Experimental Gerontology 47 (2012) 573–580

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Experimental Gerontology

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The consumption of a Jerte Valley cherry product in humans enhances mood, andincreases 5-hydroxyindoleacetic acid but reduces cortisol levels in urine

María Garrido a, Javier Espino a, David González-Gómez b, Mercedes Lozano b, Carmen Barriga a,Sergio D. Paredes c, Ana B. Rodríguez a,⁎a Department of Physiology (Neuroimmunophysiology and Chrononutrition Research Group), Faculty of Science, University of Extremadura, 06006 Badajoz, Spainb Department of Horticulture and Pomology, Technological Institute of Food and Agriculture of Extremadura (INTAEX), 06071 Badajoz, Spainc Department of Physiology, School of Medicine, Complutense University of Madrid, 28040 Madrid, Spain

Abbreviations: HPA, limbic hypothalamus–pituitary–system; aMT6-s, 6-sulfatoxymelatonin; 5-HIAA, 5-hydroal analogue scale; STAI, state–trait anxiety inventory.⁎ Corresponding author at: Department of Physiology

of Extremadura, Avda. Elvas s/n, 06006 Badajoz, Spain. Tfax: +34 924 289 388.

E-mail address: moratino@unex.es (A.B. Rodríguez).

0531-5565/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.exger.2012.05.003

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 18 October 2011Received in revised form 6 April 2012Accepted 4 May 2012Available online 11 May 2012

Section Editor: Andrzej Bartke

Keywords:CortisolMelatoninMoodSerotoninJerte Valley cherryTryptophan

Purpose: Jerte Valley cherries contain high levels of tryptophan, serotonin, and melatonin. These molecules havebeen shown to be involved in mood regulation. It has been suggested that a complex inter-relationship betweenbrain serotonin, circulating levels of cortisol (themajor stress hormone), and the hypothalamus–pituitary–adrenalaxis exists in the regulation of stress responses, where cortisol and serotonin act as markers of mood disturbances.Moreover there is growing evidence that altered HPA activity is associated with various age-related pathologies.The present study evaluated the effect of the ingestion of a Jerte Valley cherry‐based product, compared to a pla-cebo product, on urine cortisol and 5-hydroxyindoleacetic acid (5-HIAA) levels, and on mood in young, middle-aged, and elderly participants.Methods: Cortisol and 5-HIAA acid levels were measured by commercial enzyme-linked immunosorbent assaykits. The mood state profile was analysed using a visual analogue scale and the state–trait anxiety inventory.Results: Our findings showed that the ingestion of the Jerte Valley cherry product decreased urinary cortisol andincreased urinary 5-HIAA levels in all the experimental groups. Moreover, the cherry product was able to lessenanxiety status in the middle-aged and elderly participants, and enhanced subjective mood parameters, particular-ly family relationships in young participants, and frame of mind and fitness in both middle-aged and elderly sub-

jects.Conclusions: The consumption of the Jerte Valley cherry product may protect against stress and act as a mood en-hancer by increasing serotonin availability to the organism, particularly with advancing age.

© 2012 Elsevier Inc. All rights reserved.

1. Introduction

Globally, it is estimated that as many as 450 million people sufferfrom mood disturbances, with depression and anxiety being amongthe most prevalent. As an example, 13.6% of the population in six Eu-ropean countries, including Spain, reported having had an anxietydisturbance during their lifetime (Alonso et al., 2004), and morethan 150 million people suffer from depression at any given point intime.

The limbic hypothalamus–pituitary–adrenal (HPA) axis is a centralcontrol and regulatory system of the organism that connects the cen-tral nervous system (CNS) with the hormonal system, thereby

adrenal; CNS, central nervousxyindoleacetic acid; VAS, visu-

, Faculty of Science, Universityel.: +34 924 289 300x89035;

rights reserved.

conforming a stress-responsive neuroendocrine system (Kudielkaand Kirschbaum, 2005). It is widely accepted that the activated HPAaxis not only regulates body peripheral functions, but also has pro-found effects on the brain (Pariante and Lightman, 2008). Many pa-tients with depressive and/or anxiety disturbances show disruptionsin HPA axis reactivity as indexed by increased baseline plasma cortisollevels compared with healthy controls (Rajewska and Rybakowski,2003; Vreeburg et al., 2009). However, not only the HPA axis, butalso brain neuronal systems, including the monoaminergic systems,in particular the serotoninergic system, play an important role in theregulation of many physiological and behavioural processes includingmood. In this sense, a strong relationship between the HPA axis andthe serotoninergic system has been reported (Porter et al., 2004).Stress-induced elevated cortisol reduces the tonic level of serotoninin the synaptic cleft and stimulates its reuptake after a neuronal im-pulse, thereby leading to defective serotoninergic neurotransmissionin the CNS (Tafet et al., 2001) as has been reported in depression(Duval et al., 2001).

