
2Environmental and Biochemical Sciences, The James Hutton Institute, United Kingdom
The study was a randomized single-blinded controlled parallel study where participants received either 20g of LPDC or PRDC daily for a period of 4 weeks. A summary of the nutritional and chemical composition of the LPDC and the PRDC is presented in Table 1. The two types of chocolate looked identical, and contained 63.5% of cocoa solids. In order to avoid confounding factors, the two dark chocolate types were matched for macronutrient and micronutrient content, and contained similar amounts of caffeine and theobromine. They only differed in polyphenols content; with the PRDC providing 500 mg total polyphenols (400 mg flavanols) and the LPDC substantially less (< 60 mg flavanols). The products were provided by Barry-Callebaut Company, Belgium. PRDC and LPDC were produced via a method called “Acticoa” - patented trademark of Barry-Callebaut, which aims to naturally preserve polyphenols throughout the different steps of the manufacturing chain [22].
Components (per 20g daily portion) | LPDC | PRDC |
Energy (kcal) | 102 | 102 |
Total fat (g) | 7.34 | 7.34 |
Carbohydrates (g) | 7.44 | 7.44 |
Protein (g) | 1.34 | 1.34 |
Total flavanols (mg) | < 60 | 400 |
Epicatechin (mg) | 12 | 85 |
Catechin (mg) | 2 | 15 |
Caffeine (mg) | 15 | 15 |
Theobromine (mg) | 150 | 150 |
Data obtained from Barry-Callebaut company, Belgium. Total flavanols, epicatechin and catechin were analysed via LCMS method (Liquid chromatography–mass spectrometry). The amount of total polyphenols in the PRDC (500 mg) was assessed by Folin-Ciocalteu method, as provided by the company. The amount of 20g was the daily portion given to participants in the trial. The amounts of polyphenols, caffeine and theobromine provided by the company were validated by studies at the James Hutton Institute, Dundee, UK. PRDC: Polyphenol-rich dark chocolate; LPDC: Low polyphenol dark chocolate
Volunteers were recruited via University email, flyers and posters in community and sports centres, hospitals, colleges and universities in Edinburgh, as well as via word-of-mouth. Participants were studied with no restriction to race or socioeconomic status according to the following criteria 1) Adults with no history of hypertension, type 2 diabetes or CVD; 2) Participants with BMI between “18.5-24.9 kg/m2” and BMI between “25-34.9 kg/m2”; 3) Males and Females and 4) Age: 18-65 years. Exclusion criteria included history of CVD, hypertension, diabetes, intake of medications that affect insulin, glucose, lipids, and/or hs-CRP levels, intake of dietary supplements containing high doses of antioxidants (only low amounts of multivitamins were considered acceptable doses), and current smoking and heavy alcohol drinking (defined as more than 15 drinks/week for men and more than 8 drinks for women [25]). In addition, postmenopausal women receiving hormone replacement therapy, and participants with regular consumption of cocoa or DC (> 1 serving/week) [7] were not eligible for participation.
Blood samples and anthropometric measurements (weight, height, and waist circumference) were taken before the start and at the end of the intervention (after 4 weeks). Height was measured twice with person barefoot using a stadiometer to the nearest 0.5 cm. Weight was measured in the morning at fasting using an electronic scale (Tanita BF-559, Body Fat Monitor/Scale) to the nearest 0.1 Kg. Waist circumference was measured via a metal measuring tape calibrated against a steel tape, and was placed around the waist at the middle point between the lowest rib and the top of the hip bone. The tape was snug (without compressing the skin) and parallel to the floor [26]. Samples of blood were collected from the antecubital vein with minimal stasis by a butterfly needle (Vacuette®, Greiner bio-one). The blood sample drawn at each time was around or less than 20 ml. The samples were then centrifuged for 10 min at 4oC and 3000 rpm (revolutions per minute; Thermo Scientific Heraeus Primo R centrifuge). The supernatant plasma was then harvested, and stored frozen in polystyrene tubes at -80oC for subsequent analysis. Fasting serum lipids (Total cholesterol = TC, Triglyceride = TG, LDL and HDL), glucose and hs-CRP levels were measured. The analysis of glucose and lipids profile in plasma samples was undertaken at the Queen’s Medical Research Institute, University of Edinburgh using an automated platform (COBAS, UK). The technician was blinded to randomisation assignment. Hs-CRP levels were analysed at QMU lab via high sensitivity enzyme immunoassay (ELISA method) (Genway Biotech Inc, USA). Hs-CRP was considered as the test of choice when assessing inflammation in individuals with no history of CVD [27].
