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Antidepressant-like effects of a cocoapolyphenolic extract in Wistar–Unilever rats

Michaël Messaoudi1, Jean-François Bisson1, Amine Nejdi1, Pascale Rozan1,Hervé Javelot1,2

1ETAP-Applied Ethology, Centre de Recherche en Pharmacologie, Cancérologie & Pathologies Humaineset Nutrition-Santé, Vandoeuvre-lès-Nancy, France2Hôpital Psychiatrique, Etablissement Public de Santé Alsace Nord, Brumath, France

Depression is a major public health problem affecting about 12% of the world population. Drugsexist but they have many side effects. In the last few years, natural substances (e.g. flavonoids)have been tested to cure such disorders. Cocoa polyphenolic extract is a complex compoundprepared from non-roasted cocoa beans containing high levels of flavonoids. The antidepressant-likeeffect of cocoa polyphenolic extract was evaluated using the forced swimming test in rats. Cocoapolyphenolic extract significantly reduced the duration of immobility at both doses of 24 mg/kg/14 daysand 48 mg/kg/14 days, although no change of motor dysfunction was observed with the two dosestested in the open field. The results of the forced swimming test after a subchronic treatment andafter an additional locomotor activity test confirm the assumption that the antidepressant-like effectof cocoa polyphenolic extract in the forced swimming test model is specific. Further, it can bespeculated that this effect might be related to its content of active polyphenols.

Keywords: cocoa polyphenolic extract, depression, flavonoids, forced swimming test, polyphenols, rat

Introduction

In recent years, clinical depression has been recognizedas a major public health problem. According to theWorld Health Organization in its 1998 report,depression (including complications of depression)affected about 12% of the world population and wassupposed to be the second greatest cause of prematuredeath and disability world-wide by the year 2020.1

Clinical depression is characterized by episodes thatlast at least 2 weeks and can completely disruptnormal functioning. Depressed patients displayanhedonia, may be unable to perform life’s daily

functions and may experience difficulty concentratingor may have recurrent thoughts of death or suicide.Understanding how to prevent and treat depression is,therefore, an urgent subject. Although the mechanismprovoking depression has not been clearly elucidated,the main trigger is known to be exposure to chronicstress.2–4 The treatment of depression is tailored to theindividual, with the severity and cause of thedepressive episode taken into account. Antidepressantmedications are frequently prescribed; the two mostcommon type of antidepressant agents used areselective serotonin re-uptake inhibitors (SSRIs) andtricyclic antidepressants (TCAs). The SSRIs typicallyhave fewer adverse side effects than the tricyclics or themonoamine oxidase inhibitors (MAOIs), althougheffects such as hepatotoxicity, pulmonary hypertension,headache, dizziness, drowsiness, dry mouth, nervousness,anxiety, insomnia, decreased appetite, and decreasedability to function sexually may occur.5–7

Research article

Correspondence to: Michaël Messaoudi, ETAP Research Centre,Department of Psychopharmacology, 13 rue du Bois de la Champelle,54500 Vandoeuvre-lès-Nancy, France. Tel: +33 (0)383 444 635; Fax: +33(0)383 446 441; E-mail: etap@etap-lab.comReceived 4 August 2008, revised manuscript accepted 7 September 2008

© 2008 W. S. Maney and Son LtdDOI 10.1179/147683008X344165

Thus, it is important to find better antidepressantagents with as few side effects as possible. Numerousepidemiological studies indicate that dietaryflavonoids derived from fruits, vegetables, red wine,and green tea decrease the risk of death from coronaryheart disease,8,9 cancer,10 and stroke,11 and may preventneurodegenerative diseases and diabetes mellitus.12

In the last few years, several natural substanceshave been introduced with potential to fight varioushuman pathologies, particularly neuropsychiatricdisorders. This is reflected in the large number ofalternative substances assessed in preclinical models ofanxiety and depression, including medicinal plants,such as St John’s wort,13,14, Ginkgo biloba,15,16 Aloysiapolystachya,17 and Salvia elegans.18

Antidepressant-like effects of an extract of theleaves of Apocynum venetum L. (Apocynaceae),containing quercetin glycosides such as hyperoside,isoquercitrin, miquelianin and quercitrin, have beenshown in rodents using the positive results of forcedswimming test.19 In the same way, procyanidin B2from St John’s wort also displayed an antidepressantactivity in the forced swimming test in rats.20,21

