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Effects of long-term administration of a cocoa polyphenolic extract (Acticoa powder) on cognitive performances in aged rats

Published online by Cambridge University Press:  01 July 2008

Jean-François Bisson
Affiliation:
ETAP-Applied Ethology, 13 rue du Bois de la Champelle, Vandoeuvre-lès-Nancy54500, France
Amine Nejdi
Affiliation:
ETAP-Applied Ethology, 13 rue du Bois de la Champelle, Vandoeuvre-lès-Nancy54500, France
Pascale Rozan
Affiliation:
ETAP-Applied Ethology, 13 rue du Bois de la Champelle, Vandoeuvre-lès-Nancy54500, France
Sophie Hidalgo
Affiliation:
ETAP-Applied Ethology, 13 rue du Bois de la Champelle, Vandoeuvre-lès-Nancy54500, France
Robert Lalonde
Affiliation:
CHUM/St-Luc, Neuroscience Research Center, 1058 St-Denis Street, MontréalH2X 3J4, PQ, Canada
Michaël Messaoudi*
Affiliation:
ETAP-Applied Ethology, 13 rue du Bois de la Champelle, Vandoeuvre-lès-Nancy54500, France
*
*Corresponding author: Dr Michaël Messaoudi, fax +33 383 446 441, email etap@etap-lab.com
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Abstract

Numerous studies have indicated that increased vulnerability to oxidative stress may be the main factor involved in functional declines during normal and pathological ageing, and that antioxidant agents, such as polyphenols, may improve or prevent these deficits. We examined whether 1-year administration of a cocoa polyphenolic extract (Acticoa powder), orally delivered at the dose of 24 mg/kg per d between 15 and 27 months of age, affects the onset of age-related cognitive deficits, urinary free dopamine levels and lifespan in old Wistar-Unilever rats. Acticoa powder improved cognitive performances in light extinction and water maze paradigms, increased lifespan and preserved high urinary free dopamine levels. These results suggest that Acticoa powder may be beneficial in retarding age-related brain impairments, including cognitive deficits in normal ageing and perhaps neurodegenerative diseases. Further studies are required to elucidate the mechanisms of cocoa polyphenols in neuroprotection and to explore their effects in man.

Type
Full Papers
Copyright
Copyright © The Authors 2008

Because of their post-mitotic nature, neurons contend with cellular damage accumulated over many decades. The brain is more vulnerable to oxidative stress than other organs due to its low antioxidant protection system and increased exposure of target molecules to reactive oxygen species, one of the major damaging agents involved in age-associated decline(Reference Biesalski1). Increased reactive oxygen species levels produced by mitochondrial activity, inflammatory processes and excessive glutamate accelerate neurodegenerative processes(Reference Huber, Stuchbury, Burkle, Burnell and Munch2). Some parameters related to neurotransmission also decline during normal ageing(Reference Egashira, Takayama and Yamanaka3Reference Levine and Cepeda5).

