Pyranoanthocyanins (PACNs) are vibrant pigments derived from anthocyanins (ACNs) through a reaction with a cofactor, such as acetone, pyruvic acid, or caffeic acid. These pigments are found in various sources including aged wine, juice, and certain fruits, contributing to color stability and unique hues. PACNs offer improved color stability compared to their ACN precursors, exhibiting color retention across pH values and resistance to bleaching. Previous research focused primarily on malvidin-derived PACNs, while cyanidin-3-glucoside (Cy3G), being the most abundant ACN in nature, presents a readily available reactant for PACN synthesis. ACNs undergo thermal degradation via C2 hydration, forming a colorless chalcone which subsequently cleaves into phloroglucinaldehyde and protocatechuic acid. However, limited research exists on PACN thermal degradation pathways and products. This study aimed to evaluate the thermal stability and identify the degradation compounds of three PACNs derived from Cy3G, comparing them to Cy3G to understand the role of C10 substitution on stability. This knowledge is vital for the further development and application of PACNs as natural colorants.

Several studies have highlighted the enhanced stability of PACNs compared to their ACN precursors. Methyl-pyranoanthocyanidins retained significantly more absorbance after heating than their anthocyanidin counterparts. Malvidin-derived PACNs displayed degradation half-lives considerably longer than the parent anthocyanin. While most studies on PACN structural transitions do not show evidence of a hydrated PACN, a few reports indicated the presence of a C2-hydrated PACN. The literature on PACN thermal degradation pathways and resulting compounds is limited. The existing research focuses mainly on malvidin-derived PACNs; however, the vast abundance of Cy3G in nature makes it a crucial subject for studying PACN stability and degradation.

The study used commercially sourced elderberry as the Cy3G source for PACN synthesis. PACNs were formed by reacting Cy3G with pyruvic acid, acetone, or caffeic acid under conditions optimized for each cofactor. The resulting PACNs were semi-purified using solid-phase extraction and isolated through semi-preparative HPLC. Pigment purity exceeded 96%. Stability challenges during sample preparation and room temperature storage were noted, with some PACNs showing precipitation or color changes reversible by adding acidified methanol. Heat treatments involved incubating samples in a 90°C water bath for up to 15 hours at pH 3.0. Spectrophotometric measurements (absorbance and CIE-L*a*b* color parameters) were conducted at intervals, as well as UHPLC-PDA-ESI-MS/MS analysis for pigment and degradation compound identification. First-order kinetics were used to model pigment degradation. Accurate mass values were obtained via quadrupole time-of-flight (QTOF) mass spectrometry. Statistical analysis involved one-way ANOVA with Bonferroni post-hoc tests.

PACNs exhibited significantly greater thermal stability than Cy3G. 10-Methyl-pyranocyanidin-3-glucoside showed the highest stability based on UHPLC-PDA analysis, retaining ~52% of its original pigment after 12 hours at 90°C and pH 3.0. 10-Catechyl-pyranocyanidin-3-glucoside displayed the best color stability, partially attributed to a colored degradation compound (λmax = 478 nm). Cy3G degraded significantly faster, losing nearly all pigment within 15 hours. Protocatechuic acid was identified in all heated samples, suggesting a common degradation pathway involving C2 hydration. PACN degradation followed first-order kinetics, with half-lives significantly longer than Cy3G (2.1–9.4 times). The C10 substitution impacted stability; 10-carboxy-PCy3G was less stable than 10-methyl-PCy3G and 10-catechyl-PCy3G. Unique degradation compounds were identified for each PACN in addition to protocatechuic acid.

The enhanced thermal stability of PACNs compared to Cy3G likely stems from reduced C2 electrophilicity due to increased positive charge delocalization in the pyran ring, hindering ring-opening hydration. The differences in stability among the three PACNs might relate to the electronic effects of the C10 substitutions. Methyl is electron-donating, while carboxy is electron-withdrawing, altering C2 electron density and potentially influencing the rate of hydration. The colored degradation product in 10-catechyl-PCy3G contributes to its superior color stability, highlighting the influence of structural features. The detection of protocatechuic acid in all heated samples suggests that despite enhanced stability, PACNs still partially degrade via a pathway similar to ACNs, possibly requiring higher activation energy for hydration to initiate. The results provide insights into the impact of chemical structure on PACN stability, vital for their development as natural colorants.

This research demonstrated the exceptional thermal stability of PACNs, significantly exceeding that of Cy3G. The C10 substitution significantly influenced stability, with 10-methyl-PCy3G showing the highest stability by UHPLC-PDA and 10-catechyl-PCy3G exhibiting the best color stability due to a colored degradation product. Protocatechuic acid, identified as a common degradation product, indicates a similar degradation pathway to ACNs. This study highlights the importance of C10 substitution in determining PACN thermal stability and provides valuable information for their potential application as natural food colorants. Future research could focus on exploring other cofactors and ACN structures to further optimize PACN stability and color properties.

The study focused on three specific PACNs and one ACN precursor. Extrapolating these findings to other PACN structures requires further investigation. The use of a specific pH (3.0) and temperature (90°C) may limit the generalizability of the results to different conditions encountered in food processing and storage. Further characterization of the unique degradation compounds identified for each PACN is needed to fully elucidate the degradation pathways. The observed precipitation of 10-carboxy-PCy3G during sample preparation could also affect the interpretation of stability results.