The relevance of theobromine for the beneficial effects of cocoa consumption

Abstract

Cocoa consumption began in America and in the mid 16th Century it quickly spread to
Europe. Beyond being considered a pleasant habit due to its rich sweet lingering taste,
chocolate was considered a good nutrient and even a medicine. Traditionally, health
benefits of cocoa have been related with the high content of antioxidants of Theobroma
cocoa beans. However, the direct psychoactive effect due to methylxanthines in cocoa
is notable. Theobromine and caffeine in the proportions found in cocoa are responsible
for the liking of the food/beverage. These compounds influence in a positive way our
moods and our state of alertness. Theobromine, which is found in higher amounts than
caffeine, seems to be behind several effects attributed to cocoa intake. The main
mechanisms of action are inhibition of phosphodiesterases and blockade of adenosine
receptors. Further mechanisms are being explored to better understand the health
benefits associated to theobromine consumption. Unlike what happens in other
mammals –pets included-, theobromine is safe for humans and has fewer unwanted
effects than caffeine. Therefore, theobromine deserves attention as one of the most
attractive molecules in cocoa.

Full text

PERSPECTIVE ARTICLE
published: 20 February 2015
doi: 10.3389/fphar.2015.00030
The relevance of theobromine for the beneficial effects of
cocoa consumption
Eva Martínez-Pinilla1*, Ainhoa Oñatibia-Astibia2and Rafael Franco3
1Laboratory of Cell and Molecular Neuropharmacology, Department of Neuroscience, Center for Applied Medical Research, University of Navarra, Pamplona,
Navarra, Spain
2Official College of Pharmacists of Gipuzkoa, San Sebastián, Spain
3Molecular Neurobiology Laboratory, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Barcelona, Spain
Edited by:
Rabia Latif, University of Dammam,
Saudi Arabia
Reviewed by:
Leonardo Cavone, Università degli
studi di Firenze, Italy
Giovanna Cenini, Universität Bonn,
Germany
*Correspondence:
Eva Martínez-Pinilla, Laboratory of
Cell and Molecular
Neuropharmacology, Department of
Neuroscience, Center for Applied
Medical Research, University of
Navarra, Pio XII 55, 31008
Pamplona, Navarra, Spain
e-mail: martinezpinillaeva@
gmail.com
Cocoa consumption began in America and in the mid sixteenth Century it quickly spread
to Europe. Beyond being considered a pleasant habit due to its rich sweet lingering
taste, chocolate was considered a good nutrient and even a medicine. Traditionally, health
benefits of cocoa have been related with the high content of antioxidants of Theobroma
cocoa beans. However, the direct psychoactive effect due to methylxanthines in cocoa is
notable. Theobromine and caffeine, in the proportions found in cocoa, are responsible for
the liking of the food/beverage. These compounds influence in a positive way our moods
and our state of alertness. Theobromine, which is found in higher amounts than caffeine,
seems to be behind several effects attributed to cocoa intake. The main mechanisms of
action are inhibition of phosphodiesterases and blockade of adenosine receptors. Further
mechanisms are being explored to better understand the health benefits associated
to theobromine consumption. Unlike what happens in other mammals -pets- included,
theobromine is safe for humans and has fewer unwanted effects than caffeine. Therefore,
theobromine deserves attention as one of the most attractive molecules in cocoa.
Keywords: caffeine, theobromine, cocoa, adenosine receptor, neurological disease, receptor antagonist
INTRODUCTION
Caffeine present in coffee and in cola beverages is heavily con-
sumed worldwide. The reason of such high consumption relates
to its benefits for day-life activities. Caffeine actions in the central
nervous system (CNS) are fundamental to understand the interest
of the intake of caffeine-containing beverages. Beneficial actions
range from alertness to reducing the risk of neurodegenerative
diseases. Although the highest concentration of caffeine is present
in coffee, cocoa also contains this methylxanthine (Figure 1)
but at doses that probably are not enough to activate neu-
ral mechanisms. However, cocoa has elevated concentrations of
a structurally similar component, theobromine. The effects of
theobromine have been less studied than those of caffeine but
it is known that this molecule exerts some positive effects in
different human pathologies. The combination of caffeine and
theobromine in cocoa may have the expected methylxanthine-
derived benefits without the side effects reported for caffeine.
