A total of 25 overweight (BMI 28.8 ± 3.9 kg m−2), inactive office-workers to take part in two inter-related but separate studies. Characteristics for participants in each study are presented in Table 1. Participants were deemed to be inactive if they undertook < 1 h structured physical activity per week (in the preceding 6 months). All participants were absent of any other metabolic comorbidities and cardiovascular disease. Both trials were approved by the Liverpool John Moores University Research Ethics Committee. Written, informed consent was obtained following an explanation of the experimental procedures.
Screening procedures were identical for both studies. During an initial visit, height and weight were measured to determine BMI, and an assessment of body composition was conducted using bioelectrical impedance (Tanita BC 418 MA Segmental Body Composition Analyser, Tanita, Japan). Habitual dietary intake was assessed using a written diary for 72 h, with diaries analysed for total energy intake and macronutrient composition of the diet using Nutritics software (Nutritics Ltd, Dublin, Ireland). At the first visit, participants also completed a food frequency questionnaire, which listed the quantity and frequency of anthocyanin-containing foods and drinks compiled from the Phenol Explorer database . By multiplying the anthocyanin content of the portion size by the total consumption frequency of each food, daily anthocyanin intake was calculated (Table 1).
Study 1 experimental design—acute supplementation
Study 1 required participants to undertake two experimental trials separated by a washout period of ≥ 7 days. 24 h before each experimental trial participants consumed a standardised diet (50% carbohydrate, 30% fat, 20% protein) that was otherwise matched to habitual energy intake. Participants were also instructed to abstain from vigorous exercise for 48 h and alcohol and caffeine for 24 h prior. On the morning of each experimental trial, participants attended the laboratory following an overnight fast (> 10 h) and first consumed a standardised high-carbohydrate breakfast (70% CHO, 10% protein, 20% fat, and equivalent to 25% of daily caloric intake) generally consisting of Weetabix with semi-skimmed milk, orange juice, an Upbeat protein drink and a banana. After they had consumed the breakfast they worked at a computer or sat quietly for 3 h. In a randomised, double-blind, crossover design, participants then ingested 2 capsules of NZBC extract (600 mg) or a visually-identical placebo, with water, 30 min prior to lunch. Each 300 mg NZBC capsule contained 105 mg of anthocyanins, consisting of 35–50% delphinidin-3-rutinoside, 5–20% delphinidin-3-glucoside, 30–45% cyanidin-3rutinoside, and 3–10% cyanidin-3-glucoside (CurraNZ™, Health Currancy Ltd., Surrey, UK). Each placebo capsule contained 300 mg microcrystalline cellulose. Following ingestion of either the NZBC extract or placebo, an indwelling cannula was placed into the antecubital vein of one arm and an initial blood sample was obtained. Thirty min following ingestion of NZBC extract or placebo, participants consumed a high-carbohydrate, high-fat liquid test meal consisting of 75 g maltodextrin (MyProtein™, The Hut Group, Cheshire, UK) and 50 g unsaturated fatty acids (Calogen, Nutricia, Amsterdam, NL). Blood samples were subsequently collected at 15 min intervals for the first hour and 30 min intervals for the remaining two hours. Once the testing procedure was completed the cannula was removed and participants were able to leave the laboratory.
Study 2 experimental design—short-term supplementation
In a randomised, double-blinded, crossover design participants undertook 8 days supplementation with either NZBC extract (600 mg per day) or a visually-identical placebo. The supplement and placebo were identical to that used in study 1. One 300 mg capsule was ingested prior to breakfast, and one 300 mg capsule was ingested before dinner throughout the supplementation period. An overview of the experimental design for study 2 is provided in Fig. 1. On day 1 of each supplementation period a fasted blood sample was obtained from the antecubital vein of one arm. On day 5, participants were fitted with a continuous glucose monitoring system (CGMS) (described below), and provided with a standardised diet to be consumed on days 6, 7 and 8. On day 7, participants returned to the laboratory following an overnight fast (> 10 h) to undergo an oral glucose tolerance test (OGTT). Following collection of a fasted blood sample from an indwelling cannula placed in an antecubital vein, participants consumed 75 g maltodextrin (MyProtein™, The Hut Group, Cheshire, UK) diluted in 225 ml of water. Further blood samples were collected after 15, 30, 45, 60, 90 and 120 min, and collected into EDTA-containing vacutainers. Isotonic saline was used to keep the cannula patent every 15 min during the OGTT. On day 8, participants undertook their usual daily activities and the CGMS was used to examine interstitial glucose concentrations under free-living conditions. Each cross-over trial was separated by ≥ 15 days, which is based on a previous study that provided an anthocyanin dose greater than that used in the current study for 1 month and 15 days were required for antioxidant biomarkers to return to baseline levels (Alvarez-Suarez 2014).
