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Tuning a 96-Well Microtiter Plate Fluorescence-Based Assay to Identify AGE Inhibitors in Crude Plant Extracts
EA 921 SONAS, Université d'Angers, SFR QUASAV 4207, Angers 49045, France
MOLTECH Anjou, UMR 6200 CNRS, UFR Sciences, Université d'Angers, Angers 49045, France
* Author to whom correspondence should be addressed.
Received: 12 October 2013; in revised form: 7 November 2013 / Accepted: 14 November 2013 / Published: 19 November 2013
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.
Keywords: advanced glycation end-products; automation; fluorescence; natural products; pentosidine; plant extract screening; vesperlysines
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Séro, L.; Sanguinet, L.; Blanchard, P.; Dang, B.T.; Morel, S.; Richomme, P.; Séraphin, D.; Derbré, S. Tuning a 96-Well Microtiter Plate Fluorescence-Based Assay to Identify AGE Inhibitors in Crude Plant Extracts. Molecules 2013, 18, 14320-14339.
Séro L, Sanguinet L, Blanchard P, Dang BT, Morel S, Richomme P, Séraphin D, Derbré S. Tuning a 96-Well Microtiter Plate Fluorescence-Based Assay to Identify AGE Inhibitors in Crude Plant Extracts. Molecules. 2013; 18(11):14320-14339.
Séro, Luc; Sanguinet, Lionel; Blanchard, Patricia; Dang, Bach T.; Morel, Sylvie; Richomme, Pascal; Séraphin, Denis; Derbré, Séverine. 2013. "Tuning a 96-Well Microtiter Plate Fluorescence-Based Assay to Identify AGE Inhibitors in Crude Plant Extracts." Molecules 18, no. 11: 14320-14339.