Ageing can be defined as a progressive decline in physiological ef-ficiency regulated by extremely complex multifactorial processes.

574 M. Garrido et al. / Experimental Gerontology 47 (2012) 573–580

Several studies provide evidence that hyperactivity of the HPA axiscontributes to the neuronal and peripheral deterioration associatedwith ageing (Ferrari and Magri, 2008), which is reflected in decreasedconcentrations of the neurotransmitter serotonin, a modulator of age-ing in the brain (Sibille et al., 2007). Moreover, there is clear evidencethat ageing is associated with elevated basal morning levels of circu-lating glucocorticoids such as cortisol (Tizabi et al., 1992). Overall,there is increasing evidence that altered HPA activity, e.g., increasedglucocorticoid activity or a decreased brain serotonin concentration,is associated with several age-related pathologies (Aguilera, 2011).

The amino acid tryptophan, which is obtained from the diet inhumans, is the direct precursor of serotonin. It has been shown thatincreases in plasma tryptophan availability enhance positive moodand dampen the cortisol response after an acute experimental stressexposure in stress-vulnerable subjects by enhancing brain serotoninmechanisms that are involved in adaptation to stress (Firk andMarkus, 2009; Markus et al., 2000). For this reason, several studieshave focused on evaluating the efficacy of the consumption oftryptophan-enriched diets to reduce cortisol responses and improvethe ability to cope with stress, probably by way of counteracting alter-ations in brain serotonin (Lepage et al., 2002). However, most of thesestudies were carried out on animals, while available information onhumans regarding the effect of tryptophan-enriched diets on bothstress-induced cortisol responses and mood is scarce.

Cherries are an important source of phytochemicals and reported-ly have important health-promoting properties, including antioxidanteffects (McCune et al., 2011). In this regard, Jerte Valley sweetcherries contain not only high concentrations of anthocyanin pig-ments and phenolic compounds (González-Gómez et al., 2010), butalso substantial amounts of melatonin, serotonin (González-Gomezet al., 2009), and tryptophan (Cubero et al., 2010). Also, it has beenreported that both a Jerte Valley cherry-enriched diet (Garrido et al.,2010) and the ingestion of a Jerte Valley cherry-based product(Garrido et al., 2009) exhibit sleep-promoting actions, and increaseboth urinary 6-sulfatoxymelatonin (aMT6-s) and the antioxidant sta-tus in humans. Therefore, the purpose of this study was to comparethe effect of the ingestion of a Jerte Valley cherry-based product (pat-ent no. ES 2342141 B1), compared to a placebo product on cortisoland 5-hydroxyindoleacetic acid (5-HIAA) urinary levels and onmood, in young, middle-aged, and elderly subjects.

2. Material and methods

2.1. Participants

The study was carried out in young (20–30 years old, n=10; 5men and 5 women), middle-aged (35–55 years old, n=10; 5 menand 5 women) and elderly (65–85 years old, n=10; 5 men and 5women) volunteers whose weight, height, and body mass index(BMI) values are presented in Table 1. The study was approved bythe Ethics Committee of the University of Extremadura (Badajoz,Spain) in accordance with the Declaration of Helsinki, the Council ofEurope, and the Universal Declaration of UNESCO on human rights,biomedicine, and human genome. All participants were of Caucasianethnicity and were recruited through word of mouth. There were nodropouts during the study. Each participant was ascertained to be in

Table 1Weight, height, and body mass index (BMI) of young, middle-aged, and elderly participant

Young Middle-age

Men Women Men

Weight 81.00±2.70 60.5±8.22 76.00±3.9Height 1.80±0.04 1.61±0.02 1.74±0.0BMI 24.86±0.68 23.07±2.57 24.89±0.7

Each value represents the mean±SD of five participants.

good health from their medical history and a clinical examination in-cluding routine laboratory tests and screening. The participants werenon-smokers, were not using any medication, and abstained from al-cohol. Informed consent was obtained from all participants.

