Research article

Antidepressant-like effects of a cocoa polyphenolic extract in WistarUnilever rats

Michal Messaoudi1, Jean-Franois Bisson1, Amine Nejdi1, Pascale Rozan1, Herv Javelot1,2
1

ETAP-Applied Ethology, Centre de Recherche en Pharmacologie, Cancrologie & Pathologies Humaines et Nutrition-Sant, Vandoeuvre-ls-Nancy, France 2 Hpital Psychiatrique, Etablissement Public de Sant Alsace Nord, Brumath, France

Depression is a major public health problem affecting about 12% of the world population. Drugs exist 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 compound prepared from non-roasted cocoa beans containing high levels of flavonoids. The antidepressant-like effect of cocoa polyphenolic extract was evaluated using the forced swimming test in rats. Cocoa polyphenolic extract significantly reduced the duration of immobility at both doses of 24 mg/kg/14 days and 48 mg/kg/14 days, although no change of motor dysfunction was observed with the two doses tested in the open field. The results of the forced swimming test after a subchronic treatment and after an additional locomotor activity test confirm the assumption that the antidepressant-like effect of cocoa polyphenolic extract in the forced swimming test model is specific. Further, it can be speculated 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 recognized as a major public health problem. According to the World Health Organization in its 1998 report, depression (including complications of depression) affected about 12% of the world population and was supposed to be the second greatest cause of premature death and disability world-wide by the year 2020.1 Clinical depression is characterized by episodes that last at least 2 weeks and can completely disrupt normal functioning. Depressed patients display anhedonia, may be unable to perform lifes daily
Correspondence to: Michal Messaoudi, ETAP Research Centre, Department of Psychopharmacology, 13 rue du Bois de la Champelle, 54500 Vandoeuvre-ls-Nancy, France. Tel: +33 (0)383 444 635; Fax: +33 (0)383 446 441; E-mail: etap@etap-lab.com Received 4 August 2008, revised manuscript accepted 7 September 2008

functions and may experience difficulty concentrating or may have recurrent thoughts of death or suicide. Understanding how to prevent and treat depression is, therefore, an urgent subject. Although the mechanism provoking depression has not been clearly elucidated, the main trigger is known to be exposure to chronic stress.24 The treatment of depression is tailored to the individual, with the severity and cause of the depressive episode taken into account. Antidepressant medications are frequently prescribed; the two most common type of antidepressant agents used are selective serotonin re-uptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs). The SSRIs typically have fewer adverse side effects than the tricyclics or the monoamine oxidase inhibitors (MAOIs), although effects such as hepatotoxicity, pulmonary hypertension, headache, dizziness, drowsiness, dry mouth, nervousness, anxiety, insomnia, decreased appetite, and decreased ability to function sexually may occur.57

