2.1. Ochrona lipidów przed uszkodzeniem oksydacyjnym (ID 652, 1372, 1506, 3143)

The claimed effects are “antioxidant properties” and “oxidative stress reduction”. The Panel assumes that the target population is the general population.
In the context of the proposed wordings and the references provided, the Panel assumes that the claimed effect relates to the protection of body lipids from oxidative damage.
Reactive oxygen species (ROS) including several kinds of radicals are generated in biochemical processes (e.g. respiratory chain) and as a consequence of exposure to exogenous factors (e.g. radiation, pollutants). These reactive intermediates damage DNA, proteins and lipids if they are not intercepted by the antioxidant network which includes free radical scavengers such as antioxidant nutrients.
The Panel considers that the protection of lipids from oxidative damage may be a beneficial physiological effect.

2.2. Utrzymanie prawidłowego ciśnienia tętniczego (ID 1507)

The claimed effect is “vascular health”. The Panel assumes that the target population is the general population.
In the context of the proposed wording, the Panel assumes that the claimed effect refers to the maintenance of normal blood pressure.
Blood pressure is the pressure (force per unit area) exerted by circulating blood on the walls of blood vessels. Elevated blood pressure, by convention above 140 mmHg (systolic) and/or 90 mmHg (diastolic), may compromise the normal function of the arteries.
The Panel considers that maintenance of normal blood pressure is a beneficial physiological effect.

3.1. Ochrona lipidów przed uszkodzeniem oksydacyjnym (ID 652, 1372, 1506, 3143)

The majority of the references provided in the consolidated list were narrative reviews of the health effects of polyphenols in general (rather than specifically of flavanols naturally occurring in cocoa), compositional analysis of food phenolics, and human studies investigating the effects of cocoa polyphenols on health outcomes other than the claimed effect (e.g. insulin sensitivity, blood pressure, endothelial function). The Panel considers that no conclusions can be drawn from these references for the scientific substantiation of the claim.
Among the references provided, one systematic review and 11 intervention studies in humans reported on the effects of the food constituent on different markers of oxidative stress or antioxidant status.
A systematic review by Ding et al. (2006) on the effects of cocoa and chocolate on cardiovascular risk factors included nine publications reporting on LDL oxidation, other markers of lipid peroxidation, and/or antioxidant capacity of plasma among the outcomes (Fraga et al., 2005; Kondo et al., 1996; Mathur et al., 2002; Mursu et al., 2004; Osakabe et al., 2001; Serafini et al., 2003; Wan et al., 2001; Wang et al., 2000; Wiswedel et al., 2004). No statistical analysis of the effects of cocoa flavanols on any of the outcomes was provided in the review. All but one (Kondo et al., 1996) of the studies were provided in the references cited for the substantiation of the claimed effect.
Two acute studies (Wang et al., 2000; Wiswedel et al., 2004) and two chronic studies (Mathur et al., 2002; Mursu et al., 2004) reported on F2-isoprostanes, whereas one chronic study reported on in vivo LDL oxidation (Baba et al., 2007b).
Wang et al. (2000) observed no changes in plasma 8-isoprostane concentrations two hours after the ingestion of 27, 53 or 80 g of chocolate (5.3 mg of procyanidin/g of chocolate) in 20 healthy volunteers. Conversely, Wiswedel et al. (2004) observed a significant decrease in plasma concentrations of total F2-isoprostanes 2 and 4 h after the intake of a high-flavanol cocoa drink (187 mg flavan-3-ols/100 mL) versus a low-flavanol cocoa drink (14 mg/100 mL) in ten healthy subjects only when the high-flavanol cocoa drink was consumed after physical exercise.
In a three week clinical trial with parallel design, 45 healthy subjects received 75 g of either dark (365.5 mg catechins/100 g), white (0.3 mg catechins/100 g) or high-polyphenol chocolate (556.8 mg catechins/100 g). No significant differences in changes among study groups were observed with respect to plasma concentrations of F2-isoprostanes (Mursu et al., 2004). Similarly, supplementation of 25 healthy subjects for six weeks (randomised crossover design) with a chocolate bar or a cocoa powder drink (651 mg of flavanols per day) did not affect urinary F2-isoprostanes significantly (Mathur et al., 2002).
