Analyses of polyphenols in cacao liquor, cocoa, and chocolate by normal-phase and reversed-phase HPLC

The antioxidant polyphenols in cacao liquor, a major ingredient of chocolate and cocoa, have been characterized as flavan-3-ols and proanthocyanidin oligomers. In this study, various cacao products were analyzed by normal-phase HPLC, and the profiles and quantities of the polyphenols present, grouped by molecular size (monomers to approximately oligomers), were compared. Individual cacao polyphenols, flavan-3-ols (catechin and epicatechin), and dimeric (procyanidin B2), trimeric (procyanidin C1), and tetrameric (cinnamtannin A2) proanthocyanidins, and galactopyranosyl-ent-(-)-epicatechin (2alpha-->7, 4alpha-->8)-(-)-epicatechin (Gal-EC-EC), were analyzed by reversed-phase HPLC and/or HPLC/MS. The profile of monomers (catechins) and proanthocyanidin in dark chocolate was similar to that of cacao liquor, while the ratio of flavan-3-ols to the total amount of monomeric and oligomeric polyphenols in the case of pure cocoa powder was higher than that in the case of cacao liquor or chocolate.     

Flavan-3-ols from the rhizomes of Drynaria fortunei

One monomer flavan-3-ol, 4α-carboxymethyl-(+)-catechin methyl ester, two monomer flavan-3-ol glycosides, (+)-afzelechin-3-O-β-allopyranoside, (+)-afzelechin-6-C-β-glucopyranoside, two dimer flavan-3-ols, (-)-epiafzelechin-(4β→8)-4β-carboxymethyl-(-)-epicatechin methyl ester, and -(-)-epiafzelechin-(4β→8)-4α-carboxymethyl-(-)epiafzelechin ethyl ester, and one trimer flavan-3-ol, (-)-epiafzelechin-(4β→8)-(-)-epiafzelechin-(4β→8)-4β-carboxymethyl-(-)-epiafzelechin methyl ester, together with thirteen known flavan-3-ols were isolated from the rhizomes of Drynaria fortunei (Kunze) J.Sm (Polypodiaceae). The structures were established by analysis of their HRESIMS, 1D, 2D NMR spectroscopic, and CD data. In order to obtain improved resolution, the high-resolution NMR spectra of the dimers and trimer were measured at -40 °C.     

Peculiarities of polyphenolic profile of fruits of Siberian crab apple and its hybrids with <Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">Domestica</Emphasis> Borkh

For the first time, we have studied the polyphenolic profile of fruits of Siberian crabapple and its hybrids (F1, F2, and F3) with domestic apple cultivated in East Siberia. It is shown that the chemical composition of polyphenolics of Siberian crabapple fruits is overwhelmingly characteristic of the genus Malus; on the other hand, it has clearly identifiable features: low content of flavan-3-ols and derivatives of cinnamic acid; high content of procyanidin B1, phloridzin, anthocyanes, and quercetin glycosides. Procyanidin B2 was not found in both peel and flesh of Siberian crabapple. In the case of hybridization with domestic apple, the fruits of resulting cultivars show a substantial change in the flavan-3-ol content ratio because of the synthesizing of procyanidin B2 in tissues and an increase in (?)-epicatechin content.     

Influence of flavan-3-ols and procyanidins on UVC-mediated formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in isolated DNA

