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Review

Impact of Cocoa Products Intake on Plasma and Urine Metabolites: A Review of Targeted and Non-Targeted Studies in Humans

by
Ana Lucía Mayorga-Gross
1,* and
Patricia Esquivel
2
1
Centro Nacional de Ciencia y Tecnología de Alimentos, Universidad de Costa Rica, San Pedro 11501-2060, Costa Rica
2
Escuela de Tecnología de Alimentos, Universidad de Costa Rica, San Pedro 11501-2060, Costa Rica
*
Author to whom correspondence should be addressed.
Nutrients 2019, 11(5), 1163; https://doi.org/10.3390/nu11051163
Submission received: 13 February 2019 / Revised: 19 April 2019 / Accepted: 25 April 2019 / Published: 24 May 2019
(This article belongs to the Special Issue Cocoa, Chocolate and Human Health)
Versions Notes

Abstract

:
Cocoa is continuously drawing attention due to growing scientific evidence suggesting its effects on health. Flavanols and methylxanthines are some of the most important bioactive compounds present in cocoa. Other important bioactives, such as phenolic acids and lactones, are derived from microbial metabolism. The identification of the metabolites produced after cocoa intake is a first step to understand the overall effect on human health. In general, after cocoa intake, methylxanthines show high absorption and elimination efficiencies. Catechins are transformed mainly into sulfate and glucuronide conjugates. Metabolism of procyanidins is highly influenced by the polymerization degree, which hinders their absorption. The polymerization degree over three units leads to biotransformation by the colonic microbiota, resulting in valerolactones and phenolic acids, with higher excretion times. Long term intervention studies, as well as untargeted metabolomic approaches, are scarce. Contradictory results have been reported concerning matrix effects and health impact, and there are still scientific gaps that have to be addresed to understand the influence of cocoa intake on health. This review addresses different cocoa clinical studies, summarizes the different methodologies employed as well as the metabolites that have been identified in plasma and urine after cocoa intake.

1. Introduction

1.1. Theobroma cacao L. composition

Cocoa (Theobroma cacao L.) is a tree from the Malvaceae family. Its seeds are covered by a sweet and sour mucilage which contains approximately 11% of sugars, mainly sucrose, and an acidic environment with a pH of about 3.5–3.8. Citric acid is the main organic acid present in the pulp. Others such as oxalic, phosphoric, malic and tartaric acid are also present [1,2,3,4,5,6].
Fat is the main component of cocoa beans: it accounts to almost 50% of the cotyledon dry weight, where 98% corresponds to neutral lipids, and 2% to polar lipids, mainly phospholipids and glycolipids. It has been reported that the major cocoa fatty acids are palmitic, stearic and oleic [7,8].
Proteins represent 17–20% of the dried bean [9,10]. Vicilin-like globulins and albumins are the two most abundant proteins and are key factors in quality development during fermentation. Concerning the amino acid profile, glutamic acid and aspartic acid showed the highest contents while cysteine showed the lowest ones, in both fermented and non-fermented cocoa derived products [11].
From a functional point of view, cocoa has been considered an important source of different bioactive compounds. The more important groups of compounds present in cacao are flavonoids, mainly flavan-3-ols, phenolic acids, methylxanthines, peptides, N-phenylpropenoyl-L-amino acids, and stilbenes [5,12,13,14,15,16,17,18,19,20].
Polyphenols represent approximately 13% of the dried unfermented cocoa beans [21], where proanthocyanidins can reach 58% of the total polyphenol content, followed by catechins and anthocyanins with 37% and 4%, respectively [22]. Proanthocyanidins are usually (epi) catechin-based, i.e., procyanidins, and the main anthocyanins are cyanidin-3-galactoside and cyanidin-3-arabinoside [21,23,24].
The main methylxanthines are theobromine, caffeine, theophylline, and 7-methylxanthine. Theobromine is the most concentrated methylxanthine, and it could be present in concentration levels of 1–2% (dry basis) in cocoa seeds; caffeine also has an important contribution from 0–2% (dry basis) [5].
Additionally, N-phenylpropenoyl-L-amino acids have been identified in cocoa and described as polyphenol-amino acid conjugates, some of which have been related to antioxidant mechanisms, inducers of mitochondrial activity and inhibitors of pathogen adhesion in stomach tissues [20].
Cocoa and cocoa derived products suffer significant changes throughout the processing, and this has to be taken into account when studying the effects of cocoa intake on human health.
Traditionally, cocoa undergoes different processing steps after harvesting, some of which are fermentation, drying, roasting [6], and size reduction steps.
During fermentation and drying cocoa beans have extensive proteolysis, oxidation, and polymerization reactions, among others, resulting in a decrease on total flavonoids and the emergence of new metabolites [5,25,26,27,28].
Also, during roasting, high temperatures lead to glucose and sugar depletion, and to the formation of pyrazines, pyrroles, quinoxalines, pyrones, phenylalk-2-enals, lactones, diketopiperazines, and derivatives of phenylalanine, furanones and others. Compounds, such as triacylglycerols, alcohols, esters, and acids, do not go through significant changes [5]. On the other hand, flavanols and procyanidins can be affected by different temperature dependent reactions, as for example epimerization [3,29,30,31].
Roasted beans [3,5] are the starting point for chocolate and cocoa powder production, which are the cocoa derived products most commonly consumed. In both cases, formulation steps are needed, which increase the diversity of compounds present in these matrices and their interactions.

1.2. Cocoa and Health: General Aspects

A variety of health benefits have been associated to the intake of cocoa and its derived products, many of them attributed to the intake of polyphenols, particularly flavonoids [32,33,34]: improvement in insulin resistance by lowering serum insulin and in flow-mediated dilatation [35], as well as improvement in blood pressure, maintenance of normal endothelium-dependent vasodilation, vascular and platelet function, increased cerebral blood flow, potential cancer prevention, and anti-inflammatory and antioxidant activity [36,37,38,39,40]. Nonetheless, contradictory evidence has been reported indicating that more research is still needed [41,42,43,44,45,46].
It has been proposed that cocoa polyphenols can also have an influence on the gut microbiota, promoting the development of microorganisms as Lactobacillus spp. and Bifidobacterium spp. Microbiota can metabolize polyphenols into different bioactives, as valerolactones, phenylpropionic and phenylacetic derivatives some of which could activate anti-inflammatory pathways [33,47,48].
Other bioactive compounds, like methylxanthines, have shown possible beneficial effects. For example, caffeine may improve exercise performance, but more research is needed to understand the mechanisms behind this statement [49].
To understand the effects of cocoa on human health, it is fundamental to know which metabolites are produced after cocoa intake, as this can help understand the absorption, distribution, metabolism, and excretion mechanisms [47,50]. The development of different types of clinical trials contributes to this knowledge; and, in relation to cocoa, there is a particular need to promote long term research.
This review collects the results of different studies that traced several metabolites in urine and plasma after the acute or chronic cocoa intake.

2. Methodological Considerations

Data collection was done by searching individual and compound keywords in different databases, such as: cocoa, chocolate, humans, randomized, acute, health, untargeted, non-targeted, metabolomics, urine, plasma, pharmacokinetics, polyphenols, (epi) catechin, procyanidin, methylxanthines, and theobromine. The main databases used were: Google Scholar, ScienceDirect, ACS Publications, and Springer Link. The searches were done in between August 2016 and February 2019 and included studies from 1999 to February 2019.

3. Results

Table 1 and Table 2 summarize several studies that analyze the effects of the intake of different cocoa derivatives, such as chocolate (Table 1) and cocoa powder drinks or extracts (Table 2) in human plasma and urine samples using a targeted methodology. Meanwhile Table 3 summarizes non-targeted researches, including chocolate or cocoa powder drink intake studies, as well as one cross sectional study.
The methodological aspects are detailed, including the main objective of the studies, the experimental design applied [51,52,53,54], information regarding the subjects, matrices and dose employed, the biological samples studied, the analytical instrumental technique used to measure the metabolites, and the statistics applied to process data.
A total of 34 studies were reviewed, 30 of them used a targeted approach, and 4 of them were untargeted. The clinical studies employed different cocoa derived matrices. Nine of these studies included chocolate intake intervention, 24 used cocoa powders or cocoa extracts beverages, and 2 of them had a cross-sectional experimental design which did not apply dietary interventions [55,56].
Most researches followed acute single dose interventions, nonetheless only 2 of them evaluated the effect of a daily cocoa-derived products intake after 4 weeks [57,58]. Additionally, 10 studies included a non-cocoa-derived control intake in their experimental design.
Regarding the type of samples analyzed, most studies (n) investigated plasma (n = 8), urine (n = 14) or both plasma and urine (n = 12). Additionally, one study analyzed saliva [59] and another studied feces [60].
The number of volunteers (n) that finalized clinical studies was varied. In five studies only 5 volunteers participated, in ten studies 5 < n ≤ 10 were part of the clinical trial, eight studies included 10 < n ≤ 20 and twelve studies recruited and selected volunteers in a range from 21 to 80 volunteers.
Most clinical studies investigated samples from healthy non-smokers. Only two trials obtained samples from smokers [61] and from volunteers with high cardiovascular risk [57]. Ten studies investigated only men, 18 studies men and women, and one study investigated children [56].
LC-UV/Vis (liquid chromatography couple to ultraviolet or visible detectors) and FLD (fluorescence detectors), and LC-MS (liquid chromatography couple to mass spectrometers) were the predominant analytical platforms reported. In particular, when using an untargeted approach, LC-MS was present in all studies; and one study also applied NMR (nuclear magnetic resonance) [20].
Further on, Table 4, Table 5, Table 6, Table 7 and Table 8 were used to classify the metabolites identified in urine and plasma, in different studies with different approaches.
In particular, studies that mentioned acute intake of cocoa are summarized in the following tables: (a) Table 4 and Table 6 include information of different metabolites as concentrations measured in the biological samples and the corresponding sampling periods. The type of matrix analyzed is also indicated. Both tables summarize information of targeted studies, the first focused on plasma (Table 4) and the other in urine (Table 6) analysis; (b) Table 5 shows the time after intake of different chocolates, where the highest signals of the different metabolites were detected or expected in plasma samples, (c) Table 7 focuses on the main metabolites found in urine that discriminated between different periods of time after intake, with an untargeted approach.
On the other hand, discriminating metabolites in between regular/chronic consumers and non-regular/chronic consumers observed in urine and plasma are listed on Table 8. This covers both targeted and untargeted studies.
Metabolites were organized by means of the chemical group, mainly methylxanthines, flavonoids and conjugates, phenolic acids, and lignans. As for the data collected from untargeted studies, data organization was done in a way that respected as much as possible the authors´ original classification. Additionally, in each category metabolites were organized alphabetically.

4. Discussion

4.1. Methylxanthines

Theobromine showed some of the higher concentrations in plasma and urine after cocoa intake in different human intervention trials (Table 4 and Table 5). One study [81] showed that unmetabolized theobromine in urine was close to 1.5 times higher when consuming methylxanthine enriched powder compared to the non-enriched cocoa powder, which indicates that renal clearance could vary in function of exposure. Due to a possible saturation of the pathway, a lack of enzymes and/or cofactors to process the metabolite may have had affected [81,88].
There is evidence that suggests that excretion also depends on gender and age. After consumption of chocolate in a cross-over study, theobromine excretion showed by women almost doubled man´s levels and was also higher for volunteers younger than 29 years, and lower compared to the ones older than 54 years [84].
A variety of methylxanthines metabolites were also observed in non-targeted studies (Table 7), which follow characteristic metabolic pathways [89]. Some of them have been proposed as possible biomarkers of regular cocoa intake as AMMU, 3-methyluric acid, 7-methylxanthine, 3-methylxanthine, theobromine, and 3,7-dimethyluric acid [55].

