Marisol
Villalva
ab,
Esther
García-Díez
b,
María del Carmen
López de las Hazas
c,
Oreste
Lo Iacono
d,
José Ignacio
Vicente-Díez
e,
Sara
García-Cabrera
e,
Marta
Alonso-Bernáldez
c,
Alberto
Dávalos
cf,
María Ángeles
Martín
bg,
Sonia
Ramos
bg and
Jara
Pérez-Jiménez
*bg
aInstitute of Food Science Research (CIAL), Universidad Autónoma de Madrid, CEI UAM + CSIC, Madrid, Spain
bDepartment of Metabolism and Nutrition, Institute of Food Science, Technology and Nutrition, Spanish Research Council (ICTAN-CSIC), Calle Jose Antonio Novais, 6, 28040 Madrid, Spain. E-mail: jara.perez@ictan.csic.es
cLaboratory of Epigenetics of Lipid Metabolism, Madrid Institute for Advanced Studies (IMDEA)-Food, CEI UAM + CSIC, Madrid, Spain
dServicio de Aparato Digestivo, Hospital General Universitario/Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain
eMonóvar Health Center, Primary Care Management, Madrid Region Health Service, Madrid, Spain
fConsorcio CIBER de la Fisiopatología de la Obesidad y Nutrición (CIBERObn), Instituto de Salud Carlos III (ISCIII), Madrid, Spain
gCIBER Diabetes and Associated Metabolic Diseases: Diabetes and Associated Metabolic Diseases Networking Biomedical Research Centre | CIBERDEM, Carlos III Health Institute (ISCIII), Madrid, Spain
First published on 20th March 2025
Postprandial metabolic disturbances are exacerbated in type 2 diabetes (T2D). Cocoa and carob, despite showing promising effects on these alterations in preclinical studies, have not yet been jointly tested in a clinical trial. Therefore, this acute, randomised, controlled, crossover nutritional trial evaluated the postprandial effects of a cocoa–carob blend (CCB) in participants with T2D (n = 20) and overweight/obesity. The subjects followed three treatments: hypercaloric breakfast (high-sugar and high-saturated fat, 900 kcal) as the control (treatment C); the same breakfast together with 10 g of the CCB, with 5.6 g of dietary fibre and 1.6 g of total polyphenols (treatment A); and the same breakfast after consuming the CCB (10 g) the night before (treatment B). Various analyses were performed, including the determination of the clinical markers of T2D (fasting and postprandial glucose and insulin, GLP-1, and glycaemic profile), satiety evaluation, analysis of exosomal miRNA expression and ex vivo determination of inflammation modulation. No effect on glucose homeostasis (glucose, insulin, and GLP-1) was found in the study population. However, eight exosomal miRNAs were found to be significantly modified owing to CCB supplementation compared with treatment C, with three of them (miR-20A-5p, miR-23A-3p, and miR-17-5p) associated with an improvement in insulin sensitivity. Furthermore, the CCB caused a decrease in hunger feelings (0–120 min), as assessed by the visual analogue scale (VAS). Finally, treatment A caused a significant decrease in the glucose increment within 0–30 min of treatment in subjects with overweight. No significant modifications were found in the other assessed parameters. The acute intake of the CCB by subjects with T2D showed modest although significant results, which need to be validated in a long-term randomised controlled trial.
Among all the metabolic alterations caused by T2D, those present in the postprandial state are particularly relevant, being considered an independent predictor of future cardiovascular events, especially when meals are high in calories, carbohydrates and/or saturated fats, even in nondiabetic subjects.7 This is owing to the increased oxidative stress and inflammation associated with this state.8,9 Moreover, it has been observed that the altered release of incretin hormones in insulin resistance situations contributes to the decreased postprandial satiety observed in subjects with obesity.10
Owing to the complications associated with T2D and the consequent increasing use of drugs during disease development, dietary approaches may help delay those alterations, as well as the incorporation of additional pharmacological treatments. Thus, polyphenols are being investigated. These are secondary plant metabolites, which, besides other biological activities, have shown a potential benefit in the prevention or management of T2D.11 These antidiabetic activities may be mediated by several mechanisms of action, such as modulation of glucose homeostasis, thereby promoting insulin signalling pathways, or their ability to decrease oxidative stress and inflammatory processes.12 Moreover, polyphenol-rich diets have shown to be able to modulate the expression of exosomal miRNAs, potentially leading to beneficial outcomes.13 However, in order to obtain the most optimal benefits from these biological activities, their metabolic transformations after ingestion and, particularly, their interaction with the gut microbiota are crucial to ultimately generate active metabolites.14 Interestingly, an important fraction of polyphenols is linked to dietary fibre, a constituent with wide evidence for its ability to reduce the risk of T2D.15,16 Indeed, dietary fibre has a relevant property in the context of diabesity, namely, its ability to modulate satiety through complementary mechanisms, such as delaying gastric emptying and regulating the levels of satiety-related hormones.17
Cocoa and carob are two vegetal materials with high polyphenol and dietary fibre contents, along with the presence of certain bioactive compounds, i.e., methylxanthines and D-pinitol, respectively.18,19 Based on their composition and previous evidence demonstrating their T2D-modulating potential,20 we developed a new functional food based on a cocoa–carob blend (CCB) that was rich in polyphenols and dietary fibre, as well as possessed proper sensory acceptance.21 Chronic supplementation with a CCB-rich diet has shown therapeutic potential against diabetic cardiomyopathy and for improving intestinal health in a T2D animal model.22,23 However, this product has not been tested in humans. Therefore, the aim of this study was to explore the potential effects of the CCB during the postprandial state (by comparing the effects of intact compounds present in the product and those from microbial-derived metabolites) in subjects with T2D through an acute randomised crossover controlled nutritional trial. A comprehensive assessment of the potential biological modifications was carried out, including the clinical markers of T2D, satiety evaluation, analysis of exosomal miRNA expression and ex vivo determination of inflammation modulation.
Subjects were enrolled by their primary care doctors (S. G.-C. and J. I. V.-D.), in Madrid city. The inclusion criteria for the trial were: T2D diagnosed patients under treatment with metformin as the only antidiabetic drug, age 40–70 years old, and body mass index > 25 kg m−2. The exclusion criteria were: body mass index > 40 kg m−2; diagnosed or receiving medication for cardiometabolic or thyroid pathologies, Hb1Ac ≥ 7%, systolic pressure ≥ 150 mmHg or diastolic pressure ≥ 100 mmHg, fasting triglycerides ≥ 350 mg dL−1, fasting total cholesterol ≥ 280 mg dL−1, pregnant or lactating, habitual intake of dietary supplements with antioxidants or dietary fibre, adherence to a vegetarian diet, allergy or intolerance to cocoa or carob, previous bariatric surgery procedure, or current participation in any other dietary intervention trial.
