Identification of Novel Melanin Synthesis Inhibitors From Crataegus pycnoloba Using an in Vivo Zebrafish Phenotypic Assay

Abstract

Zebrafish has emerged as a powerful model organism for high throughput drug screening. Several morphological criteria, transgenic lines and in situ expression screens have been developed to identify novel bioactive compounds and their mechanism of action. Here, we used the inhibition of melanogenesis during early zebrafish embryo development to identify natural compounds that block melanogenesis. We identified an extract from the Greek hawthorn Crataegus pycnoloba as a potent inhibitor of melanin synthesis and used activity based subfractionation to identify active subfractions and eventually three single compounds of the same family (dibenzofurans). These compounds show reversible inhibition of melanin synthesis and do not act via inhibition of tyrosinase. We also showed that they do not interfere with neural crest differentiation or migration. We identified via in silico modeling that the compounds can bind to the aryl hydrocarbon receptor (AHR) and verified activation of the Ahr signaling pathway showing the induction of the expression of target genes.

Phytochemical composition and bioactivities of hawthorn ( Crataegus spp.): review of recent research advances

“…Phytochemical composition of 20 phenolic compounds quantified in C. azarolus and C. monogyna fruits were compared (Mraihi et al, 2015). Methanol and ethanol extracts of C. azarolus exerted substantial antioxidant, anti-inflammatory, and antiproliferative capacities, which were evaluated by measuring the secreted amounts of the proinflammatory mediator prostaglandin E2 (PGE2), and by assaying the mRNA levels of the proinflammatory cytokines (IL-α, IL-β, and Il-6), chemokines (CCL3 and CCL4) and inflammation-sensitive COX2 and iNOS enzymes (Kallassy et al, 2017 (Agalou et al, 2018); 9 compounds were isolated from C. oxyacantha, including 2 new natural products (Ali et al, 2017). Selected phytochemicals and antioxidant potential were studied in the C. monogyna ethanolic extracts from bark, leaves and berries: the highest TPC, radical scavenging potency as well as the levels of oleanolic acid, quercetin and lupeol were found in the bark extract, while the highest ursolic acid content was in the berries extract (Rezaei-Golmisheh et al, 2015).…”

“…The extract from the Greek hawthorn C. pycnoloba was tested as a potent inhibitor of melanin synthesis during early zebrafish embryo development. The activity based subfractionation enabled to identify 3 melanogenesis inhibiting dibenzofurans (Table 2), acting not via inhibition of tyrosinase or interfering with neural crest differentiation or migration, but via binding to the aryl hydrocarbon receptor by activating its signaling pathway and causing the induction of the target genes expression (Agalou et al, 2018). Ursolic aldehyde, isolated from the active fragments of C. pycnoloba, had no melanin synthesis inhibition activity.…”

Protoporphyrin IX Stimulates Melanogenesis, Melanocyte Dendricity, and Melanosome Transport Through the cGMP/PKG Pathway

“…Phenotype-based evaluation was conducted according to a previous study ( Choi et al., 2007 ; Agalou et al., 2018 ). Synchronized embryos were collected and arrayed in 96-well plates (three to four embryos per well with 200-μL medium).…”

Dopachrome tautomerase variants in patients with oculocutaneous albinism

“…Melanin assay was based on already described methods 14–17 and detailed in Supplementary Materials and Methods.…”