2.2. Experimental design

The study had a blind, placebo-controlled, randomised, crossoverdesign with two treatment periods of five days each, separated by awashout period of one week. Either the placebo or the Jerte Valley(Cáceres, Extremadura, Spain) cherry product was consumed twicea day, as lunch and dinner desserts. Each dose of cherry product(27.85 g) consisted of 18.85 g of pitted, freeze-dried cherries (equiv-alent to 141 g fresh cherries) in equal parts of four Jerte Valley cherrycultivars (Bourlat, Navalinda, Pico Negro, and Pico Colorado), plus7.5 g maltodextrin and 1.5 g ascorbic acid (Spanish patent no. ES2342141 B1). The freeze-dried, cherry-based product was then gro-und to a powder, and then diluted in water and bottled in 12 fl ozplastic bottles containing 125 ml (4.22 fl oz) of cherry-based productper dose. One dose of the product provided roughly 1580 mg phenoliccompounds (expressed as gallic acid equivalents), 30 mg anthocya-nins (calculated as malvidin equivalents), 690 mg total antioxidantcapacity (TAC, expressed as Trolox equivalents), 2 mg tryptophan,27 ng serotonin, and 16 ng melatonin.

The placebo was a commercial cherry-flavoured soft drink (Kool-Aid®, Kraft Foods, USA; ingredients listed: citric acid, salt, calciumphosphate, red 40, artificial flavour, ascorbic acid, artificial colour,blue 1) which was prepared by mixing it with water in the proportionrecommended by the manufacturer, followed by bottling in 12 fl ozplastic bottles to contain 125 ml (4.22 fl oz) of placebo product perdose.

2.3. Cortisol and 5-hydroxyindoleacetic acid (5-HIAA) urine levels

First-void morning urine and urine at 20:00 h were collected be-fore the trial (basal values), after a 5-day intake of the Jerte Valleycherry-based product or the placebo (trial values), and one day fol-lowing its termination (post-trial values). The samples were storedat −20 °C until biochemical assay.

Cortisol levels were measured in both urine samples and 5-HIAAlevels were measured in the 20:00 h urine using commercialenzyme-linked immunosorbent assay kits from DRG Diagnostics(Marburg, Germany) and IBL International (Hamburg, Germany), re-spectively, following the manufacturers' instructions. To adjust forvariation in the dilution of urine, cortisol and 5-HIAA were expressedas urine cortisol/creatinine or urine 5-HIAA/creatinine ratios, respec-tively. The creatinine concentration was determined by means of theJaffe's test.

2.4. Mood state profile

Changes in mood were measured on a visual analogue scale (VAS).These scales have been found to be effective tools in measuringchanges over time in response to treatment for symptoms of mooddisturbance, and their reliability and validity have been well docu-mented (Baeken et al., 2008; McCormack et al., 1988; Mosimann et

s.

d Elderly

Women Men Women

1 65.2±4.54 77±9.62 64.33±9.136 1.65±0.03 1.75±0.01 1.61±0.058 23.84±2.54 24.97±2.84 24.76±2.88

575M. Garrido et al. / Experimental Gerontology 47 (2012) 573–580

al., 2000). The mood visual analogue scale was applied for subjectiveassessment in the form of a 100-mm horizontal line oriented with an-chors placed at both poles, indicating antithetic conditions. Four suchlines were employed (4×100-mm VAS) to measure the volunteers'general mood status in terms of ‘family relationships’, ‘social rela-tions’, ‘frame of mind’, and ‘fitness’. Participants were asked to ratehow they were feeling on these 100-mm scales in which the descrip-tors ranged from ‘very bad’ (0 mm) to ‘very good’ (100 mm).

The second measurement instrument used was the state–traitanxiety inventory (STAI) (Scholey et al., 2009). This comprises twosubscales each containing 20 statements (e.g. ‘I am calm’). The stateanxiety subscale measures the anxiety at the moment of scoring,while the trait anxiety subscale measures dispositional anxiety oranxiety in general. Participants rated how much they agreed witheach statement by marking a 4-point scale ranging from ‘not at all’to ‘very much so’ on the state subscale, and from ‘hardly ever’ to ‘al-most always’ on the trait subscale. Scores ranged from 20 to 80,with higher scores indicating more anxiety.

Participants completed both tests before the beginning of the trial(basal score), after the 5-day intake of the Jerte Valley cherry productor the placebo (trial scores), and one day following its termination(post-trial scores). Up to 10 min was allowed for completion ofthese paper and pencil surveys.