Interestingly, the main action mechanism of caffeine and theo-
bromine consists of blocking adenosine receptors and inhibiting
phosphodiesterases. The present paper takes data of novel studies
that point toward alternative modes of action of theobromine.
Further research is, however, required to fully understand the
health benefits of cocoa consumption.
THEOBROMINE AND CAFFEINE CONTENT IN COCOA
The physiological effects of cocoa components and theobromine
in particular, deserve to be closely scrutinized to better under-
stand the properties of cocoa consumption. The differences
between coffee and cocoa perceived by consumers are mainly due
to their most abundant molecules: caffeine in coffee and theo-
bromine in cocoa. Moreover, the high contents of carbohydrates
in cocoa products may be a further factor to consider.
Besides the cocoa proven psychoactive potential, caffeine and
theobromine content is in full or in part responsible for the liking
of this food. Human volunteers consuming a drink plus a capsule
containing the two compounds, in amounts equivalent to those
found in 50 g of dark chocolate (19 mg caffeine and 250 mg
theobromine), liked the drink more than when the pairing was
with a capsule containing placebo (Smit and Blackburn, 2005).
These results, probably mediated by adenosine receptors, are
conclusive of reinforcing actions of methylxanthines at doses and
proportions found in cocoa. It is important to note that neither
caffeine nor theobromine are addictive substances (see National
Institute on Drug Abuse, 2014) and also they are not in the list
of doping substances provided by the World Anti-Doping Agency
(see The World Anti-Doping Agency (WADA), 2014).
THEOBROMINE STUDIES IN MAMMALS: SAFETY AND
TOXICITY
In vivo effects of xenobiotic or synthetic drugs require the use
of animal models. However, theobromine, appears to be toxic
in some mammals, including pets (Smit, 2011). Laboratory ani-
mal toxicity is a factor to consider in the extrapolation of data
to humans. Reasons for this toxicity are not well established
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Martínez-Pinilla et al. Theobromine and cocoa consumption
FIGURE 1 | Chemical structure of xanthine, caffeine, theobromine, and
adenosine.
but unequivocally suggest that the action mechanisms of theo-
bromine in humans may be different from those observed in other
mammals. Due to these facts, the molecular pharmacology of
theobromine, in particular its effect on adenosine receptors must
be revisited using human tissue samples and cells, or heterologous
systems expressing human proteins. The knowledge of adverse
effects in some animals has probably prompted a relatively high
number of clinical trials that prove that theobromine is not toxic
for humans (Pendleton et al., 2012, 2013; Baggott et al., 2013)
but has benefits in a variety of conditions (see below). It should
be noted that the link between cocoa consumption and risk of
preeclampsia in pregnant women, described previously, has not
been proven. However, recent systematic reviews suggest the ben-
efits of cocoa intake in the prevention of gestational hypertension
(Klebanoff et al., 2009; Mogollon et al., 2013).
CAFFEINE, THEOBROMINE, AND ADENOSINE RECEPTORS
The main pharmacological effects of caffeine, largely due to its
structural similarity to adenosine molecule (Figure 1), include
the inhibition of phosphodiesterases (enzymes that degrade the
second messenger, cAMP), the regulation of intracellular calcium
levels and the antagonism of adenosine receptors (Choi et al.,
1988; McPherson et al., 1991; Chen and Chern, 2011; Johnson
et al., 2012; Tazzeo et al., 2012). These primary actions result in
the well-described physiological effects of caffeine as stimulant
of CNS (Smit et al., 2004; Ciruela et al., 2006). Moreover, this
methylxanthine can also perform other peripheral processes such
as relax smooth muscles or stimulate the diuresis and cardiac
muscle contraction (Tazzeo et al., 2012). Caffeine is mainly
metabolized by the liver and, interestingly, one of its metabolites
is theobromine (Becker et al., 1984).