The flavan-3-ols (-)-epicatechin (epicatechin) and (+)-catechin (catechin) and their related oligomers (procyanidins) isolated from cocoa were assayed for their capacity to inhibit the UVC-mediated formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo(8)dG) in calf thymus DNA. The above-mentioned compounds inhibited oxo(8)dG production in a concentration- and time-dependent manner. After 30 min of irradiation (30 kJ/m(2)), 0.1, 1.0, 10, and 100 microM epicatechin inhibited oxo(8)dG formation by 20, 36, 64, and 74%, respectively. For the same dose of UVC, 0.1, 1.0, 10, and 100 microM catechin inhibited oxo(8)dG formation by 1, 23, 50, and 70%, respectively. Epicatechin was more efficient than catechin with respect to inhibiting oxo(8)dG formation (IC(50) 1.7 +/- 0.7 vs 4.0 +/- 0.7 microM). Monomer, tetramer, and hexamer fractions were equally effective in inhibiting oxo(8)dG formation when assayed at 10 microM monomer equivalent concentration. At similar concentrations (1-50 microM), the inhibition of the UVC-mediated oxo(8)dG formation by flavan-3-ols and procyanidins was in the range of that of alpha-tocopherol, Trolox, ascorbate, and glutathione. These results support the concept that flavan-3-ols and their related procyanidins can protect DNA from oxidation at concentrations that can be physiologically relevant. Both epimerism and degree of oligomerization are important determinants of the antioxidant activity of flavan-3-ols and procyanidins.     

Oxidative modification of low-density lipoprotein: lipid peroxidation by myeloperoxidase in the presence of nitrite

Oxidative modification of low-density lipoprotein (LDL) is a pivotal process in early atherogenesis and can be brought about by myeloperoxidase (MPO), which is capable of reacting with nitrite, a NO metabolite. We studied MPO-mediated formation of conjugated dienes in isolated human LDL in dependence on the concentrations of nitrite and chloride. This reaction was strongly stimulated by low concentrations (5-50 microM) of nitrite which corresponds to the reported concentration in the arterial vessel wall. Under these conditions no protein tyrosine nitration occurred; this reaction required much higher nitrite concentrations (100 microM-1 mM). Chloride neither supported lipid peroxidation alone nor was its presence mandatory for the effect of nitrite. We propose a prominent role of lipid peroxidation for the proatherogenic action of the MPO/nitrite system, whereas peroxynitrite may be competent for protein tyrosine nitration of LDL. Monomeric and oligomeric flavan-3-ols present in cocoa products effectively counteracted, at micromolar concentrations, the MPO/nitrite-mediated lipid peroxidation of LDL. Flavan-3-ols also suppressed protein tyrosine nitration induced by MPO/nitrite or peroxynitrite as well as Cu2+-mediated lipid peroxidation of LDL. This multi-site protection by (-)-epicatechin or other flavan-3-ols against proatherogenic modification of LDL may contribute to the purported beneficial effects of dietary flavan-3-ols for the cardiovascular system.     

Bioavailability of (-)-epicatechin upon intake of chocolate and cocoa in human volunteers

We evaluated the levels of (-)-epicatechin (EC) and its metabolites in plasma and urine after intake of chocolate or cocoa by male volunteers. EC metabolites were analyzed by HPLC and LC/MS after glucuronidase and/or sulfatase treatment. The maximum levels of total EC metabolites in plasma were reached 2 hours after either chocolate or cocoa intake. Sulfate, glucuronide, and sulfoglucuronide (mixture of sulfate and glucuronide) conjugates of nonmethylated EC were the main metabolites present in plasma rather than methylated forms. Urinary excretion of total EC metabolites within 24 hours after chocolate or cocoa intake was 29.8 ± 5.3% and 25.3 ± 8.1% of total EC intake. EC in chocolate and cocoa was partly absorbed and was found to be present as a component of various conjugates in plasma, and these were rapidly excreted in urine.     

Bioavailability of (-)-epicatechin upon intake of chocolate and cocoa in human volunteers

We evaluated the levels of (-)-epicatechin (EC) and its metabolites in plasma and urine after intake of chocolate or cocoa by male volunteers. EC metabolites were analyzed by HPLC and LC/MS after glucuronidase and/or sulfatase treatment. The maximum levels of total EC metabolites in plasma were reached 2 hours after either chocolate or cocoa intake. Sulfate, glucuronide, and sulfoglucuronide (mixture of sulfate and glucuronide) conjugates of nonmethylated EC were the main metabolites present in plasma rather than methylated forms. Urinary excretion of total EC metabolites within 24 hours after chocolate or cocoa intake was 29.8 ± 5.3% and 25.3 ± 8.1% of total EC intake. EC in chocolate and cocoa was partly absorbed and was found to be present as a component of various conjugates in plasma, and these were rapidly excreted in urine.     