2.5. Statistical analysis

The Kolmogorov–Smirnov test was applied to test for normality ofthe distribution of the results. When normality could be assumed(STAI and VAS scores), parametric methods were employed, i.e. anal-ysis of variance (ANOVA) for repeated measures, to examine changesin each dependent variable from baseline. If significant main effectswere detected, Tukey's post-hoc tests were performed to determinewhich points differed significantly from baseline. When data did notfulfil a normal distribution (cortisol and 5-HIAA urine levels),Friedman's non-parametric test was performed to examine changesin each dependent variable from baseline. If significant main effectswere detected, Dunn's post-hoc tests were carried out to determinewhich points differed significantly from baseline. Regarding urinarycortisol levels, since samples were assessed in the morning and inthe evening, data were also analysed taking into account time ofday (morning, evening) using a two-way [intervention (placebo,cherry product)×time-of-day] general linear model ANOVA withboth variables as repeated measure factors.

Values are expressed as mean±SEM. The significance level wasset at pb0.05. F- or χ2-values (for the main analysis), and q- or t-values (for post-hoc tests) were also reported. Additionally, effectsizes were computed for relevant change-from-baseline VAS scoresusing the Cohen's d statistic (difference between means divided bypooled standard deviation). All analyses were performed using Gra-phPad Prism (version 5.0, 2007; GraphPad Software, Inc; San Diego,CA).

3. Results

Table 2A presents the scores obtained on the VAS test. There weresignificant main effects of intervention on the majority of mood items,i.e., it was observed that the intake of the cherry product generally im-provedmood in all three age groups. The young participants in particu-lar improved their self-rated family relationships after the intake of thecherry product [92.5±4.8 mm; F(9, 18)=6.93; q(9)=5.16; pb0.05].The self-rated frame of mind was enhanced in the middle-aged partici-pants in both the trial [89.4±5.0 mm; F(9, 18)=22.97; q(9)=8.56;pb0.05] and post-trial [88.2±5.2 mm; F(9, 18)=22.97; q(9)=8.01;pb0.05] conditions with respect to their basal scores. Also, this group'sscores in relation to fitness were improved in both the trial [83.2±9.5 mm; F(9, 18)=12.15; q(9)=5.96; pb0.05] and post-trial [83.7±

9.2 mm; F(9, 18)=12.15; q(9)=6.11; pb0.05] conditionswith respectto their basal scores. Finally, the elderly participants improved theirself-rated frame of mind [92.1±4.2 mm; F(9, 18)=4.75; q(9)=3.99;pb0.05] and their fitness [93.6±3.0 mm; F(9, 18)=6.14; q(9)=4.71;pb0.05] with respect to their corresponding basal values after the in-take of the product. The enhanced fitness persisted one day after theend of the trial [88.1±7.8 mm; F(9, 18)=6.14; q(9)=3.67; pb0.05].As shown in Table 2B, the 4× VAS scores after the intake of the placeboremained unchanged in all three age groups, thus providing supportingevidence that the changes reported in the participants' moodwere like-ly to have been due to the 5-day intake of the Jerte Valley cherryproduct.

The ingestion of the cherry product had significant main effects ofintervention on the anxiety of the middle-aged and elderly partici-pants, as assessed by the STAI scales (Fig. 1). However, there was nosignificant effect of intervention on anxiety measures of young sub-jects. After the intake of the product, the state anxiety scores(Fig. 1A) were significantly lower than their corresponding basalscores in both the middle-aged [32.7±3.0; F(9, 36)=5.60; q(9)=4.44; pb0.05] and the elderly [32.4±3.2; F(9, 36)=6.35; q(9)=5.13; pb0.05] participants. The decrease was maintained 1 day afterthe end of the trial in both the middle-aged [33.6±3.2; F(9, 36)=5.60; q(9)=4.52; pb0.05] and the elderly [32.1±4.8; F(9, 36)=6.35; q(9)=4.73; pb0.05] groups. Likewise, the ingestion of theproduct caused a significant decrease (pb0.05) in trait anxiety scores(Fig. 1B) in both the middle-aged [35.2±4.4; F(9, 36)=4.76; q(9)=4.85] and elderly [32.5±4.1; F(9, 36)=6.09; q(9)=4.61] groupscompared with their corresponding basal scores. The effect continuedin post-trial conditions for middle-aged [35.6±4.6; F(9, 36)=4.76;q(9)=4.52; pb0.05] and elderly [31.8±4.3; F(9, 36)=6.09; q(9)=4.20; pb0.05] participants. Anxiety scores obtained after the intakeof the placebo remained unchanged throughout the trial, thereby pro-viding supporting evidence that the beneficial effects on anxiety werelikely to have been due to the 5-day intake of the product.

