Preparative Separation of Procyanidins from Cocoa Polyphenolic Extract: Comparative Study of Different Fractionation Techniques.

To provide further insight into the antioxidant potential of procyanidins (PCs) from cocoa beans, PC extract was fractionated by several methodologies, including solid phase extraction, Sephadex LH-20 gel permeation, and preparative HPLC using C18 and diol stationary phases. All the isolated fractions were analyzed by UHPLC-QTOF-MS to determine their relative composition. According to our results, classical techniques allowed good separation of alkaloids, catechins, dimers, and trimers, but were inefficient for oligomeric PCs. Preparative C18-HPLC method allowed the attainment of high relative composition of fractions enriched with alkaloids, catechins, and PCs with degree of polymerization (DP) < 4. However, the best results were obtained by preparative diol-HPLC, providing a separation according to the increasing DP. According to the mass spectrometry fragmentation pattern, the nine isolated fractions (Fractions II-X) consisted of exclusively individual PCs and their corresponding isomers (same DP). In summary, an efficient, robust, and fast method using a preparative diol column for the isolation of PCs is proposed. Regarding DPPH• and ABTS•+ scavenging activity, it increases according to the DP; therefore, the highest activity was for cocoa extract > PCs > monomers. Thereby, cocoa procyanidins might be of interest to be used as alternative antioxidants.

Food-Safe Process for High Recovery of Flavonoids from Cocoa Beans: Antioxidant and HPLC-DAD-ESI-MS/MS Analysis.

Considering the increasing interest in the incorporation of natural antioxidants in enriched foods, this work aimed to establish a food-grade and suitable procedure for the recovery of polyphenols from cocoa beans avoiding the degreasing process. The results showed that ultrasound for 30 min with particle sample size < 0.18 mm changed the microstructure of the cell, thus increasing the diffusion pathway of polyphenols and avoiding the degreasing process. The effect of temperature, pH, and concentration of ethanol and solute on the extraction of polyphenols was evaluated. Through a 24 full factorial design, a maximum recovery of 122.34 ± 2.35 mg GAE /g, 88.87 ± 0.78 mg ECE /g, and 62.57 ± 3.37 mg ECE /g cocoa beans, for total concentration of polyphenols (TP), flavonoids (TF), and flavan-3-ols (TF3), respectively, was obtained. Based on mathematical models, the kinetics of the solid-liquid extraction process indicates a maximum equilibrium time of 45 min. Analysis by HPLC-DAD-ESI-MS/MS showed that our process allowed a high amount of methylxanthines (10.43 mg /g), catechins (7.92 mg /g), and procyanidins (34.0 mg /g) with a degree of polymerization >7, as well as high antioxidant activity determined by Oxygen Radical Absorbance Capacity (1149.85 ± 25.10 µMTrolox eq /g) and radical scavenging activity (DPPH•, 120.60 ± 0.50 µM Trolox eq /g). Overall, the recovery method made possible increases of 59.7% and 12.8% in cocoa polyphenols content and extraction yield, respectively. This study showed an effective, suitable and cost-effective process for the extraction of bioactive compounds from cocoa beans without degreasing.

Insight of Polyphenol Oxidase Enzyme Inhibition and Total Polyphenol Recovery from Cocoa Beans.

A full factorial design (ascorbic acid/l-cysteine inhibitors, temperature, and time as factors) study was conducted to enhance inhibition of polyphenol oxidase (PPO) activity without decreasing cocoa polyphenol concentrations. The data obtained were modelled through a new equation, represented by Γ, which correlates both high polyphenol content with reduced specific PPO activity. At optimized values (70 mM inhibitory solution at 96 °C for 6.4 min, Γ = 11.6), 93.3% PPO inhibition and total polyphenol of 94.9 mg GAE/g were obtained. In addition, microscopy images confirmed the cell morphological changes measured as the fractal dimension and explained the possible cell lysis and denaturation as a result of heat treatment and chemical inhibitors. Results also showed that PPO enzyme was most suitable (higher vmax/Km ratio) for catechol, with a reduction in its affinity of 13.7-fold after the inhibition heat treatment. Overall, this work proposed a suitable and food-safe procedure for obtaining enriched polyphenol extract with low enzyme activity.

Insight of Stability of Procyanidins in Free and Liposomal Form under an in Vitro Digestion Model: Study of Bioaccessibility, Kinetic Release Profile, Degradation, and Antioxidant Activity.

