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

Abstract

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.

Figure 1

Structures and exact mass of CA, CAAE, and CAPE.

Figure 2

Modified kDPRA results for (A) CA, (B) CAAE, and (C) CAPE. The natural log of unreacted Ac-PHCKRM peptide [100—relative peptide depletion (DP) %] values are plotted against the increasing concentrations of CA, CAAE, and CAPE at specified time points.

Figure 3

CA, CAAE, and CAPE peptide adduct formation. (A) Catechol oxidation to o-quinone followed by thiol addition. (B) Thiol addition to the α,β-unsaturated carbonyl (C) Structures of CA, CAAE-, and CAPE-derived peptide adducts.

Figure 4

Tentative mechanisms for generation of caffeic acid derivative adducts through thiolate and thiyl radical pathways.

Figure 5

LC–ESI⁺–HRMS structural analysis of CA and Ac-PHCKRM peptide adducts after incubation of the peptide with 5 times molar excess of CA, in 1:1 PB (pH 7.4) ACN for 5 h prior to LC-HRMS analysis. (A,B) MS² spectrum of 1a and 1b with annotated b and y ions. (C,D). Structures of the y ions carrying the CA adduct.

Figure 6

Relative ratios of peptide adducts with CA, CAAE, and CAPE. The model peptide was incubated with five times molar excess of each compound in the presence of oxygen, in a closed system and open system (ambient air), and under an argon atmosphere prior to LC-HRMS analysis. Peak areas corresponding to each respective adduct and unreacted peptide were summed and used to calculate the relative percent compound present at each time point. (A,D,G) CA reactions. (B,E,H) CAAE reactions. (C,F,I) CAPE reactions.

Figure 7

Oxidation propensity of CA, CAAE, and CAPE. (A) Structures of CA, CAAE, and CAPE paired with their respective o-quinones CAQ, CAAEQ, and CAPEQ. (B) Normalized UV absorbance spectra of CA, CAAE, and CAPE (50 μM) alone or after a 1 h incubation with equimolar KIO₄ in a solution of 1:1 PB (pH 7.4): EtOH. (C) Absorbance at 410 nm taken at various time points of CA, CAAE, and CAPE (50 μM) solutions in 1:1 PB (pH = 7.4 or 8.8): EtOH. Data are normalized to CAPE abs values and are represented as mean ± SD from three replicates. (D) Absorbance at 525 nm taken at various time points of DPPH (0.8 mM) alone or in the presence of equimolar CA, CAAE, and CAPE in a 1:1 EtOH: H₂O solution. Data are represented as mean ± SD from three replicates.