Fig. 2 shows the urine cortisol levels obtained after the intake of aJerte Valley cherry-based product or the placebo in the young,middle-aged, and elderly participants. Since samples were assessedin the morning and in the evening, the data were analysed takinginto account time of day (morning, evening) using a two-way linealmodel ANOVA (intervention×time-of-day) with both variables as re-peated measure factors. This revealed a significant main effect of in-tensity on cortisol levels in young [F(1, 117)=42.30; pb0.05],middle-aged [F(1, 117)=15.80; pb0.05] and elderly [F(1, 117)=35.27; pb0.05] participants. To explore this effect further, within-subject Friedman tests were conducted for the morning and the eve-ning measures separately. Thus, the consumption of the product ledto decline (pb0.05) in the urine cortisol levels in both first-voidurine [morning; 0.66±0.15; χ2(8)=45.31; t(9)=4.53] and 20:00 hurine [evening; 0.64±0.12; χ2(8)=45.31; t(9)=6.98] in young par-ticipants with respect to their basal values (Fig. 2A). A similar effectwas observed 1 day after the termination of the cherry product in-take, with the decrease being statistically significant in urines collect-ed in the evening [0.70±0.14; pb0.05; χ2(8)=45.31; t(9)=5.10].Also, a significant decrease was found after the ingestion of the prod-uct in urine collected from middle-aged volunteers (Fig. 2B), in bothfirst-void [0.59±0.06; pb0.05; χ2(8)=43.59; t(9)=13.03] and20:00-h urines [0.53±0.11; pb0.05; χ2(8)=43.59; t(9)=10.15],with respect to their basal values. Thus, both morning [1.02±0.15;χ2(8)=43.59; t(9)=6.25] and evening [1.06±0.22; χ2(8)=43.59;t(9)=5.70] urine cortisol levels raised, reaching their respectivebasal values 1 day after the end of the trial (pb0.05 vs. itscorresponding trial value). In the elderly group (Fig. 2C), the inges-tion of the cherry product diminished the urine cortisol levels con-tained in both the morning [0.47±0.13; pb0.05; χ2(8)=44.80;t(9)=8.95] and the evening [0.66±0.05; pb0.05; χ2(8)=44.80;t(9)=11.87] urines. The post-trial urine cortisol values remained

Table 2BEffect of the placebo on self-rated scores obtained on a 4× VAS scale (family re onsh s, social relations, frame of mind, and fitness) in basal (before the intake of the pla o), trial (after the intake of the placebo for 5 consecutive days),and post-trial (one day after the end of the trial) conditions in young, middle d, a elderly participants. Results are expressed in millimetres (mm). Each value repre ts the mean±SEM of ten participants.

Young Middle-aged lderly

Basal Trial st-t l Basal Trial Post-trial asal Trial Post-trial

Family relationships (mm) 79.8±6.1 81.4±5.9 (0.28) a .0± .7 (0.39) 90.5±5.0 88.5±5.6 (0.40) 88.3±4.9 (0.47) 8.4±6.1 82.2±12.9 (0.45) 83.2±9.8 (0.41)Social relations (mm) 84.2±5.4 81.8±3.5 (0.41) .4± .1 (0.18) 83.3±1.7 81.6±6.1 (0.40) 82.0±3.6 (0.49) 7.0±6.8 78.8±12.6 (0.54) 79.2±10.9 (0.54)Frame of mind (mm) 84.6±5.3 82.2±10.4 (0.25) .6± .2 (0.37) 79.3±5.7 79.0±4.9 (0.06) 82.3±3.9 (0.50) 6.2±9.5 64.0±9.1 (0.25) 66.6±7.4 (0.05)Fitness (mm) 78.2±5.6 76.2±8.2 (0.30) .6± 1.0 (0.31) 78.3±1.9 76.6±3.3 (0.43) 76.6±6.0 (0.33) 7.5±13.0 69.2±12.9 (0.14) 70.7±10.6 (0.28)

a Effect sizes (Cohen's d) are shown in parentheses for change-from-basal s s.