In the study by Baba et al. (2007b), 160 subjects were randomised to consume either 13, 19.5 or 26 g/day (corresponding approximately to 140, 190, and 280 mg of flavanols) of cocoa powder as beverage or placebo for four weeks and plasma oxidised LDL concentrations were measured by the ELISA method. In this study the control beverage was adjusted to account for the theobromine content of the cocoa drink. The Panel notes that changes in LDL oxidation during the study between the intervention groups and placebo were not assessed, and therefore no conclusions can be drawn for the scientific substantiation of the claimed effect owing to the uncontrolled nature of the statistical analysis. Dose-response relationships were not reported.
The remaining human intervention studies presented reported on the effects of a single dose of flavanol-containing chocolate/cocoa on the antioxidant capacity of plasma (Serafini et al., 2003 Wang et al., 2000), TBARS (Wang et al., 2000), on the oxidation lag time of LDL ex vivo (Kondo et al., 1996; Hirano et al., 2000), on the effects of daily chocolate/cocoa consumption with different flavanol content (140-651 mg flavanols per day) on oxidation lag time of LDL ex vivo (Osakabe et al., 2001; Wan et al., 2001; Mathur et al., 2002; Mursu et al., 2004; Baba et al., 2007a) or on malondialdehyde (MDA) (Fraga et al., 2005). The Panel considers that these studies, because of the unreliability of the markers used, are not a source of data on their own for the substantiation of the claimed effect (Griffiths et al., 2002; Lykkesfeldt, 2007; Knasmüller et al., 2008).
In weighing the evidence, the Panel took into account that, although one acute study reported significant changes in plasma concentrations of total F2-isoprostanes after a single administration of cocoa flavanols, this effect was not confirmed when cocoa flavanols were consumed daily for 3-6 weeks, and that no effect of cocoa flavanols was observed on plasma concentrations of oxidised LDL particles.
The Panel concludes that a cause and effect relationship has not been established between the consumption of cocoa flavanols and protection of lipids from oxidative damage.

3.2. Utrzymanie prawidłowego ciśnienia tętniczego (ID 1507)

Some of the references provided reported on vascular outcomes other than blood pressure (e.g. flow- mediated dilation, endothelial function), had methodological weaknesses (e.g. ecological study design), were narrative reviews or reported on personal opinions that were not based on a systematic review of the literature. The Panel considers that no conclusions can be drawn from these references for the scientific substantiation of the claim.
The references cited in the list also included two meta-analysis (Taubert et al., 2007a; Hooper et al., 2008) and two systematic reviews (Ding et al., 2006; Erdman et al., 2008) addressing the effects of
cocoa flavanols on blood pressure. Furthermore, the list included nine relevant RCTs (Taubert et al., 2003, 2007b; Engler et al., 2004; Fraga et al., 2005, Grassi et al., 2005a, b, Allen et al., 2008; Davison et al., 2008; Faridi et al., 2008), of which three RCTs (Allen et al., 2008; Davison et al., 2008; Faridi et al., 2008) had not been included in the meta-analyses. Apart from RCTs, there was one observational study that was pertinent to the claim (Buijsse et al., 2006).