During the screening process, and in order to fulfill the inclusion and exclusion criteria, the subjects provided a blood analysis obtained within the last year (or specifically prescribed by their medical doctor if unavailable) with the requested parameters (glucose, Hb1Ac, and lipid profile). Since the recruitment and follow-up of the study took place between March 2021 and March 2022, with strong Covid-19 workload on the primary care services (where the medical doctors where affiliated), it was not possible to repeat the blood analysis right at baseline. Blood pressure was measured using an automated digital oscillometric device (Omron M6 Comfort from Omron Corporation, Tokyo, Japan), and the mean of two readings was taken. Anthropometric variables, such as height, body weight, and abdominal and hip circumferences were measured. Additionally, for ensuring an overview of the basal state of the volunteers, creatinine, uric acid, bilirubin, hepatic enzymes, alkaline phosphatase and basic haematological parameters were analysed, allowing subsequent determination of the Modification of Diet in Renal Disease-4 (MDR-4) values. Next, hepatic steatosis presence and progression were assessed through abdominal echography (Acuson Juniper Ultrasound, Siemens, Munich, Germany, transductor convex transductor at a frequency of 3.5–5 mHz) and FibroScan transient elastography (Echosens 530, Siemens, with M and XL probes), which can determine the degree of ultrasound attenuation due to hepatic fat.
A detailed description of the hypercaloric breakfast, which was rich in sugars and saturated fats with a total caloric value of 900 kcal, is provided in the ESI (Table S1†). Basically, it constituted full-cream milk, pineapple juice, honey and croissants. No appropriate placebo for replacing the CCB was found; nevertheless, the caloric value of the provided CCB dose was 21 kcal, which could be considered negligible. In treatments A and B, the CCB was dissolved in 250 mL of full-cream milk, while in treatment C the participants received the same volume of full-cream milk. Volunteers were instructed to follow their regular diet and daily activities between the three visits. However, and in order to limit the potential effect of high dietary polyphenol consumption close to the intervention, the subjects were required to refrain from consuming polyphenol-rich foods such as wine, coffee, tea, cocoa, whole bread, virgin oil olive, nuts, legumes and certain fruits and vegetables such as berries or artichoke 72 h prior to each visit. A detailed list of polyphenol-rich foods was provided to the volunteers, and, at their visits, they were asked questions in order to verify that they had followed those instructions.
Three subjects requested the replacement of lactose-free milk during breakfast. Four subjects were unable to complete the breakfast provided on the first day of intervention; therefore, on the remaining two visits the amount was adjusted accordingly to ensure full consumption of the breakfast.
The secondary outcomes of this study were the satiety parameters (based on validated tests) and the following blood determinations: glucose, GLP-1, triglycerides, exosomal miRNAs and several cytokines (tumour necrosis factor-α [TNF-α], interleukin-6 [IL-6] and [IL-10]) and lipopolysaccharide (LPS)-induced ex vivo levels.
Finally, in an exploratory approach, the whole group was subdivided into two subgroups based on their body mass index (BMI), or parameters related to glucose and triglyceride homeostasis, and satiety perception, (BMI ≤ 30 kg m−2 indicating overweight and BMI > 30 kg m−2 corresponding to obesity).
Fasting and postprandial blood glucose were determined by applying the enzyme electrode method, using the FreeStyle Optium Neo Meter from Abbott (Chicago, IL, USA). Plasma levels of insulin and total GLP-1 were measured using commercial ELISA kits (Merck-Millipore; Burlington, MS, USA). The areas under the curves for glucose and insulin were estimated using the trapezoidal function. Plasma triglycerides were measured using a commercial kit (Cromakit SL; Maracena, Granada, Spain).
![]() | (1) |
Results of pathway enrichment analysis were presented in circus plots using the on-line application, available at https://circos.ca/.
For parameters related to glucose and triglyceride homeostasis, as well as satiety perception, sub-groups based on BMI values (Group Ovw, BMI ≤ 30 kg m−2 indicating overweight and Group Obe, BMI > 30 kg m−2 corresponding to obesity) were established, since about half of the participants were distributed among both categories (n = 8 and n = 12, respectively) and the BMI is a relevant marker in the context of T2D. Nevertheless, it should be noted that this was just an exploratory analysis since there was not enough statistical power (the sample size calculation for the primary outcome was performed for the whole group). In particular, for assessing postprandial insulin (the primary outcome of the study), ANOVA was applied at each time point, since all specific parameters (values at different time points, maximum value, time to reach the maximum value, absolute increment and AUC) followed a normal distribution, or could be adjusted to it (by applying a decimal logarithm for the AUC 0–60 min and square root for 30–60 min increments).
For the analysis of miRNA expression, modifications between the baseline and 2 h plasma levels for each intervention were assessed by a non-parametrical paired t-test, while intra-group comparisons were performed by Wilcoxon test.
In all the cases, a two-tailed p < 0.05 was considered significant. All data are expressed as the mean ± standard error of the mean (SEM). All experimental determinations in the biological samples were performed at least in duplicate. All these data were analysed using the SPSS IBM Statistics 29 package for Windows.
![]() | ||
Fig. 2 CONSORT diagram for the clinical trial on the postprandial state effect of a cocoa–carob blend in subjects with type 2 diabetes treated with metformin. |
The basal values for the subjects are shown in Table 1. The average age was 60.5 ± 1.7 years old and three participants were women, representing 15% of the whole group. The values for the cardiometabolic markers (blood pressure, blood glucose, HbA1c, total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides) were all within normal ranges, as were the haematological and immunological basal values. Additionally, a study of the liver by FibroScan and echography was carried out, providing full characterisation for all the participants, and showing a high prevalence (90%) of steatosis in the study population with a score exceeding 238 dB/m, although no fibrosis was detected (all participants showed values < 7.0 kPa).
