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www.nature.com/scientificreports/ Figure 3. UV absorbance spectra of HSA and MG with varied concentrations of AgNP incubated at 37 °C for 6 days. UV absorbance was monitored at 280 nm. Figure 4. Fluorescence emission intensity profiles of HSA and MG with varied concentrations of AgNP. Fluorescence intensities were measured at excitation and emission wavelengths of 365 nm and 444 nm, respectively. of AGEs formation was observed when 0.27 mM AgNPs was used. No inhibition of AGEs formation was when MG-HSA mixture was incubated with Aloe vera leaf extract. Fluorescence intensities of controls and Aloe leaf extract were found to be negligible (Fig. 4; Figure S2). Our results are consistent with those of previous studies as several AGEs exhibit a characteristic fluorescence around at 370 nm/440 nm51, and fluorescence intensity can be measured in vitro to examine the formation of AGEs under different treatments52. The decrease in fluorescence and AGEs formation with increasing concentrations of AgNPs demonstrates that AgNPs act as potential antigly- cating agents in a concentration-dependant manner. HPLC elution profiles of native HSA, HSA–MG without AgNP, and HSA–MG with 0.09, 0.18 and 0.27 mM AgNPs after 6 days of incubation at 37 °C are illustrated in Fig. 5A,B,C,D,E and F. Native HSA showed a high intensity peak at a retention time of 11.14 min (Fig. 5A). On incubating the HSA and MG mixture with or with- out Aloe leaf extract, presence of prominent peaks with retention times of 9.12 (peak 2), 6.5 (peak 3), 3.7 (peak 4) and 2.5 (peak 5) min reveals formation of AGEs species (Fig. 5B,C). The diminution and disappearance of peaks was observed in HSA and MG mixtures incubated with 0.18 and 0.27 mM AgNPs, respectively (Fig. 5D,E). When the mixture of HSA and MG was incubated with 0.09 mM AgNP, only diminution of peaks was observed, whereas no such significant peaks were detected for Aloe vera alone (Fig. 5F; Figure S3). Analysis of the HPLC data revealed that AGEs generation was reduced with systemic augmentation of AgNPs concentration. Overall, assessment of the entire dataset clearly confirmed that AgNPs are a very strong antiglycating agent. The HPLC data substantiated the UV/VIS and fluorescence findings that the formation of AGEs was influenced by AgNPs in a concentration-dependent manner. Previous studies have reported anti-glycation activity of gold nanoparticles (GNP) on hemoglobin and crystalline protein53,54. In addition, AgNPs were found to decrease the cell permeabil- ity of AGEs-modified proteins by stimulating the expression of tight junction proteins55. Thus, the results of our study are consistent with previous findings of inhibition of AGEs by nanoparticles. Far-UV CD spectroscopy can be used to investigate the secondary structure of proteins and their conforma- tional changes. The far-UV CD spectrum of HSA is characterized by the presence of two strong negative bands at 208 and 222 nm, which represent the helical characteristics of HSA56. Therefore, the change in ellipticity [θ] at 208 and 222 nm was used to monitor the change in helical content of protein samples and secondary structure of HSA. The CD curve of HSA exhibited a larger divergence at 208 and 222 nm, indicating that HSA had the maxi- mum α-helical content. After incubation for 6 days, the HSA and MG mixture showed marked diminution of Scientific RepoRts | 6:20414 | DOI: 10.1038/srep20414 6PDF Image | Green synthesis of silver nanoparticles inhibitory effects on AGEs
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