As methylxanthines, caffeine and theobromine (Figure 1),
are blockers of adenosine receptors which are G-protein-coupled
receptors that sense the presence of extracellular adenosine.
Adenosine is both an intermediate metabolite and also a messen-
ger molecule that exerts its hormone-like action in the periphery
and acts as a potent neuroregulator in the CNS. Four receptor
subtypes for the compound have been identified: A1, A2A, A2B,
and A3, widely distributed in the human body although with
differential cell/tissue expression. Brain physiology is dependent
upon variations in the concentration of adenosine that impacts
on adenosine receptors in neurons. In this sense, a quick way to
start the daily activities is disrupting the effect of adenosine in
the brain by using blockers of its specific receptors. Technically
such blockers are called “antagonists” and, therefore, caffeine
and theobromine are antagonists of adenosine receptors. Grow-
ing evidence in the last decade indicates that theobromine has
psychoactive actions in humans that are qualitatively different
from those of caffeine (Mitchell et al., 2011; Baggott et al., 2013).
The effect of theobromine on blood pressure (van den Bogaard
et al., 2010) is also qualitatively different than that of caffeine
(Mitchell et al., 2011) but the reasons for these differences are not
established.
One possible explanation for the discrepancy in the effects of
caffeine and theobromine could be their different half-life. Half-
life of theobromine is higher than caffeine even in rodents, which
have a faster hepatic metabolism. Thus, half of the theobromine
administered to rats is excreted unchanged (Bonati et al., 1984).
The mean half-life in plasma from healthy volunteers is approxi-
mately 10 h and the percentage of unmodified compound present
in urine collected for 48 h after a single dose of 10 mg/Kg is
relatively high (16–18% depending on the technique for isolation
and quantitation; Tarka et al., 1983). The importance of this fact
is evidenced when methylxanthines are used as bronchodilators
in the management of asthma patients in whose serum the half-
life is also higher for theobromine than for caffeine (Becker et al.,
1984). When one of the main xenobiotic metabolizing enzymes,
cytochrome P450 1A2 (YP1A2), is expressed in heterologous cells
the rate of transformation is much lower for theobromine (5%)
than for caffeine (81%; Gu et al., 1992) thus confirming that
caffeine is more labile in terms of degradation than theobromine.
Effects of in vivo administration of caffeine are in part due to
the products of its metabolism. As relatively stable compound,
theobromine may play a crucial role in some beneficial effects
attributed to caffeine.
Theobromine is useful in asthma and in other respiratory
tract problems such as cough for which no definitive drug has
been developed. Codeine is very effective but its metabolism to
compounds acting on opioid receptors limits its use (Prieto-
Lastra et al., 2006). A safety and natural alternative could be
theobromine since it is able to prevent cough provoked by citric
acid in guinea-pigs and by capsaicin (an irritant component of
chili peppers) in humans. This double-blind placebo-controlled
study was complemented with in vitro studies using human
vagus nerve preparations in which theobromine inhibited the
depolarization effect of capsaicin (Usmani et al., 2005). Bearing
in mind these results, theobromine seems to suppress cough by
inhibiting the activation of afferent nerves. Two clinical trials
have been completed to test antitussive action of theobromine
but no results are available yet. In one of them (NCT01416480
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Martínez-Pinilla et al. Theobromine and cocoa consumption
identifier in clinicaltrials.org) 300 mg of theobromine capsules
were used for antitussive effects in acute bronchitis. In a second
study (NCT01656668 identifier in clinicaltrials.org) capsules of
300 mg theobromine were evaluated in frequent long-term cough.
Whether cocoa consumption may be helpful to prevent coughing
or to diminish cough intensity remain to be determined.
Noteworthy, van Zyl et al. (2008) reported that the diffusion
of theobromine in lung substructures is higher than that of other
drugs used in the therapy of respiratory diseases. The authors sug-
gest that not only lipophilicity but also the position of alkyl groups
in the purine ring affect the ability of caffeine and theobromine to
cross biological membranes. The differential capability of tissue
penetration and accumulation may explain why theobromine
may achieve higher effects than caffeine. Although theobromine
may have less affinity for receptors than caffeine, the efficacy of
theobromine may become higher if it readily crosses membranes
and reaches high interstitial concentrations.