Taubert et al. (2007a) performed a meta-analysis of five RCTs on cocoa intake and blood pressure (total of 173 subjects) with a median duration of two weeks and which were published before October 2006. Pooling of the five RCTs resulted in a blood pressure change estimate of -4.7 mmHg systolic and -2.8 mmHg diastolic for cocoa intake, which was statistically significant. The five RCTs included in the meta-analysis are described in more detail below. In three RCTs (Taubert et al., 2003; Grassi et al., 2005a, b) the blood pressure effect of dark versus white chocolate (100 g/d) was examined and yielded an 88 mg/d difference between groups in flavanol intake (22 mg catechin + 66 mg epicatechin). The RCT by Taubert et al. (2003) in 13 untreated hypertensive men and women (age ~59 y) showed a significant change in blood pressure of -5.1/-1.9 mmHg for dark versus white chocolate. The RCT by Grassi et al. (2005a) in 20 men and women (age ~44 y) with high-normal blood pressure showed a larger effect of dark versus white chocolate on blood pressure (-10.5/-5.9 mmHg). Another RCT by the same group in 15 normotensive men and women (age ~34 y) showed a blood pressure-lowering effect of -5.4/-3.5 mmHg following consumption of dark chocolate compared to white chocolate (Grassi et al., 2005b). In the remaining two studies, high versus low flavanol chocolates were compared (Fraga et al., 2005; Engler et al., 2004). Fraga et al. (2005) compared the blood pressure effect of high flavanol milk chocolate (105 g/d, 168 mg flavanols) with low flavanol chocolate (flavanols <5 mg/d) in 28 young normotensive men. Flavanol intake in this study was related to a blood pressure change of -4.0/-4.0 mmHg. Finally, the meta-analysis included a RCT by Engler et al. (2004) in 21 normotensive men and women (age ~32 y) who were randomised to dark chocolate with either high or low flavanol content. Subjects who consumed flavonoid-rich chocolate had a 213 mg higher daily intake of procyanidins, including 46 mg epicatechins. After 2 weeks of intake a significant increase in plasma epicatechin concentrations was seen in these subjects (from ~30 to 210 mmol/L). In contrast to other RCTs, blood pressure slightly increased (+1.8/ +1.0 mmHg) in subjects with a high compared to low flavonoid intake.
Another meta-analysis by Hooper et al. (2008) included RCTs published before June 2007. This meta- analysis included four (Taubert et al., 2003; Grassi et al., 2005a, b; Fraga et al., 2005) out of the five RCTs described above. The study by Engler et al. (2004) was excluded for reporting large differences in saturated fat intakes between the intervention and control arms. Instead, a more recent intervention study by Taubert et al. (2007b) was included. In that study, 44 men and women (age ~64 y) with untreated (pre)hypertension were randomly assigned to a daily intake of 6.3 g of polyphenol-rich dark chocolate or 5.6 g polyphenol-free white chocolate (isocaloric) for 18 weeks. The dark chocolate group had a 7.1 mg/d higher intake of catechins (catechin, epicatechin, and epicatechin-gallate) and a 21.2 mg/d higher intake of procyanidins. Dark chocolate intake significantly reduced blood pressure by -2.9/-1.9 mmHg after 18 weeks, and the prevalence of hypertension declined from 86 % to 68 %.
Overall, the meta-analysis included 97 subjects in the intervention and 97 subjects in the control groups and observed a statistically significant reduction in systolic blood pressure (by 5.88 mmHg; 95 % CI: -9.55, -2.21; 5 studies; P for heterogeneity=0.0003, I2=81 %) and in diastolic blood pressure (by 3.30 mmHg; 95 % CI: -5.77, -0.83; four studies; P for heterogeneity=0.009, I2=70 %) in the cocoa flavanol group compared to controls. The clear heterogeneity in these analyses is partly explained by dose and duration, so that sub-grouping by dose or duration reduces apparent levels of heterogeneity (effects appear greater in studies with higher doses and shorter duration; data not shown). There are only five data points, but the funnel plot suggested that small studies showing large systolic blood pressure reductions may have been over-represented.
Three recent RCTs provided in the list were not included in the above-mentioned meta-analyses (Allen et al., 2008; Faridi et al., 2008; Davison et al., 2008).
Allen et al. (2008) assigned 44 untreated normotensive men and women (aged 24-70 y) with mildly elevated serum total cholesterol to 2x4 weeks use of cocoa flavanol-containing dark chocolate with or without plant sterols, using a randomised cross-over design. The daily dose of cocoa flavanols from chocolate bars was 360 mg/d. However, intervention periods were controlled for sterol ester intake and not for cocoa flavanol intake. Therefore, the blood pressure effect of flavanols could not be adequately assessed in this RCT and no conclusions can be drawn for the scientific substantiation of the claim.
