Tuning a 96-well microtiter plate fluorescence-based assay to identify AGE inhibitors in crude plant extracts

Abstract

Advanced glycation end-products (AGEs) are involved in the pathogenesis of numerous diseases. Among them, cellular accumulation of AGEs contributes to vascular complications in diabetes. Besides using drugs to lower blood sugar, a balanced diet and the intake of herbal products potentially limiting AGE formation could be considered beneficial for patients' health. The current paper presents a simple and cheap high-throughput screening (HTS) assay based on AGE fluorescence and suitable for plant extract screening. We have already implemented an HTS assay based on vesperlysines-like fluorescing AGEs quickly (24 h) formed from BSA and ribose under physiological conditions. However, interference was noted when fluorescent compounds and/or complex mixtures were tested. To overcome these problems and apply this HTS assay to plant extracts, we developed a technique for systematic quantification of both vesperlysines (λ(exc) 370 nm; λ(em) 440 nm) and pentosidine-like (λ(exc) 335 nm; λ(em) 385 nm) AGEs. In a batch of medicinal and food plant extracts, hits were selected as soon as fluorescence decreased under a fixed threshold for at least one wavelength. Hits revealed during this study appeared to contain well-known and powerful anti-AGE substances, thus demonstrating the suitability of this assay for screening crude extracts (0.1 mg/mL). Finally, quercetin was found to be a more powerful reference compound than aminoguanidine in such assay.

Figure 1

Structures of the main fluorescent AGEs 1–9 and their fluorescent properties in neutral aqueous media [31,32,33,34,35,36].

Figure 2

(A) Total fluorescence of AGEs formed from BSA (10 mg/mL) and ribose (0.5 M). The 2D contour plot shows variations in the emission spectrum as a function of the excitation wavelength. The fluorescence intensity increased from blue to red. This graph indicates that both pentosidine-like (λ_exc 335 nm; λ_em 385 nm) and vesperlysines-like (λ_exc 370 nm; λ_em 440 nm) AGEs were the main fluorescing AGEs obtained after incubation; (B) Excitation (red) and emission (black) fluorescence spectra of vesperlysines-like (dotted, λ_exc 370 nm; λ_em 440 nm) and pentosidine-like (bold, λ_exc 335 nm; λ_em 400 nm) AGEs formed from BSA and ribose.

Figure 3

Tested NPs 10–20 and their fluorescent excitation and emission wavelengths in water.

Figure 4

Vesperlysines-like (black) and pentosidine-like (grey) AGE formation in the presence of NPs (A: 1 mg/mL; B: 0.1 mg/mL) or plant extracts (C: 1 mg/mL; D: 0.1 mg/mL).

Figure 5

Dose-effect curves for vesperlysines-like AGE (dotted) and pentosidine-like AGE (bold) formation in the presence of umbelliferone 20 (A), caffeic acid 18 (B), emetine 19 (C) or aminoguanidine (D) or quercetin 10 (E) as well as dose-effect curves for vesperlysines-like AGE (F) and pentosidine-like AGE (G) formation in the presence of Mammea neurophylla fruit methanolic extract with (dotted) or without (bold) umbelliferone 20 (10% m/m).

Scheme 1

Formation of triazine derivatives from aminoguanidine and dicarbonyl compounds. This reaction explains how aminoguanidine can prevent AGEs formation by trapping dicarbonyl compounds.

Figure 6

Dose-effect curves for vesperlysines-like AGE (dotted) and pentosidine-like AGE (bold) formation in the presence of quinquina EtOH extract (Black), sophora EtOH extract (Blue) or sophora aqueous extract (Red).