Benefits of the theobromine on cough seem to be related
with its anti-inflammatory potential as well as with modulation
of airway reactivity (Mokry et al., 2009). Non-selective phos-
phodiesterase inhibitors are already efficacious in suppression of
airway hyperreactivity. From the dozen existing enzymes cleaving
cyclic mononucleotides (cAMP/cGMP), phosphodiesterase four
is a good choice as therapeutic target in cough suppression
(Mokry and Nosalova, 2011). Cortijo et al. (1993) showed an
enrichment of phosphodiesterase four in human bronchial tissue
and a good correlation between enzyme inhibition and bron-
chorelaxation potency. Recently, Sugimoto et al. (1994, 2014)
have demonstrated that the antitumor potential effect of theo-
bromine in malignant glioblastoma proliferation results from
regulation of phosphodiesterase four, protein kinase B, extracel-
lular signal-regulated p38 mitogen-activated protein kinase and
nuclear factor-kappa B. Acting as phosphodiesterase inhibitors,
methylxanthines are able to downregulate pro-inflammatory
cytokines such as interferon-gamma or tumor necrosis factor-
alpha (Harris et al., 2010). Apart from a direct theobromine
effect on phosphodiesterases, the results are consistent with
blockade of adenosine receptors negatively affecting adenylate
cyclase activity, i.e., those coupled to Giproteins (A1and A3
receptors).
ADENOSINE RECEPTOR-INDEPENDENT EFFECTS OF
THEOBROMINE
Despite mainly acting as adenosine antagonist, theobromine may
have actions that are not mediated by the blockade of these
receptors. Theobromine and other main components of a hydro-
alcoholic guaraná extract are able to reduce cell toxicity caused by
nitric oxide generation (Bittencourt et al., 2013). It is unlikely that
reduction of oxidative stress, DNA damage and lipid peroxidation
in cells by the guaraná extract are mediated by blocking adenosine
receptors.
In recent years, theobromine is starting to be widely studied
to look for common and differential mechanisms with caffeine.
Theobromine and caffeine are methylxanthines that may form
non-covalent stacking complexes with ATP (Gattuso et al., 2011)
and affect cell metabolism and/or DNA and RNA structure
(Johnson et al., 2012). In fact, theobromine and caffeine are able
to bind to DNA at millimolar concentrations (Johnson et al.,
2012) and theobromine can also interact with RNA (Johnson
et al., 2003). However, the full physiological consequences of
these findings are not known yet. One hypothesis proposes that
sustained interaction with DNA and RNA after consumption of
methylxanthines in cocoa, might lead to induce or repress gene
expression. Oleaga et al. (2012) have shown that a polyphenolic
extract of cocoa alters the expression of genes in human breast
cancer cells. Accordingly, one attractive possibility is the impact
in the expression of genes with potential to decrease the risk of
neurodegenerative diseases. Recent reports indicated that chronic
consumption of coffee leads to reduced risk of Alzheimer’s and of
Parkinson’s disease (Maia and de Mendonça, 2002; Costa et al.,
2010; Eskelinen et al., 2011; Messerli, 2012). This beneficial effect
is totally linked to a continued consumption at mid life, i.e. intake
of methylxanthine-containing products reduces neurodegenera-
tion later in life (Pelligrino et al., 2010; Klaassen et al., 2013; Haller
et al., 2014).
The effect of theobromine in respiratory diseases is not due
to inhibition of mediators of inflammation in asthma, histamine
or slow reacting substance of anaphylaxis (Hillyard et al., 1984).
A novel differential target of methylxanthines is poly(ADP-
ribose)polymerase-1, a nuclear enzyme that is poorly inhibited by
caffeine but significantly inhibited by theobromine (Geraets et al.,
2006). In this sense, Ahmad et al. (2015) have recently shown
that inhibition of poly(ADP-ribose)polymerase-1 significantly
reduces inflammation of lungs caused by gamma-carrageenan.
