Optical absorption and fluorescence spectroscopy studies of Artepillin C, the major component of green propolis

Abstract

The bioactivity of propolis against several pathogens is well established, leading to the extensive consumption of that bee product to prevent diseases. Brazilian green propolis, collected by the species Apis mellifera, is one of the most consumed in the world. The chemical composition of green propolis is complex and it has been shown that it displays antioxidant, antimicrobial, anti-inflammatory and antitumor activities, especially due to the high content of Artepillin C. The molecule is a derivative of cinnamic acid with two prenylated groups, responsible for the improvement of the affinity of the compound for lipophilic environment. A carboxylic group (COOH) is also present in the molecule, making it a pH-sensitive compound and the pH-dependent structure of Artepillin C, may modulate its biological activity related to interactions with the cellular membrane of organisms and tissues. Molecular properties of Artepillin C on aqueous solution were examined by optical absorption, steady state and time-resolved fluorescence spectroscopies. Acid-base titration based on the spectral position of the near UV absorption band, resulted in the pKa value of 4.65 for the carboxylic group in Artepillin C. In acidic pH, below the pKa value, an absorption band raised around 350nm at Artepillin C concentration above 50μM, due to aggregation of the molecule. In neutral pH, with excitation at 310nm, Artepillin C presents dual emission at 400 and 450nm. In pH close to the pKa, the optical spectra show contribution from both protonated and deprotonated species. A three-exponential function was necessary to fit the intensity decays at the different pHs, dominated by a very short lifetime component, around 0.060ns. The fast decay resulted in emission before fluorescence depolarization, and in values of fluorescence anisotropy higher than could be expected for monomeric forms of the compound. The results give fundamental knowledge about the protonation-deprotonation state of the molecule, that may be relevant in processes mediated by biological membranes.

pH and Charge Effects Behind the Interaction of Artepillin C, the Major Component of Green Propolis, With Amphiphilic Aggregates: Optical Absorption and Fluorescence Spectroscopy Studies

“…Like the previous results for ArtC in buffer , the experimental decay profiles measured in the presence of micelles were best fitted to tri‐exponential functions (see Table ), being dominated by a very short sub‐nanosecond lifetime component, independent of pH. The value is slightly lower than those observed in buffer solutions, expressing the contribution from scattering by the micelles.…”

“…It was previously observed quite high values for steady‐state anisotropy of ArtC in buffer solution ; however, in the presence of micelles, those values decreased. That is contrary to the expected: The anisotropy would increase in micelles, due to restrictions in the molecular rotational movement imposed by the structural arrangement of the amphiphilic molecules .…”

“…In buffer solution at pH 3.0, without micelles, the fluorescence emission band of neutral ArtC shows a peak at 425 nm . Measurements made in the presence of CTAB and HPS micelles show that the emission is clearly blueshifted to wavelengths around 400 nm (Fig.…”

“…It was recently verified that the pKa of ArtC is equal to 4.65 (14) and the maximum of its absorption band, around 315 nm in the presence of the three types of micelles, corresponds to the neutral state of the carboxyl group in the structure of ArtC. The molar absorptivity in aqueous buffer at pH 3.0 is quite low (14) and the absorbance is small, compared to values in micelles. On the other side, when the bulk pH is 7.4, the local pH at the surface of CTAB micelles is 8.9 and the optical absorption spectrum of ArtC shows a peak at 291 nm, typical of deprotonated species (Fig.…”

“…ArtC also presents the carboxyl acid group (COOH), being a pH‐sensitive compound. This characteristic was recently investigated by optical absorption and fluorescence spectroscopies: it was shown that the molecule has two protonation states, and the pKa for the equilibrium between deprotonated and protonated species is 4.65 . Moreover, the protonated species is electrically neutral and may aggregate in aqueous medium at concentration above 50 μ m , which enhances the affinity of ArtC for lipophilic environment .…”