Parameter | Complete group, n = 20 | Group Ovw, n = 8 | Group Obe, n = 12 |
---|---|---|---|
Age (years) | 60.5 ± 1.7 | 62.0 ± 2.1 | 58.7 ± 2.1 |
Sex | 85% male; 15% female | 75% male; 25% female | 91.7% male; 8.3% female |
Body mass index, BMI (kg m−2) | 31.2 ± 0.7 | 27.7 ± 0.5 # | 33.4 ± 0.5 # |
Systolic pressure (mmHg) | 132.8 ± 2.0 | 128.4 ± 2.8 | 135.8 ± 2.5 |
Diastolic pressure (mmHg) | 79.4 ± 2.1 | 75.6 ± 3.8 | 81.9 ± 2.2 |
Glucose (mg dL−1) | 117.9 ± 4.2 | 103.1 ± 2.0 # | 126.5 ± 1.4 # |
HbA1c (%) | 6.3 ± 0.1 | 6.2 ± 0.2 | 6.4 ± 0.1 |
Triglycerides (mg dL−1) | 146.5 ± 14.3 | 160.8 ± 31.5 | 137.0 ± 12.3 |
HDL-Cholesterol (mg dL−1) | 43.8 ± 1.6 | 43.1 ± 3.3 | 44.3 ± 1.8 |
LDL-Cholesterol (mg dL−1) | 100.8 ± 5.8 | 104.5 ± 12.2 | 98.5 ± 5.9 |
Total Cholesterol (mg dL−1) | 172.9 ± 6.2 | 173.6 ± 14.1 | 172.3 ± 6.1 |
Creatinine (mg dL−1) | 0.91 ± 0.04 | 0.9 ± 0.1 | 0.92 ± 0.04 |
Modification of Diet in Renal Disease, MDRD-4 | 83.9 ± 2.9 | 81.2 ± 4.6 | 86.0 ± 3.9 |
Chronic Kidney Disease Epidemiology Collaboration, CKD-EPI | 83.9 ± 2.9 | 81.2 ± 4.6 | 86.0 ± 3.9 |
Uric acid (mg dL−1) | 6.7 ± 0.3 | 6.2 ± 0.4 | 6.9 ± 0.4 |
Bilirubin (mg dL−1) | 0.7 ± 0.1 | 0.5 ± 0.1 | 0.8 ± 0.1 |
Alanine aminotransferase, ALT (Ul L−1) | 36.9 ± 4.9 | 32.0 ± 7.0 | 40.1 ± 6.8 |
Gamma-glutamyl transferase, GGT (Ul L−1) | 51.8 ± 8.3 | 58.3 ± 17.4 | 48.2 ± 9.2 |
Alkaline phosphatase, ALP (Ul L−1) | 62.3 ± 3.6 | 68.0 ± 6.7 | 59.2 ± 4.2 |
Abdominal circumference (cm) | 104.7 ± 2.5 | 94.0 ± 2.6 # | 111.8 ± 1.9 # |
Hip circumference (cm) | 105.4 ± 1.8 | 98.5 ± 1.2 # | 109.9 ± 2.1 # |
Body fat (%) | 29.9 ± 1.3 | 29.4 ± 2.6 | 30.4 ± 1.4 |
Body muscle mass (kg) | 56.7 ± 2.5 | 48.5 ± 3.5 # | 62.1 ± 2.4 # |
Visceral fat (%) | 14.6 ± 1.0 | 10.6 ± 1.4 # | 17.2 ± 0.7 # |
Abdominal fat (%) | 32.7 ± 1.4 | 32.6 ± 3.0 | 32.8 ± 1.4 |
Abdominal muscle mass (kg) | 31.0 ± 1.9 | 26.5 ± 4.2 | 33.6 ± 1.3 |
Steatosis | 90% yes; 10% no | 75% yes; 25% no | 100% yes |
Portal vein size (mm) | 10.3 ± 0.2 | 9.9 ± 0.2 # | 10.5 ± 0.2 # |
Spleen size (cm) | 10.2 ± 0.2 | 9.8 ± 0.2 # | 10.5 ± 0.2 # |
FibroScan stiffness (kPa) | 5.5 ± 0.4 | 5.1 ± 0.6 | 5.8 ± 0.6 |
Controlled attenuation parameter, CAP | 302.0 ± 12.1 | 296.1 ± 20.6 | 305.9 ± 15.4 |
Fibrosis grade | 0.9 ± 0.2 | 0.7 ± 0.2 | 1.0 ± 0.3 |
The values obtained were evaluated for the entire group (n = 20) and after the participants were classified based on their BMI (kg m−2), with 31.0 ± 0.7 kg m−2 being the mean value for the whole group. Therefore, two groups were created: Group Ovw for those participants overweight (BMI ≤ 30, n = 8) and Group Obe for those with obesity (BMI > 30, n = 12). Significant differences (p < 0.05) between the sub-groups were found for anthropometric measurements, as expected, and for basal blood glucose, in terms of the portal vein size and spleen size.
No adverse or unintended effects derived from CCB consumption were reported.
Parameter | Complete group, n = 20 | Group Ovw, n = 8 | Group Obe, n = 12 | ||||||
---|---|---|---|---|---|---|---|---|---|
C | B | A | C | B | A | C | B | A | |
T max: time to reach the maximum concentration. | |||||||||
Time (min) | |||||||||
0 | 10.4 ± 1.1 | 12.7 ± 1.3 | 11.2 ± 1.3 | 8.6 ± 1.6 # | 11.6 ± 2.0 * | 8.8 ± 2.1 # | 11.7 ± 1.5 | 13.4 ± 1.7 | 12.8 ± 1.5 |
30 | 52.1 ± 6.3 | 68.5 ± 7.8 | 58.0 ± 6.5 | 48.0 ± 10.2 | 65.1 ± 13.0 | 61.1 ± 12.1 | 54.8 ± 8.4 | 70.8 ± 10.2 | 55.9 ± 7.6 |
60 | 87.7 ± 9.5 | 90.7 ± 11.2 | 90.0 ± 8.7 | 89.2 ± 17.3 | 85.8 ± 16.5 | 84.9 ± 7.0 | 86.8 ± 11.5 | 94.0 ± 15.6 | 93.4 ± 14.0 |
120 | 91.5 ± 11.3 | 110.9 ± 11.7 | 95.4 ± 11.7 | 92.3 ± 17.2 | 109.6 ± 18.7 | 89.8 ± 19.2 | 91.0 ± 15.6 | 111.8 ± 15.6 | 99.2 ± 11.4 |
Maximum value | 103.1 ± 10.3 | 120.2 ± 12.2 | 110.6 ± 9.3 | 108.4 ± 17.2 | 113.4 ± 18.5 | 111.5 ± 14.2 | 99.5 ± 13.3 | 124.8 ± 16.7 | 109.9 ± 12.8 |
T max (min) | 90.4 ± 7.6 | 95.2 ± 8.1 | 95.5 ± 7.1 | 91.4 ± 12.0 | 98.6 ± 11.2 | 91.4 ± 11.6 | 89.9 ± 10.2 | 92.8 ± 11.7 | 98.2 ± 9.4 |
Increases | |||||||||
0–30 | 41.7 ± 5.8 | 55.8 ± 6.9 | 46.8 ± 5.9 | 80.6 ± 16.2 | 76.1 ± 15.6 | 52.3 ± 10.7 | 43.2 ± 8.0 | 57.4 ± 9.1 | 43.1 ± 7.1 |
0–60 | 77.3 ± 9.2 | 78.1 ± 10.7 | 78.8 ± 8.5 | 80.6 ± 16.2 | 74.2 ± 15.6 | 76.1 ± 7.6 | 75.1 ± 11.4 | 80.7 ± 15.0 | 80.6 ± 13.6 |
0–120 | 81.1 ± 11.0 | 97.9 ± 11.2 | 84.2 ± 9.7 | 80.9 ± 16.1 | 97.0 ± 18.0 | 80.9 ± 18.8 | 79.3 ± 15.5 | 98.4 ± 15.0 | 86.4 ± 10.9 |
AUC (mg dL −1 min −1 ) | |||||||||
