Adaptation of a microbead assay for the easy evaluation of traditional anti-sickling medicines: application to DREPANOSTAT and FACA

Abstract

Context: Sickle cell disease is a common inherited blood disorder affecting millions of people worldwide. Due to lack of progress in drug discovery for a suitable treatment, sufferers often turn to traditional medicines that take advantage of the plant extracts activity used by traditional healers.

Objective: This study optimizes an anti-sickling screening test to identify preparations capable of reverting sickle cells back to the morphology of normal red blood cells. We focused on the miniaturization and practicability of the assay, so that it can be adapted to the laboratory conditions commonly found in less developed countries.

Materials and methods: We tested two traditional anti-sickling herbal medicines, FACA^® and DREPANOSTAT^®, composed of Zanthoxylum zanthoxyloides (Lam.) Zepern. & Timler (Rutaceae) and Calotropis procera (Aiton) Dryand. (Apocynaceae) at screening concentrations of hydroethanol extracts from 0.2 to 1 mg/mL. Potential bioactive molecules present in the extracts were profiled using Ultra High Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry (UHPLC-HRMS/MS) method, identified through HRMS, MS/MS spectra and in silico fragmentation tools.

Results: Hydroethanol extracts of FACA^® and DREPANOSTAT^® showed low anti-sickling activity, inhibiting less than 10% of the sickling process. The UHPLC-HRMS/MS profiles identified 28 compounds (18 in FACA^® and 15 in DREPANOSTAT^®, including common compounds) among which l-phenylalanine is already described as potential anti-sickling agent. When used as positive control, 7 mg/mL phenylalanine reduced the sickled RBC to 52%.

Discussion and conclusions: This assay has been optimized for the easy screening of plant extracts or extracted compounds from bioassay guided fractionation, valuable to laboratories from less developed countries.

Keywords: Calotropis procera; Sickle cell anemia; UHPLC-HRMS; Zanthoxylum zanthoxiloides; metabolomics; traditional medicines.

Figure 1.

Analysis of the scanned image of the bottom of a 96-well plate by ImageJ^®, the red well was obtained with normal RBC following the protocol described in the text. The white well was obtained using the same protocol with RBC-free media. The abbreviations r, g and b were used for the colors red, green and blue, respectively.

Figure 2.

Scanned image of the bottom of a 96-well plate observing color variations with different hematocrit percentages of normal RBC, ranging from 0% (RMPI only) to 100% (A). Corresponding graph representing the variation in mean values subtracted from 255 as a function of the percentage of hematocrit (B).

Figure 3.

Scanned image of the bottom of a 96-well plate obtained after Hb S RBC were incubated for different amounts of time with sodium metabisulfite (2%) (A). Variation in the rate of differentiation of sickled RBC as a function of incubation time, evaluated as mean values subtracted from 255 (B).

Figure 4.

Variation of the percentage of sickled RBC as a function of the concentration of extracts of FACA^® and DREPANOSTAT^® (A). Scanned image of the bottom of a 96-well-plate obtained with sickled RBC previously incubated with 7 mg/mL of phenylalanine (Phe) (B).