Recently, a butanolic fraction of Cecropia glaziouiSneth, rich in catechins, procyanidins (i.e. theprocyanidin B3 isomer and procyanidin B2) andflavonoids, was shown to reduce the immobility in theforced swimming test in rats.22

Polyphenols, the most abundant naturalantioxidants found in fruits, beverages (fruit juice,wine, tea, cocoa, chocolate, etc.) and, to a lesserextent, in vegetables, dry vegetables and cereals, havebeen reported to play a role in the prevention and/orthe treatment of various diseases associated withoxidative stress such as cancer, cardiovascular diseasesand inflammation.23–25

Cocoa-derived products contain high levels offlavonoids26–28 and show potent antioxidant effects.29

We have recently shown that a cocoa polyphenolicextract had protective antioxidant effects,30 protectiveeffects on prostate carcinogenesis, and preventive31

and therapeutic effects on prostate hyperplasia.32 Inaddition, it has been shown that cocoa polyphenolicextract delays the onset of age-related cognitivedeficits and prolongs life-span in aged rats.33 Duringthis last study, it was observed that agedWistar–Unilever rats treated with vehicle exhibitedsignificant resignation in the Morris water maze test incomparison with rats treated with cocoa polyphenolicextract at the dose of 24 mg/kg. Resignation expressesdepression behavior and the animals undergo, withoutfighting, the constraint of the aversive experimentalsituation.34 This lack of resignation was hypothesized

to be a stimulating effect of cocoa polyphenolicextract on locomotor activity or an antidepressanteffect of the extract.Until now, cocoa polyphenol effects have been

studied on various pathologies, for example, cancer,35,36

obesity,37 neuroprotection,30 oxidative stress and lipidoxidation,23,26,38,39 cardiovascular deseases,40–45

diabetes,46–48 and aging and cognition.33

Since oxidative stress, free radicals and nitric oxidehave been implicated in over 100 disorders includingneuropsychiatric pathologies,49–57 it is of interest tostudy the potential antidepressant-like effects of cocoapolyphenols.Even if the complex relationships between

depressive states and chocolate consumption are welldocumented, to the best of our knowledge, no studieshave been performed with cocoa polyphenols onpsychiatric disorders (i.e. anxiety and depression) andno antidepressant-like effect of cocoa polyphenols hasyet been revealed. The purpose of the present studywas to assess the effects of treatment with cocoapolyphenolic extract on locomotor activity in the openfield test and on immobility in the forced swimmingtest in rats, which has been a useful experimentalmethod for screening a wide range of antidepressantactivity (including tricyclics, MAOIs, atypicals) byreducing the duration of immobility of animals.34,58,59

Material and methods

AnimalsForty-eight male Wistar–Unilever rats (HsdCpb:WU),weighing 200–225 g at the start of the experiment,were obtained from Harlan Nederland (Horst, TheNetherlands) and were housed in groups of four ratsin polypropylene cages (48 × 27 × 20 cm; U.A.R.,Epinay-sur-Orge, France) equipped to provide foodand water. The animals were maintained in a climateroom under controlled conditions of temperature (22± 2ºC), relative humidity (50 ± 10%), with an inverted12-h light:dark cycle (lights off at 9:00 a.m.) and therats had access to standard 2016 diet (Harlan TekladEurope, Oxon, UK) and tap water ad libitum. Theanimal care unit is authorized by the FrenchMinistries of Agriculture and Research (GovernmentAuthorization no. A 54-547-1) and the presentprotocol adhered to guidelines provided by the ASABEthical Committee for the treatment of animals inbehavioral research and teaching (Animal Behavior2006; 71: 245–253), by the Canadian Council onAnimal Care (Guide to the care and use of experimentalanimals: Vol. 1, 2nd edn, 1993, vol. 2, 1984), and by the

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European Communities Council Directive of 24November 1986 (86/609/EEC).

ChemicalsSolvent-free Acticoa powder, a cocoa polyphenolicextract, was provided by Barry Callebaut France(Louviers, France). It was isolated from non-roastedbeans using the patented Acticoa process recentlydeveloped by Barry Callebaut France. The percentagesrelating to polyphenols in cocoa polyphenolic extractdetermined by HPLC and the composition of cocoapolyphenolic extract are shown in Tables 1 and 2.The reference antidepressant drug imipramine

(IMI; hydrochloride form) was purchased fromSigma-Aldrich (Saint-Quentin Fallavier, France).Both substances were stored at room temperature

protected from light and moisture.