Numerous epidemiological studies indicate that dietary flavonoids derived from fruits, vegetables, red wine and green tea decrease the risk of death from CHD(Reference Hertog, Feskens, Hollman, Martijn and Kromhout6, Reference Hertog, Kromhout and Aravanis7), cancer(Reference Steinmetz and Potter8) and stroke(Reference Keli, Hertog, Feskens and Kromhout9), and may prevent neurodegenerative diseases and diabetes mellitus(Reference Scalbert, Johnson and Saltmarsh10). Only recently have the beneficial effects of dietary polyphenols come to the attention of nutritionists(Reference Sies and Stahl11). Polyphenols are present in plants in the form of non-conjugated molecules, including ( − )-epicatechin and (+)-catechin, as well as their oligomers, also named procyanidins(Reference Adamson, Lazarus and Mitchel12Reference Natsume, Osakabe, Yamagishi, Takizawa, Nakamura, Miyatake, Hanato and Yoshida14). Cocoa-derived products contain high levels of flavonoids(Reference Adamson, Lazarus and Mitchel12, Reference Hammerstone, Lazarus, Mitchell, Rucker and Schmitz15, Reference Hammerstone, Lazarus and Schmitz16) and show potent antioxidant effects(Reference Arteel and Sies17). Biomarkers associated with CVD, such as oxidant defence molecules, LDL oxidation state and platelet function, have been assessed after acute consumption of chocolate and cocoa(Reference Holt, Lazarus, Sullards, Zhu, Schramm, Hammerstone, Fraga, Schmitz and Keen18Reference Rein, Paglieroni, Wun, Pearson, Schmitz, Gosselin and Keen20). The increase in blood epicatechin after acute procyanidin-rich chocolate consumption was associated with increased plasma antioxidant capacity and decreased plasma 2-thiobarbituric acid reactive substances(Reference Rein, Paglieroni, Wun, Pearson, Schmitz, Gosselin and Keen20). In addition, (+)-catechin and ( − )-epicatechin delayed lipid oxidation as well as depleted α-tocopherol and β-carotene levels induced by a free radical generator in human oxidized plasma(Reference Lotito and Fraga21). It was shown that consumption of green tea prevented LDL oxidation in man(Reference Miura, Chiba, Miura, Tomita, Umegaki, Ikeda and Tomita22) and that tea catechins attenuated the development of atherosclerosis in apoE-deficient mice(Reference Miura, Chiba, Tomita, Koizumi, Miura, Umegaki, Hara and Tomita23). The antioxidant properties of polyphenols and their beneficial effects on cognition have been demonstrated in animal studies(Reference Joseph, Shukitt-Hale, Denisova, Bielinski, Martin, McEwen and Bickford24). There is a possible role for ( − )-epicatechin in reducing neurodegenerative disorders such as Parkinson's and Alzheimer's diseases(Reference Pan, Jankovic and Le25, Reference Schroeter, Williams, Matin, Iverson and Rice-Evans26). Polyphenols may also possess other types of neuroprotective effects(Reference Kim, Kim, Kim, Kim and Chung27, Reference Mandel, Weinreb, Amit and Youdim28).

The main purpose of the present study was to investigate the effects of Acticoa powder, a cocoa polyphenolic extract, on cognitive function and lifespan in aged Wistar-Unilever rats, assessed with light extinction(Reference Benton, Ruffin, Lassel, Nabb, Messaoudi, Vinoy, Desor and Lang29Reference Messaoudi, Desor, Grasmuck, Joyeux, Langlois and Roman31) and water maze tests, the latter adapted from Morris(Reference Morris32) in which rats were let go from the same starting position. The rats were also evaluated for urinary free dopamine levels, susceptible to age-related decreases in Wistar rats(Reference Armando, Nowicki, Aguirre and Barontini33).

Materials and methods

Animals

Eighteen male Wistar-Unilever rats (Harlan, The Netherlands), weighing 250–275 g at reception, were housed three per cage in 48 × 27 × 20 cm polycarbonate cages (U.A.R., Epinay-Sur-Orge, France) in a regulated environment (temperature 22 ± 2°C; humidity 50 ± 10 %), provided standard food (food pellets M20; Dietex, Saint Gratien, France) and tap water ad libitum, and maintained on a 12 h light/dark cycle (lights on 20.00–08.00 hours).

After an 8-month acclimatization period in our facility, the rats were matched according to weight and randomly assigned to one of two groups (n 9): control-vehicle or Acticoa powder (cocoa polyphenolic extract) at the dose of 24 mg/kg per d (AP24), given by oral gavage. The rats were tested for cognitive functions at 9, 13 and 15 months, received AP24 or its vehicle (spring water), and were then retested at 17, 21 and 25 months for light extinction and water maze tests.

The rats used in the present study were treated according to rules provided by the ASAB Ethical Committee (1993) and the Canadian Council on Animal Care (1993). All standard operating procedures were in compliance with the European Communities Council Directive 86/609/EEC of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for scientific purposes (Official Journal L 358, 18 December 1986, pp. 0001–0028).

Tested product

The solvent-free Acticoa powder 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 general composition of AP24 was as follows (g/100 g): 3·5 g moisture, 29·4 g proteins, 13·2 g fat, 12·8 g carbohydrates, 3·8 g fibres, 2·4 g minerals and 34·9 g total polyphenols. The percentages related to the polyphenols in AP24 determined by HPLC were 88·5 % procyanidins including 0·21 % anthocyanins, 10 % epicatechin, 1 % epicatechin gallate and 0·5 % catechin. The product was freshly prepared every day; it was dissolved in spring water and administered at a dose of 24 mg/5 ml per kg body weight. AP24 and vehicle were orally administered for 12 months from the age of 15–27 months. After daily administration, the rats were allowed free access to standard food pellets and were weighed three times per week.