Enhanced oxidation of flavan-3-ols and proanthocyanidin accumulation in water-stressed tea plants

(-)-Epicatechin (EC) and (-)-epigallocatechin gallate (EGCG), two major tea flavan-3-ols, have received attention in food science and biomedicine because of their potent antioxidant properties. In plants, flavan-3-ols serve as proanthocyanidin (PA) building blocks, and although both monomeric flavan-3-ols and PAs show antioxidant activity in vitro, their antioxidant function in vivo remains unclear. In the present study, EC quinone (ECQ) and EGCG quinone (EGCGQ), the oxidation products of EC and EGCG, increased up to 100- and 30-fold, respectively, in tea plants exposed to 19 days of water deficit. Oxidation of EC and EGCG preceded PAs accumulation in leaves, which increased from 35 to 53 mg gDW(-1) after 26 days of water deficit. Aside from the role monomeric flavan-3-ols may play in PAs biosynthesis, formation of ECQ and EGCGQ strongly negatively correlated with the extent of lipid peroxidation in leaves, thus supporting a protective role for these compounds in drought-stressed plants. Besides demonstrating flavonoid accumulation in drought-stressed tea plants, we show for the first time that EC and EGCG are oxidized to their respective quinones in plants in vivo.     

Circular dichroism, a powerful tool for the assessment of absolute configuration of flavonoids

Circular dichroism is a powerful tool for establishing the absolute configuration of flavonoids and proanthocyanidin analogues. It has been utilized to study the configuration of flavanones, dihydroflavonols (3-hydroxyflavanones), flavan-3-ols, flavan-4-ols, flavan-3,4-diols, flavans, isoflavans, isoflavanones, pterocarpans, 6a-hydroxypterocarpans, rotenoids, 12a-hydroxyrotenoids, neoflavonoids, 3,4-dihydro-4-arylcoumarins, 4-arylflavan-3-ols, auronols, homoisoflavanones, proanthocyanidins, and various classes of biflavonoids. Results relevant to the correlation of circular dichroic data and the absolute configuration of the diastereoisomers of some of the above classes of compounds will be discussed.     

Changes and importance of oligomeric procyanidins during maturation of grape seeds

Flavans and procyanidins from the seeds of different grape varieties were separated and identified using HPLC techniques. The compounds identified were (+)-catechin and (?)-epicatechin, dimeric procyanidins B1, B2, B3 and B4, trimeric procyanidin C2 and gallic acid. During maturation of the grape berries, the flavan-3-ol content fell in the seeds whereas procyanidin levels increased. This suggests an interrelationship between the compounds. There was also evidence of varietal differences in the amounts of phenolic compounds in grape seeds.     

The flavan-3-ol fraction of cocoa powder suppressed changes associated with early-stage metabolic syndrome in high-fat diet-fed rats

Aims

Previous epidemiological studies have suggested that ingestion of chocolate reduces the risk of cardiovascular disease. In the present study, we examined the effects of flavan-3-ols derived from cocoa on blood pressure, lipolysis, and thermogenesis in rats fed a high-fat diet and that showed early signs of metabolic syndrome.

Main methods

The rats were divided into three groups, and fed either normal diet (normal), 60% fat high-fat diet (HFD), or HFD containing 0.2% flavan-3-ols (HFD-flavan) for 4 weeks. At the end of the feeding period, blood pressure was measured and animals were sacrificed under anesthesia. Lipolysis and thermogenesis-related protein levels were measured in several tissues by Western blotting, and mitochondrial DNA copy number was measured by RT-PCR.

