Investigation into Propolis Components Responsible for Inducing Skin Allergy: Air Oxidation of Caffeic Acid and Its Esters Contribute to Hapten Formation

[Image: see text] Propolis is a resin-like material produced by bees from the buds of poplar and cone-bearing trees and is used in beehive construction. Propolis is a common additive in various biocosmetics and health-related products, despite the fact that it is a well-known cause of contact allergy. Caffeic acid and its esters have been the primary suspects behind the sensitization potency of propolis-induced contact allergy. However, the chemical structures of the protein adducts formed between these haptens and skin proteins during the process of skin sensitization remain unknown. In this study, the reactivity of three main contact allergens found in propolis, namely, caffeic acid (CA), caffeic acid 1,1-dimethylallyl ester (CAAE), and caffeic acid phenethyl ester (CAPE), was investigated. These compounds were initially subjected to the kinetic direct peptide reactivity assay to categorize the sensitization potency of CA, CAAE, and CAPE, but the data obtained was deemed too unreliable to confidently classify their skin sensitization potential based on this assay alone. To further investigate the chemistry involved in generating possible skin allergy-inducing protein adducts, model peptide reactions with CA, CAAE, and CAPE were conducted and analyzed via liquid chromatography–high-resolution mass spectrometry. Reactions between CA, CAAE, and CAPE and a cysteine-containing peptide in the presence of oxygen, both in closed and open systems, were monitored at specific time points. These studies revealed the formation of two different adducts, one corresponding to thiol addition to the α,β-unsaturated carbonyl region of the caffeic structure and the second corresponding to thiol addition to the catechol, after air oxidation to o-quinone. Observation of these peptide adducts classifies these compounds as prehaptens. Interestingly, no adduct formation was observed when the same reactions were performed under oxygen-free conditions, highlighting the importance of air oxidation processes in CA, CAAE, and CAPE adduct formation. Additionally, through NMR analysis, we found that thiol addition occurs at the C-2 position in the aromatic ring of the CA derivatives. Our results emphasize the importance of air oxidation in the sensitization potency of propolis and shed light on the chemical structures of the resultant haptens which could trigger allergic reactions in vivo.