2008 W. S. Maney and Son Ltd DOI 10.1179/147683008X344165

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Thus, it is important to find better antidepressant agents with as few side effects as possible. Numerous epidemiological studies indicate that dietary flavonoids derived from fruits, vegetables, red wine, and green tea decrease the risk of death from coronary heart disease,8,9 cancer,10 and stroke,11 and may prevent neurodegenerative diseases and diabetes mellitus.12 In the last few years, several natural substances have been introduced with potential to fight various human pathologies, particularly neuropsychiatric disorders. This is reflected in the large number of alternative substances assessed in preclinical models of anxiety and depression, including medicinal plants, such as St Johns wort,13,14, Ginkgo biloba,15,16 Aloysia polystachya,17 and Salvia elegans.18 Antidepressant-like effects of an extract of the leaves of Apocynum venetum L. (Apocynaceae), containing quercetin glycosides such as hyperoside, isoquercitrin, miquelianin and quercitrin, have been shown in rodents using the positive results of forced swimming test.19 In the same way, procyanidin B2 from St Johns wort also displayed an antidepressant activity in the forced swimming test in rats.20,21 Recently, a butanolic fraction of Cecropia glazioui Sneth, rich in catechins, procyanidins (i.e. the procyanidin B3 isomer and procyanidin B2) and flavonoids, was shown to reduce the immobility in the forced swimming test in rats.22 Polyphenols, the most abundant natural antioxidants found in fruits, beverages (fruit juice, wine, tea, cocoa, chocolate, etc.) and, to a lesser extent, in vegetables, dry vegetables and cereals, have been reported to play a role in the prevention and/or the treatment of various diseases associated with oxidative stress such as cancer, cardiovascular diseases and inflammation.2325 Cocoa-derived products contain high levels of flavonoids2628 and show potent antioxidant effects.29 We have recently shown that a cocoa polyphenolic extract had protective antioxidant effects,30 protective effects on prostate carcinogenesis, and preventive31 and therapeutic effects on prostate hyperplasia.32 In addition, it has been shown that cocoa polyphenolic extract delays the onset of age-related cognitive deficits and prolongs life-span in aged rats.33 During this last study, it was observed that aged WistarUnilever rats treated with vehicle exhibited significant resignation in the Morris water maze test in comparison with rats treated with cocoa polyphenolic extract at the dose of 24 mg/kg. Resignation expresses depression behavior and the animals undergo, without fighting, the constraint of the aversive experimental situation.34 This lack of resignation was hypothesized
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to be a stimulating effect of cocoa polyphenolic extract on locomotor activity or an antidepressant effect 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 lipid cardiovascular deseases,4045 oxidation,23,26,38,39 diabetes,4648 and aging and cognition.33 Since oxidative stress, free radicals and nitric oxide have been implicated in over 100 disorders including neuropsychiatric pathologies,4957 it is of interest to study the potential antidepressant-like effects of cocoa polyphenols. Even if the complex relationships between depressive states and chocolate consumption are well documented, to the best of our knowledge, no studies have been performed with cocoa polyphenols on psychiatric disorders (i.e. anxiety and depression) and no antidepressant-like effect of cocoa polyphenols has yet been revealed. The purpose of the present study was to assess the effects of treatment with cocoa polyphenolic extract on locomotor activity in the open field test and on immobility in the forced swimming test in rats, which has been a useful experimental method for screening a wide range of antidepressant activity (including tricyclics, MAOIs, atypicals) by reducing the duration of immobility of animals.34,58,59

Material and methods

Animals

Forty-eight male WistarUnilever rats (HsdCpb:WU), weighing 200225 g at the start of the experiment, were obtained from Harlan Nederland (Horst, The Netherlands) and were housed in groups of four rats in polypropylene cages (48 27 20 cm; U.A.R., Epinay-sur-Orge, France) equipped to provide food and water. The animals were maintained in a climate room under controlled conditions of temperature (22 2C), relative humidity (50 10%), with an inverted 12-h light:dark cycle (lights off at 9:00 a.m.) and the rats had access to standard 2016 diet (Harlan Teklad Europe, Oxon, UK) and tap water ad libitum. The animal care unit is authorized by the French Ministries of Agriculture and Research (Government Authorization no. A 54-547-1) and the present protocol adhered to guidelines provided by the ASAB Ethical Committee for the treatment of animals in behavioral research and teaching (Animal Behavior 2006; 71: 245253), by the Canadian Council on Animal Care (Guide to the care and use of experimental animals: Vol. 1, 2nd edn, 1993, vol. 2, 1984), and by the

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Table 1

Polyphenol composition of the polyphenolic cocoa extract determined by HPLC Procyanidins Epicatechin Epicatechin gallate Catechin 88.5% 10% 1% 0.5%

Table 2 Composition of the polyphenolic cocoa extract Total polyphenols Proteins Fats Sugar Fibers Cellulose Ash Humidity Quercetin Caffeine Magnesium Phosphorus Potassium Sodium 36.9% 31.1% 3.9% 13.6% 4.0% 0.2% 4.9% 3.7% 97 mg/100 g 759 mg/100 g 242 mg/100 g 197 mg/100 g 1984 mg/100 g 21 mg/100 g

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

Chemicals

Solvent-free Acticoa powder, a cocoa polyphenolic extract, was provided by Barry Callebaut France (Louviers, France). It was isolated from non-roasted beans using the patented Acticoa process recently developed by Barry Callebaut France. The percentages relating to polyphenols in cocoa polyphenolic extract determined by HPLC and the composition of cocoa polyphenolic extract are shown in Tables 1 and 2. The reference antidepressant drug imipramine (IMI; hydrochloride form) was purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Both substances were stored at room temperature protected from light and moisture.
Treatments

Cryan et al.,60 who concluded that the effects of antidepressants were augmented following chronic treatment. IMI was also prepared fresh daily before testing. It was dissolved in spring water and stirred until homogeneous immediately before its administration at a dose of 15 mg/kg body weight, by oral gavage, in an administration volume of 10 ml/kg body weight. IMI was administered once a day in the morning and in the same conditions as the cocoa polyphenolic extract on days 13 and 14.
Forced swimming test procedure