Key findings

Mean blood pressure and epididymal adipose tissue weight of HFD-flavan were significantly lower compared with those of HFD. Uncoupling protein (UCP)1 in brown adipose tissue and UCP3 in gastrocnemius of HFD-flavan were significantly increased compared with those of HFD group. Carnitine palmitoyltransferase (CPT) 2 levels in liver and medium-chain acyl-CoA dehydrogenase (MCAD) levels in gastrocnemius and liver were significantly increased by the supplementation of flavan-3-ols.

Significance

In addition to having hypotensive effects, flavan-3-ols enhance thermogenesis and lipolysis and consequently reduce white adipose tissue weight gain in response to high-fat diet feeding.     

GC-MS detection of chiral markers in cocoa beans of different quality and geographic origin

Fermented cocoa beans (Theobroma cacao L., Sterculiaceae) from different countries of origin (Ecuador, Ghana, Trinidad) and cocoa beans roasted under defined conditions (industrial roasting; 150-220 degrees C for 20 min, dry roasting in conventional oven) were analyzed for their contents of certain chiral hydroxy acids, catechins, and amino acids. Cocoa beans are fermented, dried, and industrially transformed by roasting for the production of chocolate, cocoa powders, and other cocoa-related products. Fermentation and roasting conditions influence the contents of chiral compounds such as hydroxy acids, amino acids, and polyphenols, depending on technological procedures as well as some technical parameters. The aim of this work was to check if the content and nature of the named chiral compounds present both in fermented and roasted cocoa beans could be related to the traditional parameters used to classify the variety of seeds and the degree of fermentation. The extent of racemization of amino acids in fermented cocoa beans was low while it slowly increased during roasting, depending on the temperature applied. L-lactic acid was always higher than the D-form while citric acid was generally the most abundant hydroxy acid detected in beans. A correlation was found between polyphenol content and degree of fermentation, while epimerization of (-)-epicatechin to (+)-catechin was observed during roasting. On the whole, results showed that several chiral compounds could be considered as good quality markers for cocoa seeds and cocoa-related products of different quality and geographic origin.     

Inhibition of angiotensin converting enzyme (ACE) activity by flavan-3-ols and procyanidins

It was determined that flavan-3-ols and procyanidins have an inhibitory effect on angiotensin I converting enzyme (ACE) activity, and the effect was dependent on the number of epicatechin units forming the procyanidin. The inhibition by flavan-3-ols and procyanidins was competitive with the two substrates assayed: N-hippuryl-L-histidyl-L-leucine (HHL) and N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG). Tetramer and hexamer fractions were the more potent inhibitors, showing Ki of 5.6 and 4.7 microM, respectively. As ACE is a membrane protein, the interaction of flavanols and procyanidins with the enzyme could be related to the number of hydroxyl groups on the procyanidins, which determine their capacity to be adsorbed on the membrane surface.     

Cacao biotechnology: current status and future prospects

Theobroma cacao—The Food of the Gods, provides the raw material for the multibillion dollar chocolate industry and is also the main source of income for about 6 million smallholders around the world. Additionally, cocoa beans have a number of other nonfood uses in the pharmaceutical and cosmetic industries. Specifically, the potential health benefits of cocoa have received increasing attention as it is rich in polyphenols, particularly flavonoids. At present, the demand for cocoa and cocoa‐based products in Asia is growing particularly rapidly and chocolate manufacturers are increasing investment in this region. However, in many Asian countries, cocoa production is hampered due to many reasons including technological, political and socio‐economic issues. This review provides an overview of the present status of global cocoa production and recent advances in biotechnological applications for cacao improvement, with special emphasis on genetics/genomics, in vitro embryogenesis and genetic transformation. In addition, in order to obtain an insight into the latest innovations in the commercial sector, a survey was conducted on granted patents relating to T. cacao biotechnology.     