Small unilamellar and multilayered liposomes loaded with polymeric (epi)catechins up to pentamers were produced. The bioaccessibility, kinetic release profile, and degradation under in vitro gastrointestinal conditions were monitored by UHPLC-DAD-QTOF-MS/MS. The results show that all of the procyanidins underwent depolymerization and epimerization into small molecular oligomers and mainly to (epi)catechin subunits. Moreover, all of the liposome formulations presented higher bioaccessibility and antioxidant activity in comparison to their respective counterparts in non-encapsulated form. Similar results were obtained with procyanidins from cocoa extract-loaded liposomes. Namely, the bioaccessibility of dimer, trimer, and tetramer fractions from cocoa-loaded liposomes were 4.5-, 2.1-, and 9.3-fold higher than those from the non-encapsulated cocoa extract. Overall, the procyanidin release profile was dependent on their chemical structure and physicochemical interaction with the lipid carrier. These results confirmed that liposomes are efficient carriers to stabilize and transport procyanidins with the aim of enhancing their bioaccessibility at a controlled release rate.

Design, Fabrication, Characterization, and In Vitro Digestion of Alkaloid-, Catechin-, and Cocoa Extract-Loaded Liposomes.

Liposomes containing theobromine, caffeine, catechin, epicatechin, and a cocoa extract were fabricated using microfluidization and sonication. A high encapsulation efficiency and good physicochemical stability were obtained by sonication (75% amplitude, 7 min). Liposomes produced at pH 5.0 had mean particle diameter ranging from 73.9 to 84.3 nm. The structural and physicochemical properties of the liposomes were characterized by transmission electron microscopy, confocal fluorescence microscopy, and antioxidant activity assays. The release profile was measured by ultra-high performance liquid chromatography coupled to diode array detection. The bioaccessibility of the bioactive compounds encapsulated in liposomes was determined after exposure to a simulated in vitro digestion model. Higher bioaccessibilities were measured for all catechins-loaded liposome formulations as compared to nonencapsulated counterparts. These results demonstrated that liposomes are capable of increasing the bioaccessibility of flavan-3-ols, which may be important for the development of nutraceutical-enriched functional foods.

Characterization of secondary metabolites from green cocoa beans using focusing-modulated comprehensive two-dimensional liquid chromatography coupled to tandem mass spectrometry.

Proanthocyanidins as well as other secondary metabolites present in green cocoa beans were studied thanks to a new method involving the use of on-line comprehensive two-dimensional liquid chromatography coupled to tandem mass spectrometry (LC × LC-MS/MS). In order to enhance the performance of previously developed methodologies, the use of different modulation strategies were explored. Focusing modulation clearly allowed the attainment of higher resolving power and peak capacity compared to non-focusing modulation set-ups. Moreover, the use of active modulation by the addition of a make-up flow efficiently helped to compensate for the solvent strength mismatch produced between dimensions. The optimized method was useful to successfully describe the secondary metabolite composition of green cocoa that was characterized by the presence of 30 main compounds, including 3 xanthines, 2 flavan-3-ols and 24 oligomeric procyanidins with a degree of polymerization up to 12. The obtained results showed that the proanthocyanidins found in the cocoa beans were exclusively B-type procyanidins. The existence of (epi)catechin subunits linked to sugar or galloyl moieties was not observed. The developed method produced a good separation of secondary metabolites allowing an improvement with respect to the available methodologies for the analysis of a complex food sample such as cocoa metabolites in terms of speed of analysis, resolution and peak capacity.

Relationship between the Physiochemical Properties of Cocoa Procyanidins and Their Ability to Inhibit Lipid Oxidation in Liposomes.

The aim of this paper is to evaluate the effects of cocoa polyphenols and procyanidins with different degrees of polymerization that are encapsulated in liposome delivery systems on the inhibition of lipid oxidation at pH 3.0 and 5.0. In general, liposomes at pH 3.0 and 5.0 were physically stable in the presence of polyphenols and procyanidins with mean particle sizes of 56.56 ± 12.29 and 77.45 ± 8.67 nm and ζ-potentials of -33.50 ± 3.16 and -20.44 ± 1.98 mV at pH 3.0 and 5.0, respectively. At both pH 3.0 and pH 5.0, all the polyphenols and procyanidins inhibited lipid hydroperoxide and hexanal formation, and antioxidant activities increased with increasing polymer-chain sizes. The greater antioxidant activities of the isolated procyanidins were likely due to their increased metal-chelating capacities, as determined by ferric-reducing-ability (FRAP) assays, and their greater levels of partitioning into the lipids, as determined by their log Kow values and encapsulation efficiencies. The crude extract had the greatest antioxidant activity, which could be because other antioxidants were present, or combinations of the different polyphenols and procyanidins inhibited lipid oxidation synergistically.