Only a single dosage of AP24 was investigated on the basis of results from a chronic experiment which showed its beneficial effects on prostate cancer prevention in rats(Reference Bisson, Guardia-Llorens, Hidalgo, Rozan and Messaoudi34).

Procedures

Weight change and food and water consumption

The body weight of each rat was recorded three times per week during the whole experimental period. Food and water consumption were recorded every week from Monday to Friday, for each cage, in order to estimate the quantity of food and water ingested by rats. Then the mean quantities of food and water were calculated in g/100 g body weight for rats of each cage in the two groups.

Cognitive testing

All cognitive tests were recorded by experimenters unaware of administered treatments.

Light extinction: light extinction was tested before treatment at 9, 13 and 15 months of age and after treatment at 17, 21 and 25 months of age. Learning performance was assessed by means of an operant conditioning test based on the natural aversion of rats to bright light(Reference Benton, Ruffin, Lassel, Nabb, Messaoudi, Vinoy, Desor and Lang29Reference Messaoudi, Desor, Grasmuck, Joyeux, Langlois and Roman31). The rat was placed in a strongly illuminated (1200 lux) cage (50 × 40 × 37 cm) equipped with two levers. By pressing the active lever the rat switched off the light allowing access to a 30 s period of darkness, whereas the inactive lever had no effect on the environment. The test lasted 20 min and the number of active and inactive lever presses were recorded.

Water maze: at 8, 12 and 15 months of age, the rats were trained to find a hidden platform (1 cm beneath water surface at 23°C) in a tank (150 cm diameter) filled with water from the same starting position for five trials per test session with a maximal duration of 180 s for each trial. The platform was placed away from the wall in a constant quadrant position during the trials. On each trial, the rats were allowed to stay on the platform for 30 s and then were immediately placed back in the water for the next trial. When unable to find the platform, the rats were placed on it for the same amount of time. At 17, 21 and 25 months of age, the rats were retested under treatment or placebo with the same procedure. In our simplified procedure, the rat is proficient in learning to locate more quickly the hidden platform by memorizing its position during a single five-trial session.

Urinary dopamine levels

Rats were individually placed in metabolic cages for 7 h from 09.00 to 16.00 hours for the collection of urine samples. Urinary dopamine levels were assayed at 12, 15, 18, 21, 24 and 27 months of age by HPLC with electrochemical detection. Aliquots of 1·0 ml acidified urine were placed in 5 ml conical-base glass vials with 50 mg alumina, and the samples were adjusted to pH 8·6 by adding Tris buffer. The adsorbed dopamine was then eluted from the alumina with 200 μl of 0·2 mol/l HClO4 on Costar Spin-X microfilters. In the final phase, 50 μl of the eluate were injected into an HPLC apparatus (Gilson Medical Electronics, Villiers-le-Bel, France) with a minimal limit of detection of 4 μg/l.

Lifespan

Throughout the experiment, the date of death of each rat was noted in order to determine the mean lifespan in each group.

Statistical analysis

Due to the relatively small number of rats per group, which decreased during the course of the study, and due to unequal variances, non-parametric tests(Reference Siegel and Castellan35) were used. The Mann–Whitney U test was used to compare cognitive performances of the two groups. For each group, the Friedman test was employed to compare the repeated cognitive performances throughout the test sessions; whenever significant, the Wilcoxon test was used to compare the performances of two consecutive water maze tests and to evaluate lever discrimination in the light extinction test by comparing the numbers of active and inactive lever presses. Survival data were analysed using the Kaplan–Meier method(Reference Kaplan and Meier36). The difference between survival in the two groups was tested using the log rank test. All statistical analyses were carried out with StatView® 5 software (SAS Inc., Cary, NC, USA). The results are expressed as means and their standard errors. Differences were considered to be significant at P < 0·05.

Results

Weight change and food and water consumption

No significant differences were observed between the body weight and food and water consumption of the two groups recorded throughout the study period (data not shown).