Subjects were requested to complete a general questionnaire covering information on social characteristics, lifestyle habits and medical history. Participants were asked to maintain their life style throughout the intervention period, while avoiding flavanol-rich foods and substituting the experimental DC by another food. A diet adjustment was also considered so that DC possibly replaced another food of similar energy. The researcher helped in providing dietary advice for participants to help them achieve this goal. This happened by asking participants about the types of snacks usually consumed. Energy composition of these snacks was assessed, and substitutions based on food energy content were suggested by the researcher. To monitor changes in dietary intake, participants were asked to fill a 3-day un-weighted diet diary twice: at the run-in phase and at week 3. Energy and macronutrient intakes were then analysed using NetWISP dietary software package (V 3.0). Changes in physical activity levels were assessed via a physical activity questionnaire at the run-in phase and during the second appointment (at week 4). Physical activity levels were then transformed into MET (Metabolic equivalent task)/hour. Furthermore, an acceptability questionnaire was designed to obtain data on whether it was palatable/acceptable to consume the assigned portion of dark chocolate daily (either LPDC or PRDC). Compliance to the study protocol was assessed by: 1) through the 3-day diet diary and physical activity questionnaire and 2) Directly asking participants whether they have consumed all the samples of chocolate daily as required. A high compliance was defined as the consumption of 85% or more (equivalent to missing no more than one sample a week) of the chocolate throughout the study.
Continuous normally distributed data were expressed as mean ± SD. Data were analysed using SPSS for Windows version 19.0 (SPSS, Chicago, IL). Heterogeneity was assessed using Levene’s test for equality of variances. Differences in baseline characteristics between groups were examined using a two-tailed independent t-test. For within group comparisons, changes from baseline were analysed using a two-tailed student’s paired t-test. For between-group differences, ANCOVA was carried out to adjust for potential baseline differences in the outcomes. Non parametric data were analysed using Mann Whitney tests. Significant changes were set at p ≤ 0.05.
Study Population (n=61) | LPDC (n = 30) |
PRDC (n = 31) |
Difference Between Groups P | |
Age (years) | 28.82 ± 8.89 | 28.13 ± 8.98 | 29.48 ± 8.89 | 0.48 |
Gender (M/F) | 12/ 49 | 3/27 | 9/22 | 0.062 |
BMI (Kg/m2) | 23.92 ± 4.17 | 24.08 ± 3.78 | 23.77 ± 4.57 | 0.4 |
WC (cm) | 77.7 ± 10.52 | 76.83 ± 8.89 | 78.54 ± 11.98 | 0.14 |
There was no significant change in blood glucose levels in the PRDC group after 4 weeks. However, glucose levels increased in the LPDC group (Figure 1). In addition, TC, HDL and LDL levels did not significantly change throughout the intervention in both groups (p > 0.05), but TG levels increased significantly in the LPDC group but not significantly in the PRDC group (Table 3). No significant changes in the inflammatory marker hs-CRP in both PRDC group (from 1.35 ± 0.97 mg/L at baseline to 1.26 ± 0.88 mg/L after 4 weeks; ∆ = - 0.09 ± 0.2 mg/l, p = 0.32), and LPDC group (from 1.5 ± 1.04 mg/L at baseline to 1.54 ± 1.12 after 4 weeks; ∆ = 0.04 ± 0.8 mg/L, p = 0.57) were noted.BMI status did not have a significant impact on the outcomes: there was no difference in the response to treatment when participants were stratified according to BMI (BMI < 25 Kg/m2 or BMI > 25 Kg/m2) in the LPDC (for TC, p = 0.35; LDL, p = 0.7; HDL, p = 0.25; TG, p = 0.59) and hs-CRP (p = 0.56), and the PRDC (for TC, p = 0.73; LDL, p = 0.44; HDL, p = 0.58; TG, p = 0.13) and hs-CRP (p = 0.35) groups.

LPDC: Low polyphenol-dark chocolate, PRDC: Polyphenol-rich dark chocolate.
* Significant difference from pre intervention (baseline), p = 0.041). Data were analysed using paired-t-test. Values expressed as means mean ± SEM.
There was no significant difference in baseline glucose levels between the LPDC and PRDC groups (p = 0.27), when assessed via independent t-test.
Lipid levels (mmol /L) | Pre-PRDC Mean ± SD | Post-PRDC Mean ± SD | ∆ (Post-PRDC - Pre-PRDC) | p | Pre- LPDC Mean ± SD | Post-LPDC Mean ± SD | ∆ (Post-LPDC - Pre- LPDC) | p |
TC | 4.17±1.07 | 4.23±0.98 | 0.059±0.76 | 0.68 | 4.26 ± 0.79 | 4.5 ± 1.15 | 0.24± 0.91 | 0.18 |
LDL | 2.23±0.97 | 2.22±1.06 | -0.013±0.66 | 0.92 | 2.23 ± 0.8 | 2.35 ± 1.08 | 0.12 ± 0.73 | 0.43 |
HDL | 1.56±0.36 | 1.64±0.34 | 0.08 ± 0.27 | 0.12 | 1.66 ± 0.35 | 1.73 ± 0.4 | 0.07 ± 0.24 | 0.12 |
TG | 0.83±.0.46 | 0.69±0.23 | -0.14 ± 0.43 | 0.07 | 0.83 ± 0.32 | 0.96 ± 0.37 | 0.13 ± 0.23* | 0.008 |
*Significant difference from pre intervention, p < 0.05. Data analysed using paired-test. Mean ± SD for all values TG: Triglycerides; WC: waist circumference; LPDC: Low polyphenol-dark chocolate; PRDC: Polyphenol-rich dark chocolate.