TreatmentsAfter a 7 day-acclimatization period from their arrival,the 48 male Wistar–Unilever rats were randomized intofour groups of 12 rats each according to their bodyweight: a control group treated with vehicle (springwater), two groups treated with cocoa polyphenolicextract at doses of 24 and 48 mg/kg body weight (AP24and AP48, respectively) and a reference group treatedwith IMI at the dose of 15 mg/kg body weight (IMI).Cocoa polyphenolic extract was prepared fresh each

day before each treatment and before testing. It wasdissolved in spring water and stirred until homogeneousjust before its administration at the doses of 24 and 48mg/kg body weight (p.o.) in an administration volume of10 ml/kg body weight. Cocoa polyphenolic extract wasgiven by oral gavage during 12 days, twice a day (2 × 12and 2 × 24mg/kg bodyweight) in themorning and in theafternoon. On day 13, cocoa polyphenolic extract wasorally administered at 24 or 48 mg/kg body weightimmediately after the pretest session of the forcedswimming test which lasted 15 min. On day 14, theproduct was administered at the same two doses 5 h and1 h prior to the test session of the forced swimming test.Control vehicle treated rats received the same volume ofspring water in the same conditions as the cocoapolyphenolic extract treated rats. Administration ofcocoa polyphenolic extract was repeated, according to

Cryan et al.,60 who concluded that the effects ofantidepressants were augmented following chronictreatment.IMI was also prepared fresh daily before testing. It

was dissolved in spring water and stirred untilhomogeneous immediately before its administration ata dose of 15 mg/kg body weight, by oral gavage, in anadministration volume of 10 ml/kg body weight. IMIwas administered once a day in the morning and in thesame conditions as the cocoa polyphenolic extract ondays 13 and 14.

Forced swimming test procedureThe forced swimming test was performed according tothe method of Porsolt et al.34 with some modific-ations.19 Rats were placed in a Plexiglas cylinder (50 ×20 cm, i.d.) filled with water at 25 ± 1ºC to a depth of30 cm for 15 min (pre-test session) after 13 days oftreatment. Twenty-four hours later, the animals wereonce again exposed to the same forced swimmingconditions for 5 min (test session). Between the pre-test session and the test session, treatments were orallyadministered three times as follows: immediately afterthe pre-test session on day 13, 5 h and 1 h before thetest session on day 14. During the forced swimmingtest testing, the animals placed into the cylinder filledwith water had no possibility of escape, causingvigorous swimming and escape attempts by diving orclimbing the walls. When animals cease all move-ments, except those necessary for survival (keeping thehead above water level), they became immobile,interpreted as depression-related. The first 5 min ofthe pretest session and the 5 min of the test sessionwere recorded using a CCD-TV camera for scoring theduration of immobility.The tests were performed and the recorded

behaviors were scored by experimenters unaware ofthe administered products.

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Table 1 Polyphenol composition of the polyphenolic cocoaextract determined by HPLC

• Procyanidins 88.5%• Epicatechin 10%• Epicatechin gallate 1%• Catechin 0.5%

Table 2 Composition of the polyphenolic cocoa extract

Total polyphenols 36.9%Proteins 31.1%Fats 3.9%Sugar 13.6%Fibers 4.0%Cellulose 0.2%Ash 4.9%Humidity 3.7%Quercetin 97 mg/100 gCaffeine 759 mg/100 gMagnesium 242 mg/100 gPhosphorus 197 mg/100 gPotassium 1984 mg/100 gSodium 21 mg/100 g

Measurement of locomotor activity in the open field testThe same rats underwent an open field test to evaluatetheir general ambulation. On day 14, each rat wasindividually placed for 3 min in the centre of the openfield device composed of a circular arena 40 cm highand 60 cm in diameter with clear Plexiglas walls anddivided into 9 equal areas (8 peripheral and the centralarea), 1 h after the 14th treatment and 4 h before thetest session of the forced swimming test on day 14. Inan isolated test room, dimly lit with a single red lightand an ambient temperature of 22 ± 2ºC, a CCD-TVcamera allowed rats to be observed and recorded onVHS-videotapes from a neighbouring room. This testwas performed in order to prove the products had noeffect on the locomotor activity of the animals.