4.2. Polyphenols

Polyphenols such as (epi) catechin, and the corresponding glucuronides, sulfates, with or without methylations were the most frequently analyzed metabolites.
One example is a study that evaluated plasma and urine samples after drinking a cocoa beverage. The most abundant flavanol metabolites were (-)-epicatechin-3´-β-D-glucuronide, (-)-epicatechin-3´-sulfate, 3´-O-methyl-(-)-epicatechin-5-sulfate, and 3´-O-methyl-(-)-epicatechin-7-sulfate which represented close to 94% of the total flavanol metabolites that were measured in plasma [82].
This is consequence of the metabolic pathways that flavanols and phenolic acids follow. When absorbed, these metabolites are transferred to the liver or other tissues where phase I and phase II reactions occur. In phase I, cytochrome enzymes lead to hydroxylations, oxidations and reductions; meanwhile, phase II produces conjugates by action of glucuronosyl transferases, sulfotransferases, and catechol-O-methyl transferases [75,90,91,92].
In general terms these metabolites reached maximum concentrations in plasma in less than 4 hours after intake (Table 4). One exception was (-)-epicatechin-7´-β-D-glucuronide, that in one study required 12.8 ± 4.8 h after cocoa intake to reach maximal concentrations [66].
When these metabolites are present in plasma, they could exert particular effects. For example, one study analyzed the impact of chocolate intake on oxidative damage, and observed an increase in plasma antioxidant activity and plasma lipid oxidation products decrease as epicatechin plasma levels increased [63]. Additionally, elimination rates have an influence on the probabilities of accumulation in blood or tissues, and the subsequent effect on health [93].
In this matter, when studying urine samples, the required time to reach maximal concentration was found to be largely variable. While (epi) catechin-O-sulfate isomer required a time in between 0–2 h after cocoa powder in milk intake to reach the higher concentration [77], (-)-epicatechin-7-β-D-glucuronide required a time in between 10–24 h after dark chocolate consumption [66].
Influence of age on metabolism was analyzed in an intervention that evaluated two cocoa powder drinks. Non-significant differences or small differences were observed for the different pharmacokinetic parameters, in the renal clearance, total excreted metabolites, and apparent volume of distribution in two age groups: 18–35 years and 65–80 years. Some of the small differences observed were associated with changes in renal function due to normal aging processes [82].
Studies converge in that increasing polymerization degree (n > 2) of procyanidins hinders their absorption. Procyanidin B2 dimer has been detected in plasma after consuming a cocoa beverage, showing a maximum concentration in the period between 0.5–2 h (Table 5). In this same study, B5 dimer was not detected [68]. Similar results were observed in another study where volunteers drank samples with cocoa extracts that contained procyanidins from 2–10 units. In this study, B2 dimer was detected in plasma in approximately 80% of the volunteers, indicating maximum concentrations at 2 h after intake, and with concentrations close to the limit of detection. Additionally, B5 dimer was not detected in any of the plasma samples [80].
Polyphenol absorption and excretion can depend on the food matrix, but contradictory information exists. For example, excretion patterns of microbial phenolic acids metabolites measured in urine differed depending on the liquid vehicle of cocoa powder consumed: water or milk [78]. One randomized study compared the levels of (-)-epicatechin glucuronide in plasma after the intake of cocoa powder with milk or water, and reported no statistical differences in the results from each group [73]. Also, milk protein did not affect the bioavailability of polyphenols present in a chocolate drink [74]. In another study the excretion of different flavan-3-ol metabolites within 24 h after cocoa intake was close to 20% of the ingested dose if cocoa was provided as a water-based beverage, but only 10% if ingested as a milk-based cocoa drink [77]. When consuming two cocoa beverages, either prepared with water or with milk, the total excretion was not affected, but kinetics was different. In the same period of time after intake (-)-epicatechin glucuronide was the most concentrated metabolite when water was the vehicle—doubling the concentration of total excreted (-)-epicatechin sulfates- but when milk was used, glucuronides and total sulfates reached similar concentrations in urine [75]. No significant differences in excretion patterns were found when comparing a variety of metabolites in urine after consuming cocoa powder in milk or in water [86]. It is suggested that polyphenol absorption and metabolism might be affected because of the interactions between milk polypeptides and polyphenols, which can decrease the bioaccessibility [94,95]. In addition, polyphenol absorption could also be affected by carbohydrates, as it has been reported that flavanol uptake could increase when simultaneously consuming these macronutrients [50].
Flavanol absorption and metabolism has also been demonstrated to be dependent on the stereochemistry. Differences in absorption and metabolism were detected after the consumption of equal amounts of (-)-epicatechin, (-)-catechin, (+)-catechin, and (+)-epicatechin in a cocoa based milk drink [79].
Non-absorbed flavonoids and phenolic acids could follow metabolic pathways that are carried out by the colonic microbiota [66,90,91,92]. Some epicatechin metabolites can return to small intestine including non-absorbed epicatechin pass to the colon, which can account a total close to 70 % of the total ingested [96].
Microbial metabolism can lead to the production of phenolic acids, sulfated and glucuronidated valerolactones [57]. Some examples are ferulic acid, phenylacetic acid, hippuric acid (Table 6), 4-hydroxy-5-(dihydroxyphenyl)-valeric acid glucuronide, and methoxyhydroxyphenyl valerolactone (Table 6 and Table 7).
In general terms, these types of metabolites require longer times to reach mean concentrations in urine and plasma than the metabolites following phase II metabolism (Table 4 and Table 6, Table 7 and Table 8). To illustrate, ferulic and m-hydroxyphenyl acetic acids showed maximum concentrations in between 24 and 48 h after chocolate intake, and vanillic acid was the only metabolite that returned to basal level concentrations in the 48 h of observation [65].
Research suggests that consumption of cocoa and derivatives could modulate human metabolism. A randomized controlled cross-over intervention trial analyzed the acute consumption of three types of chocolates in 42 healthy volunteers and concluded that several endogenous metabolites were affected (Table 5 and Table 7). In particular, several amino acids, organic acids, creatinine, lactate, and N1-methylnicotinamide decreased meanwhile pyruvate, tyrosine, and p-hydroxyphenylacetate increased after the intervention. Flavan-3-ols, methylxanthines and their metabolites were suggested to modulate endogenous and colonic microbial metabolism [84].
Studies with longer periods of intervention detected additional metabolome changes due to regular cocoa consumption (Table 8). For example, different glucuronide and sulfate conjugates of (-)-epicatechin, O-methyl-epicatechin, dihydroxyphenyl valerolactones, and methoxy hydroxyphenyl valerolactones increased their levels in plasma after regular consumption of cocoa powder in a non-healthy group of volunteers. Hydroxyphenylacetic acids, AMMU isomers, 5-(3´,4´-dihydroxyphenyl)-γ-valerolactone, and its glucuronides and sulfates have been proposed as biomarkers of regular consumption of cocoa [55,57]. Further on, one study reported that methylglutaryl carnitine, derived from the acylcarnitine pathway, decreased after a long-term cocoa intake [55].
More recent studies have supported this information as 5-(3´,4´-dihidroxyphenyl)-γ-valerolactone has also been proposed as a specific biomarker of flavan-3-ols consumption, deriving only from (epi) catechin-based mono and oligomeric cocoa flavanols [83].
There is also evidence that polyphenol consumption from cocoa products might change the gut microbiota, exerting prebiotic effects, and which could be related to the activation of anti-inflammatory pathways with benefits in the host and alter the obtained profile of metabolites. One study observed an increase of Lactobacillus spp. and Bifidobacterium spp. in contrast with a decrease of Clostridium spp. populations after daily consumption during one month of a flavanol-rich cocoa beverage, compared to a low flavanol cocoa beverage [33,97,98].

4.3. Other Metabolites

Metabolites different than methylxanthines and polyphenols metabolism have also been detected after cocoa intake. For example, trigonelline (N-methylnicotinic acid) and hydroxynicotinic acid may be a result of nicotinic acid metabolism. They were detected in urine only within 6 h after cocoa powder consumption. Also, diketopiperazines such as cyclo(ser-tyr) and cyclo(pro-pro) were reported for the first time associated to cocoa powder intake and are considered flavor and taste metabolites [86].
N-phenylpropenoyl-L-amino acids have also been metabolites of interest as there is some preliminary evidence of their biological activity. Thirteen of them have been reported in urine after cocoa intake [20].
Additionally, significant changes in plasma metabolites after 6 h of chocolate intake have been reported for β-hydroxybutyrate, acetone, acetoacetate, and aspartate. (Table 5) [84].
One study observed that daily dark chocolate intake for 2 weeks modulated energy and hormone metabolism of humans with low and high anxiety traits, and also modulated their microbial gut metabolism. Urine analysis showed a reduction of stress hormone metabolites, and other stress-related metabolites presented a trend to change towards the low anxiety profile. Some of these metabolites are glycine, citrate, trans-aconitate, proline, DOPA, β-alanine, hippurate, and p-cresol sulfate [99]. This can eventually help understand the impact of cocoa intake on health.

5. Conclusions

A diversity of metabolites has been identified in urine and plasma after consumption of cocoa or cocoa derived products. Theobromine and caffeine have been some of the main methylxanthines identified together with their metabolites. Polyphenolic phase I and phase II metabolism leads mainly to (epi)catechin sulphates, glucuronides, and sulfoglucuronides. Non absorbed polyphenols are transformed by colonic microorganisms yielding a diversity of metabolites that range from phenolic acids to different valerolactones, and more complex metabolites.
Biomarkers of consumption of cocoa have been proposed: AMMU, 3-methyluric acid, 7-methylxanthine, 3-methylxanthine, theobromine, 3,7-dimethyluric acid, hydroxyphenylacetic acid, and 5-(3´,4´-dihydroxyphenyl)-γ-valerolactone; the latter with its correspondant glucuronides and sulfates.
Evidence shows that absorption, metabolism, and excretion of cocoa metabolites depend on the food matrix, the dose, age, gender, overall health status and other factors such as the polymerization degree (e.g., procyanidins), and stereochemistry (e.g., flavanols).
The development of clinical studies is fundamental to understand the metabolic pathways of different metabolites present in cocoa and further on the effects on health. Special attention must be given to properly design the experiments, and instrumental methodologies for the extraction and quantification of metabolites in biological samples.

Author Contributions

Writing—original draft preparation: A.L.M.-G.; review and editing: P.E.

