NMR structural elucidation of dehydrodimers resulting from oxidation of 5-O-caffeoylquinic acid in an apple juice model solution.

During apple juice and cider-making processes, phenolic compounds undergo enzymatic oxidation. 5-O-caffeoylquinic acid (CQA) is one of the major hydroxycinnamic acid derivatives and it is the preferential substrate for polyphenol oxidase (PPO) in apple juices. Consequently, CQA dehydrodimers (MW 706 Da) are among the main products resulting from CQA oxidation. CQA dehydrodimers were previously synthesized in a biomimetic apple juice model solution. Following their purification and characterization using UV-Visible spectra and mass spectrometry, the structures of seven CQA dehydrodimers were elucidated using 1H and 13C one- and two-dimensional NMR spectroscopy. Six of them exhibited dihydrobenzofuran, benzodioxane, or dihydronaphtalene skeletons, which are caffeicin-like structures. Interestingly, a new dehydrodicaffeoyldiquinic acid molecule was also characterised for which two novel structures showing a symmetric dicatechol skeleton were also proposed.

Interactions between polyphenol oxidation products and salivary proteins: Specific affinity of CQA dehydrodimers with cystatins and P-B peptide.

Throughout the apple juice and cider making process, polyphenols undergo enzymatic oxidation which generates a great variety of polyphenol oxidation products. Since 5'-O-Caffeoylquinic acid (CQA) is one of the major phenolic compounds and the preferential substrate for polyphenoloxidase in apple juice, its oxidation leads to the formation of newly formed molecules by which dehydrodimers (MW 706 Da) are included. Interactions of salivary proteins (SP) with native polyphenols is a well-known phenomenon, but their interactions with polyphenol oxidation products has not been studied yet. In this work, we decided to decipher the interactions between CQA dehydrodimers and SP (gPRPs, aPRPs, statherins/P-B peptide, and cystatins) using HPLC-UV and fluorescence. These results showed that contrary to what was expected, CQA dehydrodimers presented a low interaction with PRPs, but revealed a specific interaction with statherins/P-B peptide and cystatins. This work settles for the first time the interactions between SP and polyphenol oxidation products.

Preparative fractionation of 5'-O-caffeoylquinic acid oxidation products using centrifugal partition chromatography and their investigation by mass spectrometry.

When apples are processed into juices or ciders, a great variety of neoformed molecules are generated by enzymatic oxidation of polyphenols. These phenolic oxidation products could be responsible of specific organoleptic properties in apple juices and ciders. 5'-O-Caffeoylquinic acid (CQA) is the major hydroxycinnamic acid in apple and the preferential substrate of apple polyphenoloxidase (PPO). Its main oxidation products were synthetized and purified at the multi-milligrams scale to decipher their structures using mass spectrometry. CQA oxidation products were first synthetized in model solution by oxidizing CQA, in the presence of oxygen and PPO. Then, a specific method involving centrifugal partition chromatography (CPC) was developed to fractionate the main oxidation products corresponding to CQA dehydrodimers (MW 706 Da). For this purpose, CPC was performed in Elution-Extrusion Countercurrent Chromatography (EECCC) mode using a two-phase solvent system precisely selected according to the partition coefficient of the targeted compounds. After a last purification step using semi-preparative reversed phase HPLC, ten CQA dehydrodimers resulting from oxidative coupling were successfully purified, with a UV 280 nm chromatographic purity superior to 85%. Hypothetical structures were formulated for all CQA dehydrodimers based on their UV-vis, MS and MSn spectra. According to this study, centrifugal partition chromatography in combinaison with semi-preparative HPLC was a promising tool to fractionate or purify phenolic oxidation products.