Statistical analysisThe effects of treatments were analyzed with a one-way analysis of variance (ANOVA). Unpairedcomparisons between control group and treatedgroups were checked with the Dunnett’s t-test. In eachgroup, repeated measures were compared using a pairedt-test (2-tailed). The results were expressed as mean ±SEM. For all the comparisons, differences wereconsidered to be significant at the level of P < 0.05.All statistical analyses were carried out using the

StatView® v.5 statistical package (SAS, Institute, Inc.,Cary, NC, USA).

Results

Effects of treatments on body weightAs shown in Table 3, there were no statisticaldifferences between the body weights of rats in thefour treatment groups at the beginning of the study onday 1, prior to treatments [F(3,44) = 0.05;P= 0.99]. On day13, before the pretest session of the forced swimming test,and on day 14, before the test session of the forcedswimming test, the body weight of rats in the four groupswere statistically different [F(3,44) = 6.12; P = 0.0014 andF(3,44) = 6.89; P = 0.0007, respectively].

On day 13, the body weight of IMI-treated rats wassignificantly lower than those of vehicle, AP24- andAP48-treated rats (t = 4.02; P = 0.0006: t = 3.38; P =0.003: and t = 3.18; P = 0.004, respectively). Nostatistical differences were observed between the bodyweight of control, AP24- and AP48-treated animals.On day 14, the body weight of IMI-treated rats was

significantly lower than those of control, AP24- andAP48-treated rats (t = 4.20; P = 0.0004: t = 3.61; P =0.002: and t = 3.63; P = 0.002, respectively). Nostatistical differences were observed between the bodyweight of control, AP24- and AP48-treated rats.Between days 1–14, the mean body weight gains of

the four groups of rats were statistically different [F(3,44)= 27.71; P < 0.0001]. During this period, the mean bodyweight gain of IMI-treated rats was significantly lowerthan those of control, AP24- and AP48-treated animals(t = 8.13; P < 0.0001: t = 7.11; P < 0.0001: and t = 7.86;P < 0.0001, respectively). No statistical differences wereobserved between the mean body weight gains of thecontrol, AP24 and AP48 groups.

Locomotor activityAs seen in Table 4, administration of IMI (at a dose of15 mg/kg body weight), and cocoa polyphenolicextract (at doses of both 24 and 48 mg/kg bodyweight) did not result in any behavioral changes in thelocomotor activity of rats performed in the open fieldtest on day 14.No significant differences were observed in the

number of squares crossed between control, IMI-,AP24- and AP48-treated rats [F(3,44) = 1.97; P = 0.13].No significant differences were observed in the

number of rearings between the control, IMI, AP24and AP48 groups [F(3,44) = 0.80; P = 0.50].The fours groups did not differ in terms of

defecations [F(3,44) = 0.08; P = 0.97].

Forced swimming testAs shown in Figure 1, a significant difference wasobserved between the immobility times of the fourgroups during the pretest session performed on day 13

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Table 3 Effects of the polyphenolic cocoa extract (AP) and imipramine (IMI), orally administered for 14 days, on body weightand mean body weight gain

Body weight (g) Mean body weight gain (g)

Treatments Day 1 Day 13 Day 14 Day 1 – day 14

Control 262.0 ± 3.0 326.9 ± 4.1 329.8 ± 4.4 +67.8 ± 2.3AP24 (24 mg/kg body weight) 262 .3 ± 3.5 324.9 ± 4.8 328.1 ± 5.0 +65.8 ± 2.4AP48 (48 mg/kg body weight) 260.7 ± 3.1 321.8 ± 4.0 326.1 ± 4.2 +65.4 ± 2.0IMI (15 mg/kg body weight) 261.5 ± 3.4 304.1 ± 3.9*** 305.8 ± 4.4*** +43.7 ± 1.9***

Data are expressed as mean ± SEM, n = 12 in each group . Dunnett’s t-test: ***P <0.001 (versus control).

[F(3,44) = 3.45; P = 0.02]. During this session, only theimmobility time of IMI-treated rats was significantlyreduced after a 13-day period of treatment incomparison with that of control rats.During the test session performed on day 14, a

significant difference was observed between theimmobility times of the four treatment groups [F(3,44) =13.73; P < 0.0001]. After a 14-day treatment period, IMIand cocoa polyphenolic extract at the two doses,significantly reduced the immobility time in comparisonwith that of controls (t = 4.32; P = 0.0003: t = 3.48; P =0.002: and t = 3.53; P = 0.002, respectively).In addition, the immobility time significantly

increased in the control group, while it significantlydecreased in the AP24, AP48 and IMI groups betweenthe pretest and the test sessions (t = 3.22; P = 0.008: t =5.16; P = 0.0003: t = 2.66; P = 0.02: and t = 2.75; P =0.02, respectively).