Funding

This research was done within the project 735-B5-050 “Valorización de matrices alimentarias mediante la evaluación del impacto del procesamiento sobre el perfil metabolómico”, funded by the University of Costa Rica, Costa Rica.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Dand, R. 2-Agronomics of international cocoa production. In The International Cocoa Trade, 3rd ed.; Woodhead Publishing Limited: Cambridge, UK, 2011; pp. 23–64. [Google Scholar]
  2. Integrated Taxonomic Information System (ITIS) Report: Theobroma cacao L. Available online: https://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=505487#null (accessed on 30 December 2017).
  3. Afoakwa, E.O. Cocoa Production and Processing Technology; CRC Press, Taylor & Francis: Boca Raton, FL, USA, 2014; pp. 173–178. [Google Scholar] [CrossRef]
  4. Nair, K.P.P. 5–Cocoa (Theobroma cacao L.). In The Agronomy and Economy of Important Tree Crops of the Developing World; Elsevier: Burlington, MA, USA, 2010; pp. 131–180. [Google Scholar]
  5. Biehl, B.; Ziegleder, G. Cococa: Chemistry of processing. In Encyclopedia of Food Sciences and Nutrition, 2nd ed.; Caballero, B., Trugo, L., Finglas, P.M., Eds.; Elsevier: Amsterdam, The Netherlands, 2003; pp. 1436–1448. [Google Scholar]
  6. Biehl, B.; Ziegleder, G. Cocoa: Production, products and use. In Encyclopedia of Food Sciences and Nutrition, 2nd ed.; Caballero, B., Trugo, L., Finglas, P.M., Eds.; Elsevier: Amsterdam, The Netherlands, 2003; pp. 1448–1463. [Google Scholar]
  7. Hernandez, B.; Castellote, A.I.; Permanyer, J.J. Triglyceride analysis of cocoa beans from different geographical origins. Food Chem. 1991, 41, 269–276. [Google Scholar] [CrossRef]
  8. Parsons, J.G.; Keeney, P.G.; Patton, S. Identification and Quantitative Analysis of Phospholipids in Cocoa Beans. J. Food Sci. 1969, 34, 497–499. [Google Scholar] [CrossRef]
  9. Aremu, C.Y.; Agiang, M.A.; Ayatse, J.O.I. Nutrient and antinutrient profiles of raw and fermented cocoa beans. Plant Foods Hum. Nutr. 1995, 48, 217–223. [Google Scholar] [CrossRef]
  10. Afoakwa, E.O.; Quao, J.; Takrama, J.; Budu, A.S.; Saalia, F.K. Chemical composition and physical quality characteristics of Ghanaian cocoa beans as affected by pulp pre-conditioning and fermentation. J. Food Sci. Technol. 2013, 50, 1097–1105. [Google Scholar] [CrossRef] [PubMed]
  11. Adeyeye, E.I.; Akinyeye, R.O.; Ogunlade, I.; Olaofe, O.; Boluwade, J.O. Effect of farm and industrial processing on the amino acid profile of cocoa beans. Food Chem. 2010, 118, 357–363. [Google Scholar] [CrossRef]
  12. Jerkovic, V.; Bröhan, M.; Monnart, E.; Nguyen, F.; Nizet, S.; Collin, S. Stilbenic profile of cocoa liquors from different origins determined by RP-HPLC-APCI(+)-MS/MS: Detection of a new resveratrol hexoside. J. Agric. Food Chem. 2010, 58, 7067–7074. [Google Scholar] [CrossRef] [PubMed]
  13. Cádiz-Gurrea, M.L.; Lozano-Sanchez, J.; Contreras-Gámez, M.; Legeai-Mallet, L.; Fernández-Arroyo, S.; Segura-Carretero, A. Isolation, comprehensive characterization and antioxidant activities of Theobroma cacao extract. J. Funct. Foods 2014, 10, 485–498. [Google Scholar] [CrossRef]
  14. Cooper, K.A.; Campos-Giménez, E.; Alvarez, D.J.; Nagy, K.; Donovan, J.L.; Williamson, G. Rapid reversed phase ultra-performance liquid chromatography analysis of the major cocoa polyphenols and inter-relationships of their concentrations in chocolate. J. Agric. Food Chem. 2007, 55, 2841–2847. [Google Scholar] [CrossRef] [PubMed]
  15. Hurst, W.J.; Glinski, J.A.; Miller, K.B.; Apgar, J.; Davey, M.H.; Stuart, D.A. Survey of the trans-resveratrol and trans-piceid content of cocoa-containing and chocolate products. J. Agric. Food Chem. 2008, 56, 8374–8378. [Google Scholar] [CrossRef]
  16. Marseglia, A.; Palla, G.; Caligiani, A. Presence and variation of γ-aminobutyric acid and other free amino acids in cocoa beans from different geographical origins. Food Res. Lnt. 2014, 63, 360–366. [Google Scholar] [CrossRef]
  17. Ortega, N.; Romero, M.P.; Macià, A.; Reguant, J.; Anglès, N.; Morelló, J.R.; Motilva, M.J. Comparative study of UPLC–MS/MS and HPLC–MS/MS to determine procyanidins and alkaloids in cocoa samples. J. Food Compost. Anal. 2010, 23, 298–305. [Google Scholar] [CrossRef]
  18. Patras, M.A.; Milev, B.P.; Vrancken, G.; Kuhnert, N. Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn. Food Res. Lnt. 2014, 63, 353–359. [Google Scholar] [CrossRef]
  19. Sánchez-Rabaneda, F.; Jáuregui, O.; Casals, I.; Andrés-Lacueva, C.; Izquierdo-Pulido, M.; Lamuela-Raventós, R.M. Liquid chromatographic/electrospray ionization tandem mass spectrometric study of the phenolic composition of cocoa (Theobroma cacao). J. Mass. Spectrom. 2003, 38, 35–42. [Google Scholar] [CrossRef] [PubMed]
  20. Stark, T.; Lang, R.; Keller, D.; Hensel, A.; Hofmann, T. Absorption of N-phenylpropenoyl-L-amino acids in healthy humans by oral administration of cocoa (Theobroma cacao). Mol. Nutr. Food Res. 2008, 52, 1201–1214. [Google Scholar] [CrossRef]
  21. Kim, J.; Lee, K.W.; Lee, H.J. Cocoa (Theobroma cacao) seeds and phytochemicals in human health. In Nuts and Seeds in Health and Disease Prevention; Preedy, R.V., Watson, R.R., Patel, B.V., Eds.; Academic Press, Elsevier: Burlington, MA, USA, 2011; pp. 351–360. [Google Scholar]
  22. Wollgast, J.; Anklam, E. Review on polyphenols in Theobroma cacao: Changes in composition during the manufacture of chocolate and methodology for identification and quantification. J. Food Res. Int. 2000, 33, 423–447. [Google Scholar] [CrossRef]
  23. Niemenak, N.; Rohsius, C.; Elwers, S.; Omokolo-Ndoumou, D.; Lieberei, R. Comparative study of different cocoa (Theobroma cacao L.) clones in terms of their phenolics and anthocyanins contents. J. Food Compost. Anal. 2006, 19, 612–619. [Google Scholar] [CrossRef]
  24. Ortega, N.; Romero, M.-P.P.; Macià, A.; Reguant, J.; Anglès, N.; Morelló, J.-R.R.; Motilva, M.-J.J. Obtention and characterization of phenolic extracts from different cocoa sources. J. Agri. Food Chem. 2008, 56, 9621–9627. [Google Scholar] [CrossRef]
  25. Ardhana, M.; Fleet, G. The microbial ecology of cocoa bean fermentations in Indonesia. Int. J. Food Microbiol. 2003, 86, 87–99. [Google Scholar] [CrossRef]
  26. Mozzi, F.; Ortiz, M.E.; Bleckwedel, J.; De Vuyst, L.; Pescuma, M. Metabolomics as a tool for the comprehensive understanding of fermented and functional foods with lactic acid bacteria. Food Res. Int. 2003, 54, 1152–1161. [Google Scholar] [CrossRef]
  27. Nazaruddin, R.; Seng, L.K.; Hassan, O.; Said, M. Effect of pulp preconditioning on the content of polyphenols in cocoa beans (Theobroma cacao) during fermentation. Ind. Crops Prod. 2006, 24, 87–94. [Google Scholar] [CrossRef]
  28. Hii, C.L.; Law, C.; Suzannah, S.; Cloke, M. Polyphenols in cocoa (Theobroma cacao L.). As. J. Food Ag-Ind. 2010, 2, 702–722. [Google Scholar]
  29. Gutiérrez, T.J. State-of-the-art chocolate manufacture: A review. Compr. Rev. Food Sci. Food Saf. 2017, 16, 1313–1344. [Google Scholar] [CrossRef]
  30. Ioannone, F.; Di Mattia, C.D.; De Gregorio, M.; Sergi, M.; Serafini, M.; Sacchetti, G. Flavanols, proanthocyanidins and antioxidant activity changes during cocoa (Theobroma cacao L.) roasting as affected by temperature and time of processing. Food Chem. 2015, 174, 256–262. [Google Scholar] [CrossRef]
  31. Kothe, L.; Zimmermann, B.F.; Galensa, R. Temperature influences epimerization and composition of flavanol monomers, dimers and trimers during cocoa bean roasting. Food Chem. 2013, 141, 3656–3663. [Google Scholar] [CrossRef]
  32. Steinberg, F.M.; Bearden, M.M.; Keen, C.L. Cocoa and chocolate flavonoids: Implications for cardiovascular health. J. Am. Diet Assoc. 2003, 103, 215–223. [Google Scholar] [CrossRef]
  33. Magrone, T.; Russo, M.A.; Jirillo, E. Cocoa and dark chocolate polyphenols: From biology to clinical applications. Front Immunol. 2017, 8, 677. [Google Scholar] [CrossRef]
  34. Aprotosoaie, A.C.; Miron, A.; Trifan, A.; Simon, L.V.; Costache, I.J. The cardiovascular effects of cocoa polyphenols–an overview. Diseases 2016, 4, 39. [Google Scholar] [CrossRef]
  35. Hooper, L.; Kay, C.; Abdelhamid, A.; Kroon, P.A.; Cohn, J.S.; Rimm, E.B.; Cassidy, A. Effects of chocolate, cocoa, and flavan-3-ols on cardiovascular health: A systematic review and meta-analysis of randomized trials. Am. J. Clin. Nutr. 2012, 95, 740–751. [Google Scholar] [CrossRef]
  36. Corti, R.; Flammer, A.J.; Hollenberg, N.K.; Lüscher, T.F. Cocoa and cardiovascular health. Circulation 2009, 119, 1433–1441. [Google Scholar] [CrossRef]
  37. Latif, R. Chocolate/cocoa and human health: A review. The Neth. J. Med. 2013, 71, 63–68. [Google Scholar]
  38. Martin, M.A.; Goya, L.; Ramos, S. Potential for preventive effects of cocoa and cocoa polyphenols in cancer. Food Chem. Toxicol. 2013, 56, 336–351. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  39. Decroix, L.; Soares, D.D.; Meeusen, R.; Heyman, E.; Tonoli, C. Cocoa flavanol supplementation and exercise: A systematic review. Sports Med. 2018, 48, 867–892. [Google Scholar] [CrossRef]
  40. EFSA. Scientific Opinion on the modification of the authorisation of a health claim related to cocoa flavanols and maintenance of normal endothelium-dependent vasodilation pursuant to Article 13 (5) of Regulation (EC) No 1924/2006 following a request in accordance with article 19 of Regulation (EC) No 1924/2006. EFSA J. 2014, 12, 3654. [Google Scholar] [CrossRef]
  41. Dicks, L.; Kirch, N.; Gronwald, D.; Wernken, K.; Zimmermann, B.; Helfrich, H.-P.; Ellinger, S. Regular intake of a usual serving size of flavanol-rich cocoa powder does not affect cardiometabolic parameters in stably treated patients with type 2 diabetes and hypertension—a double-blinded, randomized, placebo-controlled trial. Nutrients 2018, 10, 1435. [Google Scholar] [CrossRef]
  42. Vlachojannis, J.; Erne, P.; Zimmermann, B.; Chrubasik-Hausmann, S. The impact of cocoa flavanols on cardiovascular health. Phytother. Res. 2016, 30, 1641–1657. [Google Scholar] [CrossRef] [PubMed]
  43. Ellinger, S.; Stehle, P. Impact of cocoa consumption on inflammation processes—a critical review of randomized controlled trials. Nutrients 2016, 8, 321. [Google Scholar] [CrossRef]
  44. Hollman, P.C.; Cassidy, A.; Comte, B.; Heinone, M.; Richelle, M.; Richling, E.; Serafini, M.; Scalbert, A.; Sies, H.; Vidry, S. The biological relevance of direct antioxidant effects of polyphenols for cardiovascular health in humans is not established. J. Nutr. 2011, 141, 989S–1009S. [Google Scholar] [CrossRef] [PubMed]
  45. Morze, J.; Schwedhelm, C.; Bencic, A.; Hoffmann, G.; Boeing, H.; Przybylowics, K.; Schwingshackl, L. Chocolate and risk of chronic disease: A systematic review and dose-response meta analysis. Eur. J. Nutr. 2019. [Google Scholar] [CrossRef]
  46. González-Sarrías, A.; Combet, E.; Pinto, P.; Mena, P.; Dall´Asta, M.; García-Aloy, M.; Rodríguez-Mateos, A.; Gibney, E.R.; Dumont, J.; Massaro, M.; et al. A systematic review and meta-analysis of the effects of flavanol-containing tea, cocoa and apple products on body composition and blood lipids: Exploring the factors responsible for variability in their efficacy. Nutrients 2017, 9, 746. [Google Scholar] [CrossRef]
  47. Tomás-Barberán, F.A.; Espín, J.C. Effect of food structure and processing on (poly)phenol-gut microbiota interactions and the effects on human health. Annu. Rev. Food Sci. Technol. 2019, 10. [Google Scholar] [CrossRef]
  48. Fraga, C.G.; Croft, K.D.; Kennedy, D.O.; Tomás-Barberán, F.A. The effects of polyphenols and other bioactives on human health. Food Funct. 2019, 10, 514–528. [Google Scholar] [CrossRef] [Green Version]
