New tyrosinase inhibitors from a nitrogen-enriched chemically engineered extract via hydroxylamine treatment

RAMALLO, I AYELEN; CASALONGUE, MATIAS; FURLAN, RICARDO L E

Rosario

Congreso; 7ma Reunión Internacional de Ciencias Farmacéuticas RICIFA; 2023

Comité Ejecutivo RICiFa 2023

The continued success of natural product scaffolds as platforms for drug discovery has heightened interest in developing strategies for diversifying natural products. Chemical engineering of natural extracts is one such strategy aimed at transforming a significant portion of the natural components found in crude extracts into bioactive semi-synthetic compounds.Tyrosinase is the key enzyme involved in the biosynthesis of melanin, making its inhibitors promising candidates for the treatment of hyperpigmentary disorders. Additionally, this enzyme plays a role in the enzymatic browning of fruits and vegetables, making its inhibitors potential agents for preventing food browning.A set of 40 modified extracts (MEs) were generated by subjecting commercial essential oils (EOs) with hydroxylamine hydrochloride treatment under reflux, 7 h. After complexion, the excess reagent was removed using water/dichloromethane extractions. To address the search for new tyrosinase inhibitors, solutions of the 40 MEs were prepared in DMSO. The samples were then seeded in microplates, resulting in a final concentration per well of 200 μg/mL. Kojic acid was used as reference drug. From the entire set of extracts, approximately 48% exhibited enzyme inhibition equal to or greater than 60%.Out of the MEs that exhibited inhibitory capacity in the microplate assay, the respective bioautographic analysis was conducted. The AK (1 μg/spot), the MEs (100 μg/spot) and the starting EOs were seeded. The modified EO of Pimenta racemosa (Mill.) J.W.Moore (Myrtaceae) (PRM) exhibited the most favorable bioactivity profile. Using silica gel as the stationary phase and dichloromethane: methanol (97:3) as the mobile phase, five halos were located: Rf= 0.19, 0.27, 0.46, 0.50 and 0.66. Two of them, Rf = 0.46 and 0.66, correspond to halos present in the starting EO, indicating that they are not molecules generated by the reaction. The purification of the active compounds was initiated by chromatographically separating 1 g of PRM through a normal-phase column using DCM:MeOH (99:1) as the mobile phase. The process was monitored using UV254nm and tyrosinase bioautography. The halos in Rf= 0.46 and 0.66 were identified as eugenol and methyl chavicol. The halo in Rf= 0.19 was identified as 5,5'-diallyl-3,3'-dimethoxy-2,2'-dimethyl-1,1'-biphenyl. The halo in Rf= 0.27 decomposes in the halo Rf= 0.50, as could be concluded from a 2D TLC experiment. They were respectively identified as (Z)-4-hydroxy-3-methoxybenzaldehyde oxime and 4-hydroxy-3-methoxybenzonitrile.In summary, the application of a simple procedure to complex natural mixtures, such as herbal extracts, resulted in the generation of complex semi-synthetic mixtures with significant alterations in their tyrosinase inhibition properties.