Cognitive testing

Light extinction

Total lever presses: as shown in Fig. 1, no significant group differences were apparent for total number of lever presses prior to Acticoa powder or vehicle administration at 9, 13 and 15 months of age (Mann–Whitney U test: U 29·50, NS; U 24·00, NS; U 31·50, NS, respectively). But at 17, 21 and 25 months of age, total lever pressing activity was significantly more elevated in AP24-treated rats (Mann–Whitney U test: U 13·0, P < 0·03; U 4·50, P < 0·004; U 5·00, P < 0·04, respectively).

Fig. 1 Total number of lever presses (active+inactive lever presses) in the light extinction test before (9, 13 and 15 months) and after the start of treatments (17, 21 and 25 months) in control rats (□) and in rats fed Acticoa powder (24 mg/kg per d; AP24; ). The numbers in parentheses are the number of rats remaining in each group. Values are means with their standard errors depicted by vertical bars. Mean values were significantly different from those of the control group (Mann–Whitney U test): *P < 0·05, **P < 0·01.

Lever discrimination: as shown in Fig. 2, control rats (treated with the vehicle) failed to show a significant discrimination between the two levers during baseline at 9, 13 and 15 months (Wilcoxon test: z 0·95, NS; z 1·27, NS; z 0·68, NS, respectively) and during the treatment period at 17, 21 and 25 months (Wilcoxon test: z 0·43, NS; z 0·65, NS; z 0·38, NS, respectively). Although AP24-treated rats did not discriminate between the two levers at baseline (Wilcoxon test: z 0·34, NS; z 0·57, NS; z 1·02, NS, respectively), lever discrimination was observed at 17 and 21 months (Wilcoxon test: z 2·32, P < 0·02; z 2·68, P < 0·008, respectively). But at 25 months, presses on the active lever only tended to be higher than those on the inactive one (Wilcoxon test: z 1·70, P < 0·09).

Fig. 2 Discrimination between active () and inactive (□) levers in the light extinction test before (9, 13 and 15 months) and after the start of treatments (17, 21 and 25 months) in control rats (A) and in rats fed Acticoa powder (24 mg/kg per d; AP24; B). Values are means with their standard errors depicted by vertical bars. Mean values were significantly different from those of the inactive levers (Wilcoxon test): TP < 0·10, *P < 0·05, **P < 0·01.

Water maze

Global performances: during baseline at 8, 12 and 15, and during treatment at 17 months of age, no intergroup differences were found for escape latencies (Mann–Whitney U test: U 34·50, NS; U 32·50, NS; U 35, NS; U 20·5, NS, respectively; Fig. 3). But at 21 and 25 months, escape latencies of AP24-treated rats were significantly lower than those of control rats (Mann–Whitney U test: U 6·00, P = 0·007; U 0·00, P = 0·006, respectively).

Fig. 3 Water maze performances before (8, 12 and 15 months) and after the start of treatments (17, 21 and 25 months) in control rats (□) and in rats fed Acticoa powder (24 mg/kg per d; AP24; ). Results are given as mean latencies over five trials before finding the hidden platform in each test session. Values are means with their standard errors depicted by vertical bars. Mean values were significantly different from those of the AP24-treated rats (Mann–Whitney U test): **P < 0·01.

Long-term memory: long-term memory was evaluated by comparing group performances over months using the Wilcoxon test (Fig. 3). Both groups improved their performances from month 8 to 12 (z 2·67, P < 0·008 for AP24-treated rats; z 2·52, P < 0·02 for controls) and from month 8 to 15 (z 2·67, P < 0·008 for AP24-treated rats; z 2·52, P < 0·02 for controls). The performance of control rats was still stable from month 15 to 17 (z 0·154, NS), but significantly deteriorated at 21 and 25 months (z 2·37, P = 0·018; z 2·02, P = 0·043, respectively). In contrast, the performance of AP24-treated rats was stable from 15 to 17 (z 0·48, NS), from 15 to 21 (z 0·42, NS) and from 15 to 25 (z 0·31, NS) months.