  49. Stellingwerff, T.; Godin, J.-P.; Chou, C.J.; Grathwohl, D.; Ross, A.B.; Cooper, K.A.; Williamson, G.; Actis-Goretta, L. The effect of acute dark chocolate consumption on carbohydrate metabolism and performance during rest and exercise. Appl. Physiol. Nutr. Metab. 2014, 39, 173–182. [Google Scholar] [CrossRef]
  50. Schramm, D.D.; Karim, M.; Schrader, H.R.; Holt, R.R.; Kirkpatrick, N.J.; Polagruto, J.A.; Ensunsa, J.L.; Schmitz, H.H.; Keen, C.L. Food effects on the absorption and pharmacokinetics of cocoa flavanols. Life Sci. 2003, 73, 857–869. [Google Scholar] [CrossRef]
  51. Machin, D.; Campbell, M.J.; Walters, S.J. Medical Statistics: A Textbook for the Health Sciences, 4th ed.; Wiley: West Sussex, UK, 2007; pp. 217–239. [Google Scholar]
  52. Grimes, D.A.; Schulz, K.F. An overview of clinical research: The lay of the land. Lancet 2002, 359, 57–61. [Google Scholar] [CrossRef]
  53. Friedman, L.M.; Furberg, C.D.; DeMets, D.L.; Reboussin, D.M.; Granger, C.B. Fundamentals of clinical trials, 5th ed.; Springer International Publishing: Cham, Switzerland, 2015. [Google Scholar]
  54. Ferrara, M.; Sébédio, J.-L. 1- Challenges in nutritional metabolomics. In Metabolomics as a Tool in Nutrition Research; Elsevier and Woodhead Publishing: Cambridge, UK, 2015; pp. 3–16. [Google Scholar] [CrossRef]
  55. Garcia-Aloy, M.; Llorach, R.; Urpi-Sarda, M.; Jáuregui, O.; Corella, D.; Ruiz-Canela, M.; Salas-Salvadó, J.; Fitó, M.; Estruch, R.; Andres-Lacueva, C. A metabolomics-driven approach to predict cocoa product consumption by designing a multimetabolite biomarker model in free-living subjects from the PREDIMED study. Mol. Nutr. Food Res. 2015, 59, 212–220. [Google Scholar] [CrossRef]
  56. Rodriguez, A.; Costa-Bauza, A.; Saez-Torres, C.; Rodrigo, D.; Grases, F. HPLC method for urinary theobromine determination: Effect of consumption of cocoa products on theobromine urinary excretion in children. Clin. Biochem. 2015, 48, 1138–1143. [Google Scholar] [CrossRef]
  57. Urpi-Sarda, M.; Monagas, M.; Khan, N.; Llorach, R.; Lamuela-Raventós, R.M.; Jáuregui, O.; Estruch, R.; Izquierdo-Pulido, M.; Andrés-Lacueva, C. Targeted metabolic profiling of phenolics in urine and plasma after regular consumption of cocoa by liquid chromatography-tandem mass spectrometry. J. Chromatogr. 2009, 1216, 7258–7267. [Google Scholar] [CrossRef]
  58. Davinelli, S.; Corbi, G.; Zarrelli, A.; Arisi, M.; Calzavara-Pinton, P.; Grassi, D.; De Vivo, I.; Scapagnini, G. Short-term supplementation with flavanol-rich cocoa improves lipid profile, antioxidant status and positively influences the AA/EPA ratio in healthy subjects. J. Nutr. Biochem. 2018, 61, 33–39. [Google Scholar] [CrossRef]
  59. Ptolemy, A.S.; Tzioumis, E.; Thomke, A.; Rifai, S.; Kellogg, M. Quantification of theobromine and caffeine in saliva, plasma and urine via liquid chromatography-tandem mass spectrometry: A single analytical protocol applicable to cocoa intervention studies. J. Chromatogr. B Analyt. Thecnol. Biomed. Life Sci. 2010, 878, 409–416. [Google Scholar] [CrossRef]
  60. Wiese, S.; Esatbeyoglu, T.; Winterhalter, P.; Kruse, H.-P.; Winkler, S.; Bub, A.; Kulling, S.E. Comparative biokinetics and metabolism of pure monomeric, dimeric, and polymeric flavan-3-ols: A randomized cross-over study in humans. Mol. Nutr. Food Res. 2015, 59, 610–621. [Google Scholar] [CrossRef]
  61. Heiss, C.; Kleinbongard, P.; Dejam, A.; Perré, S.; Schroeter, H.; Sies, H.; Kelm, M. Acute consumption of flavanol-rich cocoa and the reversal of endothelial dysfunction in smokers. J. Am. Coll. Cardiol. 2005, 46, 1276–1283. [Google Scholar] [CrossRef]
  62. Richelle, M.; Tavazzi, I.; Enslen, M.; Offord, E. Plasma kinetics in man of epicatechin from black chocolate. Eu. J. Clin. Nutr. 1999, 53, 22–26. [Google Scholar] [CrossRef]
  63. Wang, J.F.; Schramm, D.D.; Holt, R.R.; Ensunsa, J.L.; Fraga, C.G.; Schmitz, H.H.; Keen, C.L. A dose-response effect from chocolate consumption on plasma epicatechin and oxidative damage. J. Nutr. 2000, 130, 2115S–2119S. [Google Scholar] [CrossRef]
  64. Baba, S.; Osakabe, N.; Yasuda, A.; Natsume, M.; Takizawa, T.; Nakamura, T.; Terao, J. Bioavailability of (-)-epicatechin upon intake of chocolate and cocoa in human volunteers. Free Radic. Res. 2000, 33, 635–641. [Google Scholar] [CrossRef]
  65. Rios, L.Y.; Gonthier, M.-P.; Remesy, C.; Mila, I.; Lapierre, C.; Lazarus, S.A.; Williamson, G.; Scalbert, A. Chocolate intake increases urinary excretion of polyphenol-derived phenolic acids in healthy human subjects. Am. J. Clin. Nutr. 2003, 77, 912–918. [Google Scholar] [CrossRef] [Green Version]
  66. Actis-Goretta, L.; Lévèques, A.; Giuffrida, F.; Romanov-Michailidis, F.; Viton, F.; Barron, D.; Duenas-Paton, M.; González-Manzano, S.; Santos-Buelga, C.; Williamson, G.; et al. Elucidation of (-)-epicatechin metabolites after ingestion of chocolate by healthy humans. Free Radic. Biol. Med. 2012, 53, 787–795. [Google Scholar] [CrossRef]
  67. Costa-Bauza, A.; Grases, F.; Calvó, P.; Rodríguez, A.; Prieto, R.M. Effect of consumption of cocoa-derived products on uric acid crystallization in urine of healthy volunteers. Nutrients 2018, 10, 1516. [Google Scholar] [CrossRef]
  68. Holt, R.R.; Lazarus, S.A.; Sullards, M.C.; Zhu, Q.Y.; Schramm, D.D.; Hammerstone, J.F.; Fraga, C.G.; Schmitz, H.H.; Keen, C.L. Procyanidin dimer B2 [epicatechin-(4β -8)-epicatechin] in human plasma after the consumption of a flavanol-rich cocoa. Am. J. Clin. Nutr. 2002, 76, 798–804. [Google Scholar] [CrossRef]
  69. Ito, H.; Gonthier, M.-P.; Manach, C.; Morand, C.; Mennen, L.; Rémésy, C.; Scalbert, A. Polyphenol leveles in human urine after intake of six different polyphenol-rich beverages. Br. J. Nutr. 2005, 94, 500–509. [Google Scholar] [CrossRef]
  70. Roura, E.; Andrés-Lacueva, C.; Jáuregui, O.; Badia, E.; Estruch, R.; Izquierdo-Pulido, M.; Lamuela-Raventós, R.M. Rapid liquid chromatography tandem mass spectrometry assay to quantify plasma (-)-epicatechin metabolites after ingestion of a standard portion of cocoa beverage in humans. J. Agric. Food Chem. 2005, 53, 6190–6194. [Google Scholar] [CrossRef]
  71. Schroeter, H.; Heiss, C.; Balzer, J.; Kleinbongard, P.; Keen, C.L.; Hollenberg, N.K.; Sies, H.; Kwik-Uribe, C.; Schmitz, H.H.; Kelm, M. (-)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans. Proc. Natl. Acad. Sci. USA 2006, 103, 1024–1029. [Google Scholar] [CrossRef]
  72. Roura, E.; Almajano, M.P.; Bilbao, M.L.M.; Andrés-Lacueva, C.; Estruch, R.; Lamuela-Raventós, R.M. Human urine: Epicatechin metabolites and antioxidant activity after cocoa beverage intake. Free Radical. Res. 2007, 41, 943–949. [Google Scholar] [CrossRef]
  73. Roura, E.; Andrés-Lacueva, C.; Estruch, R.; Mata-Bilbao, M.L.; Izquierdo-Pulido, M.; Waterhouse, A.L.; Lamuela-Raventós, R.M. Milk does not affect the bioavailability of cocoa powder flavonoid in healthy human. Ann. Nutr. Metab. 2007, 51, 493–498. [Google Scholar] [CrossRef]
  74. Keogh, J.B.; McInerney, J.; Clifton, P.M. The effect of milk protein on the bioavailability of cocoa polyphenols. J. Food Sci. 2007, 72, S230–S233. [Google Scholar] [CrossRef]
  75. Roura, E.; Andrés-Lacueva, C.; Estruch, R.; Lourdes Mata Bilbao, M.; Izquierdo-Pulido, M.; Lamuela-Raventós, R.M. The effects of milk as a food matrix for polyphenols on the excretion profile of cocoa (-)-epicatechin metabolites in healthy human subjects. Br. J. Nutr. 2008, 100, 846–851. [Google Scholar] [CrossRef]
  76. Urpi-Sarda, M.; Monagas, M.; Khan, N.; Lamuela-Raventos, R.M.; Santos-Buelga, C.; Sacanella, E.; Castell, M.; Permanyer, J.; Andres-Lacueva, C. Epicatechin, procyanidins, and phenolic microbial metabolites after cocoa intake in humans and rats. Anal. Bioanal. Chem. 2009, 394, 1545–1556. [Google Scholar] [CrossRef]
  77. Mullen, W.; Borges, G.; Donovan, J.L.; Edwards, C.A.; Serafini, M.; Lean, M.E.J.; Crozier, A. Milk decreases urinary excretion but not plasma pharmacokinetics of cocoa flavan-3-ol metabolites in humans. Am. J. Clin. Nutr. 2009, 89, 1784–1791. [Google Scholar] [CrossRef] [Green Version]
  78. Urpi-Sarda, M.; Llorach, R.; Khan, N.; Monagas, M.; Rotches-Ribalta, M.; Lamuela-Raventos, R.; Estruch, R.; Tinahones, F.J.; Andres-Lacueva, C. Effect of milk on the urinary excretion of microbial phenolic acids after cocoa powder consumption in humans. J. Agric. Food Chem. 2010, 58, 4706–4711. [Google Scholar] [CrossRef]
  79. Ottaviani, J.I.; Momma, T.Y.; Heiss, C.; Kwik-Uribe, C.; Schroeter, H.; Keen, C.L. The stereochemical configuration of flavanols influences the level and metabolism of flavanols in humans and their biological activity in vivo. Free Radic. Biol. Med. 2011, 50, 237–244. [Google Scholar] [CrossRef]
  80. Ottaviani, J.I.; Kwik-Uribe, C.; Keen, C.L.; Schroeter, H. Intake of dietary procyanidins does not contribute to the pool of circulating flavanols in humans. Am. J. Clin. Nutr. 2012, 95, 851–858. [Google Scholar] [CrossRef] [Green Version]
  81. Martínez-López, S.; Sarriá, B.; Gómez-Juaristi, M.; Goya, L.; Mateos, R.; Bravo-Clemente, L. Theobromine, caffeine, and theophylline metabolites in human plasma and urine after consumption of soluble cocoa products with different methylxanthine contents. J. Food Res. Int. 2014, 63, 446–455. [Google Scholar] [CrossRef] [Green Version]
  82. Rodriguez-Mateos, A.; Cifuentes-Gomez, T.; Gonzalez-Salvador, I.; Ottaviani, J.I.; Schroeter, H.; Kelm, M.; Heiss, C.; Spencer, J.P.E. Influence of age on the absorption, metabolism, and excretion of cocoa flavanols in healthy subjects. Mol. Nutr. Food Res. 2015, 59, 1504–1512. [Google Scholar] [CrossRef]
  83. Ottaviani, J.I.; Fong, R.; Kimball, J.; Ensunsa, J.L.; Britten, A.; Lucarelli, D.; Luben, R.; Grace, P.B.; Mawson, D.H.; Tym, A.; et al. Evaluation at scale of microbiome-derived metabolites as biomarker of flavan-3-ol intake in epidemiological studies. Sci. Rep. 2018, 8, 9859. [Google Scholar] [CrossRef]
  84. Ostertag, L.M.; Philo, M.; Colquhoun, I.J.; Tapp, H.S.; Saha, S.; Duthie, G.G.; Kemsley, E.K.; De Roos, B.; Kroon, P.A.; Le Gall, G. Acute consumption of flavan-3-ol-enriched dark chocolate affects human endogenous metabolism. J. Proteome Res. 2017, 16, 2516–2526. [Google Scholar] [CrossRef]
  85. Ostertag, L.M.; Kroon, P.A.; Wood, S.; Horgan, G.W.; Cienfuegos-Jovellanos, E.; Saha, S.; Duthie, G.G.; De Roos, B. Flavan-3-ol-enriched dark chocolate and white chocolate improve acute measures of platelet function in a gender-specific way-a randomized-controlled human intervention trial. Mol. Nutr. Food Res. 2013, 57, 191–202. [Google Scholar] [CrossRef]
  86. Llorach, R.; Urpi-Sarda, M.; Jauregui, O.; Monagas, M.; Andres-Lacueva, C. An LC-MS-based metabolomics approach for exploring urinary metabolome modifications after cocoa consumption. J. Proteome Res. 2009, 8, 5060–5068. [Google Scholar] [CrossRef]
  87. Llorach-Asunción, R.; Jauregui, O.; Urpi-Sarda, M.; Andres-Lacueva, C. Methodological aspects for metabolome visualization and characterization: A metabolomic evaluation of the 24 h evolution of human urine after cocoa powder consumption. J. Pharm. Biomed. Anal. 2010, 51, 373–381. [Google Scholar] [CrossRef]
  88. Smart, R.C.; Hodgson, E. Molecular and Biochemical Toxicology, 5th ed.; Wiley: Raleigh, NC, USA, 2018. [Google Scholar]
  89. Caffeine metabolism—Reference pathway. Available online: http://www.genome.jp/kegg-bin/show_pathway?org_name=rn&mapno=00232&mapscale=&show_description=hide (accessed on 11 January 2017).
  90. Kohlmeier, M. Nutrient Metabolism; Academic Press: London, UK, 2003. [Google Scholar]