0–30 | 966 ± 109 | 1266 ± 144 | 1095 ± 113 | 876 ± 180 | 1204 ± 248 | 1061 ± 213 | 1027 ± 141 | 1308 ± 182 | 1117 ± 131 |
0–60 | 2990 ± 307 | 3170 ± 367 | 3076 ± 278 | 2948 ± 553 | 2957 ± 523 | 2856 ± 225 | 3018 ± 376 | 3311 ± 518 | 3222 ± 443 |
0–120 | 6241 ± 722 | 7621 ± 753 | 6553 ± 664 | 6111 ± 1116 | 7404 ± 1185 | 5989 ± 1207 | 6328 ± 985 | 7747 ± 1008 | 6929 ± 788 |
30–60 | 2102 ± 238 | 2404 ± 256 | 2152 ± 203 | 2015 ± 397 | 2229 ± 367 | 2234 ± 258 | 2160 ± 310 | 2520 ± 359 | 2098 ± 299 |
60–120 | 5499 ± 606 | 6312 ± 605 | 5746 ± 519 | 5487 ± 952 | 6180 ± 789 | 5287 ± 590 | 5507 ± 820 | 6389 ± 865 | 6052 ± 778 |
Parameter | Complete group, n = 20 | Group Ovw, n = 8 | Group Obe, n = 12 | ||||||
---|---|---|---|---|---|---|---|---|---|
C | B | A | C | B | A | C | B | A | |
T max: time to reach the maximum concentration. | |||||||||
Time (min) | |||||||||
0 | 116.8 ± 4.0 | 117.1 ± 4.0 | 111.9 ± 3.7 | 111.5 ± 4.0 | 109.6 ± 5.0 | 107.1 ± 48 | 120.3 ± 6.0 | 122.0 ± 5.4 | 115.0 ± 5.1 |
30 | 163.8 ± 5.6 | 166.5 ± 4.3 | 156.1 ± 5.1 | 164.5 ± 8.6 | 161.8 ± 5.3 | 145.8 ± 5.7 | 163.3 ± 7.6 | 169.6 ± 6.1 | 162.9 ± 7.1 |
60 | 161.1 ± 6.8 | 164.2 ± 6.7 | 161.7 ± 6.1 | 158.5 ± 9.6 | 155.0 ± 6.1 | 153.8 ± 11.6 | 162.8 ± 9.7 | 170.3 ± 10.3 | 167.0 ± 6.6 |
120 | 136.6 ± 7.2 | 144.2 ± 7.2 | 141.7 ± 7.9 | 124.9 ± 10.0 | 128.4 ± 5.1 | 132.8 ± 11.6 | 144.4 ± 9.6 | 154.7 ± 10.6 | 148.0 ± 10.6 |
180 | 116.9 ± 7.1 | 118.5 ± 6.4 | 117.3 ± 6.7 | 105.1 ± 8.3 | 105.3 ± 6.2 | 107.6 ± 8.0 | 124.8 ± 10.0 | 127.3 ± 9.3 | 123.8 ± 9.6 |
240 | 104.7 ± 5.6 | 107.1 ± 4.2 | 106.7 ± 5.7 | 96.5 ± 7.7 | 103.1 ± 6.6 | 103.0 ± 5.3 | 110.2 ± 7.7 | 109.7 ± 5.5 | 109.1 ± 9.0 |
Maximum value | 175.4 ± 5.8 | 180.8 ± 4.1 | 170.2 ± 5.0 | 169.4 ± 7.8 | 171.0 ± 2.8 | 164.4 ± 8.6 | 179.3 ± 8.3 | 187.3 ± 6.0 | 174.1 ± 6.1 |
T max (min) | 53.7 ± 6.1 | 54.0 ± 6.1 | 62.0 ± 7.3 | 46.5 ± 5.7 | 46.1 ± 6.0 | 49.4 ± 5.3 | 58.5 ± 9.3 | 59.3 ± 9.3 | 70.4 ± 11.2 |
Increases | |||||||||
0–30 | 47.0 ± 4.7 | 49.4 ± 4.1 | 44.2 ± 3.9 | 53.0 ± 8.8 * | 52.1 ± 5.4 #, * | 38.6 ± 4.8 # | 42.9 ± 5.2 | 47.6 ± 5.9 | 47.9 ± 5.5 |
0–60 | 44.3 ± 7.3 | 47.1 ± 6.5 | 49.9 ± 6.4 | 47.0 ± 11.8 | 45.4 ± 9.6 | 46.6 ± 13.6 | 42.5 ± 9.7 | 48.3 ± 9.2 | 52.0 ± 6.1 |
0–120 | 19.8 ± 6.9 | 27.1 ± 7.0 | 29.9 ± 7.2 | 13.4 ± 11.6 | 18.8 ± 6.1 | 25.1 ± 12.1 | 24.1 ± 8.7 | 32.7 ± 10.9 | 33.0 ± 9.2 |
0–180 | 0.1 ± 6.4 | 1.4 ± 6.2 | 5.5 ± 6.2 | −6.4 ± 8.5 | −4.4 ± 8.0 | 0.5 ± 9.9 | 4.4 ± 9.1 | 5.3 ± 8.8 | 8.8 ± 8.1 |
0–240 | −12.1 ± 5.4 | −10.0 ± 4.5 | −5.2 ± 5.4 | −15.0 ± 7.2 | −6.5 ± 6.3 | −4.1 ± 5.6 | −10.2 ± 7.8 | −12.3 ± 6.3 | −5.9 ± 8.4 |
30–60 | −2.7 ± 6.2 | −2.3 ± 7.0 | 5.7 ± 5.9 | −6.0 ± 7.2 | −6.8 ± 10.0 | 8.0 ± 13.5 | −0.4 ± 9.4 | 0.7 ± 9.9 | 4.1 ± 4.6 |
60–120 | −24.5 ± 7.3 | −20.0 ± 5.4 | −20.0 ± 5.7 | −33.6 ± 9.9 | −26.6 ± 6.9 | −21.5 ± 7.0 | −18.4 ± 10.1 | −15.6 ± 7.7 | −19.0 ± 8.5 |
120–180 | −19.7 ± 4.2 | −25.7 ± 4.5 | −24.4 ± 6.1 | −19.8 ± 7.8 | −23.1 ± 6.0 | −24.6 ± 8.7 | −19.7 ± 5.0 | −27.4 ± 6.5 | −24.3 ± 8.7 |
180–240 | −12.2 ± 4.3 | −11.4 ± 5.5 | −10.7 ± 4.8 | −8.6 ± 3.9 | −2.1 ± 7.0 | −4.6 ± 7.1 | −14.6 ± 6.8 | −17.4 ± 7.5 | −14.7 ± 6.4 |
Maximum increase | 58.6 ± 4.9 | 63.7 ± 4.0 | 58.4 ± 5.1 | 57.9 ± 6.4 | 61.4 ± 5.6 | 57.3 ± 9.9 | 59.0 ± 6.3 | 65.3 ± 5.6 | 59.1 ± 5.6 |
AUC (mg dL −1 min −1 ) (/10) | |||||||||
0–30 | 436 ± 15 | 440 ± 12 | 43 ± 17 | 433 ± 20 | 416 ± 12 | 384 ± 20 | 438 ± 21 | 455 ± 18 | 457 ± 21 |
0–60 | 845 ± 26 | 868 ± 29 | 832 ± 25 | 819 ± 29 | 816 ± 23 | 793 ± 38 | 863 ± 39 | 903 ± 44 | 860 ± 33 |
0–120 | 1564 ± 74 | 15960 ± 62 | 1540 ± 62 | 1433 ± 71 | 1445 ± 50 | 1454 ± 83 | 1651 ± 109 | 1640 ± 88 | 1598 ± 87 |
0–180 | 2114 ± 87 | 2133 ± 80 | 2085 ± 83 | 1954 ± 89 | 1949 ± 74 | 1939 ± 80 | 2218 ± 125 | 2256 ± 112 | 2182 ± 123 |
0–240 | 2680 ± 101 | 2707 ± 82 | 26![]() |
1509 ± 126 | 2570 ± 121 | 2521 ± 107 | 2793 ± 140 | 2798 ± 105 | 2695 ± 145 |
30–60 | 484 ± 18 | 508 ± 16 | 461 ± 15 | 473 ± 25 | 489 ± 16 | 457 ± 20 | 492 ± 26 | 521 ± 25 | 463 ± 21 |
60–120 | 930 ± 5 | 931 ± 43 | 931 ± 44 | 858 ± 57 | 844 ± 21 | 867 ± 68 | 979 ± 860 | 988 ± 67 | 973 ± 56 |
120–180 | 724 ± 31 | 776 ± 40 | 784 ± 44 | 681 ± 43 | 701 ± 31 | 707 ± 48 | 753 ± 43 | 826 ± 60 | 835 ± 64 |
180–240 | 679 ± 40 | 680 ± 27 | 650 ± 33 | 6160 ± 58 | 628 ± 34 | 625 ± 38 | 720 ± 52 | 715 ± 37 | 666 ± 50 |
Glycemic profile | 2.4 ± 0.3 | 2.1 ± 0.2 | 2.6 ± 0.3 | 2.3 ± 0.5 | 2.3 ± 0.3 | 2.4 ± 0.3 | 2.5 ± 0.3 | 2.1 ± 0.3 | 2.7 ± 0.5 |