Table 2AEffect of the Jerte Valley cherry product on self-rated scores obtained on a 4× V scal family relationships, social relations, frame of mind, and fitness) in basal (before th take of the cherry product), trial (after the intake of the cherryproduct for 5 consecutive days), and post-trial (one day after the end of the l) c ditions in young, middle-aged, and elderly participants. Results are expressed in llimetres (mm). Each value represents the mean±SEM of tenparticipants.

Young Middle-aged lderly

Basal Trial ost- al Basal Trial Post-trial asal Trial Post-trial

Family relationships (mm) 85.2±6.8 92.5±4.8⁎ (0.81)a 0.8± .1 (0.62) 86.5±8.9 89.8±6.1 (0.26) 91.2±5.9 (0.37) 5.9±8.4 84.5±7.6 (0.13) 89.8±5.6 (0.33)Social relations (mm) 83.3±5.4 84.8±7.1 (0.19) 7.2± .8 (0.51) 86.1±6.6 84.5±9.0 (0.12) 87.5±7.6 (0.12) 8.8±4.7 88.9±6.5 (0.01) 91.7±6.0 (0.33)Frame of mind (mm) 76.8±13.4 82.0±12.8 (0.24) 2.2± .5 (0.29) 72.0±11.2 89.4±5.0⁎ (1.21) 88.2±5.2⁎ (1.12) 8.2±10.2 92.1±4.2⁎ (1.08) 90.1±7.6 (0.72)Fitness (mm) 70.3±14.0 79.4±8.7 (0.47) 0.8± .8 (0.58) 70.8±10.4 83.2±9.5⁎ (0.80) 83.7±9.2⁎ (0.82) 9.2±12.4 93.6±3.0⁎ (0.96) 88.1±7.8⁎ (0.81)

⁎ pb0.05 with respect to their corresponding basal scores.a Effect sizes (Cohen's d) are shown in parentheses for change-from-basal s s.

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Fig. 1. Effect of the Jerte Valley cherry product and the placebo on state anxiety scores (A) and trait anxiety scores (B) in young, middle-aged, and elderly participants. Basal scoreswere taken before the intake of the cherry product or the placebo; trial scores were taken on the last day of a 5-day cherry product intake or placebo intake, and post-trial scoreswere obtained 1 day after the termination of the trial. Bars indicate mean scores±SEM from ten participants. ⁎pb0.05 with respect to their corresponding basal scores.

577M. Garrido et al. / Experimental Gerontology 47 (2012) 573–580

lower than the basal levels, although not significantly. Interestingly,the placebo intake did not modify cortisol levels from either themorning or the evening urines in any experimental group (Fig. 2A,B, and C).

Fig. 3 shows the 5-HIAA levels found in the 20:00 h urine collectedafter a 5-day intake of the product or the placebo and 1 day afterwardsin each experimental group. Once again, analyses of changes from base-line values indicated that the intervention was generally effective. Infact, the Jerte Valley cherry product intake produced a rise in 5-HIAAlevels in the young [1.39±0.10; pb0.05 χ2(5)=28.60; t(9)=9.60],middle-aged [2.35±0.42; pb0.05; χ2(5)=28.40; t(9)=6.20] and el-derly [1.62±0.06; pb0.05; χ2(5)=29.92; t(9)=24.75] participantswith respect to their corresponding basal values. Regarding the post-trial values, the 5-HIAA levels diminished in all groups of age, the effectbeing particularly significant in elderly participants whose 5-HIAAlevels substantially decreased [0.87±0.06; pb0.05; χ2(5)=29.92;t(9)=22.87] with respect to their trial values. The placebo intake didnot change the 5-HIAA levels in any group.

4. Discussion

In the present work, it was observed that the intake of a Jerte Val-ley cherry product had beneficial effects on the mood of healthy par-ticipants. Compared with the placebo, which did not have any effecton the parameters evaluated, the cherry product reduced anxiety(as measured in terms of both state and trait anxieties) and had apositive effect on subjective mood (as measured in terms of family re-lationships, social relations, frame of mind, and fitness). This was es-pecially notable in the middle-aged and elderly participants. Thesefindings were supported by the decrease in urine cortisol and the in-crease in 5-HIAA concentrations found after the cherry productconsumption.