  91. Urpi-sarda, M.; Llorach, R.; Monagas, M.; Khan, N.; Rotches-Ribalta, M.; Roura, E.; Lamuela-Raventós, R.; Estruch, R.; Andres-Lacueva, C. 5. Effect of cocoa powder in the prevention of cardiovascular disease: Biological, consumption and inflammatory biomarkers. A metabolomic approach. In Recent Advances in Pharmacutical Sciences; Muñoz-Torrero, D., Ed.; Transworld Research Network: Kerata, India, 2011; pp. 121–132. [Google Scholar]
  92. Scalbert, A.; Morand, C.; Manach, C.; Rémésy, C. Absorption and metabolism of polyphenols in the gut and impact on health. Biomed. Pharmacother. 2002, 56, 276–282. [Google Scholar] [CrossRef]
  93. Cifuentes-Gomez, T.; Rodriguez-Mateos, A.; Gonzalez-Salvador, I.; Alañon, M.E.; Spencer, J.P.E. Factors affecting the absorption, metabolism, and excretion of cocoa flavanols in humans. J. Agric. Food Chem. 2015, 63, 7615–7623. [Google Scholar] [CrossRef]
  94. Serafini, M.; Testa, M.F.; Villaño, D.; Pecorari, M.; Van Wieren, K.; Azzini, E.; Brambilla, A.; Maiani, G. Antioxidant activity of blueberry fruit is impaired by association with milk. Free Radic. Biol. Med. 2009, 46, 769–774. [Google Scholar] [CrossRef]
  95. Patton, S.; Keenan, T.W. The Milk fat globule membrane. Biochim. Biophys. Acta Biomembranes. 1975, 415, 273–309. [Google Scholar] [CrossRef]
  96. Borges, G.; Ottaviani, J.I.; Van der Hooft, J.J.J.; Schroeter, H.; Crozier, A. Absorption, metabolism, distribution and excretion of (−)-epicatechin: A review of recent findings. Mol. Aspects Med. 2018, 16, 18–30. [Google Scholar] [CrossRef]
  97. Tzounis, X.; Vulevic, J.; Kuhnle, G.G.C.; George, T.; Leonczak, J.; Gibson, G.R.; Kwik-Uribe, C.; Spencer, J.P.E. Flavanol monomer-induced changes to the human faecal microflora. Br. J. Nutr. 2008, 99, 782–792. [Google Scholar] [CrossRef] [PubMed]
  98. Tzounis, X.; Rodriguez-Mateos, A.; Vulevic, J.; Gibson, G.R.; Kwik-Uribe, C.; Spencer, J.P.E. Prebiotic evaluation of cocoa-derived flavanols in healthy humans by using a randomized, controlled, double-blind, crossover intervention study. Am. J. Clin. Nutr. 2011, 93, 62–72. [Google Scholar] [CrossRef]
  99. Martin, F.-P.J.; Rezzi, S.; Peré-Trepat, E.; Kamlage, B.; Collino, S.; Leibold, E.; Kastler, J.; Rein, D.; Fay, L.B.; Kochhar, S. Metabolic effects of dark chocolate consumption on energy, gut microbiota, and stress-related metabolism in free-living subjects. J. Proteome. Res. 2009, 8, 5568–5579. [Google Scholar] [CrossRef] [PubMed]
Table
Table 1. Summary of the main aspects of targeted cocoa studies, involving chocolate intake.
Table 1. Summary of the main aspects of targeted cocoa studies, involving chocolate intake.
ObjectivesExperimental DesignSubjectsMatrices Description and DoseBiological SamplesSampling periodAnalytical TechniqueStatistical AnalysisYearReference
Analyze plasma kinetics of epicatechin after dark chocolate intake.Non-randomized cross-over. 1-week washout.8 males. Average age and BMI of 40 years and 23.9 kg/m2.40 g and 80 g of dark chocolate with bread.Plasma.0, 1, 2, 3, 4, and 8 h.HPLC-UV or fluorescence.Student´s and Wilcoxon tests.1999[62]
Evaluate changes in plasma epicatechin levels, antioxidant capacity and plasma lipid oxidation products after chocolate intake.Randomized, cross-over. 1-week washout.14 women and 6 men. From 20 to 56 years, with average BMI of 23.8 kg/m2.0, 27, 53, and 80 g of semi-sweet chocolate rich on procyanidins, and 47 g of bread.Plasma.0, 2, and 6 h.HPLC coupled with a coulometric detector.ANOVA with control for multiple measurements, and Tukey–Kramer test.2000[63]
Evaluate (-)-epicatechin and its metabolites in plasma and urine after cocoa and chocolate intervention.Cross-over. 6-day washout.5 males. Ages from 30 to 33 years and BMI from 20.4 to 23.9 kg/m2.Chocolate or cocoa.Urine and plasma.Plasma: Baseline, 1, 2, 4, 8, and 24 h.Urine: 0–8 h, 8–24 h.HPLC and LC-MS negative mode.Student´s t-test.2000[64]
Quantify in urine, different phenolic acids formed in the colon after the consumption of chocolate.Uncontrolled.7 men and 4 women. Mean age of 24, mean height of 172 cm and mean weight of 67 kg.80 g of chocolate.Urine.Baseline, 0–3, 3–6,6–9, 9–24, and 24–48 h.GC-MS, HPLC-DAD, HPLC-ESI-MS in negative ionization mode.ANOVA and Tukey test.2003[65]
Determine theobromine and caffeine in saliva, plasma and urine after dark chocolate intake.Uncontrolled.5 healthy volunteers with no dietary restrictions.41 g of dark chocolate bars.Urine, saliva, and plasma.Baseline and 90 min.UPLC-DAD triple quadrupole MS/MS.No tests were applied. Data was expressed as averages with confidence intervals.2010[59]
Identify and quantify (-)-epicatechin conjugates in plasma and urine after chocolate intake.Uncontrolled.5 healthy volunteers. Average age of 23 years and average BMI of 22 kg/m2.100 g of 70% chocolate having 79 mg of (-)-epicatechin, 26 mg of (±)-catechin, and 49 mg of procyanidin B2.Urine and plasma.Plasma: Baseline, 15, 30, 45 min, and 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 3, 4, 6, 8, 10, 14, 18, and 24 h.
Urine: Baseline, 0–5 h, 5–10 h, 10–24 h, and 0–24 h.		UPLC-ESI-Quattro Micro API.One-way ANOVA with Tukey test.2012[66]
Analyze the relation between chocolate consumption, glucose metabolism, and exercise performance.Single blinded, randomized, cross-over.16 male cyclists (from 4 to 20 h/week of training).Cocoa enriched dark 70% chocolate and cocoa depleted control.Plasma.−10, 0, 15, 30, 45, 60, 90, and 120, 140, 180, 210, 240, 300, and 360 min.HPLC-UV/VisMixed model with F-test, Hidges-Lehmann, Wilcoxonsigned-rank and Student´s t-test.2014[49]
Evaluate the effects of intake of milk chocolate, cocoa powder or dark chocolate consumption on uric acid crystallization.Cross-over.11 males and 9 females with ages from 22 to 65 years.2 × 20 g of a cocoa derived product/day (milk chocolate, chocolate powder and dark chocolate).Urine.Baseline and urine obtained after 12 h overnight at the end of the intervention.HPLC-UV/Vis.ANOVA, Bonferroni and Wilcoxon signed-rank tests.2018[67]
Notes: The abbreviation BMI corresponds to body mass index, and BW to body weight.
Table
Table 2. Summary of the main aspects of targeted cocoa studies, involving cocoa powder or cocoa extracts-based drinks intake.
Table 2. Summary of the main aspects of targeted cocoa studies, involving cocoa powder or cocoa extracts-based drinks intake.
ObjectivesExperimental DesignSubjectsMatrices Description and DoseBiological SamplesSampling periodAnalytical TechniqueStatistical AnalysisYearReference
Determine the presence of specific procyanidins in human plasma after consumption of cocoa flavanol extract.Uncontrolled.3 men and 2 women. Age: 23–34 years, average body weight 70.5 kg.0.375 g of cocoa extract/kg BW in 300 mL of water (average of 26.4 g of cocoa).Plasma.Baseline, 0.5, 2 and 6 h.HPLC-coulometric electrochemical multiple-array detection; HPLC-MS.Kruskal–Wallis one-way ANOVA and Tukey or Dunn tests.2002[68]
Evaluate the effect of a flavanol-rich cocoa beverage on the circulating pool of nitric oxide and of endothelial dysfunction.Randomized, double-blinded, cross-over.6 males and 5 females, with mean age of 31, and mean BMI of 21.8 kg/m2. They smoked an average of 17 cigarettes/day.High and low flavanol content cocoa drink in water.Plasma.Baseline and 2 h.HPLC-FLD.ANOVA, pairwise tests with Bonferroni correction for multiple comparisons.2005[61]
Measure different metabolites in urine after polyphenol-rich beverages intake, with a fast method.Randomized, cross-over. 14-day washout.5 women and 4 men, with ages in between 20–32 years and BMI in between 18.9–24.8 kg/m2.10 g of cocoa powder in 200 mL of water (other non-cocoa beverages were tested in the research), or hot water.Urine.0–24 h.HPLC-ESI-MS/MSMann–Whitney U test.2005[69]
Developing a rapid and reproducible method for analysis of (-)-epicatechin metabolites in plasma and urine.Randomized, cross-over.2 women and 3 men in a range of 18–49 years.250 mL of milk or 40 g of cocoa powder in 250 mL of milk.Plasma and urine.Plasma: 0 and 2 h Urine: 0 and 6 h.HPLC coupled to an API-QQQ-MS.Student´s t-test.2005[70]
Evaluate if dietary flavanols and their metabolites can function as vasoactive mediators.Randomized, double-blinded, cross-over. Minimum 2-day washout.16 males within 25–32 years and with a BMI of 19–23 kg/m2.High and low flavanol content cocoa powder with 300 mL of water.Plasma.Baseline, 1, 2, 3, 4, and 6 h.HPLC-MSANOVA, pairwise tests with Bonferroni correction for multiple comparisons.2006[71]
Analyze (-)-epicatechin metabolites and total antioxidant activity after cocoa beverage intake.Randomized and cross-over.9 women and 12 men, within 18 and 50 years, and with a BMI of 19.1–27.7 kg/m2.40 g of cocoa powder with 250 mL of water and 250 mL of milk as control.Urine.Baseline, 0, 6, 12, and 24 h.API-QQQ-MS/MS.ANCOVA and Student´s t-test; Pearson´s correlation.2007[72]
Analyze the effect of milk in the bioavailability of (-)-epicatechin from a cocoa powder.Randomized and cross-over. 1-week washout.9 women, 12 men, within 18 and 50, and with a BMI of 19.1–27.7 kg/m2.250 mL of milk used as a control, 40 g of cocoa powder dissolved in 250 mL of water or milk.Plasma.Baseline, 2 and 6 h.LC-MS.ANCOVA.2007[73]
Determine the effect of milk protein addition on the uptake of cocoa polyphenols by analyzing metabolites in plasma samples, after a cocoa drink intervention.Randomized, controlled, double blind, and cross-over. 1-week washout.10 men and 14 women. Age in the range of 52–65 years with BMI in the range of 18–30 kg/m2. 13 volunteers were taking medications or dietary supplements.200 mL of dairy and non-dairy chocolate drink.Plasma.Baseline, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 h.Analytical: HPLC-fluorescence.Paired t-test with Bonferroni correction for multiple tests.2007[74]
Quantify and evaluate human metabolism of N-phenylpropenoyl-L-amino acids present in a cocoa drink.Uncontrolled.4 males and 4 females, from 24 to 30 years.Cocoa powder-based beverage.Urine.Baseline, 1, 2, 3, 4, 6 and 8 h.LC-MS/MS multiple reaction monitoring and NMR.Not specified.2008[20]
Evaluate the impact of milk addition on the (-)-epicatechin metabolites after intake of a cocoa drink.Randomized cross-over. 1-week washout.9 women and 12 men with ages between 18–50 and BMI from 19.1 to 27.7 kg/m2.Cocoa beverage with 40 g of cocoa powder and: (a) 250 mL of milk; (b) 250 mL of water or (c) 250 mL of milk without cocoa powder.Urine.0–6, 6–12 and 12–24 h.HPLC coupled to an API-QQQ-MS.ANCOVA and Student´s t-test.2008[75]
Develop an analytical method for determining cocoa metabolites in human and rat urine.Uncontrolled.9 women and 12 men, within 18 and 50 years and a mean BMI of 21.6 kg/m2.40 g of cocoa powder in 250 mL of water.Urine.Baseline and 24 h after intake.SPE and LC-MS/MS.Wilcoxon test.2009[76]
Determine the effect of milk on the bioavailability of cocoa flavan-3-ol metabolites by evaluating plasma and urine samples after cocoa powder intervention.Controlled, cross-over. 4-week washout.6 man and 3 women. Ages in the range of 20–43. Average BMI of 24.7 kg/m2.10 g of cocoa powder in 250 mL of milk or water with 1 g of paracetamol and 5 g of lactulose.Urine and plasma.Plasma: baseline 0.5, 1, 2, 3, 4, 6, 8, and 24 h. Urine: baseline, 0–2, 2–5, 5–8 and 8–24 h.HPLC-PDA-MS2.2-factor repeated measures ANOVA and Student´s t-test.2009[77]
Evaluate plasma and urine metabolites after cocoa powder intake in high cardiovascular risk subjects.Randomized, controlled and cross-over.High CVD risk patients: 19 men and 23 women. Average age of 69.7 years.2 × 20 g of cocoa powder/day with 250 skimmed milk or only 500 mL/day of skimmed milk for 4 weeks.Urine and plasma.0–24 h.LC-MS/MS.Wilcoxon test.2009[57]