Parameter | Complete group, n = 19 | Group Ovw, n = 7 | Group Obe, n = 12 | ||||||
---|---|---|---|---|---|---|---|---|---|
C | B | A | C | B | A | C | B | A | |
T max: time to reach the maximum concentration. | |||||||||
Time (min) | |||||||||
0 | 99.4 ± 8.0 | 100.1 ± 7.9 | 104.9 ± 9.5 | 103.0 ± 9.7 | 103.4 ± 13.7 | 115.4 ± 15.7 | 97.3 ± 12.3 | 98.2 ± 25.8 | 98.7 ± 12.1 |
30 | 98.0 ± 7.7 | 92.6 ± 7.8 | 97.0 ± 9.0 | 108.3 ± 11.6 | 101.6 ± 16.6 | 102.3 ± 11.0 | 92.0 ± 14.6 | 87.3 ± 30.2 | 94.0 ± 13.0 |
60 | 105.9 ± 8.3 | 101.5 ± 8.8 | 112.0 ± 9.2 | 118.6 ± 11.9 | 112.3 ± 17.4 | 126.4 ± 13.3 | 98.6 ± 15.0 | 95.3 ± 25.4 | 103.6 ± 12.0 |
120 | 128.1 ± 8.9 | 122.2 ± 9.4 | 139.3 ± 12.3 | 141.5 ± 15.5 | 131.6 ± 18.3 | 164.2 ± 19.9 | 120.3 ± 22.0 | 116.8 ± 27.8 | 124.8 ± 14.6 |
180 | 138.0 ± 10.0 | 132.2 ± 11.2 | 142.7 ± 11.4 | 153.8 ± 19.2 | 148.2 ± 25.8 | 165.8 ± 22.2 | 128.8 ± 24.4 | 122.9 ± 24.2 | 129.2 ± 11.7 |
240 | 142.9 ± 10.9 | 133.4 ± 13.0 | 146.5 ± 14.0 | 159.7 ± 20.8 | 141.7 ± 26.5 | 170.3 ± 26.1 | 133.1 ± 29.5 | 128.6 ± 26.4 | 132.7 ± 15.5 |
270 | 141.5 ± 11.0 | 137.4 ± 14.5 | 137.4 ± 13.0 | 158.0 ± 20.0 | 155.2 ± 33.6 | 164.7 ± 26.2 | 131.9 ± 30.0 | 127.6 ± 24.7 | 121.4 ± 12.5 |
Maximum value | 151.3 ± 10.8 | 163.6 ± 13.8 | 163.6 ± 13.2 | 167.0 ± 19.2 | 164.8 ± 31.6 | 189.2 ± 23.2 | 142.1 ± 29.4 | 144.9 ± 27.7 | 148.6 ± 15.0 |
T max (min) | 224.0 ± 11.5 * | 227.7 ± 12.2 * | 182.8 ± 14.6 # | 228.1 ± 20.8 | 223.6 ± 23.2 | 176.6 ± 25.6 | 221.6 ± 14.2 * | 230.2 ± 14.7 * | 186.4 ± 18.4 # |
Increases | |||||||||
0–30 | −1.3 ± 3.5 | −7.5 ± 5.0 | −7.8 ± 3.0 | 5.3 ± 6.9 | −1.8 ± 4.5 | −13.2 ± 5.8 | −5.2 ± 2.9 | −10.9 ± 5.9 | −4.7 ± 3.2 |
30–60 | 7.9 ± 3.1 | 8.9 ± 3.9 | 14.9 ± 3.9 | 10.2 ± 4.9 | 10.7 ± 5.6 | 24.1 ± 8.5 | 6.5 ± 6.8 | 7.9 ± 5.6 | 9.6 ± 3.1 |
60–120 | 22.2 ± 3.2 | 20.7 ± 3.2 | 27.4 ± 5.5 | 22.9 ± 5.2 | 19.3 ± 5.6 | 37.9 ± 9.2 | 21.8 ± 9.0 | 21.5 ± 7.1 | 21.2 ± 6.5 |
120–180 | 9.9 ± 3.6 | 10.0 ± 5.6 | 3.5 ± 5.5 | 12.3 ± 7.5 | 16.7 ± 12.9 | 1.5 ± 8.8 | 8.5 ± 5.9 | 6.2 ± 6.4 | 4.8 ± 7.4 |
180–240 | 4.9 ± 3.8 | 1.2 ± 5.6 | 3.9 ± 6.4 | 5.9 ± 5.1 | −6.5 ± 6.9 | 4.6 ± 10.4 | 4.3 ± 7.5 | 5.7 ± 10.0 | 3.4 ± 8.5 |
240–270 | −1.4 ± 2.6 | 4.3 ± 4.7 | −9.2 ± 5.1 | −1.8 ± 2.9 #, * | 13.4 ± 7.7 * | −5.6 ± 8.1 # | −1.2 ± 7.3 | −1.0 ± 5.2 | −11.2 ± 6.7 |
Maximum increase | 51.9 ± 6.0 | 56.7 ± 9.5 | 58.7 ± 9.7 | 64.0 ± 12.3 | 61.4 ± 21.3 | 73.8 ± 16.4 | 44.9 ± 17.8 | 53.9 ± 13.8 | 49.9 ± 11.8 |
AUC (mg dL −1 min −1 ) (/10) | |||||||||
0–30 | 305 ± 24 | 298 ± 22 | 323 ± 32 | 328 ± 32 | 314 ± 45 | 330 ± 42 | 292 ± 47 | 288 ± 92 | 319 ± 45 |
0–60 | 622 ± 47 | 625 ± 49 | 661 ± 56 | 669 ± 60 | 667 ± 90 | 737 ± 86 | 594 ± 81 | 601 ± 153 | 616 ± 72 |
0–120 | 1397 ± 100 | 1353 ± 98 | 1484 ± 126 | 1475 ± 139 | 1415 ± 185 | 1700 ± 207 | 1352 ± 199 | 1316 ± 323 | 1358 ± 154 |
0–180 | 2146 ± 154 | 2104 ± 157 | 2247 ± 176 | 2321 ± 251 | 2282 ± 343 | 2533 ± 319 | 2044 ± 322 | 1999 ± 440 | 2080 ± 202 |
0–240 | 2929 ± 217 | 2820 ± 224 | 3026 ± 258 | 3166 ± 350 | 2958 ± 464 | 3424 ± 453 | 2790 ± 493 | 2739 ± 601 | 2794 ± 306 |
0–270 | 3490 ± 613 | 3211 ± 268 | 3270 ± 268 | 3523 ± 385 | 3049 ± 663 | 3782 ± 493 | 3094 ± 562 | 3049 ± 663 | 2971 ± 294 |
30–60 | 302 ± 22 | 300 ± 24 | 303 ± 25 | 333 ± 31 | 333 ± 47 | 350 ± 36 | 284 ± 37 | 281 ± 77 | 276 ± 33 |
60–120 | 730 ± 59 | 668 ± 53 | 767 ± 63 | 784 ± 78 | 714 ± 108 | 879 ± 93 | 698 ± 105 | 641 ± 166 | 693 ± 81 |
120–180 | 773 ± 60 | 755 ± 59 | 847 ± 67 | 890 ± 113 | 853 ± 123 | 969 ± 122 | 705 ± 134 | 698 ± 148 | 776 ± 76 |
180–240 | 855 ± 62 | 802 ± 72 | 855 ± 80 | 945 ± 116 | 869 ± 156 | 1001 ± 140 | 802 ± 167 | 762 ± 152 | 770 ± 93 |
240–270 | 401 ± 35 | 390 ± 43 | 418 ± 42 | 458 ± 69 | 426 ± 90 | 513 ± 92 | 368 ± 94 | 369 ± 74 | 363 ± 33 |
Regarding the primary outcome of the study, i.e. postprandial insulin, CCB supplementation could not modify insulinaemia at the different assessed times after the breakfast provided to the participants (Table 2). Besides, insulin levels showed significant differences (p < 0.05) at 0 min when the CCB was consumed the night before the visit (intervention B), compared with the control and treatment A.