Continuous glucose monitoring
A Dexcom G4 Platinum CGM probe (Dexcom, San Diego, CA, USA) was inserted subcutaneously into the lower abdominal region on day 5 of each supplementation period. This provided adequate time for “bedding in” and for the participants to become accustomed to using the CGMS. Participants were trained how to use the device and instructed to calibrate the device a minimum of four times daily using capillary blood samples. The monitor remained in place for the next 4 days, during which participants were provided with a standardised diet to consume that was matched to habitual energy intake but with a set macronutrient content (see Table 2 for overview of energy and macronutrient composition). On day 8, free-living glucose responses were assessed. On this day, participants were instructed to undertake their habitual daily activities, but consume their meals at pre-defined time points; 7–9 am breakfast, 12–2 pm lunch, and 5–7 pm evening meal. These times were chosen to ensure that there was a minimum 3 h postprandial period between meals.
Blood sample analysis
Across both studies, plasma samples for each time point were obtained following centrifugation (10 min at 1000 g at 4 °C) and stored at − 80 °C for subsequent analyses. Plasma glucose and triglyceride concentrations were determined spectrophotometrically using a semiautomatic analyser in combination with commercially available kits (Randox Laboratories, Antrim, UK). Plasma insulin, high-sensitivity interleukin-6 (hsIL-6), C-reactive protein (CRP) and hsTNF-ɑ concentrations were determined using commercially available ELISA kits (Invitrogen, Thermo Fisher Scientific, UK). For all assays the intra-assay coefficient of variation was ≤ 8.5% and the inter-assay coefficient of variation was ≤ 9.8%. The analytical sensitivity of the assays for insulin, CRP, hsIL-6 and hsTNF-ɑ was 0.17 µIU mL−1, < 10 pg mL−1, 0.03 pg mL−1, and 0.13 pg mL−1, respectively. Each sample was analysed in duplicate.
Calculations and statistical analysis
Area under the curve (AUC) for plasma glucose, insulin and triglycerides was calculated using the conventional trapezoid rule. Insulin sensitivity was assessed using the homeostatic model assessment (HOMA) index and Matsuda  insulin sensitivity index. CGMS data were downloaded from the device using Dexcom Studio™ software (220.127.116.11) and first the glucose responses to each meal were investigated. In this regard, the 3 h postprandial period was evaluated for mean, peak and end glucose concentrations, and the area under the curve for the entire postprandial period was also calculated. All statistical analyses were performed using SPSS (v26.0, Chicago, IL, USA). Results are expressed as means ± S.D, and significance was set at the 0.05 level of confidence. For both study 1 and 2, time-dependent variables were assessed using a two-factor repeated-measures ANOVA, with the within-subject factors ‘condition’ (NZBC vs. placebo) and ‘time’. Significant main effects and interactions were assessed using Bonferroni adjustment post-hoc analysis. All other variables were investigated using a paired t-test. Both studies were powered to detect differences in glucose AUC between conditions (NZBC vs. placebo), with G*Power 3.1 software (G*Power Software Inc., Kiel, Germany) used to calculate the required sample size. A medium effect size (f = 0.30) was adopted and deemed to be physiologically-relevant, based on the data from two previous studies [7, 15], and used alongside an alpha of 0.05 and power of 0.80, to calculate the required sample size.