Evaluate the effect of milk on the urinary excretion of colonic microbial-derived phenolic acids after cocoa powder intake.Randomized and cross-over.12 men and 9 women. Age in the range of 18–50 years with BMI of 21.6 kg/m2.40 g of cocoa powder in 250 mL of water or milk.Urine.Baseline, 0–6, 6–12, and 12–24 h.LC-MS/MS and LC-PAD.Wilcoxon test for related samples.2010[78]
Study the stereochemical configuration of four different flavanols on their absorption, metabolism, and biological activity.Randomized, double-blinded, cross-over.7 males within 18 and 35 years old, with average BMI of 24 kg/m2.1.5 mg/kg BW of (-)-epicatechin, (+)-epicatechin, (+)-catechin, (-)-catechin, with 0.5 g/kg BW of low-flavanol cocoa based dairy drink.Urine and plasma.Urine: collected over 24 h. Plasma:baseline, 2 and 4 h.HPLC-UV/VisAnd fluorescence.Two-way repeated measures ANOVA and Tukey´s test.2011[79]
Quantify different metabolites in plasma and urine, after the consumption of a cocoa drink with added flavanols and procyanidins.Randomized, double-masked, cross-over.12 males in between 18 and 35 years old, with average BMI of 24 kg/m2.0.48 g/kg BW of flavanol (F) and procyanidin (P) free mimetic cocoa drink powder reconstituted in 4 g/kg of BW of milk with 1% fat with (a) cocoa extract with monomeric F and P, (b) cocoa extract high in P or (c) (-)-epicatechin isolated from cocoa.Urine and plasma.Urine: 0–7 h and 7–24 h. Plasma: Baseline, 1, 2, and 4 h.HPLC-UV and coulometric electrochemical detector, and HPLC-diode array detector.Two-way repeated measures ANOVA and Tukey´s test.2012[80]
Study the bioavailability of methylxanthines in two soluble cocoa products by evaluating plasma and urine samples the after intervention.Randomized, controlled, single-blind, cross-over. 10-day washout.3 males and 10 females. Ages in the range of 18–45 years. Average BMI of 22.5 kg/m2 for men and 23.4 kg/m2 for women.15 g of the powder without enrichment and 25 g of methylxanthine-enriched powder, with 200 mL of semi-skimmed milk.Urine and plasma.Plasma: baseline, 0.5, 1, 2, 3, 4, 6, and 8 h. Urine: baseline, 0–4, 4–8, 8–12, and 12–24 h.HPLC-DAD, HPLC-Q-ToF in positive ionization mode.Mixed model.2014[81]
Analyze the bioavailability, metabolism and microbial breakdown of (-)-epicatechin, procyanidin B1, and a cocoa procyanidin fraction.Randomized, double-blinded, cross-over. 1-week washout.7 healthy male volunteers with ages in between 24 and 31 years and with a BMI of 22 and 26 kg/m2.Pure (-)-epicatechin, pure procyanidin B1 and a purified cocoa polymeric procyanidin fraction.Plasma, urine and feces.Plasma: baseline, 1, 2, 4, 8, 24 and 48 h. Urine: 0–4, 4–8, 8–24 h, and 48 h. Feces: 0–24 h.GC-MS/MS and HPLC-ESI-Q-MS.Not specified.2015[60]
Validate and HPLC method for measuring theobromine in urine and evaluating theobromine urinary levels in children with different cocoa intake patterns.Cross-sectional.80 healthy children from 8–17 years: 26 did not consume cocoa derived products, 19 of them did not consumed cocoa powder but did consume 1 cocoa derived product daily, 12 children just consumed cocoa powder in breakfast and 23 consumed cocoa derived products > once a day.No dietary intervention or recommendations.Urine.12 h in the day and 12 h in the night.HPLC-UV.Kruskal–Wallis and Wilcoxon signed-rank tests.2015[56]
Compare absorption, metabolism, and excretion after a cocoa drink intake.Randomized cross-over. 1-week washout.40 males. Group 1 (young): 18–35 years and average BMI of 24 kg/m2; Group 2 (elderly): 65–80 years and average BMI of 27 kg/m2.Fruit-flavored cocoa powder drinks with 5.3 mg or 10.7 mg of cocoa flavanols/kg BW.Plasma and urine.Plasma: 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, and 24-h.
Urine: 0–2, 2–4, 4–6, 6–12, 12–24 h.		HPLC-FLD/UV and electrochemical detection, and HPLC-UV.Two-way ANOVA with Bonferroni and Tukey tests.2015[82]
Evaluate the effects of milkand dark chocolate and cocoa powder intake on uric acid crystallization.Cross-over.11 males and 9 females with ages from 22 to 65 years.2 × 20 g/day of a cocoa derived product: milk chocolate, chocolate powder and dark chocolate.Urine.Baseline and urine obtained after 12 h overnight at the end of the intervention.HPLC-UV/Vis.ANOVA, Bonferroni and Wilcoxon signed-rank test.2018[67]
Analyze the use of gVL-3´/4´-sulphate and gVL-3´/4´-O-glucuronide as biomarkers of flavan-3-ols intake.Randomized, double-masked, cross-over.I. 8 males, from 25–60 years.
II. 14 males, from 25–40 years.		I. 8 different nondairy drinks with flavan-3-ols (F) with 34.8 mg of (-)-epicatechin or the equivalent concentration of one of the following: (-)-epigallocatechin, (-)-epicatechin-3-O-gallate, (-)-epigallocatechin-3-O-gallate (isolated from green tea), 1:1:1 of theaflavin-3-O-gallate, theaflavin-3‘-O-gallate and theaflavin-3,3‘-O-digallate, thearubigins, (isolated from black tea), procyanidin B2 isolated from cocoa, and a control without added F.
II. Four different nondairy drinks with different amounts of flavan-3-ols ranging from 95 mg to 1424 mg per volunteer.		Urine.I. 0–6 and 6–24 h.
II. 0–4, 4–8, 8–12, and 12–24 h.		UPLC-MS.ANOVA and Student‘s t-test.2018[83]
Evaluate the effect of cocoa intake on lipid profiles and biomarkers of oxidative stress, and arachidonic acid/ eicosapentaenoic acid ratio.Randomized, three-arm parallel group.48 healthy subjects divided in three groups: low-cocoa (n = 16), middle-cocoa (n = 16), and high-cocoa group (n = 16). Average age and BMI were close to 44 years and 23 kg/m2.Cocoa capsules dosed to different groups: a) low-cocoa group: 1 g of cocoa (55 mg/day), middle-cocoa group: 2 g of cocoa (110 mg flavanols/day), and high-cocoa group: 4 g of cocoa (220 mg flavanols/day), for 4 weeks.Plasma and urine.0, 1, 2, and 4 (at the beginning of the study and after 4 weeks of intervention).HPLC-FLD-UVANOVA and Tukey´s test.2018[58]
Notes: The abbreviation BMI corresponds to body mass index, and BW to body weight.
Table
Table 3. Summary of the main aspects of untargeted cocoa intervention studies.
Table 3. Summary of the main aspects of untargeted cocoa intervention studies.
ObjectivesExperimental DesignSubjectsMatrices and DoseBiological SamplesSampling periodAnalytical TechniqueStatistical AnalysisYearReference
Study the chemical profile of plasma and urine after flavan-3-ol enriched dark chocolate, standard dark chocolate and white chocolate intake.Randomized, controlled and cross-over. Minimum 2-wk washout.16 males and 26 females with average BMI of 24.5 kg/m2.60 g of: flavan-3-ol (FLA) enriched dark chocolate, standard dark chocolate low in FLA and white chocolate with no FLA.Plasma and urine.Baseline, 2 and 6 h. (1)1H NMR (600 MHz) and HPLC-ToF-MS.PCA and PLS-DA. Kruskal–Wallis and Dunn tests.2017, 2013[84,85]
Evaluate the changes in humane urine metabolome after cocoa powder intake.Randomized, controlled and cross-over. 1-week washout.5 men and 5 women. Ages in the range of 18–50 and average BMI of 21.6 kg/m2.40 g of cocoa powder in 250 mL of water or milk and 250 mL of milk as a control.Urine.Baseline, 0–6, 6–12 and 12–24 h.HPLC-Q-ToF. Positive ionization mode.PCA, PLS-DA, (OSC) PLS and OSC-PLS-DA.2009[86]
Analyze the changes in urine metabolome after cocoa powder intervention.Uncontrolled.5 women and 5 men between 18–50 years with BMI 21.6 kg/m2.40 g of cocoa powder with 250 mL of milk.Urine.Baseline, 0–6, 6–12, and 12–24 h.HPLC-Q-ToF-MS in positive mode.PLS-DA, OSC-PLS-DA and two-way hierarchical clustering (HCA) applying Bonferroni correction.2010[87]
Apply an untargeted metabolomics approach to propose a model that can discriminate the urinary metabolome of regular cocoa product consumers and non-consumers, revealing dietary biomarkers, in a free-living population.Randomized, controlled, parallel group, multicenter, and cross-sectional.64 high risk (2) free-living subjects. 32 were classified as cocoa consumers (at least 3 servings/week of chocolate and/or cocoa powder) and 32 as non-cocoa consumers (0 g/day) (3).No dietary interventions or recommendations.Urine.Baseline.Analytical: HPLC-Q-ToF-MS. Positive and negative ionization modes.OSC-PLS-DA, Mann–Whitney and Student´s t-test.2015[55]
Notes: (1) Samples taken 2 h after intake were analyzed only by NMR, and not by LC-MS. (2) At least with type 2 diabetes mellitus or with at least three conventional cardiovascular risk factors. (3) Baseline data and urine samples were obtained from a PREDIMED study of 275 volunteers.
Table
Table 4. Metabolites analyzed in plasma after different acute cocoa intake interventions, by targeted methodologies.
Table 4. Metabolites analyzed in plasma after different acute cocoa intake interventions, by targeted methodologies.
Chemical CategoryMetaboliteTreatment 1Treatment 2Reference
Type of MatrixConcentrationSampling Period (h)Type of MatrixConcentrationSampling Period (h)
Flavonoids and conjugates(-)-epicatechin 3´-sulfateN.R.N.R.N.R.Dark chocolate233 ± 60 nmol/L3.2 ± 0.2[66]
Drink with 5.3 mg cocoa flavanols/kg body weight (young)98 ± 12 nmol/L1.8 ± 0.2Drink with 5.3 mg cocoa flavanols/kg body weight (elderly)125 ± 13 nmol/L1.7 ± 0.2[82]
Drink with 10.7 mg cocoa flavanols/kg body weight (young)251 ± 20 nmol/L1.5 ± 0.1Drink with 10.7 mg cocoa flavanols/kg body weight (elderly)212 ± 13 nmol/L1.7 ± 0.1[82]
(-)-epicatechin 4´-sulfateN.R.N.R.N.R.Dark chocolate11 ± 3 nmol/L3.5 ± 0.3[66]
(-)-epicatechin-3´-β-D-glucuronideN.R.N.R.N.R.Dark chocolate290 ± 49 nmol/L3.2 ± 0.2[66]
Drink with 5.3 mg cocoa flavanols/kg body weight (young)309 ± 41 nmol/L1.1 ± 0.1Drink with 5.3 mg cocoa flavanols/kg body weight (elderly)371 ± 34 nmol/L1.3 ± 0.1[82]
Drink with 10.7 mg cocoa flavanols/kg body weight (young)551 ± 67 nmol/L1.2 ± 0.1Drink with 10.7 mg cocoa flavanols/kg body weight (elderly)645 ± 66 nmol/L1.2 ± 0.1[82]
(-)-epicatechin-4´-β-D-glucuronideN.R.N.R.N.R.Dark chocolate44 ± 11 nmol/L3.4 ± 0.3[66]
(-)-epicatechin-7´-β-D-glucuronideN.R.N.R.N.R.Dark chocolate22 ± 6 nmol/L12.8 ± 4.8[66]
(-)-epicatechin glucuronideCocoa powder with water330 ± 156 nmol/L2Cocoa powder with milk273 ± 138 nmol/L2[73]
(4-hydroxyphenyl) acetic acidN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate120 ± 40 ng/mL2 ± 1[60]
epicatechin-O-sulfateCocoa powder with water83 ± 8 nmol/L1.4 ± 0.2Cocoa powder with milk77 ± 14 nmol/L1.3 ± 0.2[77]
3´-O-methyl-(-)-epicatechin 4´-sulfateN.R.N.R.N.R.Dark chocolate49 ± 14 nmol/L3.6 ± 0.3[66]
3´-O-methyl-(-)-epicatechin 7-sulfateN.R.N.R.N.R.Dark chocolate40 ± 10 nmol/L3.8 ± 0.2[66]
Drink with 5.3 mg cocoa flavanols/kg body weight (young)76 ± 6 nmol/L1.7 ± 0.2Drink with 5.3 mg cocoa flavanols/kg body weight (elderly)63 ± 6 nmol/L1.8 ± 0.1[82]
Drink with 10.7 mg cocoa flavanols/kg body weight (young)167 ± 19 nmol/L1.7 ± 0.1Drink with 10.7 mg cocoa flavanols/kg body weight (elderly)109 ± 11 nmol/L1.9 ± 0.2[82]
Flavonoids and conjugates3´-O-methyl-(-)-epicatechin 5-sulfateN.R.N.R.N.R.Dark chocolate153 ± 43 nmol/L3.8 ± 0.2[66]
Drink with 5.3 mg cocoa flavanols/kg body weight (young)75 ± 7 nmol/L1.4 ± 0.1Drink with 5.3 mg cocoa flavanols/kg body weight (elderly)72 ± 7 nmol/L1.4 ± 0.1[82]
Drink with 10.7 mg cocoa flavanols/kg body weight (young)176 ± 14 nmol/L1.6 ± 0.1Drink with 10.7 mg cocoa flavanols/kg body weight (elderly)128 ± 11 nmol/L1.8 ± 0.1[82]
4´-methyl-(-)-epicatechinLow-flavanol cocoa drinkN.R.N.R.High-flavanol cocoa drinkN.R.N.R.[71]
4´-O-methyl-(-)-epicatechinLow-flavanol cocoa drink25 ± 5 nmol/L2High-flavanol cocoa drink41 ± 10 nmol/L2[61]
4´-O-methyl-(-)-epicatechin 5-sulfateN.R.N.R.N.R.Dark chocolate18 ± 6 nmol/L3.8 ± 0.2[66]
4´-O-methyl-(-)-epicatechin 7-sulfateN.R.N.R.N.R.Dark chocolate13 ± 4 nmol/L3.8 ± 0.2[66]
4´-O-methyl-(-)-epicatechin-O-β-D-glucuronideLow-flavanol cocoa drinkN.R.N.R.High-flavanol cocoa drinkN.R.N.R.[71]