Short-term memory: short-term memory was assessed by comparing group performances over daily trials (Fig. 4). During the first water maze test at baseline, both groups improved their escape latencies from trial 1 to trial 5 (Friedman test: χ2(4 df ) 20·56, P = 0·0004 for AP24-treated rats; χ2(4 df ) 10·58, P = 0·03 for controls). During 12- and 15-month test sessions (baseline), escape latencies of both groups remained low throughout every trial to the extent that short-term memory assessment became meaningless (month 12: χ2(4 df ) 7·17, NS for AP24-treated rats; χ2(4 df ) 10·80, P = 0·03 for controls; month 15: χ2(4 df) 14·92, P = 0·005 for AP24-treated rats; χ2(4 df ) 7·52, NS for controls) (Figs. 4 (A), (B) and (C)). At the 17-month test session, AP24-treated and control rats improved their latency to find the platform from trial 1 to trial 2 (z 2·67, P = 0·008; z 2·37, P = 0·018, respectively). Between trial 2 and trial 5, latencies remained low and stable in the two groups (Friedman test: χ2(3 df ) 2·87, NS; χ2(3 df ) 0·79, NS, respectively). However, the performance of AP24-treated rats was better than that of controls for trials 2, 3 and 4 (Fig. 4 (D)). At the 21- and 25-month test sessions, AP24-treated rats improved their latencies on the first two trials (Wilcoxon test: z 2·08, P = 0·04; z 2·07, P = 0·03, respectively) and between trial 2 and trial 5, their latencies remained low and stable (Friedman test: χ2(3 df ) 4·01, NS; χ2(3 df ) 2·50, NS, respectively). In contrast, latencies of control rats showed no improvement between trial 1 and trial 5 (Friedman test: χ2(4 df ) 2·84, NS; χ2(4 df ) 2·26, NS, respectively). Over the five trials of these sessions, the performance of AP24-treated rats was significantly better than that of control rats (Figs. 4 (E) and (F)).

Fig. 4 Water maze performances at 8 (A), 12 (B), 15 months (C) (before treatment), and 17 (D), 21 (E) and 25 (F) (period of treatment) months of age in control rats (○) and in rats fed Acticoa powder (24 mg/kg per d; AP24; ●). Results are given as mean latencies from the five trials of the test session (T1–T5). Mean values were significantly different from those of the AP24-treated rats (Mann–Whitney U test): TP < 0·10, *P < 0·05, **P < 0·01.

Urinary free dopamine

As seen in Fig. 5, urinary free dopamine concentrations remained stable in the two groups from 12 to 15 months of age and no group difference was discerned (Mann–Whitney U test: U 10, NS; U 6, NS, respectively). Urinary free dopamine concentrations of AP24-treated rats were significantly lower than those of control rats at 18 months of age (U 2, P < 0·02), but significantly higher at 24 and 27 months of age (Mann–Whitney U test: U 0, P < 0·002; U 0, P < 0·008, respectively). While dopamine levels remained stable in AP24-treated rats from 18 to 27 months (Friedman test: χ2(3 df) 0·80, NS), values of control rats tended to decline (Friedman test: χ2(3 df) 7·46, P < 0·06).

Fig. 5 Urinary free dopamine concentrations before (12 and 15 months) and after the start of treatments (18, 21, 24 and 27 months) in control rats (□) and in rats fed Acticoa powder (24 mg/kg per d; AP24; ). The numbers in parentheses are the number of rats remaining in each group. Values are means with their standard errors depicted by vertical bars. Mean values were significantly different from those of the control group (Mann–Whitney U test): *P < 0·05, **P < 0·01.

Lifespan

The log-rank statistical test showed a significant increase of survival times in the AP24-treated group in comparison to control rats (χ2(1 df) 5·169, P = 0·025), corresponding to a longer lifespan of about 11 % over the 27-month period (Fig. 6).

Fig. 6 Survival rates of control rats () and rats fed Acticoa powder (24 mg/kg per d; AP24; ●). Survival times of rats were analysed using the Kaplan–Meier method and the log-rank test.

