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www.nature.com/scientificreports/ Sample Lysine reacted (%) Arginine reacted (%) Carbonyl nmol/mg protein CML nM/ml HSA Native HAS (Control) HSA+MG HSA+MG+Aloe vera Leaf extract HSA+MG+AgNP 0.09 mM HSA+MG+AgNP 0.18 mM HSA+MG+AgNP 0.27 mM — — 66.70 59.90 63.73 53.46 50.25 40.60 35.30 29.75 28.85 21.26 3.75 ± 0.28 25.83 ± 1.16 23.61 ± 1.23 19.33 ± 1.10 14.58 ± 1.02 8.36 ± 0.08 0 1.85 ± 0.30 1.79 ± 0.44 1.46 ± 0.15 0.96 ± 0.13 0.58 ± 0.08 Table 1. Effect of AgNPs on various parameters and AGEs. intra-molecular cross-links of proteins known as AGEs41. On the basis of high pervasiveness of AGEs and oxi- dative stress in numbers of diseases; it has become quite imperative to explore the new inhibitors which can be utilized for the prevention of AGEs formation. The formation of AGEs may be prevented by inhibiting glycoxi- dation process or by sequestering the reactive α-dicarbonyl compounds. Aminoguanidine and other inhibitors (Pimagedine1) were initially found to be potential inhibitors of Maillard reaction42. However, later their use was abandoned due to the side effect observed during phase III clinical trials in diabetic patients43. Discovery of new molecules and manipulation of those available that are naturally nanosized has great potential to improve health care44. Nanosize particles showing anti-glycating properties may also have important clinical significance. Keeping the above explanation in mind, we attempted to discern the role of AgNPs in the inhibition of AGEs formation. Epsilon-amino groups in native and MG-glycated HSA were determined by fluorescamine assay. The percent- age of reacted lysine residues in the MG-glycated HSA mixture without AgNPs was found to be 66.70% relative to the control. A decrease in the reactivity of lysine residues (50.25, 35.30 and 28.85%) was observed with increasing concentrations (0.09, 0.18 and 0.27 mM) of AgNPs. A similar trend was also observed in the reactivity of arginine residues with increasing concentrations 0.09, 0.18 and 0.27 mM of AgNPs. The MG-glycated HSA mixture with or without Aloe vera leaf extract, the arginine and lysine residues showed almost same reactivity (Table 1). These results show that the reactivity of both lysine and arginine residues of HSA with MG gradually decreases with increasing concentration of AgNPs, indicating that AgNPs are capable of inhibiting the glycation reaction at the initial stage in a concentration dependant-manner. MG-mediated oxidation may lead to modification of amino acid side chains. Carboxylation of lysine, arginine, threonine and proline residues is a typical marker of protein oxidation. The carbonyl content in MG-glycated HSA mixture with or without Aloe vera leaf extract was (25.83 ± 1.23 and 23.61 ± 1.23 nmol/mg protein respec- tively) approximately same, but 19.33 ± 1.10, 14.58 ± 1.02 and 8.36 ± 0.08 nmol/mg protein carbonyl contents was observed in the reaction mixture with increasing concentration (0.09, 0.18 and 0.27 mM) of AgNPs, respec- tively (Table 1). The decreasing carbonyl content with increasing levels of AgNPs was caused by the antiglycating activity of AgNPs. The presence of carbonyl contents in vivo and in vitro are considered biomarkers of oxidative stress that predict irreversible oxidative modifications in proteins during the glycation process45. The CML con- tent in native and MG-glycated HSA protein was determined by ELISA. In native HSA, no CML was observed, while a significant and almost same amount of CML was detected in MG-glycated HSA mixture with or without Aloe vera leaf extract (1.86 ± 0.30 mol/mol HSA and 1.79 ± 0.44 respectively). However, the MG-HSA mixture showed 1.46 ± 0.15, 0.96 ± 0.13 and 0.58 ± 0.08 nmol/ml HSA CML content upon treatment with increasing con- centrations of 0.09, 0.18 and 0.27 mM AgNPs, respectively (Table 1). Several types of AGEs and their structures have been described in vivo and in vitro, among which CML and pentosidine are considered important AGEs46,47. The oxidatively formed AGEs CML and pentosidine are closely related to oxidative stress-induced damage48. Our study showed that formation of CML and carbonyl content decreased with increasing levels of AgNPs, suggesting that AgNPs has substantial ability to inhibit AGEs formation. UV-Visible spectra of native HSA showed the maximum absorbance at 280 nm (Fig. 3). Upon modification with MG, an increase in absorbance (hyperchromicity) was observed at 280 nm. When compared to native HSA, the MG-glycated HSA mixture with or without Aloe vera leaf extract showed approximately 71.90% hyperchro- micity at 280 nm, as well as an increase in the absorbance at 300–400 nm. However, when the MG-HSA mix- ture was incubated with 0.09, 0.18 and 0.27 mM of AgNPs, the hyperchromicity was 58.0%, 35.93% and 25.0%, respectively. Incubation of the MG-HSA mixture with increasing concentrations of AgNPs also led to a signif- icant decrease in absorbance between 300 and 400 nm. Aloe leaf extract did not show significant difference in absorbance at 200–400 nm (Figure S1). The hyperchromicity observed at 280 nm in modified-HSA may be due to exposure of aromatic amino acids or formation of new chromophoric groups resulting from unfolding of the protein helix upon glycation. A previous study attributed the hyperchromicity of protein as a result of glycation49, whereas the increase in absorbance from 300 to 400 nm suggests the generation of AGEs45,49. A number of studies have also explained the usefulness of increases in absorbance in this range (300–400 nm) in determining the for- mation of proteins-AGEs or DNA-AGEs50. Native HSA and MG mixtures with various concentrations of AgNPs were excited at λex 365 nm, and the emission (λem = 445 nm) profiles were recorded to determine the inhibitory effects of AgNPs in advanced glyca- tion end products formation. MG-glycated HSA mixture with or with without Aloe leaf extract showed almost same and comprehensively elevated emission fluorescence intensity (84%) at λmax 445 nm. When HSA and MG mixtures were incubated with 0.09, 0.18 and 0.27 mM AgNPs, the fluorescence intensity was observed to be 44.6, 29.3 and 13.7%, respectively, compared to native HSA (Fig. 4). In addition, AgNPs at 0.09 and 0.18 mM inhibited the formation of AGEs induced by MG by 19.36% and 46.31%, respectively, while the highest inhibition (64.0%) Scientific RepoRts | 6:20414 | DOI: 10.1038/srep20414 5PDF Image | Green synthesis of silver nanoparticles inhibitory effects on AGEs
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