The Arabidopsis MATE transporter TT12 acts as a vacuolar flavonoid/H+ -antiporter active in proanthocyanidin-accumulating cells of the seed coat

Phenotypic characterization of the Arabidopsis thaliana transparent testa12 (tt12) mutant encoding a membrane protein of the multidrug and toxic efflux transporter family, suggested that TT12 is involved in the vacuolar accumulation of proanthocyanidin precursors in the seed. Metabolite analysis in tt12 seeds reveals an absence of flavan-3-ols and proanthocyanidins together with a reduction of the major flavonol quercetin-3-O-rhamnoside. The TT12 promoter is active in cells synthesizing proanthocyanidins. Using translational fusions between TT12 and green fluorescent protein, it is demonstrated that this transporter localizes to the tonoplast. Yeast vesicles expressing TT12 can transport the anthocyanin cyanidin-3-O-glucoside in the presence of MgATP but not the aglycones cyanidin and epicatechin. Inhibitor studies demonstrate that TT12 acts in vitro as a cyanidin-3-O-glucoside/H(+)-antiporter. TT12 does not transport glycosylated flavonols and procyanidin dimers, and a direct transport activity for catechin-3-O-glucoside, a glucosylated flavan-3-ol, was not detectable. However, catechin-3-O-glucoside inhibited TT12-mediated transport of cyanidin-3-O-glucoside in a dose-dependent manner, while flavan-3-ol aglycones and glycosylated flavonols had no effect on anthocyanin transport. It is proposed that TT12 transports glycosylated flavan-3-ols in vivo. Mutant banyuls (ban) seeds accumulate anthocyanins instead of proanthocyanidins, yet the ban tt12 double mutant exhibits reduced anthocyanin accumulation, which supports the transport data suggesting that TT12 mediates anthocyanin transport in vitro.     

Analysis of cocoa products for ochratoxin A and aflatoxins

Eighty-five samples of cocoa products sampled in Canada were analysed for ochratoxin A (OTA) and aflatoxins in 2011–2012. Inclusion of the aflatoxins in this survey required additional method development. Chocolate was extracted with methanol–water plus NaCl, while for cocoa two successive extractions with methanol and methanol–water were made. Extracts were cleaned on an AflaOchra immunoaffinity column (IAC). Determination was by reversed phase high performance liquid chromatography (HPLC). Detection of the aflatoxins was with a post-column photochemical reactor and of OTA by fluorescence detection. Mean limits of quantification (LOQ) of chocolate and cocoa powders were 0.16 ng/g (OTA) and 0.07 ng/g (aflatoxin B₁), respectively. Survey results showed that the incidences of OTA above the LOQ in natural cocoa were 15/15 (mean 1.17 ng/g), 20/21 for alkalized cocoa (mean 1.06 ng/g), 9/9 for baking chocolate (mean 0.49 ng/g), 20/20 for dark chocolate (mean 0.39 ng/g), 7/10 for milk chocolate (mean 0.19 ng/g), 5/5 for cocoa liquor (mean 0.43 ng/g), and 0/5 for cocoa butter. These results confirm our previous work with OTA. In the same samples, incidences of aflatoxin B₁ above the LOQ were 14/15 for natural cocoa (mean 0.86 ng/g), 20/21 for alkalized cocoa (mean 0.37 ng/g), 7/9 for baking chocolate (mean 0.22 ng/g), 16/20 for dark chocolate (mean 0.19 ng/g), 7/10 for milk chocolate (mean 0.09 ng/g), 4/5 for cocoa liquor (mean 0.43 ng/g), and 0/5 for cocoa butter. Both aflatoxins and OTA were confirmed by HPLC-MS/MS when OTA or aflatoxin levels found were above 2 ng/g in cocoa.     