Recent evidence demonstrates neovascularization in an animal
model of asthma (Wagner et al., 2015). Interestingly, theobromine
may reduce neovascularization accompanying tumor growth and
metastasis (Gil et al., 1993) and, therefore, it may reduce both
acute symptoms and angiogenesis in asthma.
Exposure to nitrogen mustards causes lung inflammation and
upregulation of oxidative stress proteins in macrophages. The
analog of theobromine, pentoxifylline, is effective in reducing
inflammation and increasing the number of macrophages with
wound repair anti-inflammatory properties (Sunil et al., 2014).
The concentration of adenosine at inflammation sites is notable
(Cronstein et al., 1999) and, consequently, it can activate adeno-
sine receptors present in lung cells and in macrophages. Blockade
of adenosine receptors and/or inhibition of phosphodiesterases
may underlie the phenotypic changes caused by methylxanthines
in macrophages activated after the mustard inhalation.
A pilot study was developed to test whether theobromine
was able to protect the enamel surface of human molars. The
results of this in vitro study showed that two different concen-
trations of theobromine were able to preserve the structure of
the teeth treated three days with acidic hydroxyl-ethyl-cellulose
for demineralization (Kargul et al., 2012). This protective effect
may not be due to adenosine receptors since they are not present
on enamel surfaces. Theobromine benefits at this level were
attained at relatively high concentrations. Actually, cocoa contains
carbohydrates that may be metabolized by bacteria in the mouth
and causing dental caries so caution may be taken to consider
cocoa intake as protector for teeth. Sugar-free cocoa alternative
could result in benefits to reduce caloric intake and preventing
dental caries.
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Martínez-Pinilla et al. Theobromine and cocoa consumption
Other adenosine receptor-independent effect of theobromine
is demonstrated in cardiovascular protection by significant
increases in HDL cholesterol plasma levels and decreases in LDL
ones. Clinical trials have been undertaken in volunteers taking
cocoa to assess the effect of this substance on plasma lipoprotein
levels (Kris-Etherton et al., 1994; Mursu et al., 2004; Wang-
Polagruto et al., 2006; Baba et al., 2007; Mellor et al., 2010;
Khan et al., 2012). The results of the clinical trial NCT01481389
(clinicaltrials.org) suggest that theobromine but not flavonoids is
the responsible for the increase in HDL levels in individuals taking
cocoa products (Neufingerl et al., 2013). The mechanism of HDL-
increasing effect is probably multifactorial and non-necessarily
related to the blockade of adenosine receptors. Likely based on
a diuretic effect in dogs (Macnider, 1917), theobromine has been
considered useful for weight loss and it is supplemented to herbal
tea preparations (Khazan et al., 2014). However, there is neither
enough data to confirm weight-loss potential in humans nor the
putative underlying mechanism.
CONCLUSION
Over the last decades, a remarkable progress has allowed under-
standing some of the molecular mechanisms that are behind
the proved health benefits of cacao consumption in man. Apart
from the high content of antioxidants, solid evidence points to
methylxanthines as key players in the beneficial effects. Caffeine
has been classically considered with higher potential than other
methylxanthines. Recent studies have highlighted the potential of
theobromine, which may act as antitumoral, anti-inflammatory
or cardiovascular protector molecule without the undesirable side
effects described for caffeine. The main mechanisms of action of
theobromine are inhibition of phosphodiesterases and blockade
of adenosine receptors but, interestingly, it exhibits other impor-
tant adenosine receptor-independent effects as the reduction of
cellular oxidative stress or regulation of gene expression. In this
sense, theobromine could be considered a safe and natural alter-
native in the treatment of some human diseases and may serve as
lead compound for the development of novel drugs
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Conflict of Interest Statement: The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Received: 14 November 2014; paper pending published: 01 December 2014; accepted:
03 February 2015; published online: 20 February 2015.
Citation: Martínez-Pinilla E, Oñatibia-Astibia A and Franco R (2015) The relevance
of theobromine for the beneficial effects of cocoa consumption. Front. Pharmacol. 6:30.
doi: 10.3389/fphar.2015.00030
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