Considering glycaemia (Table 3), CCB supplementation, either at breakfast or the night before, did not significantly modify either glucose levels at different times nor the other parameters evaluated when the whole group was assessed. On the contrary, when the subjects were classified according to their BMI, a significant decrease (p < 0.05) in the glycaemia increment from 0 to 30 min was observed in those with overweight (BMI ≤ 30, n = 8) when consuming the CCB at breakfast compared with the control breakfast (Table 3). No significant differences were found between the treatments for any group in terms of GLP-1 values (Table S2†).
Regarding triglycerides (Table 4), the time to reach their maximum concentration was diminished in the complete group when the intake of CCB took place together with breakfast (intervention A) (p < 0.05), compared to treatments C (control) and B. This was also observed in the group with obesity (group Obe, BMI ≤ 30, n = 12). Moreover, in the group who were overweight (group Ovw, BMI ≤ 30, n = 8), the triglyceride increment from 240 to 270 min was significantly higher during intervention B compared to intervention A (p < 0.05), but it was not different with respect to the control treatment.
Questions (no.) | Complete group, n = 20 | Group Ovw, n = 8 | Group Obe, n = 12 | ||||||
---|---|---|---|---|---|---|---|---|---|
C | B | A | C | B | A | C | B | A | |
Question 1: How strong is your desire to eat something sweet? 2: How full do you feel? 3: How hungry do you feel? 4: How strong is your desire to eat something salty? 5: How strong is your desire to eat? 6: How much food do you think you could eat? | |||||||||
Satiety perception t = 0 | |||||||||
1 | 3.8 ± 0.4 | 2.9 ± 0.4 | 3.3 ± 0.4 | 4.0 ± 0.5 | 2.9 ± 0.7 | 2.9 ± 0.6 | 3.7 ± 0.6 | 2.9 ± 0.5 | 3.6 ± 0.5 |
2 | 2.7 ± 0.4 | 2.6 ± 0.4 | 3.5 ± 0.5 | 3.1 ± 0.6 | 2.8 ± 0.7 | 3.1 ± 0.8 | 2.4 ± 0.5 | 2.4 ± 0.4 | 3.8 ± 0.7 |
3 | 4.6 ± 0.5 | 3.6 ± 0.3 | 3.9 ± 0.3 | 3.6 ± 0.7 | 3.1 ± 0.4 | 3.8 ± 0.6 | 5.2 ± 0.6 | 3.8 ± 0.4 | 4.0 ± 0.5 |
4 | 4.7 ± 0.5 | 2.7 ± 0.4 | 3.4 ± 0.4 | 3.3 ± 0.6 # | 1.9 ± 0.4 * | 3.3 ± 0.8 # | 4.0 ± 0.6 | 3.2 ± 0.4 | 3.4 ± 0.6 |
5 | 4.9 ± 0.4 | 3.9 ± 0.3 | 4.1 ± 0.3 | 4.3 ± 0.6 | 3.4 ± 0.6 | 4.0 ± 0.5 | 5.3 ± 0.5 | 4.2 ± 0.4 | 4.1 ± 0.4 |
6 | 5.1 ± 0.3 | 4.7 ± 0.4 | 4.9 ± 0.3 | 4.8 ± 0.4 | 4.4 ± 0.5 | 4.5 ± 0.4 | 5.2 ± 0.4 | 4.9 ± 0.5 | 5.2 ± 0.4 |
CSS | 80.1 ± 0.2 | 80.8 ± 0.2 | 80.7 ± 0.2 | 80.5 ± 0.3 | 81.2 ± 0.3 | 80.8 ± 0.3 | 79.8 ± 0.4 | 80.6 ± 0.3 | 80.6 ± 0.4 |
AUC (0–60 min) | |||||||||
1 | 177 ± 17 | 143 ± 13 | 153 ± 16 | 188 ± 17 | 135 ± 17 | 128 ± 18 | 170 ± 24 | 148 ± 18 | 170 ± 21 |
2 | 246 ± 25 | 239 ± 22 | 281 ± 30 | 274 ± 26 | 244 ± 31 | 255 ± 35 | 228 ± 36 | 235 ± 29 | 298 ± 42 |
3 | 189 ± 14 | 161 ± 9 | 168 ± 11 | 158 ± 20 | 146 ± 13 | 158 ± 11 | 210 ± 16 | 170 ± 11 | 175 ± 15 |
4 | 179 ± 20 | 135 ± 15 | 174 ± 15 | 165 ± 30 | 109 ± 15 | 176 ± 16 | 188 ± 24 | 153 ± 20 | 173 ± 21 |
5 | 210 ± 12 * | 167 ± 11 # | 176 ± 11 #, * | 184 ± 13 | 154 ± 16 | 161 ± 12 | 228 ± 14 * | 175 ± 12 # | 185 ± 14 #, * |
6 | 240 ± 16 | 231 ± 19 | 228 ± 17 | 221 ± 21 | 206 ± 20 | 191 ± 17 | 253 ± 20 | 248 ± 28 | 253 ± 23 |
AUC (0–120 min) | |||||||||
1 | 336 ± 33 | 288 ± 37 | 312 ± 30 | 330 ± 28 | 285 ± 37 | 270 ± 33 | 340 ± 51 | 290 ± 36 | 340 ± 41 |
2 | 456 ± 52 | 414 ± 42 | 504 ± 51 | 488 ± 64 | 375 ± 34 | 475 ± 58 | 435 ± 71 | 440 ± 64 | 525 ± 73 |
3 | 402 ± 34 * | 327 ± 17 # | 348 ± 19 # | 338 ± 42 | 300 ± 21 | 323 ± 16 | 445 ± 41 | 345 ± 21 | 365 ± 29 |
4 | 342 ± 36 | 282 ± 28 | 339 ± 31 | 278 ± 41 | 2323 ± 25 | 345 ± 40 | 385 ± 46 | 315 ± 40 | 335 ± 41 |
5 | 417 ± 29 | 360 ± 23 | 369 ± 21 | 360 ± 31 | 3223 ± 36 | 354 ± 27 | 455 ± 39 | 385 ± 25 | 380 ± 27 |
6 | 489 ± 33 | 474 ± 40 | 486 ± 34 | 420 ± 36 | 428 ± 44 | 413 ± 31 | 535 ± 42 | 505 ± 56 | 535 ± 46 |
Regarding the miRNA expression levels, among the 87 miRNAs analysed, 32 showed significant changes in at least one of the three interventions when comparing baseline to 2 h-later levels (data not shown). Of these 32, 9 miRNAs whose fold change varied |0.5| between both time points were selected for further analysis: miR-15b-5p, miR-17-5p, miR-20a-5p, miR-20b-5p, miR-23a-3p, miR-146a-5p, miR-369-3p, miR-223-3p, and miR-483-5p (Fig. 3). The results showed that the exosomal miR-15b-5p, miR-20a-5p, miR-20b-5p, and miR-23a-3p plasma levels were upregulated after the hypercaloric breakfast taken together with a supplementation of cocoa and carob-derived polyphenols (intervention A). At the same time, the miR-17-5p, miR-146a-5p, miR-369-3p, and miR-483-5p circulating levels were significantly downregulated when the participants consumed the polyphenols the night before having the hypercaloric breakfast (intervention B). Also, miR-17-5p was commonly modulated by both interventions with polyphenols (A and B), showing a more significant change when taken at the same time (intervention A). None of these miRNAs were significantly altered in the control intervention (treatment C), while miR-223-3p was significantly modified in all three interventions, which could reflect its susceptibility to change under dietary intervention, and for this reason, it was excluded for further analysis.