Faridi et al. (2008) randomly assigned 45 normotensive men and women (age ~53 y) who were overweight/obese (BMI ~30 kg/m2) to a dark chocolate bar or a cocoa-free placebo bar per day. The cocoa bar contained 821 mg flavanols (of which 32 mg catechin + epicatechin) versus 0 mg in the placebo bar. The trial was repeated using sugar-containing versus sugar-free cocoa. Postprandial blood pressure was assessed two hours after intake. This study did not address the effects of chronic administration of cocoa flavonols on blood pressure and therefore no conclusions can be drawn for the scientific substantiation of the claimed effect.
In another RCT by Davison et al. (2008), 49 normotensive men and women (age ~49 y) were randomised to a high-flavanol cocoa drink (451 mg flavanols) or a low-flavanol cocoa drink (18 mg flavanols) daily for 12 weeks. Within these groups, subjects were additionally randomised to an exercise programme. Changes in blood pressure were found of approximately +1.5/+1.2 mmHg for the low flavanol group and -0.2/-1.5 mmHg for the high flavanol group when combining data from both exercise groups at the end of the study. Differences between groups were only statistically significant for diastolic blood pressure when measurements at 6 and 12 weeks were combined in a nested analysis. No significant differences between groups were observed for systolic blood pressure at any time point.
Three recently published RCTs on the effects of cocoa flavanols on blood pressure were also considered by the Panel (Muniyappa et al., 2008; Crews et al., 2008; Grassi et al., 2008).
Muniyappa et al. (2008) randomly assigned 29 untreated men and women (age ~51 y) with mild-to-moderate hypertension and obesity (BMI ~33 kg/m2) to daily consumption of cocoa drinks (900 mg/d flavanols) or placebo drinks (28 mg/d flavanols) in a double-blind manner. Drinks were matched for calories and content of other nutrients and appearance (calories, fat, macronutrients, mineral content, theobromine, caffeine, colour, taste and packaging) to test the effects of cocoa flavanols per se and to eliminate ascertainment or expectation bias. A 2x2 week cross-over design was used, interrupted by a one week wash-out period. A sample size of 20 is sufficient to detect a 5 mmHg change in systolic blood pressure with > 90 % power and a 2 mmHg change in diastolic blood pressure with >90 % power and a 2-sided α=0.05. No intention-to-treat analysis was applied, and only subjects who completed the study (n=20) were included in the data-analysis. No significant differences in blood pressure were observed between the cocoa and placebo periods. There was no evidence of a carryover effect of either cocoa or placebo.
Crews et al. (2008) performed a large RCT in 101 untreated men and women (age ~69 y) with normal to high-normal blood pressure who randomly received a 37 g dark chocolate bar and 237 mL of aspartame-sweetened cocoa drink daily, or low-polyphenol matched (for appearance, smell, taste, and caloric content) placebo products, for 6 weeks. The chocolate bar (11 g of natural cocoa) contained 397.3 mg of total proanthocyanins. The cocoa beverage (12 g dry weight, of which 11 g of natural cocoa) contained 357.4 mg proanthocyanins. Matching placebo products contained 0.2 mg and 40.9 mg proanthocyanins, respectively. Pre-treatment systolic blood pressure was on average 2 mmHg higher in the cocoa group (128.6 mmHg) than in the placebo group (126.7 mmHg). During the
intervention, blood pressure decreased both in the cocoa group (-3.6/-0.5 mmHg) and in the placebo group (-3.1/-0.6 mmHg), with no statistical differences between groups.
In another randomised, controlled cross-over study by Grassi et al. (2008), 19 subjects with hypertension and impaired glucose tolerance who were not on pharmacological treatment for hypertension consumed 100 g/d of dark (147 mg flavanols) versus white (flavanol-free) chocolate for 15 days each with one week wash-out period in between. Systolic and diastolic blood pressure significantly decreased during the dark chocolate consumption compared to the white chocolate after 15 days of intervention (-3.8 and -3.9 mmHg, respectively).
