2.1 Setting

This is a human nutrition intervention study with a parallel group design [25] were twenty-four healthy non-diabetic adults with ages between 18 and 35 years were recruited from the local community.

Subjects were randomly assigned in 2 groups (n=12 each): group A (reference meal) and group B (test meal). The participants were required to have an 8 hour fasting before the day of the study and were ask not to ingest any cinnamon at the day before the intervention. For both A and B groups, intervention consisted of the ingestion the reference meal either with or without cinnamon respectively, in the morning (with a duration of proximally 5 minutes). Both interventions were employed under supervision by an investigator.

2.2 Inclusion and Exclusion Criteria

Inclusion criteria included subjects aged 18 or more, both gender with non-diabetic condition (fasting blood glucose level <126 mg/dL) [26]. The exclusion criteria were impaired fasting glucose, pregnancy and lactating or menopausal conditions. Moreover, subjects should not take medication for glycaemia control or antibiotics, nor have gastrointestinal symptoms or diseases. The study also excluded subjects who have altered medication, pregnancy, lactation and allergy to cinnamon.

2.3 Data collection

Following overnight fasting blood glucose level was measured using a capillary drop blood, before intervention (t₀). In group A, subjects ingested of 100g of mousse without cinnamon and in group B subjects ingested 100g of mousse with cinnamon. Blood samples were then collected, for each participant, at 30 (t₃₀), 60 (t₆₀), 90 (t₉₀) and 120 (t₁₂₀) minute after ingestion reference and test meal. Sterilized lancet, glucosemeter equipment and strips for glucosemeter (FreeStyle_Abbott Diabetes Care) were used for blood glucose level measurement. Blood glucose concentrations were measured with the OneTouch Vita^® glucometer, which has an accuracy of 98%.

The blood glucose area under the curve (AUC) was calculated above zero and are presented as means. The AUC of each subject was calculated using GRAPH PAD PRISM software (version 5.01).

The anthropometric data was recorded through bio-impedance body fat analyzer InBody® 230 and the height was measured by the stadiometer Craveira Jofre.

No specific diet was imposed and the participants consumed their usual diet. A 24-hours dietary recall was taken preceding each intervention to compare food intake at the day before the intervention between groups. The Food Processor SQL (version 10.5.0) program was used to analyze the food nutritional composition.

All data collection was registered on Excel_® file where a coded number was attributed to each participant.

2.4 Ethical approval

This study was approved by a Portuguese state recognized Ethics Committee (Ethics Committee of Cooperativa de Ensino Superior Egas Moniz) and was carried out in accordance with the Helsinki Declaration of 1975 as revised in 2000. A written informed consent was obtained from each participant after given written and oral information about the study.

2.5 Test meal preparation and constitution

The meal consisted in a mousse prepared by the investigator, with the following ingredients and amounts (for a total of 25 doses): 10 gelatin sheets, 1 L of skim milk, 12 egg yolks (204,1 g), 323 g of white sugar, 82,3 g of butter and 963,8 g of sour cream. The test meal consisted of 100 g of mousse mixed with 3 g cinnamon, which was milled with the coffee grinder Taurus Aromatic 150 W. The reference meal consisted of 100 g of the same receipt mousse without cinnamon.

2.6 Chemical analysis (Total phenolic content and antioxidant activity)

Ferric Chloride (III) hexahydrate (FeCl₃.6H₂O), Folin-Ciocalteu (2,2-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid)), Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), TPTZ 2,4,6-tri(2-piridil)-s-triazine, methanol (CH3OH), nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium (NBT) 2-Amino-2-hydroxymethyl-propane-1,3-diol (tris) and Phenazine methosulfate (PMS) were purchased in Sigma-Aldrich, gallic acid-1- hydrate (C₆H₂(OH)₃COOH.H₂O) was purchased in Acros Organics and sodium carbonate (Na₂CO₃) was purchased in ICS Science group. All reagents were _{pro analysis} grade.

All the absorbance measurements were performed in a Perkin- Elmer Lambda 25. The regents were weight in an analytical balance (Sartorius, ±0.0001g) and all the solutions were done with distilled water.

2.7 Extract preparation

The mousse was subjected to a hydro-ethanolic extract (20:80). Posteriorly, the mixture was filtered using the Whatmanpaper filter. Homogeneous samples were obtained and subjected to chemical analysis.