Inhibitory effects of cocoa flavanols and procyanidin oligomers on free radical-induced erythrocyte hemolysis

Excessive peroxidation of biomembranes is thought to contribute to the initiation and progression of numerous degenerative diseases. The present study examined the inhibitory effects of a cocoa extract, individual cocoa flavanols (-)-epicatechin and (+)-catechin, and procyanidin oligomers (dimer to decamer) isolated from cocoa on rat erythrocyte hemolysis. In vitro, the flavanols and the procyanidin oligomers exhibited dose-dependent protection against 2,2'-azo-bis (2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis between concentrations of 2.5 and 40 microM. Dimer, trimer, and tetramer showed the strongest inhibitory effects at 10 microM, 59.4%, 66.2%, 70.9%; 20 microM, 84.1%, 87.6%, 81.0%; and 40 microM, 90.2%, 88.9%, 78.6%, respectively. In a subsequent experiment, male Sprague-Dawley rats (approximately 200 g; n = 5-6) were given a 100-mg intragastric dose of a cocoa extract. Blood was collected over a 4-hr time period. Epicatechin and catechin, and the dimers (-)-epicatechin-(4beta>8)-epicatechin (Dimer B2) and (-)-epicatechin-(4beta>6)-epicatechin (Dimer B5) were detected in the plasma with concentrations of 6.4 microM, and 217.6, 248.2, and 55.4 nM, respectively. Plasma antioxidant capacity (as measured by the total antioxidant potential [TRAP] assay) was elevated (P < 0.05) between 30 and 240 min following the cocoa extract feeding. Erythrocytes obtained from the cocoa extract-fed animals showed an enhanced resistance to hemolysis (P < 0.05). This enhanced resistance was also observed when erythrocytes from animals fed the cocoa extract were mixed with plasma obtained from animals given water only. Conversely, plasma obtained from rats given the cocoa extract improved the resistance of erythrocytes obtained from rats given water only. These results show cocoa flavanols and procyanidins can provide membrane protective effects.     

Effect of flavan-3-ols on in vitro egg hatching, larval development and viability of infective larvae of Trichostrongylus colubriformis

The effects of flavan-3-ols (the monomer units of condensed tannins (CT)) and their galloyl derivatives on the viability of eggs, the development of first stage (L1) larvae, and the viability of the infective larvae of Trichostrongylus colubriformis were investigated under in vitro conditions. Each of the flavan-3-ol gallates showed some inhibition of egg hatching at 100 μg/ml, and 100% inhibition at 1000 μg/ml, with epigallocatechin gallate being the most effective in the egg hatch (EH) assay. In contrast, none of the flavan-3-ols were able to completely inhibit egg hatching. The flavan-3-ols and galloyl derivatives dose-dependently inhibited the development of infective larvae as assessed by the larval development (LD) assay. A larval migration inhibition (LMI) assay was used to assess the effect of flavan-3-ols and their galloyl derivatives on the motility of the infective third-stage (L3) larvae of T. colubriformis. In general, the flavan-3-ol gallates were more effective than the flavan-3-ols at immobilising the infective larvae as evidenced by their ability to inhibit more (P<0.05–0.01) larvae from passing through the LMI sieves. At 500 μg/ml, epigallocatechin gallate inhibited significantly more (P<0.1) larvae from passing through the sieves than did catechin gallate, epicatechin gallate, or gallocatechin gallate. Comparisons were made between the flavan-3-ols and their galloyl derivatives with the in vitro effects of CT extracts from several forage legumes, which have exhibited effects on parasites in vivo. The forage legumes tested at 200–500 μg/ml reduced the proportion of eggs that hatch, with comparable results to those obtained using the flavan-3-ols. The activities may be influenced by the prodelphinidin: procyanidin (PD:PC) ratios: CT extracts from Lotus pendunculatus and sainfoin have PD:PC ratios of 70:30 and 77:23, respectively, whereas the less active CT extract from Lotus corniculatus has a PD:PC ratio of 27:73. The active CT extracts from forage legumes have epigallocatechin as the dominant flavan-3-ol extender unit, and epigallocatechin is the most active flavan-3-ol in both the EH and LD assays.