Preparative isolation of apple Flavan-3-ols by pH-zone-refining centrifugal partition chromatography combined with reversed-phase liquid chromatography

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Inglés. MAIN CONCLUSION A new procedure combining centrifugal partition chromatography (CPC) and preparative reversed phase chromatography (Prep-RP-HPLC) allowed recovering a highly purified flavan-3-ol fraction from a crude cider apple polyphenol extract. Our methodological development demonstrated the relevance of using CPC combined with a pH-displacement mode to efficiently discard hydroxycinnamic acid esters on a preparative scale. This step was followed by Prep-RP-HPLC to remove dihydrochalcones and flavonols. INTRODUCTION Cider apples are naturally rich in phenolic compounds that are divided in several classes including flavan-3-ols, hydroxycinnamic acid esters (HCA), flavonols (FO), and dihydrochalcones (DHC). Among them, flavanols (i.e. catechin monomers and procyanidin oligomers) are of particular interest exhibiting numerous properties in food matrix such as tanning properties, organoleptic (bitterness and astringency), nutritional effects, and biological activity (antimicrobial, antioxidant, etc.). In general, their fractionation, mainly based on liquid chromatography on solid phases, is tedious, time-consuming, and solvent-wasting, with the risk of losing a part of the tannins through irreversible adsorption. Moreover, reversed-phase HPLC with a classical gradient using a water/organic solvent mixture elutes apple procyanidins and catechin monomers throughout the chromatogram without any clear separation from HCA, FO, or DHC phenolic classes. In this study, a gentle strategy was developed to recover a highly purified flavan-3-ol fraction. Firstly, pH-zone-refining centrifugal partition chromatography (pH-ZRCPC) was optimized to discard HCA using their ionisation property. Finally, preparative reversed-phase liquid chromatography (Prep-RPLC) was used to remove the others unwanted compounds (DHC and FO). MATERIALS & METHODS A crude extract from a polyphenol-rich French cider apple cultivar was obtained from a juice according to the method described by Millet et al. [1]. Biphasic solvent systems adapted from OKA [2] were tested and composed of ethyl acetate:n-butanol:water with 5:0:5, 3:2:5, 2:3:5 and 1:4:5 (v:v). The partition coefficients were determined using HPLC-UV-MS quantification of the target compounds in the upper and lower phases, for both conventional CPC and pH-ZRCPC by addition of a base (eluter) or an acid (retainer). Then, the suitable solvant system was selected to remove HCA using pH-ZRCPC, starting from 1 g of crude extract, in descending mode at a flow rate of 5 mL/min and a constant rotation speed of 1200 rpm. Several eluter and retainer concentrations were tested. CPC fractions were pooled according to analyses results. An ultimate fractionation on the intermediate extract was realized by Prep-RP-HPLC, using an isocratic mode (20% Acetonitrile), to recover the flavan-3-ol fraction.RESULTS & DISCUSSION Centrifugal partition chromatography combined with a pH shift technique during elution (pH ZRCPC) was used to fractionate apple polyphenols and the fractionation was refined by reversed- Polyphenols Communications 2021 165 P1.1 phase preparative HPLC. A crude polyphenol extract prepared from a polyphenol-rich cider apple juice was used as starting material. First, several biphasic solvent systems, also including variations in TFA (retainer) and NaOH (eluter) concentrations, were tested on an analytical scale for the liquid/liquid fractionation of this extract, to determine the partition coefficients (K) of the phenolic compounds. K values measured using conventional CPC showed that none of the solvent systems were appropriate to remove HCA. However, for pH-ZRCPC, K values of HCA were sufficiently different at acid and alkaline pHs to discard them, and recover the others phenolic compounds. The suitable solvent system for pH-ZRCPC was composed of ethyl acetate:n-butanol:water (3:2:5, v :v). The used retainer (TFA) concentration was 10 mM and high enough to ensure the protonation of all ionizable phenolic compounds (including HCA) and therefore to promote their trapping in the organic stationary phase. In addition, the eluter (NaOH) in the aqueous mobile phase was adjusted to 10 mM also, to enable the progressive neutralization of the retainer (H+) and the displacement of HCA only from the stationary phase to the aqueous mobile phase [3]. Finally, the elution-extrusion mode, which consists in replacing the aqueous mobile phase with the organic phase, was applied to allow the recovery of more hydrophobic compounds, including the procyanidins and catechin monomers. Their degradation was limited because the pH was not too alkaline, and the CPC fractions were acidified to pH 3 just after their collection. Finally, one single pH-ZRCPC run performed with one gram of crude extract followed by Preparative HPLC, allowed to obtained 409 mg flavan-3-ols fraction with a purity of 83% and a recovery of 73%.