison of fluorographic and autoradiographic images indicated that glutathione had incorporated 35S-methionine demonstrating that functional transsulfuration occurs in mammary cells. Pathway inhibitors reduced incorporation by roughly 80%. Measurement of glutathione production in HMEC's treated with and without hydrogen peroxide and/or pathway inhibitors indicates that the transsulfuration pathway plays a significant role in providing cysteine for glutathione production both normally and under conditions of oxidant stress.

Glycation of human erythrocyte glutathione peroxidase: effect on the physical and kinetic properties.

BACKGROUND: Glutathione peroxidase (GPx) is a significant antioxidant enzyme that plays a key role in protecting the body from reactive oxygen species (ROS) and their toxicity. As a biocatalyst, the enzyme has been shown to reduce hydrogen peroxide to water and lipid hydroperoxides to their respective alcohols. The increased levels of ROS in patients with diabetes have been speculated to arise, in part, from alterations in the activity of glutathione antioxidant enzymes, perhaps, by mechanisms such as the glycation of the protein, in vivo. METHODS: Under physiological conditions of temperature and pH, we investigated the susceptibility of human glutathione peroxidase to glycation, determined the effects of glycation on the physical and kinetic properties of the enzyme, and identified the protein's vulnerable amino acid sites of glycation. RESULTS: Circular dichroism, UV and mass spectrometry studies revealed that methylglyoxal and DL-glyceraldehyde are potent glycators of glutathione peroxidase; destabilizing its structure, altering its pH activity and stability profiles and increasing its Km value. CONCLUSIONS: In comparison to DL-glyceraldehyde, methylglyxol was a more potent glycator of the enzyme and was found to nonenzymatically condense with Arg-177, located near the glutathione binding site of GPx.

Non-enzymatic glycation of melamine with sugars and sugar like compounds.

Melamine (1,3,5-triazine-2,4,6-triamine) is employed in the manufacture of plastics, laminates and glues, yet, it has been found sometimes added illegally to dairy products to artificially inflate foods' protein content. In 2008, dairy products adulterated with melamine were blamed for the death of several infants in China, a situation that forced Beijing to introduce stricter food safety measures. The objectives of this study were threefold: (1) to investigate the susceptibility of the amine groups of melamine to glycation with D-galactose, D-glucose and lactose, sugars commonly found in milk, (2) to study the rate and extent of melamine's glycation with methylglyoxal, glyoxal and DL-glyceraldehyde, three highly reactive metabolites of D-galactose, D-glucose and lactose, and (3) to characterize, using mass spectrometry, the Advanced Glycation Endproducts (AGEs) of melamine with sugars found commonly in milk and their metabolites. Incubation of D-galactose, D-glucose and lactose with melamine revealed that D-galactose was the most potent glycator of melamine, followed by D-glucose, then lactose. Methylglyoxal, glyoxal, and DL-glyceraldehyde glycated melamine more extensively than D-galactose, with each yielding a broader range of AGEs. The non-enzymatic modification of melamine by sugars and sugar-like compounds warrants further investigation, as this process may influence melamine's toxicity in vivo.

In vitro galactation of human serum albumin: analysis of the protein's galactation sites by mass spectrometry.

The posttranslational modification of proteins by sugars has been demonstrated in diabetes and classical galactosemia. In diabetes, the glycation process occurs as a result of d-glucose nonenzymatically reacting with proteins such as albumin and hemoglobin, used today as important tools to monitor the efficiency of dietary control and therapy during treatment of diabetes. In classical galactosemia, d-galactose contributes to the formation of glycated proteins as well, suggesting that, akin to diabetes with glucated proteins, the monitoring of galactated proteins may facilitate management of patients with galactosemia. The objectives of this study were (i) to galactate human serum albumin (HSA) in vitro; (ii) to determine, by a sodium borohydride-dependent mass peptide mapping method, the galactation sites in HSA; and (iii) to compare HSA's galactation sites with the protein's reported glucation sites. Treatment of galactated HSA with sodium borohydride stabilized the condensed sugars on the protein and yielded discrete fragmentation patterns by tandem mass spectrometry, allowing reliable identification of HSA's galactation sites. Liquid chromatography/electrospray ionization/mass spectrometry, in combination with tandem mass spectrometry, revealed that the principal sites of galactation in HSA were the ε-amino groups of lysine residues 12, 233, 281/276, 414, and 525. Lysyl residues 12, 233, 276, and 525 were previously reported as privileged sites for the nonenzymatic binding of d-glucose with HSA.