Discussion

Cognitive testing

The main purpose of the present work was to determine whether a daily dose of Acticoa powder ingested by aged rats over a 12-month period retards the onset of age-related cognitive deficits. In the light extinction test, AP24-treated rats had more total lever pressing activity than control rats. Moreover, unlike placebo-treated controls, AP24-treated rats exhibited significant discrimination between active and inactive levers at 17 and 21 months of age. The reason why control rats failed to discriminate is uncertain. We previously observed significant lever discrimination in 3-month-old male Wistar rats(Reference Benton, Ruffin, Lassel, Nabb, Messaoudi, Vinoy, Desor and Lang29Reference Messaoudi, Desor, Grasmuck, Joyeux, Langlois and Roman31). We suspect that age level is critical and it should be interesting to evaluate younger Wistar-Unilever rats in the same paradigm. It remains to be determined whether the elevated lever activity of AP24-treated rats is generalizable to exploratory activity in the open-field and hole-board, susceptible to decline as a result of advanced ageing in rats and mice(Reference Lalonde and Badescu37). It also remains to be determined why the AP24 group discriminates. One possiblity is faciliation of cognitive processes. A second possibility is that light is more aversive to them. There is a need to assess the effects of this substance on photophobia and other anxiety tests.

While water maze performance declined in control rats at 21 and 25 months of age, it remained stable in AP24-treated rats. At 21 and 25 months, but not at 17 months, escape latencies of AP24-treated rats were lower than those of control rats. Both long- and short-term memory processes were improved by the cocoa polyphenolic extract, as evaluated by month-to-month or trial-to-trial performances. The present results demonstrate beneficial effects of Acticoa powder on spatial learning.

Many studies indicate that age-associated neurobehavioural deficits are attenuated by dietary supplementation with potent antioxidant activity, such as Ginkgo biloba flavonoids, including memory(Reference Rai, Shovlin and Wesnes38), attention(Reference Kleijnen and Knipschild39) and calcium-induced increases in oxidative metabolism(Reference Oyama, Fuchs, Katayama and Noda40). It may be possible to reduce the deleterious effects of Alzheimer's disease with such supplements(Reference Kanowski, Herrmann, Stephan, Wierich and Horr41) or at least delay the physiological impairment associated with normal ageing(Reference Sastre, Lloret, Borras, Pereda, Garcia-Sala, Droy-Lefaix, Pallardo and Vita42). In particular, Moriguchi et al. (Reference Moriguchi, Saito and Nishiyama43) reported that a garlic extract prevented brain atrophy, as well as learning and memory impairments in the senescence accelerated mouse(Reference Nishiyama, Morigushy and Saito44).

The present results are in accordance with those of Joseph et al. (Reference Joseph, Shukitt-Hale, Denisova, Bielinski, Martin, McEwen and Bickford24) concerning the beneficial action of dietary supplementation with spinach, strawberry and blueberry extracts high in flavonoid levels as well as antioxidant activity(Reference Cao, Sofic and Prior45) on neuronal and cognitive functions in aged rats. Moreover, age-related declines in spatial memory tasks and hippocampal plasticity parameters were improved by antioxidant-rich diets containing blueberries(Reference Casadesus, Shukitt-Hale, Stellwagen, Zhu, Lee, Smith and Joseph46, Reference Lau, Shukitt and Joseph47). Likewise, tea polyphenols immediately after ischaemia improved memory impairment and reduced hippocampal damage in mice(Reference Matsuoka, Kitamura, Tsukahara, Terai, Tooyama, Kimura and Taniguchi48) and in gerbils(Reference Lee, Suh and Kim49). In the mouse hippocampal model system for oxidative stress, many flavonoids protected HT-22 cells from glutamate-induced toxicity as well as other oxidative insults(Reference Ishige, Shubert and Sagara50).