After a 7 day-acclimatization period from their arrival, the 48 male WistarUnilever rats were randomized into four groups of 12 rats each according to their body weight: a control group treated with vehicle (spring water), two groups treated with cocoa polyphenolic extract at doses of 24 and 48 mg/kg body weight (AP24 and AP48, respectively) and a reference group treated with 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 was dissolved in spring water and stirred until homogeneous just before its administration at the doses of 24 and 48 mg/kg body weight (p.o.) in an administration volume of 10 ml/kg body weight. Cocoa polyphenolic extract was given by oral gavage during 12 days, twice a day (2 12 and 2 24 mg/kg body weight) in the morning and in the afternoon. On day 13, cocoa polyphenolic extract was orally administered at 24 or 48 mg/kg body weight immediately after the pretest session of the forced swimming test which lasted 15 min. On day 14, the product was administered at the same two doses 5 h and 1 h prior to the test session of the forced swimming test. Control vehicle treated rats received the same volume of spring water in the same conditions as the cocoa polyphenolic extract treated rats. Administration of cocoa polyphenolic extract was repeated, according to

The forced swimming test was performed according to the method of Porsolt et al.34 with some modifications.19 Rats were placed in a Plexiglas cylinder (50 20 cm, i.d.) filled with water at 25 1C to a depth of 30 cm for 15 min (pre-test session) after 13 days of treatment. Twenty-four hours later, the animals were once again exposed to the same forced swimming conditions for 5 min (test session). Between the pretest session and the test session, treatments were orally administered three times as follows: immediately after the pre-test session on day 13, 5 h and 1 h before the test session on day 14. During the forced swimming test testing, the animals placed into the cylinder filled with water had no possibility of escape, causing vigorous swimming and escape attempts by diving or climbing the walls. When animals cease all movements, except those necessary for survival (keeping the head above water level), they became immobile, interpreted as depression-related. The first 5 min of the pretest session and the 5 min of the test session were recorded using a CCD-TV camera for scoring the duration of immobility. The tests were performed and the recorded behaviors were scored by experimenters unaware of the administered products.

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Measurement of locomotor activity in the open field test

The same rats underwent an open field test to evaluate their general ambulation. On day 14, each rat was individually placed for 3 min in the centre of the open field device composed of a circular arena 40 cm high and 60 cm in diameter with clear Plexiglas walls and divided into 9 equal areas (8 peripheral and the central area), 1 h after the 14th treatment and 4 h before the test session of the forced swimming test on day 14. In an isolated test room, dimly lit with a single red light and an ambient temperature of 22 2C, a CCD-TV camera allowed rats to be observed and recorded on VHS-videotapes from a neighbouring room. This test was performed in order to prove the products had no effect on the locomotor activity of the animals.
Statistical analysis

The effects of treatments were analyzed with a oneway analysis of variance (ANOVA). Unpaired comparisons between control group and treated groups were checked with the Dunnetts t-test. In each group, repeated measures were compared using a paired t-test (2-tailed). The results were expressed as mean SEM. For all the comparisons, differences were considered 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).

On day 13, the body weight of IMI-treated rats was significantly lower than those of vehicle, AP24- and AP48-treated rats (t = 4.02; P = 0.0006: t = 3.38; P = 0.003: and t = 3.18; P = 0.004, respectively). No statistical differences were observed between the body weight 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- and AP48-treated rats (t = 4.20; P = 0.0004: t = 3.61; P = 0.002: and t = 3.63; P = 0.002, respectively). No statistical differences were observed between the body weight of control, AP24- and AP48-treated rats. Between days 114, 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 body weight gain of IMI-treated rats was significantly lower than 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 were observed between the mean body weight gains of the control, AP24 and AP48 groups.
Locomotor activity

Results
Effects of treatments on body weight

As shown in Table 3, there were no statistical differences between the body weights of rats in the four treatment groups at the beginning of the study on day 1, prior to treatments [F(3,44) = 0.05; P = 0.99]. On day 13, before the pretest session of the forced swimming test, and on day 14, before the test session of the forced swimming test, the body weight of rats in the four groups were statistically different [F(3,44) = 6.12; P = 0.0014 and F(3,44) = 6.89; P = 0.0007, respectively].
Table 3

As seen in Table 4, administration of IMI (at a dose of 15 mg/kg body weight), and cocoa polyphenolic extract (at doses of both 24 and 48 mg/kg body weight) did not result in any behavioral changes in the locomotor activity of rats performed in the open field test 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, AP24 and 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 test