Finally, Fig. 4 shows the potential targets and pathways involved in the miRNAs modulated by the CCB. We found 96 target genes for treatment A and 83 genes for treatment B; these were involved in biological processes (BPs), molecular functions (MFs) and associated KEGG pathways. The overrepresented pathways for treatment A included the PI3K-Akt, FoxO or MAPK signalling pathways. While for treatment B, these included monocyte chemotaxis, inflammatory response and the chemokine-mediated signalling pathway.
In contrast to our hypothesis for this randomised controlled nutritional trial, supplementation with 10 g of the CCB during breakfast or the night before did not cause an overall significant variation in the primary outcome of the study, i.e. on postprandial insulin. This hypothesis was based on the existing evidence on the beneficial effects of polyphenols and dietary fibre in postprandial insulinaemia, as shown in reviews on clinical trials on the topic.28,29 Although, at the same time, the same analysis of the literature revealed other studies with null effects in the same variable, which evidences the complexity of the involved factors in nutrition studies.
Similarly to the observed results for insulin, moreover, it did not affect other parameters of glucose homeostasis (postprandial glucose and GLP-1 levels), compared to the control intervention. Nevertheless, a significant decrease in the increase in glucose from 0 to 30 min was shown in the overweight subgroup when consuming the product at the same time as breakfast (intervention A). The physiological relevance of this reduction is that postprandial glucose peaks are considered an independent risk factor for cardiovascular diseases due to different mechanisms of action; this is particularly important in the context of T2D, with a high prevalence of associated cardiovascular complications. This CCB effect may be associated with its polyphenol profile. In this sense, some randomised controlled trials have reported a reduction of glucose levels after supplementation with polyphenol-rich chocolate in young participants30 or supplements enriched in cocoa flavonoids in older subjects,31 although other studies did not find such results.32 In the case of carob, although the evidence is still limited, a trial conducted with 40 healthy volunteers aimed to evaluate the effect of consuming a drink enriched with bioactive compounds from carob. After 12 weeks of supplementation and under a normocaloric diet, the authors found that there was a decrease in glucose levels in subjects after breakfast, lunch, and dinner.33
Regarding other parameters involved in glucose homeostasis, the CCB did not significantly modify postprandial insulin levels, in contrast to a trial where the intake of cagaita (Eugenia dysenterica DC) fruit juice by dysglycaemic subjects with metabolic syndrome, together with breakfast, led to a significant decrease in plasma insulin levels compared with only breakfast.34 Interestingly, another study on blackcurrant extract did not observe modifications in postprandial sensitivity after acute intake by subjects with overweight/obesity, but a significant improvement in insulin sensitivity was observed after consuming the product for eight days.35 Therefore, a similar effect after chronic CCB supplementation could not be excluded. In our trial, no modification was found in GLP-1 levels after CCB supplementation. Other postprandial studies with acute supplementations in healthy adults with flavanol-rich products have reported both beneficial and null effects on active GLP-1.36,37 Herein, total GLP-1 was measured but the discrepant results may be mostly linked to the fact that the postprandial regulation of GLP-1 in subjects with overweight/obesity is not yet completely understood, as evidenced from the results in different cohorts.