2.8 Total phenolic content determination

The extracts total phenolic concentration was determined according Folin-Ciocalteu method [4] employing gallic acid as standard. The samples were analysed in triplicate: a volume of 312.5 μl were pipette to an ethanol: water 80: 20 (V/V) solution, to which was added 187.5 μl of water, 5 ml of solution Folin-Ciocalteu reagent (1:10 diluted with water) and 4 ml of aqueous solution Na₂CO₃ 1M. After 15 min the absorbance was measured at 765 nm. Gallic acid was used as the standard solution (Y=0.0034X+0.018 (R²=0.9966)) and the results were expressed in mg of gallic acid equivalents (GAE) / L.

2.9 Characterization of the antioxidant activity: Ferric Reducing Antioxidant Potential (FRAP) assay

This method was adapted from the method described by [27] and is based on the activity of the antioxidants to reduce Fe³⁺, a colorless ferric complex to a blue-colored ferrous complex, Fe²⁺ in the presence of 2,4,6-tri(2-piridil)-s-triazine (TPTZ).

The FRAP solution was previously prepared, adding 25 ml of sodium acetate 300 mM pH 3.6,to2.5 ml of 10 mM TPTZ in HCl 40mM, and 2,5ml of FeCl₃.6H₂O 20 mM. 150 μl of the samples were added with 2850 μl of FRAP solution. The mixture was kept in the dark for 30 minutes and then the absorbance was read at 593 nm. A blank solution was prepared, in which the sample was replaced by 150 μl of water, in the same conditions. The Trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid), a vitamin E analogous, was used as standard (Y=2,17x10-3X+2,32x10-2 (R²=0,998)) and results were expressed as μM TE.

2.10 Statistical analysis

Statistical analysis was performed with SPSS for Windows (version v19.0; SPSS Institute, Chicago, IL). Data are presented as mean ± SEM. Mann-Whitney U test was used to assess the food intake in the day prior to the intervention between 2 groups. Repeated Measures ANOVA of mixed type was used to assess the postprandial blood glucose level at different times, between 2 groups. Independent sample t-test was used for blood glucose levels area under the curve (AUC) and overnight fasting statistical analysis. All statistical tests were performed at the 5% level of significance.

3.1 Sample characterization

General characteristics of sample regarding age, weight (Kg), body mass index (BMI), fat mass (FM), muscular mass (MM), waist to hip ratio (WHR) and waist circumference (WC) are represented in table 1.

Table 1: General characteristics of the participants.(N= 12 for each group).
SEM: standard error of the mean;BMI: Body mass index; SMM: Skeletal muscle mass; WHR: Waist to hip ratio.

Total calories, macronutrient and dietary fiber intake at the day before the intervention were estimated for both groups. Statistical comparison with Mann-Whitney test revealed homogeneity between groups (p>0.05), table 2.

Table 2: Dietary analysis at the day prior to the intervention (N=12 for each group).
SEM: standard error of the mean.

3.2 Postprandial blood glucose response

The addition of 3 g of C. burmannii result in a slightly increased postprandial blood glucose response at different postprandial times (30, 60, 90 and 120 min), compared with reference meal (without cinnamon), table 3. However, the statistical analysis revealed no interaction between the independent and repeated measures factors (p=0.250), which means that it is not possible to infer about differences in different times.

Table 3: Blood glucose levels (mg/dl) obtained on OGTT (N=12 for each group).
OGTT: oral glucose tolerance test.

Regarding to the postprandial blood glucose levels AUCs the addition of cinnamon to the mousse slightly increased this value. However, the statistical analysis (Independent sample t-test) revealed that cinnamon addition did not significantly altered the AUC values (p=0.081), table 4.

Table 4: Postprandial blood glucose area under the curve (AUC) (N=12 for each group).

3.3 Antioxidant characterization of each meal

The results of the chemical analysis of the reference meal (mousse without cinnamon) and the test meal (mousse with cinnamon) are represented in Table 5 and revealed that addition of C. burmannii (3g) to the mousse, increased the total phenolic content and the antioxidant activity (assessed by FRAP test).

Table 5: Total phenolic content and antioxidant activity (FRAP test) of meals.