Mental health is crucial to the overall well-being of individuals, soci-eties, and countries, but the human brain is exposed to frequent chal-lenges as a consequence of stress. Although the stress response of thebody ismeant tomaintain stability or homeostasis, long-term activation

of the stress system can have a harmful or even lethal effect on the body,increasing the risk of depression, anxiety, and a variety of other disrup-tions. A major focus of investigation has been the role of the HPA axis,both as amarker of the stress response and as amediator of addition-al downstream pathophysiological changes in the brain (Klok et al.,2011; Mello et al., 2003). Several studies in humans and experimen-tal animals provide evidence that hyperactivity of the HPA axis con-tributes to the neuronal and peripheral deterioration associated withageing (Aguilera, 2011). In advancing age, the inability to limit thestress response, especially during chronic stress, is likely to enhancethe effects of ageing due to the damaging effects of prolonged expo-sure to stress hormones (Aguilera, 2011).

Mental ageing itself may be due to loss of neuronal and synapticfunction and a decrease in concentrations of various neurotransmit-ters (Slotkin et al., 2005). Indeed, it has been reported that the ageingprocess results in a progressive decline in brain serotonin levels, andin changes in serotonin metabolism and in the expression of many se-rotonin receptors (Duncan and Hensler, 2002; Duncan et al., 2000).This could be responsible for alterations in many of the organism'sphysiological and behavioural functions, including mood and sleep(Ciarleglio et al., 2011).

In recent years, there has been great interest in determining therole of compounds of plant origin, i.e., phytonutrients, in improvinghealth. Despite the mechanisms of such protection not being fully un-derstood, and the plethora of potentially beneficial ingredients thatexist in plant foods, there is relatively consistent evidence that the an-tioxidant and anti-inflammatory properties of phytonutrients play arole in this protection (Heber, 2004; Mayorga et al., 2004). In partic-ular, both sweet and tart cherries are an excellent source of nutrientand bioactive food components for the human diet (Garrido et al.,2009; Garrido et al., 2010), and several studies have shown their ben-eficial effects on cancer, cardiovascular disease, diabetes, and inflam-mation (McCune et al., 2011). Some experiments have been carriedout to evaluate the effects of the consumption of tart cherry juice onhealth (Connolly et al., 2006; Kuehl et al., 2010; Traustadottir et al.,2009), since it had been reported that natural antioxidants found in

Fig. 2. Effect of the Jerte Valley cherry product and the placebo on cortisol levelsobtained in first-void (morning) and 20:00 h (evening) urines, in young (A), middle-aged (B) and elderly (C) participants. Basal values are urine samples obtained beforethe intake of the cherry product or the placebo, trial values are urine samples takenon the last day of a 5-day cherry product intake or placebo intake, and post-trial valuesare urine samples obtained 1 day after the termination of the trial. Data are expressedas fold increase over the basal levels. Each value represents the mean±SEM from tenparticipants. ⁎pb0.05 with respect to their corresponding basal values. #pb0.05 withrespect to their corresponding trial values.

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these fruits may be used as ingredients of functional foods (Blando etal., 2004). However, none of these studies attributed the aforemen-tioned health-promoting effects to the presence of tryptophan, sero-tonin, and/or melatonin in the cherries.

In the present work, the improvement of anxiety and mood statefound after the intake of the Jerte Valley cherry product was likely dueto the elevation in the participants' circulating levels of tryptophan, se-rotonin, andmelatonin. Indeed, the supplementation of tryptophan, se-rotonin, and/or melatonin in the diet has been reported to havebeneficial effects on mood, and to present antidepressant-like actions(Rios et al., 2010; Silber and Schmitt, 2010). These findings are also co-herent with previous work which demonstrated that improving thetryptophan supply in the diet or applying amelatonin treatment resultsin an increase of serotonin andmelatonin circulating levels in both diur-nal and nocturnal animals (Mateos et al., 2009; Paredes et al., 2009).

There has been a clinical interest in the measurement of cortisolbecause disturbances of cortisol levels are evident in many patholog-ical states. Cortisol has long been used in human psychobiologicalstudies as a biological marker of stress, anxiety, and depression(Levine et al., 2007). Its effects on mood appear to be dose-dependent, since a transient moderate elevation in cortisolaemiadue to acute stress has a protective effect on mood in critical situa-tions (Het and Wolf, 2007), although permanent hypercortisolismmay induce behavioural, psychic, and cognitive disturbances due tofunctional and, over time, structural alterations in specific targetareas of the brain (Fietta et al., 2009). Thus, several studies havereported increased baseline plasma cortisol levels in depressed patientscompared with healthy controls (Rajewska and Rybakowski, 2003),while others have found that more than half the cases of major depres-sion are associated with hypercortisolism (Pitts et al., 1995). The presentwork has shown decreased urine cortisol levels after the intake of theJerte Valley cherry product in all the participants (young, middle-aged,and elderly). This finding is particularly relevant for older populationssince high basal levels of cortisol and loss of circadian rhythmicity havebeen associated with greater cognitive decline as age advances, andmay have damaging effects that contribute to pathologies associatedwith advancing age, including depression, anxiety, and immune andmetabolic disturbances (Aguilera, 2011). Although circulating cortisolwas heremeasured indirectly by a non-invasivemethod, urinary cortisollevels have been extensively evaluated in both developmental (Diego etal., 2004) and affective research (Maes et al., 1998), and its clinical rele-vance has been clearly demonstrated (Lin et al., 1997). Likewise, it hasbeen reported that urine cortisol excretion resulting from glomerular fil-tration is a useful index of integrated 24-h plasma free cortisol (Levine etal., 2007).

