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 (λexc 370 nm; λem 440 nm) and pentosidine-like (λexc 335 nm; λem 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

New Isolinariins C, D and E, Flavonoid Glycosides from <i>Linaria japonica</i>

“…The mixture of hexane and ethyl acetate layers of a MeOH extract of Linaria japonica was fractionated by various types of chromatography to afford eight compounds (1)(2)(3)(4)(5)(6)(7)(8).…”

“…Therefore, inhibitors of AGEs formation are demanded as potential therapeutic remedy. The inhibitory effects of the isolated compounds (1-8) on AGEs formation were evaluated using a fluorescent method 8,9) (Table 2). The results showed that new compounds 1, 2 and 3 showed stronger inhibitory activity (IC 50 =34.8, 35.0 and 19.5 µM, respectively) than that of a reference compound, aminoguanidine (1.29 mM).…”

“…3) The present study on the same non-polar fraction has demonstrated the presence of three new flavonoid glycosides (1-3) along with linariin (4), pectolinarin (5), pectolinarigenin (6), apigenin (7) and luteolin (8) (Fig. 1) through the isolation by various chromatographic techniques such as silica gel, octadecylsilyl (ODS) and HPLC.…”

“…Measurements were performed in triplicate and the IC 50 of the intensity of fluorescent were determined graphically. 8) Collagenase Inhibition Assay Collagenase inhibitory activity was examined using the modified method described by Teramachi et al 10) Briefly, the test compounds, 10 µg/mL of enzyme (collagenase from Clostridium histolyticum (SigmaAldrich Japan)) and 50 mM Tricine buffer (pH 7.5) were added to a 96-well microtiter plate and preincubated for 10 min at 37°C. Afterwards, the substrate solution ((7-methoxycoumarin-4-yl) acetyl-L-prolyl-L-leucylglycyl-L-leucyl-[N β -(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-alanyl-L-arginine amide) (PEPTIDE INSTITUTE, Osaka, Japan) at a final concentration of 10 µM was added to initiate the reaction.…”

Inhibition of Advanced Glycation End-Product Formation by Quercetin and Catechin: An Alternative Therapy for Treating Diabetic Complications

“…Polyphenols such as genistein, naringin, and ellagic acid are reported to control aldehyde-induced glycation and prevention of the formation of AGE precursors [15]. This work adapts a cost-effective high-throughput screening-based assay to identify the potency of aldose reductase inhibitors (ARIs), quercetin, and catechin in the prevention of early AGEs [16].…”