Low-flavanol cocoa drink151 ± 46 nmol/L2High-flavanol cocoa drink287 ± 58 nmol/L2[61]
Epicatechin-O-β-D-glucuronideLow-flavanol cocoa drinkN.R.N.R.High-flavanol cocoa drinkN.R.N.R.[71]
Epicatechin-7-β-D-glucuronideLow-flavanol cocoa drink9 ± 3 nmol/L2High-flavanol cocoa drink39 ± 13 nmol/L2[61]
CatechinChocolate drink milk-free0.21 ± 0.2 µmol/L1–1.5Chocolate drink with milk0.20 ± 0.02 µmol/L0.5–1[74]
No cocoa extract0.00 µmol/L0Cocoa extract in water0.16 ± 0.03 µmol/L0.5–2[68]
N.R.N.R.N.R.Cocoa based dairy drink149 ± 18 nmol/L2[79]
Low-flavanol cocoa drinkN.R.N.R.High-flavanol cocoa drinkN.R.N.R.[71]
Low-flavanol cocoa drink9 ± 3 nmol/L2High-flavanol cocoa drink18 ± 3 nmol/L2[61]
EpicatechinN.R.N.R.N.R.Cocoa based dairy drink889 ± 114 nmol/L2[79]
Chocolate drink milk-free12.89 ± 0.95 µmol/L1–2Chocolate drink with milk12.42± 0.97 µmol/L0.5–1[74]
No cocoa extract0.08 ± 0.46 µmol/L0Cocoa extract in water5.96 ± 0.60 µmol/L0.5–2[68]
Bread19 ± 14 nmol/L280 g of chocolate and 47 g of bread355 ± 49 nmol/L2[63]
Chocolate0.15 ± 0.04 µmol/L1–2Cocoa0.22 ± 0.06 µmol/L0–1[64]
Flavonoids and conjugatesEpicatechin40 g of chocolate103 ± 29 ng/mL2.00 ± 0.0080 g of chocolate196 ± 57 ng/mL2.57 ± 0.50[62]
MilkN.D.2Milk and cocoa powder625.7 ± 198.3 nmol/L2[70]
Low-flavanol cocoa drinkN.R.N.R.High-flavanol cocoa drinkN.R.N.R.[71]
Low-flavanol cocoa drink3 ± 0 nmol/L2High-flavanol cocoa drink19 ± 6 nmol/L2[61]
Low-cocoa group578 ± 61 nmol/L2High-cocoa groupN.R.N.R.[58]
Epicatechin glucuronideChocolate0.78 ± 0.28 µmol/L1–2Cocoa0.91 ± 0.21 µmol/L1–2[64]
Epicatechin sulfateChocolate1.11 ± 0.43 µmol/L0–1Cocoa1.14 ± 0.21 µmol/L1–2[64]
Epicatechin sulfoglucuronideChocolate1.19 ± 0.44 µmol/L0–1Cocoa1.28 ± 0.76 µmol/L0–1[64]
Methylated epicatechin sulfateChocolate0.95 ± 0.27 µmol/L1–2Cocoa1.00 ± 0.34 µmol/L1–2[64]
Methylated epicatechin sulfoglucuronideChocolate0.71 ± 0.14 µmol/L1–2Cocoa0.78 ± 0.51 µmol/L4–8[64]
O-methyl-(epi)-catechin-O-sulfateCocoa powder with water60 ± 8 nmol/L1.0 ± 0.2Cocoa powder with milk50 ± 8 nmol/L1.3 ± 0.2[77]
Procyanidin B2No cocoa extractDetected0Chocolate extract in water41 ± 4 nmol/L0.5–2[68]
N.R.N.R.N.R.Cocoa dairy drink4.0 ± 0.6 nmol/L2[80]
Methylxanthines3-methylxanthineCocoa powder with milk0.6 ± 1.4 µmol/L3.4 ± 3.3Cocoa powder enriched with methylxanthines with milk1.8 ± 0.9 µmol/L2.2 ± 2.5[81]
7-methylxanthineCocoa powder with milk2.1 ± 1.4 µmol/L3.3 ± 1.6Cocoa powder enriched with methylxanthines with milk4.6 ± 1.8 µmol/L4.5 ± 1.5[81]
CaffeineCocoa powder with milk2.1 ± 1.3 µmol/L2.1 ± 2.0Cocoa powder enriched with methylxanthines with milk12.1 ± 2.6 µmol/L2.0 ± 1.1[81]
No chocolate dose<2.5–21.4 µmol/L0Dark chocolate4.6–25.3 µmol/L1.5[59]
ParaxanthineCocoa powder with milk9.5 ± 1.3 µmol/L4.5 ± 2.9Cocoa powder enriched with methylxanthines with milk12.0 ± 2.2 µmol/L3.5 ± 2.5[81]
TheobromineNo chocolate dose<2.5–7.1 µmol/L0Dark chocolate43.2–67.5 µmol/L1.5[59]
Cocoa powder with milk15.8 ± 3.3 µmol/L1.9 ± 1.0Cocoa powder enriched with methylxanthines with milk51.9 ± 13 µmol/L2.2 ± 1.1[81]
MethylxanthinesTheobromineCocoa depleted controlN.R.N.R.Dark chocolate70 µmol/L3[49]
40 g of chocolate6.365 ± 0.894 µg/mL2.25 ± 0.8880 g of chocolate11.414 ± 1.190 µg/mL3.25 ± 0.45[62]
Non-cocoa consumers0.04–0.17 mg/kg0–24High cocoa consumers0.33–0.66 mg/kg0–24[56]
Baseline2.3 ± 2.4 mg/L0–12Milk chocolate7.6 ± 5.4 mg/L0–12[67]
Baseline2.3 ± 2.4 mg/L0–12Cocoa powder19.3 ± 5.9 mg/L0–12[67]
Baseline2.3 ± 2.4 mg/L0–12Dark chocolate30.6 ± 10.3 mg/L0–12[67]
TheophyllineCocoa powder with milk11.5 ± 2.6 µmol/L3.1 ± 3.2Cocoa powder enriched with methylxanthines with milk12.3 ± 4.3 µmol/L1.9 ± 2.3[81]
Phenolic acidsFerulic acidN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate15 ± 2 ng/mL3 ± 1[60]
Notes: N.R. means non reported or non-cocoa matrix was used.
Table
Table 5. Metabolites detected in plasma samples, that discriminated the baseline from treatments (enriched dark chocolate, dark chocolate and white chocolate), and times after intake where increase in measured signal was the highest, in an untargeted study [84].
Table 5. Metabolites detected in plasma samples, that discriminated the baseline from treatments (enriched dark chocolate, dark chocolate and white chocolate), and times after intake where increase in measured signal was the highest, in an untargeted study [84].
MetaboliteSampling Period (h)
β-hydroxybutyrate6
Acetone6
Acetoacetate6
Aspartate6
Lactate2
Table
Table 6. Metabolites analyzed in urine after acute cocoa interventions and studied with targeted methodologies.
Table 6. Metabolites analyzed in urine after acute cocoa interventions and studied with targeted methodologies.
Chemical CategoryMetaboliteTreatment 1Treatment 2Reference
Type of MatrixConcentrationSampling Period (h)Type of MatrixConcentrationSampling Period (h)
Flavonoids and conjugates(-)-catechinN.R.N.R.N.R.Dairy cocoa drink4.0 ± 0.4 µmol0–24[79]
(-)-epicatechinN.R.N.R.N.R.Dairy cocoa drink13 ± 2 µmol0–24[79]
N.R.N.R.N.R.Non dairy cocoa drinkN.R.0–24[76]
N.R.N.R.N.R.Cocoa polymeric procyanidin concentrate0.6 ± 0.2 ng/mg creatintine0–4[60]
(-)-epicatechin glucuronideNon-dairy cocoa drink194.95 µg/g creatinine0–6Dairy cocoa drink112.79 µg/g creatinine0–6[75]
(-)-epicatechin sulfate (isomer 1)Non-dairy cocoa drink48.83 µg/g creatinine0–6Dairy cocoa drink30.86 µg/g creatinine0–6[72,75]
(-)-epicatechin sulfate (isomer 2)No- dairy cocoa drink195.29 µg/g creatinine6–12Dairy cocoa drink128.47 µg/g creatinine6–12[72,75]
(-)-epicatechin sulfate (isomer 3)Non-dairy cocoa drink5.07 µg/g creatinine0–6Dairy cocoa drink72.45 µg/g creatinine0–6[72,75]
(-)-epicatechin-3´-sulfateN.R.N.R.N.R.Dark chocolate5.80 ± 1.78 µmol5–10[66]
(-)-epicatechin-3´-β-D-glucuronideN.R.N.R.N.R.Dark chocolate8.74 ± 2.92 µmol5–10[66]
(-)-epicatechin-4´-sulfateN.R.N.R.N.R.Dark chocolate0.37 ± 0.13 µmol5–10[66]
(-)-epicatechin-4´-β-D-glucuronideN.R.N.R.N.R.Dark chocolate0.56 ± 0.14 µmol5–10[66]
(-)-epicatechin-5-sulfateN.R.N.R.N.R.Dark chocolate0.72 ± 0.36 µmol5–10[66]
(-)-epicatechin-7-β-D-glucuronideN.R.N.R.N.R.Dark chocolate4.59 ± 0.74 µmol10–24[66]
Flavonoids and conjugates(-)-epicatechin-O-glucuronideCocoa powder with water405 ± 44 µmol/L0–2Cocoa powder with milk136 ± 24 µmol/L2–5[77]
(-)-epicatechin-O-sulfate (isomer 2)Cocoa powder with water1127 ± 196 µmol/L0–2Cocoa powder with milk737 ± 118 µmol/L2–5[77]
(+)-catechinN.R.N.R.N.R.Dairy cocoa drink9.8 ± 1.4 µmol0–24[79]
(+)-epicatechinN.R.N.R.N.R.Dairy cocoa drink10 ± 1 µmol0–24[79]
EpicatechinHot waterN.D.0–24Cocoa powder with water4.3 ± 4.2 µmol0–24[69]
(epi) catechin-O-sulfate (isomer 1)Cocoa powder with water928 ± 110 µmol/L0–2Cocoa powder with milk476 ± 75 µmol/L0–2[77]
3´-O-methyl-(-)-epicatechinN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate0.2 ± 0.3 ng/mg creatinine0–4[60]
3´-O-methyl-(-)-epicatechin 4´-sulfateN.R.N.R.N.R.Dark chocolate1.27 ± 0.39 µmol5–10[66]
3´-O-methyl-(-)-epicatechin 5-sulfateN.R.N.R.N.R.Dark chocolate8.87 ± 3.05 µmol5–10[66]
3´-O-methyl-(-)-epicatechin 7-sulfateN.R.N.R.N.R.Dark chocolate1.55 ± 0.55 µmol5–10[66]
3´-O-methyl-(-)-epicatechin-3´-β-D-glucuronideN.R.N.R.N.R.Dark chocolate0.69 ± 0.12 µmol5–10[66]
4´-O-methyl-(-)-epicatechin 5-sulfateN.R.N.R.N.R.Dark chocolate0.92 ± 0.34 µmol5–10[66]
4´-O-methyl-(-)-epicatechin 7-sulfateN.R.N.R.N.R.Dark chocolate0.55 ± 0.29 µmol5–10[66]
O-methyl-(epi)-catechin-O-sulfateCocoa powder with water1146 ± 231 µmol/L0–2Cocoa powder with milk823 ± 131 µmol/L0–2[77]
Flavonoids and conjugatesNaringeninHot water0.1 ± 0.2 µmol0–24Cocoa powder with water0.3 ± 0.4 µmol0–24[69]
Procyanidin B2N.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
LignansEnterodiolN.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
Hot water0.5 ± 1.1 µmol0–24Cocoa powder with water0.2 ± 0.3 µmol0–24[69]
EnterolactoneN.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
Hot water3.8 ± 2.8 µmol0–24Cocoa powder with water3.6 ± 2.9 µmol0–24[69]
Methylxanthines1,3,7-trimethyluric acidCocoa powder with milk0.7 ± 0.3 µmol/L0–2Cocoa powder with milk, enriched with methylxanthines1.8 ± 0.6 µmol/L9.7 ± 6.9[81]
1,3-dimethyluric acidCocoa powder with milk1.3 ± 1.1 µmol/L21.5 ± 6.0Cocoa powder with milk, enriched with methylxanthines3.4 ± 1.9 µmol/L18.0 ± 7.5[81]
1,7-dimethyluric acidCocoa powder with milk3.9 ± 2.7 µmol/L20.0 ± 7.7Cocoa powder with milk, enriched with methylxanthines13.5 ± 7.8 µmol/L12.7 ± 8.7[81]
1-methyluric acidCocoa powder with milk9.2 ± 4.1 µmol/L19.7 ± 8.2Cocoa powder with milk, enriched with methylxanthines45.2 ± 16.4 µmol/L12.4 ± 7.5[81]
1-methylxanthineCocoa powder with milk5.5 ± 2.7 µmol/L21.2 ± 6.8Cocoa powder with milk, enriched with methylxanthines15.3 ± 7.3 µmol/L13.7 ± 7.7[81]
Methylxanthines3,7-dimethyluric acidCocoa powder with milk2.7 ± 1.1 µmol/L21.2 ± 6.8Cocoa powder with milk, enriched with methylxanthines7.3 ± 2.9 µmol/L14.3 ± 8.6[81]
3-methylxanthineCocoa powder with milk34.8 ± 8.9 µmol/L14.8 ± 9.1Cocoa powder with milk, enriched with methylxanthines98.1 ± 21.9 µmol/L12.0 ± 7.4[81]
7-methylxanthineCocoa powder with milk110.1 ± 40.1 µmol/L16.3 ± 8.7Cocoa powder with milk, enriched with methylxanthines187.4 ± 82.1 µmol/L12.0 ± 7.2[81]
CaffeineCocoa powder with milk2.1 ± 0.7 µmol/L14.5 ± 9.3Cocoa powder with milk, enriched with methylxanthines9.9 ± 3.5 µmol/L7.7 ± 5.5[81]
Baseline<2.5–23.4 µmol/L0Dark chocolate4.2–24.7 µmol/L1.5[59]
ParaxanthineCocoa powder with milk3.5 ± 1.8 µmol/L15.1 ± 9.0Cocoa powder with milk, enriched with methylxanthines10.7 ± 5.8 µmol/L11.7 ± 7.5[81]
TheobromineCocoa powder with milk50.4 ± 18.4 µmol/L14.2 ± 9.6Cocoa powder with milk, enriched with methylxanthines177.4 ± 45.0 µmol/L7.7 ± 5.5[81]
Baseline2.5–94.9 µmol/L0Dark chocolate131.9–449.4 µmol/L1.5[59]
TheophyllineBaseline1.0 ± 1.4 µmol/L14.9 ± 8.9Dark chocolate2.5 ± 1.3 µmol/L7.6 ± 5.8[81]
N-phenylpropenoyl-L-amino acidsN-[3´,4´-dihydroxy-(E)-cinnamoyl]-L-aspartic acidBaselineN.D.0Non-dairy cocoa drink4.76 ± 4.01 µg2[20]
N-[3´,4´-dihydroxy-(E)-cinnamoyl]-L-dopaBaselineN.D.0Non-dairy cocoa drink1.25 ± 1.19 µg2[20]
N-[3´,4´-dihydroxy-(E)-cinnamoyl]-L-glutamic acidBaselineN.D.0Non-dairy cocoa drinkN.D.N.D.[20]
N-[3´,4´-dihydroxy-(E)-cinnamoyl]-L-tyrosineBaselineN.D.0Non-dairy cocoa drink0.30 ± 0.39 µg2[20]
N-[4´-hydroxy-(E)-cinnamoyl]-L-aspartic acidBaselineN.D.0Non-dairy cocoa drink64.37 ± 20.22 µg2[20]
N-[4´-hydroxy-(E)-cinnamoyl]-L-dopaBaselineN.D.0Non-dairy cocoa drink0.85 ± 0.96 µg2[20]
N-phenylpropenoyl-L-amino acidsN-[4´-hydroxy-(E)-cinnamoyl]-L-glutamic acidBaselineN.D.0Non-dairy cocoa drink22.17 ± 12.57 µg2[20]
N-[4´-hydroxy-(E)-cinnamoyl]-L-tryptophaneBaselineN.D.0Non-dairy cocoa drink<0.1 µg1–4[20]
N-[4´-hydroxy-(E)-cinnamoyl]-L-tyrosineBaselineN.D.0Non-dairy cocoa drink18.91 ± 5.53 µg2[20]
N-[4´-hydroxy-3´-methoxy-(E)-cinnamoyl]-L-aspartic acidBaselineN.D.0Non-dairy cocoa drink9.70 ± 2.93 µg2[20]
N-[4´-hydroxy-3´-methoxy -(E)-cinnamoyl]-L-tyrosineBaselineN.D.0Non-dairy cocoa drink1.91 ± 0.59 µg2[20]
N-[cinnamoyl]-L-aspartic acidBaselineN.D.0Non-dairy cocoa drink0.43 ± 0.33 µg2[20]
Phenolic acids and others3-hydroxybenzoic acidN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate1 ± 1 ng/mg creatinine0–4[66]
N.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
3,4-dihydroxyphenyl acetic acidBaseline15.8 ± 4.4 nmol/mg creatinine−24–0Chocolate38.8 ± 12.3 nmol/mg creatinine0–24[65]
Cocoa powder with water1.60 ± 0.37 nmol/mg creatinine6–12Cocoa powder with milk0.45 ± 0.10 nmol/mg creatinine12–24[78]
N.R.N.R.N.R.Cocoa polymeric procyanidin concentrate10 ± 10 ng/mg creatinine0–4[60]
N.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