Urinary free dopamine

Dopamine is an essential neurotransmitter enabling smooth, controlled movements as well as efficient memory, attention and problem-solving function. Three months after the start of the treatment period, urinary free dopamine levels were paradoxically much higher in control than AP24-treated rats. At 21 months of age (6 months of treatment), no significant difference was observed between the dopamine levels of the two groups. At 24 and 27 months of age, urinary dopamine concentration declined in control rats, but remained relatively high and stable in AP24-treated rats. On the basis of the present results, the variability of urinary dopamine is probably not due to the salt content in the extract. Urinary dopamine levels exhibited by control rats are concordant with the age-related decrease found in Wistar rats(Reference Armando, Nowicki, Aguirre and Barontini33). Some studies directly associate the level of free dopamine in urine with the severity of the Parkinsonian syndrome(Reference Crowley, Hoehn, Rutledge, Stallings, Heaton, Sundell and Stilson51, Reference Hoehn, Crowley and Rutledge52). Hoehn et al. (Reference Hoehn, Crowley and Rutledge52) concluded that, although many peripheral sources contribute to urinary free dopamine, a small decrease in the level may actually reflect the severity of the disturbance of central dopamine metabolism and the known deficiency of dopamine in the neurons of the Parkinsonian brain. Green tea polyphenols inhibited the uptake of [3H]dopamine and 1-methyl-4-phenylpyridinium by dopamine transporters and partially protected embryonic rat mesencephalic dopaminergic neurons from 1-methyl-4-phenylpyridinium-induced injury(Reference Pan, Fei, Zhou, Jankovic and Le53). Similarly, potent neuroprotective properties of green tea polyphenols were demonstrated with respect to striatal dopamine depletion and substantia nigra dopaminergic neuronal loss caused by 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine in mice(Reference Levites, Weinreb, Maor, Youdim and Mandel54). Reactive oxygen species are involved in the decline of functions associated with ageing(Reference Biesalski1) and flavonoid administration attenuated apoptotic injury of mesencephalic dopamine neurons caused by oxidative stress(Reference Mercer, Kelly, Horne and Beart55). Joseph et al. (Reference Joseph, Shukitt-Hale, Denisova, Bielinski, Martin, McEwen and Bickford24) examined the brain tissues of blueberry-supplemented rats and found that dopamine levels were significantly higher than those of control rats.

Despite distinct chemical structures, cocoa antioxidant effects are similar to those of tea or blueberry, providing antioxidant defences against reactive oxygen species(Reference Adamson, Lazarus and Mitchel12, Reference Osakabe, Yamagishi, Sanbongi, Natsume, Takizawa and Osawa56). Cocoa polyphenol extracts may therefore protect vulnerable structures such as the nigro-striatal system against dopamine depletion in psychopathological and neurological disorders(Reference Cropley, Fujita, Innis and Nathan57Reference Shook, Franz, Higginson, Wheelock and Sigvardt61).

Lifespan

The lifespan of AP24-treated rats was prolonged relative to placebo by approximately 11 % over the 27-month test period. The mechanisms underlying prolongation of the lifespan of the Acticoa powder remain to be fully established. It was demonstrated that cocoa powder enhances the level of antioxidative activity in rat plasma(Reference Baba, Osakabe, Natsume, Yasuda, Takizawa, Nakamura and Terao62, Reference Rozan, Hidalgo, Nejdi, Bisson, Lalonde and Messaoudi63) and among human subjects, a flavonoid-rich chocolate increased plasma antioxidant capacity and reduced the amounts of plasma 2-thiobarbituric acid-reactive substances(Reference Wang, Schramm, Holt, Ensunsa, Fraga, Schmitz and Keen64). Lee et al. (Reference Lee, Kim, Lee and Lee65) showed that cocoa powder and cocoa extracts exhibit greater antioxidant capacity than many other flavanol-rich foods and food extracts, such as green and black tea, and red wine.

However, the beneficial effects of the cocoa extract are attributable not only to antioxidant properties of its polyphenol constituents (procyanidins and methylxanthines), but also via inhibition of nitric oxide-stimulated protein kinase C activity, since flavanols improved nitric oxide-dependent vasodilatation even in the presence of pre-existing endothelial dysfunction(Reference Heiss, Dejam, Kleinbongard, Schewe, Sies and Kelm66, Reference Sies, Schewe, Heiss and Kelm67). Cocoa polyphenols may improve vasodilatation, dependent on epicatechin(Reference Schroeter, Heiss, Balzer, Kleinbongard, Keen, Hollenberg, Sies, Kwik-Uribe, Schmitz and Kelm68). The cocoa polyphenolic extract may also act on the cardiovascular system through a variety of mechanisms including reduced LDL oxidation(Reference Kondo, Hirano, Matsumoto, Igarashi and Itakura19, Reference Baba, Osakabe, Natsume, Yasuda, Takizawa, Nakamura and Terao62, Reference Pearson, Schmitz, Lazarus and Keen69, Reference Wan, Vinson, Etherton, Proch, Lazarus and Kris-Etherton70), improved endothelium-dependent relaxation(Reference Karim, McCormick and Kappagoda71), modulation of cytokines and eicosanoids involved in the inflammatory response(Reference Mao, Powell, Van de Water, Keen, Schmitz, Hammerstone and Gershwin72Reference Schramm, Wang, Holt, Ensunsa, Gonsalves, Lazarus, Schmitz, German and Keen74), and inhibition of platelet activation(Reference Rein, Paglieroni, Wun, Pearson, Schmitz, Gosselin and Keen20, Reference Murphy, Chronopoulos, Singh, Francis, Moriarty, Pike, Turner, Mann and Sinclair75, Reference Vita76).

