Study Design and Participants
This study was a randomized, double-blind, single-center, parallel-group, controlled clinical trial. The research protocol was approved by the Ethics Committee of the National Nutrition and Food Technology Research Institute at Shahid Beheshti University of Medical Sciences (IR.SBMU.nnftri.Rec.1398.009). This trial was registered at Clinicaltrial.gov under the identification number: NCT04163757 and has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). Participants were recruited from the Imam Hossein Hospital in Tehran, Iran, from January to June 2019. The study protocol was explained for eligible subjects, and written informed consent was signed by all patients if they agreed to participate in the study. Participants could withdraw from the study by their own decision without any penalty.
This trial was performed in patients aged between 30 and 70 years, with a clinical diagnosis of diabetes mellitus (1-10 years) according to Standards of Medical Care in Diabetes Guidelines (American Diabetes Association, 2019) (27), body mass index (BMI) between 18.5 and 30 kg/m2, and also the use of oral hypoglycemic agents for controlling diabetes. Participants who took insulin, herbal and/or nutritional supplements, glucocorticoids, and non-steroid anti-inflammatory drugs within 3 months before the start of the study and during the study were excluded from the study. Other major criteria for exclusion were clinically diagnosed chronic diseases such as renal, hepatic, cardiovascular, autoimmune or any kind of inflammatory diseases, being pregnant or lactating, being on a weight loss diet within last 6 months, and having uncontrolled diabetes (HbA1c ≥ 8.5%).
Randomization and Treatment
A total of 112 patients were assessed for eligibility for inclusion in the trial. Fifty-seven of 112 subjects met the inclusion criteria, seven of whom were reluctant to attend (consort diagram, Fig 1). Patients were selected using a simple sampling procedure and stratified (1:1) into two groups based on their sex and age, they were randomly allocated to receive either the crocin supplement (n=25) or the placebo supplement (n=25) for 3 months. Randomization sequence was computer-generated by a blinded biostatistician who was not involved with recruitment, using permuted block randomization (block size 4) and given to the interviewer. Patients were randomly assigned to the trial groups in accordance with the randomization list (code letters A or B) in chronological order. All investigators, staff related to the care of the patients, and participants were blinded to the treatment assignment until the final statistical analysis was completed.
The intervention group was administered orally two tablets of 15 mg crocin (Samisaz CO., Mashhad, Iran), and the control group was given two tablets of placebo (starch), with main meals (breakfast and dinner), for 12 weeks. Placebo tablets were similar to the crocin supplements in terms of the size, color, shape, smell and distribution bottles. Crocin was extracted from saffron stigmas using crystallization method with a purify more than 97%.
According to standard formula suggested for parallel clinical trials, the sample size was calculated for the glucose level, which was based on detection of 25 mg/dL difference in the mean glucose levels and standard deviation 29 mg/dL with a power of 80% (β = 20%) and a significance level of 0.05, yielding an estimated sample size of 23 for each group (28). Due to the potential loss of samples, 25 patients were included in each treatment group.
Follow-up Assessments and Compliance
Follow-up assessments were performed at every 4 weeks following initiation of the study. Anthropometric measurements, food records (3 days), physical activity assessment and tablets counts were assessed at each follow-up visit. Adherence to study treatment and adverse events were also ascertained at each visit. Compliance with consumption of tablets during the study was determined by counting the remaining tablets in each visit and weekly telephone call. Participants who had not consumed at least 90% of the expected tablets were regarded as noncompliance, which resulted in exclusion from the trial. The subjects were instructed to maintain their usual lifestyle and dietary habits during the study.
Clinical, Para-clinical and Dietary Intake Assessment
At baseline, a general questionnaire was completed during a personal interview for every patient about demographics, medical history, medication use and health status. Height, body weight, waist and hip circumferences, and body fat percentage (BFP) were measured by an expert nutritionist at baseline, each follow-up visit, and the end of the study. Body weight and height were measured using a calibrated Seca balance measuring scale with stadiometer while participants wearing light clothes and no footwear. Waist and hip circumferences were measured using constant tension measuring tape and according to a standardized method. BFP was determined by Bioelectrical Impedance Analysis (BIA) method using a portable electrical micro-current monitor. Body mass index (BMI) and waist-to-hip ratio (WHR) were calculated according to WHO recommendation (29, 30).
Blood pressure measurements were also taken from each patient in a sitting position, twice with a 10-min interval, and the mean systolic and diastolic blood pressure measurements were used for analysis. Dietary intake of each patient was collected using 3-day 24-h dietary record (two weekdays and one weekend) in months 0 and 3 (31). These records were verified by a dietitian, and then analyzed by Nutritionist IV (First Databank, Hearst Corp, San Bruno, CA, USA). Physical activity was also evaluated by using a validated semi-quantitative questionnaire, based on metabolic equivalent (MET)-min/day values (32).
Biochemical testing was performed on each patient at the beginning and end of the study, after 12-h overnight fasting. All blood samples divided two parts, 1mL for HbA1c determination and the second remained parts were immediately centrifuged (3500 rpm:10 min at room temperature) and the separated sera was stored at -80˚C for subsequent biochemical analysis. All biochemical parameters were assessed in same laboratory by using standard laboratory methods. Fasting glucose concentration was measured by using GOD/POD method. Fasting insulin concentrations were determined using the enzyme-linked immunosorbent assay (ELISA) (Demeditec Diagnostics, Germany) with a lower sensitivity limit of 1.76 µIU/mL. The hemoglobin A1c (HbA1c) was measured on the whole blood sample by direct enzymatic HbA1c assay (Diazyme Laboratories, Inc., CA, USA). HOMA-IR, QUICKI, and HOMA-β were calculated to estimate insulin resistance, insulin sensitivity, and β-cell homeostasis, respectively (33, 34). Phosphorylated AMP-activated protein kinase (P-AMPK) was measured in peripheral blood mononuclear cell by using an ELISA kit (ZellBio, Ulm, Germany) according to the manufacturer’s protocol.
In this study, statistical analysis of data was performed by SPSS software version 24. Normality of data distribution was checked through the Kolmogorov-Smirnov test. Results of categorical variables were presented as frequency, and continuous data were shown as mean ± SD. Student’s t test was done to detect differences between groups. For within-group comparison, paired t test was used. Parameters with skewed distribution were natural logarithm-transformed (ln) to normalize distribution.
To remove the effects of confounding factors, analysis of covariance (ANCOVA) was used to determine any differences at the end of the study with adjusting for baseline values and height. The data were analyzed according to the intention-to-treat (ITT) principle. Statistical significance was defined at P<0.05, based on two-sided tests.