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.