The VAS and STAI tests are psychological scales used as reliabletools for investigating changes in mood (Schaller et al., 2011;Scholey et al., 2009). The VAS test is by itself a suitable and validmethod for measuring anxiety, and compares well with the state anx-iety scores of the STAI test (Kindler et al., 2000). In the present study,lower cortisol levels were accompanied by an improvement in thesubjective mood and anxiety scores as measured by both the VASand the STAI tests, thereby underlining the potential of this cherry-based drink for use as a natural product with mood-enhancing prop-erties, especially in middle-aged and elderly individuals. Further-more, the 5-HIAA levels, the main urine metabolite of serotonin,were increased after the ingestion of the cherry product in all the ex-perimental groups. Consequently, this indicates that brain and/or cir-culating serotonin levels were likely to have been elevated, sincetryptophan supplementation produces an increase of these levels(Mateos et al., 2009; Paredes et al., 2009). In this respect, the elevated5-HIAA levels may also be related to the mood improvement ob-served after the intake of the cherry product, since the neurotrans-mitter serotonin is involved in mood regulation (Murphy et al.,2008). For instance, studies of untreated depressed patients suggestthat serotonin function is reduced in depression, and studies of treat-ed patients indicate that the mechanism of action of antidepressantsis mediated by the enhancement of serotonin and/or noradrenalineneurotransmission (Toker et al., 2010). Also, brain serotonin synthe-sis is considered to be proportional to tryptophan transport into thebrain (Pardridge, 1998), and tryptophan depletion is found to reducethe rate of serotonin synthesis by 90% of baseline values (Young et al.,1999).

Since humans cannot synthesise tryptophan, it is considered as anessential amino acid that can only be obtained in the diet. In this re-spect, the ingestion of foodstuffs able to enhance not only tryptophan,but also serotonin and/or melatonin availability in the organism couldinduce an increase in the circulating levels of the amino acid, the neu-rotransmitter, and the indole, subsequently improving those func-tions in which deficiencies in these compounds have been reported,such as age-related sleep or behavioural disturbances.

Fig. 3. Effect of the Jerte Valley cherry product and the placebo on 5-HIAA levels obtained in 20:00 h urine, in young (A), middle-aged (B) and elderly (C) participants. Basal valuesare urine samples obtained before the intake of the cherry product or the placebo, trial values are urine samples taken on the last day of a 5-day cherry product intake or placebointake, and post-trial values are urine samples obtained 1 day after the termination of the trial. Data are expressed as fold increase over the basal levels. Each value represents themean±SEM from ten participants. ⁎pb0.05 with respect to their corresponding basal values. #pb0.05 with respect to their corresponding trial values.

579M. Garrido et al. / Experimental Gerontology 47 (2012) 573–580

Therefore, the consumption of a Jerte Valley cherry product (Span-ish patent no. ES 2342141 B1) may protect against stress and act as amood enhancer by improving serotonin availability in the organism.

Acknowledgements

We are indebted to the volunteers in this study for their outstandingcommitment and cooperation. This investigation was supported by theConsejería de Economía, Comercio e Innovación-Fondo Social Europeo(Junta de Extremadura, PDT0A008). M Garrido was a beneficiary of agrant from the University of Extremadura (Plan de iniciación a lainvestigación, desarrollo tecnológico e innovación 2010). J Espino wasa beneficiary of a grant from the Ministerio de Ciencia e Innovación(AP2009-0753). SD Paredes was a beneficiary of a grant from the Con-sejería de Economía, Comercio e Innovación‐Fondo Social Europeo(Junta de Extremadura, REI09009).

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