As shown in Figure 1, a significant difference was observed between the immobility times of the four groups during the pretest session performed on day 13

Effects of the polyphenolic cocoa extract (AP) and imipramine (IMI), orally administered for 14 days, on body weight and mean body weight gain Body weight (g) Mean body weight gain (g) Day 14 329.8 4.4 328.1 5.0 326.1 4.2 305.8 4.4*** Day 1 day 14 +67.8 2.3 +65.8 2.4 +65.4 2.0 +43.7 1.9***

Treatments Control AP24 (24 mg/kg body weight) AP48 (48 mg/kg body weight) IMI (15 mg/kg body weight)

Day 1 262.0 3.0 262 .3 3.5 260.7 3.1 261.5 3.4

Day 13 326.9 4.1 324.9 4.8 321.8 4.0 304.1 3.9***

Data are expressed as mean SEM, n = 12 in each group . Dunnetts t-test: ***P <0.001 (versus control).

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Table 4 Treatment

Effects of the polyphenolic cocoa extract (AP) and imipramine (IMI), orally administered for 14 days, on locomotor activity and defecation in the open field test Crossing squares (n) 56.4 2.5 58.2 2.4 65.3 3.6 57.4 2.8 Rearings (n) 17.5 0.9 16.3 1.5 18.3 0.9 19.1 1.8 Defecations (n) 1.9 0.5 1.8 0.7 1.8 0.8 1.5 0.7

Control AP24 (24 mg/kg body weight) AP48 (48 mg/kg body weight) IMI (15 mg/kg body weight)

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

[F(3,44) = 3.45; P = 0.02]. During this session, only the immobility time of IMI-treated rats was significantly reduced after a 13-day period of treatment in comparison with that of control rats. During the test session performed on day 14, a significant difference was observed between the immobility times of the four treatment groups [F(3,44) = 13.73; P < 0.0001]. After a 14-day treatment period, IMI and cocoa polyphenolic extract at the two doses, significantly reduced the immobility time in comparison with 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 significantly decreased in the AP24, AP48 and IMI groups between the 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 the antidepressant-like effects of a cocoa polyphenolic

extract in rats, using a modified version of the forced swimming test developed by Porsolt et al.34 In the forced swimming test, rats display a range of behaviors, in particular immobility which is facilitated by the exposure to the inescapable water situation during the pretest session. The immobility displayed by rodents when subjected to unavoidable stress such as forced swimming is thought to reflect a state of despair or lowered mood, which, in turn, are thought to reflect depressive disorders in humans. Rats treated with IMI showed a significant lower body weight gain than those of rats treated with cocoa polyphenolic extract at both doses, or with vehicle. This side-effect of IMI has been previously observed in rats61 and is expressed in an opposite way in human patients, in whom IMI induces a significant body weight gain during chronic administration.62 In this experimental paradigm, the immobility time has been shown to be reduced by treatment with antidepressant drugs. Moreover, a significant correlation was found between the clinical efficacy of antidepressant drugs and their potency in this model.59 In the present study, cocoa polyphenolic extract significantly reduced immobility time in the forced

