Chemicals
Glucose, fructose, and 2,4-dinitrophenyl hydrazine (DNPH) were purchased from Ajax Finechem (Taren Point, Australia). Catechin, gallic acid, sodium azide, Nitroblue tetrazolium (NBT), aminoguanidine hydrochloride (AG), guanidine hydrochloride, Thioflavin T (ThT), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and l-cysteine were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Trichloroacetic acid (TCA) was purchased from Merck (Darmstadt, Germany). OxiSelectTM CML ELISA kit was purchased from Cell Biolabs (San Diego, CA, USA). All other reagents used were of analytical grade.
Preparation of Moringa oleifera aqueous leaf extract
The leaves of Moringa oleifera were obtained from local areas of Bangkok in Nongkhame district, Thailand. The herbarium number of A014172 (BCU) was authenticated by a Taxonomist at Department of Botany, Faculty of Science, Chulalongkorn University, Thailand. The dried leaves (250 g) were extracted with distilled water twice (3 L) for 3 h at 100 °C. The extraction was filtered through Whatman No. 1 filter paper under the vacuum. The filtrate was further subjected to a spray dryer SD-100 (Eyela world, Tokyo Rikakikai Co., LTD, Japan) to obtain the extract powder. The spray drying conditions, inlet and outlet air temperature was set at 160 and 89–99 °C, respectively.
Determination of total phenolic content
Total phenolic content of Moringa oleifera leaf extract (MOE) was determined by the Folin-Ciocalteu method (Verma et al. 2009). The extract powder was dissolved in distilled water (1.25 mg/mL). The freshly prepared Folin-Ciocalteu reagent was gently mixed with 10 µL of sample. Then, 75 µL of 7.5 % sodium carbonate (Na2CO3) was added and allowed to stand for 30 min at room temperature in the dark. The mixture was measured at 725 nm by a spectrophotometer. Gallic acid (0.025–0.4 mg/mL) was used as a standard and the content of total phenolics was expressed as mg gallic acid equivalents/g dried extract.
Preparation of glycated bovine serum albumin (BSA)
Glycated BSA was performed according to a previously described method (Povichit et al. 2010) with slight modifications. Briefly, BSA (10 mg/mL) was incubated with glucose or fructose (0.5 M) in 0.1 M phosphate buffer (pH 7.4) containing 0.02 % sodium azide (NaN3) with or without (MOE) (0.5–2.0 mg/mL) and aminoguanidine (AG, 1.0 mg/mL) at 37 °C for 4 weeks. Samples were kept at −20 °C until analysis.
Determination of advanced glycation end product (AGE) formation
The fluorescent AGEs, the irreversible products at the end stage of non-enzymatic glycation, were determined by a spectrofluorometer (Wallac 1420 Victor3 V, PerkinElmer, Santa Clara, CA, USA) at excitation and emission wavelengths of 355 nm and 460 nm, respectively (Povichit et al. 2010).
Determination of N
ε-(carboxymethyl) lysine (CML)
Non-fluorescent AGEs, N
ε-(carboxymethyl) lysine (N
ε-CML), is the most abundant product of glycation reaction. Commercially available ELISA kit was used for measurement of N
ε-CMLformation (Cell Biolabs, CA, USA).
Determination of fructosamine
The levels of fructosamine was analyzed by nitroblue-tetrazolium (NBT) assay with minor modification (Armbruster 1987). Briefly, 90 µL of 2.5 mM nitrobluetetrazolium (NBT) reagent was added to 10 µL of glycated BSA in carbonate buffer (pH 10.3). After 10 and 15 min of incubation, the mixture was measured at 590 nm. The concentration of fructosamine was calculated by using the different absorption at 10 and 15 min time points compared with the standard 1-deoxy-1-morpholino-fructose (1-DMF) curve.
Determination of protein carbonyl
Protein carbonyl content were determined according to a previously published method with minor modifications (Levine et al. 1990). In brief, 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2.5 M HCl (400 µL) was added to 100 µL of glycated BSA and incubated in the dark at room temperature for 60 min. Then, 20 % (w/v) trichloroacetic acid (500 µL) was added and kept on ice for 5 min. Protein precipitation was centrifuged and the protein pellet was then washed with 1:1 (v/v) ethanol/ethyl acetate and dissolved in 6 M guanidine hydrochloride. The absorbance was determined at 370 nm. The concentration of protein carbonyl content was calculated using an absorption coefficient of 22,000 M−1cm−1. The results were expressed as nmol carbonyl/mg protein.
Determination of protein thiol groups
The determination of free thiol groups were performed according to Ellman’s assay using 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) (Ellman 1959). Glycated BSA (10 µL) was incubated with 6 mM DTNB in 0.1 M PBS (pH 7.4) for 15 min at room temperature. The absorbance was determined at 410 nm. The free thiol concentration was calculated for the standard curve of l-cysteine (0.3–10 µM) and expressed as nmol/mg protein.
Statistical analysis
Data are expressed as mean ± standard error of mean (SEM) of triplicate determination (n = 3). Differences among groups were analyzed for statistical significance by one-way ANOVA followed by Duncan as post hoc comparison. P value < 0.05 was considered statistically significant.