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

Molecules. 2013 Nov 19;18(11):14320-39. doi: 10.3390/molecules181114320.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arginine / analogs & derivatives
  • Arginine / analysis
  • Fluorescence*
  • Glycation End Products, Advanced / analysis*
  • Lysine / analogs & derivatives
  • Lysine / analysis
  • Plant Extracts / chemistry*

Substances

  • Glycation End Products, Advanced
  • Plant Extracts
  • Arginine
  • pentosidine
  • Lysine