Participants aged 40-75 years were recruited from local community at outpatient clinic of a hospital in Guangzhou city, China. Potential participants were initially screened by their recent fasting blood glucose records, and volunteers were further screened by a face-to-face interview and were given a 3-hour 75 g oral glucose tolerance test (OGTT) to confirm their eligibility. Patients with prediabetes or newly diagnosed diabetes were eligible for this study. According to the diagnostic criteria of the American Diabetes Association (ADA) , prediabetes is an intermediate state in which subjects meet one of the following criteria: impaired fasting glucose (IFG, 5.6~6.9 mmol/L), impaired glucose tolerance (IGT, 2-hour glucose 7.8~11.0 mmol/L), or a glycated hemoglobin A1c (HbA1c) level of 5.7~6.4%. Newly diagnosed diabetes was diagnosed in subjects who exceeded the upper limit of the above criteria (fasting glucose >6.9 mmol/L, 2-hour glucose>11.0 mmol/L, or HbA1c >6.4%), and had not taken any diabetic medications before the screening.
Participants were excluded if they had pre-existing diabetes, had a history of hypoglycemic medical treatment, acute or chronic infectious diseases, untreated thyroid disease, serious liver or kidney dysfunction, the use of glucocorticoids, or suffering from traumatic injury or undergoing surgery within 6 months before enrollment; were lactating or pregnant women; or were individuals with polycystic ovarian syndrome. This trial was approved by the Ethics Committee of Sun Yat-Sen University, and written informed consent was obtained from each participant before enrollment.
This study was a 12-week randomized placebo-controlled trial that was registered at ClinicalTrials.gov as NCT02689765. The primary outcome was change in serum adiponectin. Sample size calculation were based on the previous study using purified anthocyanins as supplements , in which the change in serum adiponectin was 1.2 µg/mL (SD 1 µg/mL). We calculated that 60 participants (prediabetes or newly diagnosed diabetes) per group, would be enough to detect a significant change in adiponectin (1 µg/mL, SD 2 µg/mL) at 80% power and an alpha level of 0.05. Considering a 20% dropout rate, we planned to recruit 80 participants per group and a total of 160 participants in this study.
Eligible participants were randomly assigned to either the anthocyanins group (n = 80) or the placebo group (n = 80). The allocation sequences were determined by a computer-generated random-numbers table. The anthocyanins group consumed two anthocyanin capsules (Medox; Polyphenols AS, Norway) twice daily for a total of 320 mg anthocyanins. The dose of anthocyanins was determined based on our previous trials that were performed in patients with T2DM  and dyslipidemia . The control group consumed two identically packaged placebo capsules twice daily.
The participants were asked to maintain their habitual diet and physical activities, and to avoid consuming anthocyanin-rich foods, such as berries and grapes, during the whole study. Dietary analyses were conducted at baseline and 12 weeks of the study. Subjects were asked to provide detailed three-day food records, and they were guided by a trained investigator. Scheduled capsules were dispensed to the participants, and participants were asked to return any remaining capsules to the clinic every 2 weeks. Compliance was assessed by using a short questionnaire and counting their remaining capsules. When the subjects consumed fewer than 80% of the dispensed capsules, they were excluded from the trial.
At baseline and at the end of intervention, height and weight were measured by trained staff, and body mass index (BMI) was calculated as weight divided by height squared (kg/m2). Waist circumference, hip circumference and blood pressure were measured by the same staff. Fasting blood samples were collected after fasting overnight, and a 3-hour OGTT was performed (blood samples were collected at 30, 60, 120, and 180 minutes after a 75 g glucose challenge).
Serum samples were centrifuged (15 min, 3000×g, 4 °C) within 30 minutes after blood collection and were stored at -80℃. Fasting serum adiponectin was measured using an ELISA kit with a solid phase two-site enzyme immunoassay (Mercodia, Uppsala, Sweden). The average intra- and inter-assay coefficients of variation for adiponectin were 3.1 and 5.4%, respectively.
HbA1c was analyzed by high-pressure liquid chromatography (HPLC) (Bio-Rad Laboratories, USA). Insulin and C-peptide were assayed by electrochemical luminescence (Roche Diagnostics, Indianapolis, USA). Laboratory analyses of blood glucose, lipid profiles (total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, apolipoprotein A-1 and apolipoprotein B), C-reactive protein (CRP), and safety variables (liver enzymes) were performed by using an automatic biochemical analyzer (Mindray BS600, Shenzhen, China) following standard protocols.
SPSS 22.0 (IBM Inc., Chicago, IL, USA) was used for statistical analyses, and R 3.5.3 (R Foundation, Vienna, Austria) was used for graphing. P < 0.05 was considered significant. The per-protocol data set without the imputation of missing data was used for data analysis. We used the unpaired Student’s t-test (for continuous variables) and Chi-squared test (for categorical variables) to evaluate the baseline differences between the anthocyanins and the placebo groups. An independent Student’s t-test was used to compare the differences between the two groups after intervention. We assessed the differences before and after the intervention within each group by using a paired t-test. Multiple comparisons were statistically corrected by using the false discovery rate.