Figure 1 Effects of the polyphenolic cocoa extract (AP) and imipramine (IMI), orally administered for 14 days, on the immobility time (s) during pretest and test sessions in the forced swimming test. Data are expressed as mean SEM, n = 12 in each group. Dunnetts 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)
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swimming test after repeated administration at both doses of 24 and 48 mg/kg body weight to rats for 14 days. The intensity of immobility reduction was similar at both doses. The efficacy of the decreasing immobility in the forced swimming test was also shown previously with psychostimulants, which exert an indiscriminate motor stimulating activity.59 In order to exclude this falsepositive result, we performed an open field test to check the motor stimulating activity of cocoa polyphenolic extract. Administration of cocoa polyphenolic extract at both doses of 24 and 48 mg/kg body weight for 14 days (doses active in the forced swimming test) resulted in no behavioral changes or motor dysfunction in the open field test, indicating that the reduction in the duration of immobility could be attributed to an inherent antidepressant-like effect of cocoa polyphenolic extract. IMI, the reference antidepressant substance tested, also significantly reduced the immobility time in the forced swimming test at a dose of 15 mg/kg body weight after a repeated administration for 14 days. Conversely, control rats daily treated with vehicle significantly increased their immobility time in the forced swimming test. As described in the introduction, several natural substances have been shown to possess antidepressantlike effects in the forced swimming test in rats.14,19 The positive effects observed with these substances is probably due to their content of flavonoids, quercetin glycosides, procyanidins and catechins. It was demonstrated that the butanolic fraction of Cecropia glazioui Sneth BFCGS, rich in catechins, procyanidins and flavonoids, the most active compounds, inhibited the uptake of [3H]-serotonin, [3H]-dopamine and [3H]noradrenaline by synaptosomes from different brain regions in vitro, implying that the antidepressant effect of this compound was most likely due to the blockade of monoamine uptake in the central nervous system.22 Indeed, in addition to the immobility displayed by rats in the forced swimming test, it was demonstrated that the stress related to the inescapable water exposure induced increases in brain MAO-A and B activities, CRF levels, as well as a decrease in brain monoamine neurotransmitter levels.63 Cocoa polyphenolic extract contains all the active constituents referred to above, and is likely to show all the beneficial effects related to polyphenols of the medicinal plants evaluated in neuropsychiatric disorders. Indeed, cocoa polyphenolic extract is a complex product prepared from cocoa beans and cocoa-derived products that contain high levels of flavonoids.2628 Procyanidins in cocoa and dark chocolate are mainly homodimers and homotrimers 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 plasma as early as 30 min after the consumption of a flavanol-rich cocoa.64 It was also observed that the increase in blood epicatechin after acute procyanidin-rich chocolate consumption was associated with increased plasma antioxidant capacity and decreased plasma 2thiobarbituric acid reactive substances.65 In a clinical study assessing well-being and health among elderly men, variables related to psychological well-being (i.e. feeling loneliness, feeling of happiness, having plans for the future and the Zung depression score) were significantly better in those preferring chocolate.66 There is a possible role for ()epicatechin in reducing neurodegenerative disorders such as Parkinsons and Alzheimers diseases.67,68 Polyphenols may also possess other types of neuroprotective effects.69,70 In addition to containing ()-epicatechin, (+)-catechin and procyanidins, cocoa and chocolate contain other flavonoids, including other catechins and the flavanol quercetin and its glycosides.71,72 Cocoa polyphenolic extract contains a high concentration of quercetin (97 mg/100 g), while cocoa powder contains only about 20 mg/100 g. A preliminary study indicated that quercetin dose-dependently reduced the immobility time in diabetic mice and this effect was similar to that of fluoxetine and imipramine in the forced swimming test. However, in naive mice, quercetin failed to induce any antidepressant activity.73 In another study, it was shown that quercetin interacts with presynaptic 2-adrenoreceptors to induce a behavioral despair in the forced swimming test.74 However, a recent study assessed the effect of the quercetin-rich vegetable, onion, on depression in the rat and the results suggested that onion displayed antidepressant-like activity in the forced swimming test model that acted independently of the hypothalamic pituitaryadrenal axis.75 Cocoa polyphenolic extract also contents a high amount of magnesium (242 mg/100 g). This mineral has been shown to be effective in depression-like behavior in mice.76 In a clinical study, it was reported that a recovery from major depression was observed in less than 7 days using 125300 mg of magnesium with each meal and at bed-time.77 The antioxidant potential of cocoa polyphenolic extract and the properties of some of its constituents are probably the origin of its antidepressantlike effect.

Conclusions
Cocoa polyphenolic extract, through its active constituents, exerts antidepressant-like effects in the forced swimming test in rats, which seems to be mediated, at least partially, via reversal of brain oxidative damage, since depression is associated with

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elevated antioxidative enzyme activities and lipid peroxidation78 and/or via its possible role on the uptake of brain monoamine neurotransmitters. The results of the forced swimming test after a subchronic treatment and after an additional locomotor activity test confirm the assumption that the antidepressantlike effect of cocoa polyphenolic extract in the behavioral despair model is specific. Further, it can be speculated that this effect might be related to its active polyphenols. However, as cocoa polyphenolic extract contains caffein, magnesium and other minerals, it would be interesting to understand their synergystic effects with cocoa polyphenols. This is the first investigation of cocoa polyphenols on depression. Other studies are now necessary to discover the mechanism of action of cocoa polyphenolic extract. Further work is required to identify the active components with antidepressant-like activity in the cocoa polyphenol extract that might be useful in the prevention and therapy of mood disorders.

Acknowledgements
The authors would like to thank the Barry Callebaut Group for sponsoring this study and for the supply of the polyphenolic cocoa extract samples produced according to the Acticoa process. The authors also thank Valrie Demade, Sophie Hidalgo and Hlne Massinet, preclinical research assistants, for their skilful technical assistance.

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