Phenolic acids and others3,4-dihydroxyphenyl propionic acidN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate6 ± 5 ng/mg creatinine0–4[60]
Baseline3.1 ± 0.7 nmol/mg creatinine−24–0Chocolate2.6 ± 0.6 nmol/mg creatinine0–24[65]
Cocoa powder with water16.84 ± 2.81 nmol/mg creatinine0–6Cocoa powder with milk16.9 ± 4.0 nmol/mg creatinine0–6[78]
N.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
3-hydroxyphenyl acetic acidCocoa powder with water10.1 ± 3.42 nmol/mg creatinine12–24Cocoa powder with milk7.36 ± 1.61 nmol/mg creatinine12–24[78]
N.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
N.R.N.R.N.R.Cocoa polymeric procyanidin concentrate30 ± 20 ng/mg creatinine0–4[60]
3-hydroxyphenyl propanoic acidN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate10 ± 7 ng/mg creatinine0–4[60]
3-methoxy-4-hydroxy-phenylacetic acidCocoa powder with water11.30 ± 1.63 nmol/mg creatinine6–12Cocoa powder with milk9.30 ± 0.74 nmol/mg creatinine6–12[78]
N.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
4-hydroxy-5-(3´,4´-dihydroxyphenyl) valeric acidN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate0.2 ± 0.3 ng/mg creatinine0–4[60]
4-hydroxybenzoic acidN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate16 ± 6 ng/mg creatinine0–4[60]
N.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
4-hydroxyphenyl acetic acidN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate210 ± 50 ng/mg creatinine0–4[60]
Phenolic acids and others4-hydroxyphenyl propanoic acidN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate0.02 ± 0.04 ng/mg creatinine0–4[60]
4-O-methylgallic acidN.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
Hot waterN.D.0–24Cocoa powder with water0.6 ± 1.1 µmol0–24[69]
5-(3´,4´-dihydroxyphenyl) valerolactoneN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate2 ± 4 ng/mg creatinine48[60]
Caffeic acidN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate1.2 ± 0.5 ng/mg creatinine0–4[60]
N.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
Hot water0.1 ± 0.2 µmol0–24Cocoa powder with water0.4 ± 0.4 µmol0–24[69]
Dihydroxyphenyl valeric acidN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate0.01 ng/mg creatintine-[60]
Ferulic acidN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate21 ± 4 ng/mg creatinine0–4[60]
Baseline131 ± 59 nmol/mg creatinine−24–0Chocolate321 ± 99 nmol/mg creatinine24–48[65]
N.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
Hippuric acidCocoa powder with water193.16 ± 27.89 nmol/mg creatinine0–6Cocoa powder with milk122.82 ± 18.83 nmol/mg creatinine12–24[78]
N.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
Baseline2943 ± 1923 nmol/mg creatinine−24–0Chocolate974± 115 nmol/mg creatinine0–24[65]
m-coumaric acidN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate1 ± 1 ng/mg creatinine0–4[60]
Hot water0.5 ± 0.9 µmol0–24Cocoa powder with water1.4 ± 1.4 µmol0–24[69]
Phenolic acids and othersm-coumaric acidN.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
Methyl-5-(3´, 4´-dihydroxyphenyl)-valerolactoneN.R.N.R.N.R.Cocoa polymeric procyanidin concentrate0.5 ± 0.8 ng/mg creatinine48[60]
m-hydroxybenzoic acidBaselineNon detected−24–0Chocolate8.93 ± 3.9 nmol/mg creatinine24–48[65]
Cocoa powder with water0.56 ± 24 nmol/mg creatinine6–12Cocoa powder with milk0.60 ± 0.28 nmol/mg creatinine12–24[78]
m-hydroxyphenyl acetic acidBaseline21.2 ± 3.5 nmol/mg creatinine−24–0Chocolate156 ± 54 nmol/mg creatinine24–48[65]
m-hydroxyphenyl propionic acidBaseline5.4 ± 2.4 nmol/mg creatinine−24–0Chocolate13.4 ± 4.1 nmol/mg creatinine24–48[65]
p-coumaric acidN.R.N.R.N.R.Cocoa procyanidin concentrate1 ± 1 ng/mg creatinine48[60]
N.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
Phenylacetic acidCocoa powder with water163.77 ± 22.10 nmol/mg creatinine0–6Cocoa powder with milk167.72 ± 23.80 nmol/mg creatinine0–6[78]
Baseline70.1 ± 11.3 nmol/mg creatinine−24–0Chocolate36.8 ± 8.1 nmol/mg creatinine24–48[65]
p-hydroxybenzoic acidBaseline54.2 ± 8.1 nmol/mg creatinine−24–0Chocolate41.2 ± 10.4 nmol/mg creatinine24–48[65]
Cocoa powder with water6.39 ± 0.66 nmol/mg creatinine0–6Cocoa powder with milk3.01 ± 0.97 nmol/mg creatinine12–24[78]
Phenolic acids and othersp-hydroxyhippuric acidCocoa powder with water3.13 ± 0.60 nmol/mg creatinine0–6Cocoa powder with milk1.76 ± 0.30 nmol/mg creatinine6–12[78]
Baseline71.1 ± 13.6 nmol/mg creatinine−24–0Chocolate90.5 ± 21.3 nmol/mg creatinine24–48[65]
N.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
Protocatechuic acidCocoa powder with water11.07 ± 1.19 nmol/mg creatinine0–6Cocoa powder with milk8.8 ± 2.2 nmol/mg creatinine0–6[78]
N.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
N.R.N.R.N.R.Cocoa polymeric procyanidin concentrate40 ± 20 ng/mg creatinine8–24[60]
Vanillic acidCocoa powder with water5.96 ± 1.15 nmol/mg creatinine0–6Cocoa powder with milk9.85 ± 1.27 nmol/mg creatinine0–6[78]
Baseline64.6 ± 25.5 nmol/mg creatinine−24–0Chocolate228 ± 33 nmol/mg creatinine0–24[65]
N.R.N.R.N.R.Non-dairy cocoa drinkN.R.0–24[76]
N.R.N.R.N.R.Cocoa polymeric procyanidin concentrate14 ± 4 ng/mg creatinine0–4[60]
Notes: N.R. refers to non-reported or non-cocoa matrix was used, and N.D. to non-detected.
Table
Table 7. Metabolites analyzed in urine after acute cocoa intervention studied with non-targeted methodologies.
Table 7. Metabolites analyzed in urine after acute cocoa intervention studied with non-targeted methodologies.
Period (h)Purine MetabolitesPolyphenol MetabolitesNicotinic Acid MetabolitesAmino Acid MetabolitesOthersReference
BaselineN-methylguanine----[87]
0–63-methyluric acid
3-methylxanthine
3,7-dimethyluric acid
7-methyluric acid
7-methylxanthineAMMU (4)
Caffeine
Theobromine		3´-methoxy-4´-hydroxyphenyl valerolactone
3´-methoxy-4´-hydroxyphenyl valerolactone glucuronide
4-hydroxy-5-(3,4-dihydroxyphenyl)-valeric acid
5-(3´,4´-dihydroxyphenyl)-γ-valerolactone glucuronide
Epicatechin-O—sulfate
O-methyl epicatechin
Vanillic acid
Vanilloylglycine		Hydroxynicotinic acid TrigonellineTyrosine3,5-diethyl-2-methyl pyrazine
Cyclo(Ser-Tyr)
Cyclo(Pro-Pro)
Hydroxyacetophenone		[86]
3-methylxanthine
7-methylxanthine
Theobromine		Vanilloylglycine---[87]
3-methyluric acid
3-methylxanthine
3,7- dimethyluric acid
7-methyluric acid
7-methylxanthine
AMMU
Caffeine
Theobromine		3´-methoxy-4´-hydroxyphenyl valerolactone
3´-methoxy-4´-hydroxyphenyl valerolactone glucuronide
4-hydroxy-5-(3,4-dihydroxyphenyl)-valeric acid
5-(3´,4´-dihydroxyphenyl)--valerolactone sulfate
5-(3´,4´-dihydroxyphenyl)-valerolactone glucuronide
Hippurate
Epicatechin-O-sulfate
Vanilloylglycine 		Hydroxynicotinic acid
N1-methylnicotinamide		Alanine
Arginine
Glycine
Tyrosine
Valine		2-hydroxyisobutyrate
3-hydroxyisobutyrate
3-hydroxyisovalerate
4-hydroxyphenyl acetate
Creatinine
Dimethylamine
Lactate
O-feruoylquinate
Pyruvate		[84]
6–123-methyluric acid
3-methylxanthine
3,7-dimethyluric acid
7-methyluric acid
7-methylxanthine
AMMU
Caffeine
Theobromine 		3´-methoxy-4´-hydroxyphenyl valerolactone
3´-methoxy-4´-hydroxyphenyl valerolactone glucuronide
4-hydroxy-5-(3,4-dihydroxyphenyl)-valeric acid
5-(3´,4´-dihydroxyphenyl)-g-valerolactone glucuronide
5-(3´,4´-dihydroxyphenyl)-γ-valerolactone sulfate
5-(3´,4´-dihydroxyphenyl)-γ-valerolactone glucuronide		--Cyclo(Ser-Tyr)
3,5-diethyl-2-methyl pyrazine		[86]
3-methylxanthine
7-methylxanthine
Theobromine		Dihydroxyphenyl valerolactone glucuronide--Furoylglycine[87]
12–243-methyluric acid
3-methylxanthine
3,7-dimethyluric acid
7-methyluric acid
7-methylxanthine
AMMU
Theobromine		3´-methoxy-4´-hydroxyphenyl valerolactone
3´-methoxy-4´-hydroxyphenyl valerolactone glucuronide
4-hydroxy-5-(3,4-dihydroxyphenyl)-valeric acid
5-(3´,4´-dihydroxyphenyl)-g-valerolactone glucuronide
5-(3´,4´-dihydroxyphenyl)-γ-valerolactone glucuronide		--Cyclo(Ser-Tyr)[86]
3-methylxanthine
7-methylxanthine
Theobromine		--Xanthurenic acid-[87]
Notes: (4) AMMU means 6-amino-5-[N-methylformylamino]-1-methyluracil.
Table
Table 8. Metabolites analyzed in urine and plasma after acute, short-term, or regular cocoa intake interventions, studied with non-targeted and targeted methodologies.
Table 8. Metabolites analyzed in urine and plasma after acute, short-term, or regular cocoa intake interventions, studied with non-targeted and targeted methodologies.
Type of MetaboliteMetabolites in UrineMetabolites in Plasma (Targeted)
Non-Targeted [55]Targeted [57][57][58]
Polyphenols metabolites(DHPV) 5-(3´,4´-dihydroxphenyl)-valerolactone sulfoglucuronide
(HDHPVA) 4-hydroxy-5-(dihydroxyphenyl)-valeric acid glucuronide
(HHMPVA) 4-hydroxy-5-(hydroxyl-methoxyphenyl)-valeric acid glucuronide
(HHPVA) 4-hydroxy-5-(hydroxyphenyl)-valeric acid sulphate
(HPV) hydroxyphenyl-valerolactone glucuronide
(HPVA) 4-hydroxy-5-(phenyl)-valeric acidsulphate
(MHPV) methoxyhydroxyphenyl
valerolactone
DHPV glucuronide (1) and (2)
DHPV sulphate
(epi) catechin glucuronide
(epi) catechin sulphate
HDHPVA
HDHPVA sulphate
HHPMVA sulphate
HPV sulphate
MHPV glucuronide
Vanillic acid
Vanillin sulphate 		(-)-epicatechin
3-hydroxybenzoic acid
3-hydroxyhippuric acid
3-hydroxyphenyl acetic acid
3-hydroxyphenyl propionic acid
3-methoxy-4-hydroxyphenyl acetic acid
3,4-dihydroxyphenyl acetic acid
3,4-dihydroxyphenyl propionic acid4-hydroxybenzoic acid
4-hydroxyhippuric acid5-(3´,4´-dihydroxyphenyl)-γ-valerolactone
5-(3´-methoxy, 4´-hydroxyphenyl)-γ-valerolactone O-glucuronide
5-(3´-methoxy, 4´-hydroxyphenyl)-γ-valerolactone O-sulfate
5-(3´,4´-dihydroxyphenyl)-γ-valerolactone O-glucuronide isomers
Caffeic acid
Epicatechin-3´-O-glucuronide
Epicatechin-7-O-glucuronide
Epicatechin-O-glucuronide isomers
Epicatechin-O-sulfate isomers
Ferulic acidm-coumaric acidO-methyl-epicatechin-O-glucuronide isomersO-methyl-epicatechin-O-sulfate isomersp-coumaric acid
Phenylacetic acid
Protocatechuic acid
Vanillic acid		3-hydroxyhippuric acid
3-hydroxyphenyl acetic acid
3-hydroxyphenyl propionic acid
3,4-dihydroxyphenyl acetic acid
3,4-dihydroxyphenyl propionic acid
4-hydroxybenzoic acid
4-hydroxyhippuric acid
5-(3´,4´-dihydroxyphenyl)-γ-valerolactone
5-(3´-methoxy, 4´-hydroxyphenyl)-γ-valerolactone O-glucuronide
5-(3´,4´-dihydroxyphenyl)-γ-valerolactone O-glucuronide (1) and (2)
Caffeic acid
Ferulic acid
p-coumaric acid
Phenylacetic acid
Protocatechuic acid
Vanillic acid		3´-O-methyl-(-)-epicatechin
4´-O-methyl-(-)-epicatechin
Epicatechin
Purine metabolites3-methyluric acid
3,7-dimethyluric acid
7-methylxanthine
AMMU 1 and 2 (5)
Theobromine
Xanthine		---
OthersAspartyl-phenylalanine
Cyclo(aspartyl-phenylalanyl)
Furoylglycine
Methylglutaryl carnitine		---
Notes: (5) 1 and 2 correspond to isomers AMMU= 6-amino-5-[N-methylformylamino]-1-methyluracil.

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MDPI and ACS Style

Mayorga-Gross, A.L.; Esquivel, P. Impact of Cocoa Products Intake on Plasma and Urine Metabolites: A Review of Targeted and Non-Targeted Studies in Humans. Nutrients 2019, 11, 1163. https://doi.org/10.3390/nu11051163

AMA Style

Mayorga-Gross AL, Esquivel P. Impact of Cocoa Products Intake on Plasma and Urine Metabolites: A Review of Targeted and Non-Targeted Studies in Humans. Nutrients. 2019; 11(5):1163. https://doi.org/10.3390/nu11051163

Chicago/Turabian Style

Mayorga-Gross, Ana Lucía, and Patricia Esquivel. 2019. "Impact of Cocoa Products Intake on Plasma and Urine Metabolites: A Review of Targeted and Non-Targeted Studies in Humans" Nutrients 11, no. 5: 1163. https://doi.org/10.3390/nu11051163

APA Style

Mayorga-Gross, A. L., & Esquivel, P. (2019). Impact of Cocoa Products Intake on Plasma and Urine Metabolites: A Review of Targeted and Non-Targeted Studies in Humans. Nutrients, 11(5), 1163. https://doi.org/10.3390/nu11051163

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