Discussion

The purpose of this study was to evaluate theantidepressant-like effects of a cocoa polyphenolic

extract in rats, using a modified version of the forcedswimming test developed by Porsolt et al.34 In theforced swimming test, rats display a range ofbehaviors, in particular immobility which is facilitatedby the exposure to the inescapable water situationduring the pretest session. The immobility displayedby rodents when subjected to unavoidable stress suchas forced swimming is thought to reflect a state ofdespair or lowered mood, which, in turn, are thoughtto reflect depressive disorders in humans.Rats treated with IMI showed a significant lower

body weight gain than those of rats treated with cocoapolyphenolic extract at both doses, or with vehicle.This side-effect of IMI has been previously observedin rats61 and is expressed in an opposite way in humanpatients, in whom IMI induces a significant bodyweight gain during chronic administration.62

In this experimental paradigm, the immobility timehas been shown to be reduced by treatment withantidepressant drugs. Moreover, a significantcorrelation was found between the clinical efficacy ofantidepressant drugs and their potency in this model.59

In the present study, cocoa polyphenolic extractsignificantly reduced immobility time in the forced

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Table 4 Effects of the polyphenolic cocoa extract (AP) and imipramine (IMI), orally administered for 14 days, on locomotoractivity and defecation in the open field test

Treatment Crossing squares (n) Rearings (n) Defecations (n)

Control 56.4 ± 2.5 17.5 ± 0.9 1.9 ± 0.5AP24 (24 mg/kg body weight) 58.2 ± 2.4 16.3 ± 1.5 1.8 ± 0.7AP48 (48 mg/kg body weight) 65.3 ± 3.6 18.3 ± 0.9 1.8 ± 0.8IMI (15 mg/kg body weight) 57.4 ± 2.8 19.1 ± 1.8 1.5 ± 0.7

Data are expressed as mean ± SEM, n = 12 in each group.

Figure 1 Effects of the polyphenolic cocoa extract (AP) and imipramine (IMI), orally administered for 14 days, on theimmobility time (s) during pretest and test sessions in the forced swimming test. Data are expressed as mean ± SEM,n = 12 in each group. Dunnett’s t-test: *P < 0.05; ***P < 0.005 (versus control in each session). Paired t-test (2-tail): #P< 0.05; ##P < 0.01; ###P < 0.005 (pretest session versus test session in each treatment group)

swimming test after repeated administration at bothdoses of 24 and 48mg/kg bodyweight to rats for 14 days.The intensity of immobility reduction was similar atboth doses. The efficacy of the decreasing immobility inthe forced swimming test was also shown previously withpsychostimulants, which exert an indiscriminate motorstimulating activity.59 In order to exclude this false-positive result, we performed an open field test to checkthe motor stimulating activity of cocoa polyphenolicextract. Administration of cocoa polyphenolic extract atboth doses of 24 and 48 mg/kg body weight for 14 days(doses active in the forced swimming test) resulted in nobehavioral changes or motor dysfunction in the openfield test, indicating that the reduction in the duration ofimmobility could be attributed to an inherentantidepressant-like effect of cocoa polyphenolic extract.IMI, the reference antidepressant substance tested, alsosignificantly reduced the immobility time in the forcedswimming test at a dose of 15 mg/kg body weight after arepeated administration for 14 days. Conversely, controlrats daily treated with vehicle significantly increased theirimmobility time in the forced swimming test.As described in the introduction, several natural

substances have been shown to possess antidepressant-like effects in the forced swimming test in rats.14,19 Thepositive effects observed with these substances isprobably due to their content of flavonoids, quercetinglycosides, procyanidins and catechins. It was demon-strated that the butanolic fraction of Cecropia glaziouiSneth BFCGS, rich in catechins, procyanidins andflavonoids, the most active compounds, inhibited theuptake of [3H]-serotonin, [3H]-dopamine and [3H]-noradrenaline by synaptosomes from different brainregions in vitro, implying that the antidepressant effect ofthis compound was most likely due to the blockade ofmonoamine uptake in the central nervous system.22