Conclusion

Since AP24 administration did not show any influence on weight and on food and water consumption of treated rats throughout the experimental period, the benefits observed with the AP24 cocoa extract are not due to dietary restriction. The present results suggest that Acticoa powder may be beneficial in retarding age-related brain impairments, including cognitive deficits in normal ageing and perhaps neurodegenerative diseases. On the basis of the present results, it is of interest to carry out further in-depth preclinical and clinical studies on the neurobehavioural actions of Acticoa powder in order to determine whether age-related dysfunctions may be prevented and to elucidate the mechanisms of cocoa polyphenols in neuroprotection.

Acknowledgements

We are grateful to the Barry Callebaut Group (France) for supplying Acticoa powder samples. J.-F. B., M. M. and S. H. were responsible for performing the study. M. M., A. N. and P. R. were responsible for data management and statistical expertise. J.-F. B., M. M., A. N. and P. R. were also responsible for data interpretation and manuscript writing. R. L. contributed to data interpretation and manuscript writing. There were no conflicts of interest.

References

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Figure 0

Fig. 1 Total number of lever presses (active+inactive lever presses) in the light extinction test before (9, 13 and 15 months) and after the start of treatments (17, 21 and 25 months) in control rats (□) and in rats fed Acticoa powder (24 mg/kg per d; AP24; ). The numbers in parentheses are the number of rats remaining in each group. Values are means with their standard errors depicted by vertical bars. Mean values were significantly different from those of the control group (Mann–Whitney U test): *P < 0·05, **P < 0·01.

Figure 1

Fig. 2 Discrimination between active () and inactive (□) levers in the light extinction test before (9, 13 and 15 months) and after the start of treatments (17, 21 and 25 months) in control rats (A) and in rats fed Acticoa powder (24 mg/kg per d; AP24; B). Values are means with their standard errors depicted by vertical bars. Mean values were significantly different from those of the inactive levers (Wilcoxon test): TP < 0·10, *P < 0·05, **P < 0·01.

Figure 2

Fig. 3 Water maze performances before (8, 12 and 15 months) and after the start of treatments (17, 21 and 25 months) in control rats (□) and in rats fed Acticoa powder (24 mg/kg per d; AP24; ). Results are given as mean latencies over five trials before finding the hidden platform in each test session. Values are means with their standard errors depicted by vertical bars. Mean values were significantly different from those of the AP24-treated rats (Mann–Whitney U test): **P < 0·01.

Figure 3

Fig. 4 Water maze performances at 8 (A), 12 (B), 15 months (C) (before treatment), and 17 (D), 21 (E) and 25 (F) (period of treatment) months of age in control rats (○) and in rats fed Acticoa powder (24 mg/kg per d; AP24; ●). Results are given as mean latencies from the five trials of the test session (T1–T5). Mean values were significantly different from those of the AP24-treated rats (Mann–Whitney U test): TP < 0·10, *P < 0·05, **P < 0·01.

Figure 4

Fig. 5 Urinary free dopamine concentrations before (12 and 15 months) and after the start of treatments (18, 21, 24 and 27 months) in control rats (□) and in rats fed Acticoa powder (24 mg/kg per d; AP24; ). The numbers in parentheses are the number of rats remaining in each group. Values are means with their standard errors depicted by vertical bars. Mean values were significantly different from those of the control group (Mann–Whitney U test): *P < 0·05, **P < 0·01.

Figure 5

Fig. 6 Survival rates of control rats () and rats fed Acticoa powder (24 mg/kg per d; AP24; ●). Survival times of rats were analysed using the Kaplan–Meier method and the log-rank test.

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