2.2. In Vitro Assays of SME Anti-Glycation Activity

The BSA-AGEs assay was carried out, as described previously [31]. Briefly, 10 mg/mL BSA (fraction V; Sigma-Aldrich, St. Louis, MO, USA) was added either in D-glucose 0.5 M, D-fructose 0.5 M, or methylglyoxal 2.5 mM, and made in a solution 0.1 M PBS (pH 7.4) and 0.02% sodium azide. SME concentrations (5, 10, 25, 50, 100, and 200 mg/mL) were added to each mixture and then incubated at 37 °C for 4 weeks. Afterward, the unreacted sugars were removed by dialysis against distilled water for two days at 4 °C. BSA-AGEs levels were determined by fluorescence spectrophotometry (Ex 370/Em 440 nm; model LS 30, PerkinElmer LAS Ltd., Buckinghamshire, UK) [32]. The glycation of BSA protein by reducing sugars and MGO were the positive controls (BSA glycated), while the incubation of BSA glycated with aminoguanidine (1 mg/mL) was used as the negative control (BSA glycated-AG). The assays were performed in triplicate.

The fructosamine level was determined by the nitro blue tetrazolium (NBT) assay [33], which is based on the ability of fructosamine to reduce NBT, forming formazan, a colored end-product under alkaline conditions. Ten microliters either of the controls or glycated BSA-incubated SME concentrations (BSA-SME) were added in 90 µL of NBT at 2.5 mM, prepared in a carbonate buffer (0.1 M; pH 10.3); all were incubated at 37 °C for 30 min. The absorbance at 530 nm was measured. Fructosamine concentrations (nmol/mg) were calculated according to a standard curve of formazan.

Carbonyl group concentration was measured in BSA-SME and controls samples, according to Levine et al. [34]. A solution of 2,4-dinitrophenylhydrazine (DNPH; 10 mM) in 400 μL of 2.5 M HCl was added to 100 μL each sample and incubated in darkness for 60 min at room temperature. Then, 500 μL of trichloroacetic acid (20% w/v) was added and kept on ice for 5 min. The samples were centrifuged at 4000 rpm for 15 min, and the protein pellet was washed three times with ethyl acetate/ethanol (1:1 v/v). Afterward, the samples were suspended in 250 μL guanidine hydrochloride to 6 M. The concentration of carbonyl groups (nmol/mg protein) was calculated by spectrophotometry at 370 nm absorbance (EON, BioTek Instruments, Inc., Winooski, VT, USA) using the absorption coefficient of DNPH (22,000 M⁻¹ cm⁻¹).

According to Ellman’s method, the determination of thiol group depletion in BSA-SME and control samples was performed. Briefly, 10 µL of 5,5′-Disulfanediylbis(2-nitrobenzoic acid) (DNTB; 10 mM, prepared in PBS) with 50 µL of glycated samples was incubated for 15 min at room temperature, then the absorbance at 410 nm was measured. The level of free thiol groups was calculated using a standard curve of L-cysteine (0.4–11 μM) and expressed in nmol/mg protein [34].