Indeed, in addition to the immobility displayed by rats inthe forced swimming test, it was demonstrated that thestress related to the inescapable water exposure inducedincreases in brain MAO-A and B activities, CRF levels,as well as a decrease in brain monoamine neuro-transmitter levels.63

Cocoa polyphenolic extract contains all the activeconstituents referred to above, and is likely to show allthe beneficial effects related to polyphenols of themedicinal plants evaluated in neuropsychiatric disorders.Indeed, cocoa polyphenolic extract is a complex productprepared from cocoa beans and cocoa-derived productsthat contain high levels of flavonoids.26–28 Procyanidins incocoa and dark chocolate are mainly homodimers andhomotrimers of (–)-epicatechin or heterodimers of (–)-epicatechin and (+)-catechin. (–)-Epicatechin and (+)-catechin belong to the flavan-3-ol class of flavonoids.

Dimeric procyanidin B2 can be detected in human plasmaas early as 30min after the consumption of a flavanol-richcocoa.64 It was also observed that the increase in bloodepicatechin after acute procyanidin-rich chocolateconsumption was associated with increased plasmaantioxidant capacity and decreased plasma 2-thiobarbituric acid reactive substances.65 In a clinical studyassessing well-being and health among elderly men,variables related to psychological well-being (i.e. feelingloneliness, feeling of happiness, having plans for the futureand the Zung depression score) were significantly better inthose preferring chocolate.66 There is a possible role for (–)-epicatechin in reducing neurodegenerative disorders suchas Parkinson’s and Alzheimer’s diseases.67,68 Polyphenolsmay also possess other types of neuroprotective effects.69,70

In addition to containing (–)-epicatechin, (+)-catechin andprocyanidins, cocoa and chocolate contain otherflavonoids, including other catechins and the flavanolquercetin and its glycosides.71,72 Cocoa polyphenolicextract contains a high concentration of quercetin (97mg/100 g), while cocoa powder contains only about 20mg/100 g. A preliminary study indicated that quercetindose-dependently reduced the immobility time in diabeticmice and this effect was similar to that of fluoxetine andimipramine in the forced swimming test.However, in naivemice, quercetin failed to induce any antidepressantactivity.73 In another study, it was shown that quercetininteracts with presynaptic α2-adrenoreceptors to induce abehavioral despair in the forced swimming test.74 However,a recent study assessed the effect of the quercetin-richvegetable, onion, on depression in the rat and the resultssuggested that onion displayed antidepressant-like activityin the forced swimming test model that acted indep-endently of the hypothalamic– pituitary–adrenal axis.75

Cocoa polyphenolic extract also contents a highamount of magnesium (242 mg/100 g). This mineral hasbeen shown to be effective in depression-like behavior inmice.76 In a clinical study, it was reported that a recoveryfrom major depression was observed in less than 7 daysusing 125–300 mg of magnesium with each meal and atbed-time.77 The antioxidant potential of cocoapolyphenolic extract and the properties of some of itsconstituents are probably the origin of its antidepressant-like effect.

Conclusions

Cocoa polyphenolic extract, through its activeconstituents, exerts antidepressant-like effects in theforced swimming test in rats, which seems to bemediated, at least partially, via reversal of brainoxidative damage, since depression is associated with

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elevated antioxidative enzyme activities and lipidperoxidation78 and/or via its possible role on theuptake of brain monoamine neurotransmitters. Theresults of the forced swimming test after a subchronictreatment and after an additional locomotor activitytest confirm the assumption that the antidepressant-like effect of cocoa polyphenolic extract in thebehavioral despair model is specific. Further, it can bespeculated that this effect might be related to its activepolyphenols. However, as cocoa polyphenolic extractcontains caffein, magnesium and other minerals, itwould be interesting to understand their synergysticeffects with cocoa polyphenols.This is the first investigation of cocoa polyphenols on

depression. Other studies are now necessary to discoverthe mechanism of action of cocoa polyphenolic extract.Further work is required to identify the activecomponents with antidepressant-like activity in thecocoa polyphenol extract that might be useful in theprevention and therapy of mood disorders.

Acknowledgements

The authors would like to thank the Barry CallebautGroup for sponsoring this study and for the supply ofthe polyphenolic cocoa extract samples producedaccording to the Acticoa process. The authors alsothank Valérie Demade, Sophie Hidalgo and HélèneMassinet, preclinical research assistants, for theirskilful technical assistance.

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