Another significant effect associated with CCB supplementation was an improvement in the postprandial satiety as assessed by the validated VAS, an approach that, in an earlier meta-analysis, provided a consistently significant (although of low magnitude) correlation with further energy intake.38 It is likely that this effect was mostly due to the high dietary fibre content of CCB since, for instance, a previous trial with two nutraceuticals containing only polyphenols or a combination of polyphenols and dietary fibre found that the second one was more efficient for satiety regulation in subjects who were overweight/obese, by both objective and subjective determinations.39 Nevertheless, a trial where healthy subjects were supplemented with a polyphenol-rich extract from pomegranate (lacking dietary fibre) found significant improvements in satiety parameters as measured by VAS, although the mechanism of action was not assessed.40 At the same time, the obtained results for the subjective assessment of satiety perception seem to be in contradiction with the lack of effect in GLP-1 levels. In this sense, it should be mentioned that other studies based on a supplementation with polyphenol-rich or dietary fibre-rich products also reported significant effects in VAS not accompanied by modifications in GLP-1.39,41 Due to the complexity of all the signals involved in appetite regulation,42 more research is needed to ascertain how dietary polyphenols and dietary fibre may contribute to the neuroendocrine regulation of hunger.
Another relevant aspect regarding the satiety effects obtained here is the fact that the modulating effects took place not only when the product was consumed together with breakfast but also when it was consumed the night before, which highlights the contribution of the derived metabolites, resulting from dietary fibre and/or from polyphenols. In this way, a previous study on bread or porridge from rye kernel, compared with white bread, found a decrease in satiety not only 3 h after but even 7 h after intake.43 Moreover, another study focused on supplementation with several pulses compared with macaroni and cheese found that the effects on subjective appetite led to a significant decrease in energy intake in the next meal.44 Besides, a study on brown beans compared with white bread found that their consumption the night before not only produced a significant decrease in subjective hunger the next morning, but also in the circulating levels of ghrelin and in clinical markers such as postprandial glucose, postprandial insulin and IL-8.45
Regarding ex vivo analysis, cytokine release after LPS stimulation was not modified either after treatment A or B. Similar previous ex vivo studies where subjects were supplemented with bioactive-rich samples such as extra virgin olive oil (in patients undergoing coronary angiography) and a spice blend (in subjects with overweight/obesity) observed significant modifications in the final levels of IL-10 and IL-6, respectively, compared with control groups, even when the LPS treatment did not induce significant modifications over time in the control group.46,47 Since consistent evidence supports the role of polymeric flavanols as anti-inflammatory agents, the results observed here would be likely associated with an insufficient dose; indeed, the mentioned trial with a spice blend found an ex vivo anti-inflammatory effect with a dose of 6 g of the blend but not with a 2 g dose.46
This trial also aimed to evaluate the underlying molecular modifications that may occur due to CCB supplementation beyond just the clinical markers. In this context, miRNA modulation has been suggested as an additional mechanism of action of polyphenol,48 mostly after chronic intake, although a recent trial also found differences during the postprandial state.49 The results observed here indicated that circulating miRNAs transported in exosomes could be both induced and repressed in response to CCB consumption. In particular, a tendency towards an improvement in insulin sensitivity was observed. Thus, among the miRNAs significantly increased by treatment A, miR-20a-5p has been found to be downregulated in T2D and its overexpression associated with an enhancement in insulin sensitivity and hepatic glycogen synthesis, as well as a decrease in hepatic steatosis.50,51 Similarly, miR-23a-3p has been reported to be decreased in patients with T2D compared with people with normal glucose tolerance test results,52 while the CCB induced its release into the circulation. Furthermore, miR-17-5p, with increased expressions after treatments A and B compared with after treatment C, has been reported to improve insulin sensitivity and decrease steatosis.53 It should be mentioned that all three of them were significantly modified after treatment A, while only miR-17-5p was significantly modified (and to a lesser extent) after treatments A and B. Thus, the effect of CCB supplementation on miRNA expression seems to be more connected with the phenolic compounds directly observed after intake than to the metabolites derived from prolonged transformations after intake. Nevertheless, further studies are needed in order to confirm these effects, since the overexpression of miR-15b-5p detected after treatment A has been connected with a decrease in insulin sensitivity.54 Additionally, it is remarkable that miR-146a-5p, increased after treatment B, has been reported to be highly expressed in the milk-derived extracellular vesicles from pregnant rats receiving a diet enriched in resistant starch, a type of dietary fibre; thus, there may be a connection with this dietary constituent.55 The fact that these modifications in miRNA expression were not connected with changes in the markers of glucose homeostasis may be due with the fact that miRNA expression may express subtle modifications that may be present before the clinical markers are affected.56
The main strengths of this trial were that the composition of the supplemented product was fully characterized and that a robust crossover design was established, with comprehensive assessment of baseline subject characteristics, including a study of the liver status performed via echography and FibroScan (indeed, most participants exhibited NAFLD, although they were unaware, according to other evidence on the underdiagnosis of this pathology).57 Furthermore, we explored the molecular mechanism underlying clinical modifications, based on exosomal miRNA analyses, which has been scarcely assessed in previous postprandial studies. The main limitation is the acute design, based on the effect of a single serving of the product; for this reason, despite the randomised controlled design, it should be considered a pilot approach. Moreover, it was not possible to ascertain whether the observed effects were due to polyphenols, dietary fibre or both. Furthermore, future studies should include objective measurements of satiety and hunger, which would greatly complement the subjective results reported here. Some population adjustments in future studies are also recommended, including a replication study, specifically for a female population, considering the high proportion of male participants in this trial, and a replication study with enough statistical data, for validating the specific effects observed in individuals with overweight/obesity.
In summary, this acute randomised crossover controlled nutritional trial on the postprandial effects of CCB in subjects with T2D showed promising significant differences regarding postprandial hyperglycaemia in subjects who were overweight (when CCB was consumed together with a hypercaloric breakfast); satiety perception in the whole group and in subjects who were obese (when CCB was consumed together with a hypercaloric breakfast or the night before); and significant upregulations or downregulations of several miRNAs, which are mostly involved in pathways related to insulin sensitivity. At the same time, no adverse effect was reported. These results may be attributed to a combination of the mechanisms of action reported for both polyphenols and dietary fibre as bioactive constituents of CCB. A chronic intervention randomised controlled trial with this product should be guaranteed in order to confirm the observed results, as well as the potential sustained metabolic modifications.
The data for this article, including all the raw data used in the generation of tables and figures (also those provided as ESI†) are available at the public repository digital.csic.es of the Spanish Research Council (CSIC) at https://digital.csic.es/handle/10261/367662.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4fo04498c |
This journal is © The Royal Society of Chemistry 2025 |