Analysis of differences from baseline showed that weight, BMI and WC did not significantly change in the PRDC group, whereas four weeks of daily DC low in polyphenols consumption increased weight in the LPDC group (Table 4). Subgroup analysis based on BMI (BMI< 25 Kg/m2 or BMI> 25 Kg/m2) did not show a difference in gained weight between normal weight and overweight participants in the LPDC group, when assessed via Mann Whitney test (p=0.36).In the LPDC group, the mean difference (post-pre) (IQR) was 0.12 Kg/m2 (0.41) for the normal weight population and 0.24 Kg/m2 (0.48) for the overweight population.
Pre-PRDC Mean ± SD | Post-PRDC Mean ± SD | ∆ (Post-PRDC - Pre-PRDC) | P | Pre-LPDC Mean ± SD | Post-LPDC Mean ± SD | ∆ (Post-LPDC - Pre-LPDC) | P | |
BMI (Kg/m2) | 23.77± 4.57 | 23.76±4.65 | -0.01±0.3 | 0.82 | 24.08±3.78 | 24.25±3.87 | 0.17 ±0.32 | 0.007* |
WC (cm) | 78.54±11.98 | 78.75±11.8 | 0.21± 1.7 | 0.5 | 76.83±8.89 | 76.96±8.98 | 0.13 ±0.84 | 0.42 |
* Significant difference from pre intervention, p < 0.05. Data analysed using paired-test. Mean ± SD for all values. LPDC: Low polyphenol-dark chocolate; PRDC: Polyphenol-rich dark chocolate.
Run-in period | Week 3 | Significance (p=) | ||
Energy (Kcal) | LPDC (N=30) | 1799 ± 522 | 1746 ± 416 | 0.54 |
PRDC (N=31) | 1868 ± 564 | 1998 ± 644 | 0.1 | |
Carbohydrate (g) | LPDC (N=30) | 226.9 ± 64 | 208.3 ± 58 | 0.28 |
PRDC (N=31) | 233 ± 61 | 234 ± 62 | 0.93 | |
Protein (g) | LPDC (N=30) | 64 ± 22.2 | 63.2 ± 19.5 | 0.99 |
PRDC (N=31) | 73 ± 41.7 | 84 ± 49.5 | 0.07 | |
Fat (g) | LPDC (N=30) | 73.2 ± 32 | 73.4 ± 25 | 0.96 |
PRDC (N=31) | 72.6 ± 29.3 | 82.7 ± 34.3 | 0.08 |
Differences between run-in period and week 3 were not significant for energy and macronutrient, p > 0.05.
Data expressed as mean ± SD. Results were analysed using paired tests on the NetWISP dietary software package (V3).
LPDC: Low polyphenol-dark chocolate; PRDC: Polyphenol-rich dark chocolate.
With regard to acceptability, it was found that LPDC was more acceptable to consume than PRDC (Figure 2). The reasons given for unacceptability were mainly the bitterness and intense flavour, the texture, and few subjects reported some side effects: nausea, bloating, winds, stomach pain, and to a less extent the dark colour of the chocolate which made it less appealing. These complaints, although noteworthy, did not result in any dropout.

LPDC: Low polyphenol dark chocolate, PRDC: Polyphenol-rich dark chocolate.
Some limitations are inherent to this study. The short study duration did not elucidate whether the effects noted are purely short term or might persist for a longer term. Women represented the largest proportion of both arms of the study, which may limit the generalization of the results. Furthermore, an inability to control the diet of participants resulted from the inclusion of free-living individuals who commonly exhibit a large variability in their dietary habits [39]. Therefore, we cannot exclude the possibility that they consumed other polyphenol-rich foods. Moreover, the compliance to intervention was not assessed by analysing total polyphenols in a 24-hour urine samples as a biochemical marker for compliance. Despite the limitations related to this test, it might have provided a better indication of adherence [40]. Also, body fat percentage was not assessed to indicate whether the increase in weight was caused by an increase in fat mass. In addition, differences in abiding to the dietary instructions provided by the researcher prior to the study can constitute an important limitation. The results of this study may not apply to regular DC consumers, who were among the exclusion criteria; and due to the small changes observed, it cannot be ruled out that the inter individual differences in the bioavailability of polyphenols might have affected the results. Importantly, including a third arm in this study (blank control with no experimental chocolate) would have been helpful in providing a more valid explanation for the results obtained. Lastly, the effects obtained in the LPDC group might be attributed to the restriction of polyphenols intake in the LPDC group (as instructed) that might caused a change in their usual diet. Therefore, these results constitute a preliminary finding and justify carrying out further studies to elucidate the effect of low polyphenol DC and other chocolate products on anthropometric and biochemical markers.
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