One observational, epidemiological study was cited in relation to the claim. In 470 elderly Dutch men the intake of cocoa-containing foods (among others) was repeatedly assessed between 1985 and 1995, and blood pressure was monitored (Buijsse et al., 2006). In 1985, one-third of the men did not consume cocoa, one-third had a median intake of 0.9 g/d and one-third had an intake of 4.2 g/d. After adjustment for potential confounders, the mean systolic blood pressure in the top tertile of cocoa intake was -3.7 mmHg lower (95 % confidence interval, -7.1 to -0.3 mmHg) and the mean diastolic blood pressure was -2.1 mmHg lower (-4.0 to -0.2 mmHg) compared with the bottom tertile. Data on the actual intake of cocoa flavanols, however, were not available and residual confounding by other dietary and lifestyle factors inherent to the observational study design cannot be excluded.
The Panel notes that 10 of the above-mentioned RCTs allowed conclusions to be drawn in relation to the substantiation of the claimed effect (Taubert et al., 2003, 2007b; Grassi et al., 2005a, b, 2008; Fraga et al., 2005, Engler et al., 2004; Davison et al., 2008; Muniyappa et al., 2008; Crews et al., 2008). Of these, five small RCTs by two different research groups (Taubert et al., 2003, 2007b, Grassi et al., 2005a, b, 2008) were performed in normotensive and untreated hypertensive subjects who consumed either dark or white chocolate during 2-18 weeks. Flavanol doses in these five RCTs were low (<150 mg/d). Significant blood pressure reductions, ranging from -3/-2 mmHg to -11/-6 mmHg, were found which were not clearly related to pre-treatment blood pressure levels. The Panel notes that these low-dose RCTs suffer from lack of blinding, which could have influenced the results. In three other small RCTs in normotensive subjects, higher flavanol doses (168-433 mg/d) were administered via flavanol-rich chocolate or beverages. These studies, which aimed to be double-blinded, showed either a reduction in systolic and diastolic blood pressure (-2/-3 mmHg and -4/-4 mmHg; Fraga et al., 2005), a decrease in diastolic blood pressure only (-2.5 mmHg; Davison et al., 2008), or an increase in blood pressure (+2/+1 mmHg; Engler et al., 2004) associated with cocoa flavanol consumption. Another small but well controlled and adequately powered double-blind RCT (Muniyappa et al., 2008) was performed in untreated hypertensive subjects with a relatively high flavanol dose (872 mg/d), and showed no effect on blood pressure (-1/+1 mmHg). Finally, a large RCT by Crews et al. (2008) with very high daily doses of cocoa flavanols (>750 mg proantocyanidins) showed no effect on blood pressure.
Thus, double-blind RCTs on the effects of cocoa flavanols on blood pressure are inconsistent, showing a tendency to reduce blood pressure at lower levels of flavanol intake (<500 mg/d) and showing no effect on blood pressure at higher intakes. The Panel notes that, at low doses of cocoa flavanol intake, small and un-blinded studies tend to show an effect on blood pressure (Taubert et al., 2003, 2007b, Grassi et al., 2005a, b, 2008), whereas studies aiming to be double-blinded show conflicting results (Fraga et al., 2005; Davison et al., 2008; Engler et al., 2004). The Panel also notes that adequately powered and well controlled intervention studies at higher doses (>750 mg proantocyanidins) do not show an effect of cocoa flavanols on blood pressure (Muniyappa et al., 2008; Crews et al., 2008), and that these studies were not considered in the meta-analyses by Taubert et al. (2007) and Hooper et al. (2008). The Panel considers that the evidence from RCTs for a blood pressure-lowering effect of cocoa flavanols is inconsistent and notes that adequately powered and well controlled studies with higher doses did not confirm the effect observed in small and un-blinded
studies with lower doses, and that the findings from blinded studies with lower doses were conflicting.
In weighing the evidence the Panel took into account that evidence from 10 RCTs for a blood pressure-lowering effect of cocoa flavanols was inconsistent, that evidence from small and un-blinded studies with lower doses in favour of an effect was in conflict with evidence from adequately powered and well controlled studies with higher doses, and that evidence from blinded studies with lower doses was conflicting.
The Panel concludes that the evidence provided is insufficient to establish a cause and effect relationship between the consumption of cocoa flavanols and maintenance of normal blood pressure.