-Study Design and subjects:
The ethics committee of Zahedan University of Medical Sciences approved this double-blind randomized placebo-controlled clinical trial under the code of IR.ZAUMS.REC.1397.332. The study was registered to Iranian Registry of Clinical Trials as IRCT20181207041876N1 on 18/01/2019. The participators were overweight or obese T2DM patients referring to the diabetic clinic of Bu-Ali Hospital of Zahedan. This trial lasted from 23 June 2019 until 22 October 2019.
Inclusion criteria were T2DM diagnosed, age 30–65 years, and 25≤BMI<40 kg/m2. On the other hand, Exclusion criteria were Suffering from cognitive disease or other psychotic illnesses, depression, Acute systemic disease, cystic fibrosis disease, muscular dystrophy, protein malnutrition, a history of gastrointestinal surgery, neurological disorders, structural abnormalities of the gastrointestinal tract, disability, uncontrolled hypertension (>140/90 mmHg), Treatment with statins, antihypertensive, probiotics, multivitamin-mineral and antioxidant supplements during the three past months, pregnancy or lactation, expert athlete, intake of statins, caraway interacting drugs, losing weight during the past 3-months and not taking more than 10% of the intervention supplement.
This trial adhered to CONSORT guidelines and included a completed CONSORT checklist as an additional file.
-Randomization and Intervention:
According to the block randomization method, participants were divided into two equal groups by an assistant (BP [n=30] or placebo [n=30] groups). The stratified randomization method was utilized for matching age (30-45 and 46-65 yrs) and gender. The ratio of the two groups was 1:1. Three patients from the PB group and 2 patients from the placebo group declined to participate after randomization and before the beginning of the study (Figure 1).
The blinding of intervention allocation was done for both the participants and investigators as A and B packages. The PB and placebo capsules were made by the Herbarium center of the Kerman Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran. The shape, size, and color of the capsules were similar. The contents of capsules were 1 g of whole PB or Starch powder. Before supplementation, the capsules were weekly placed alongside each other to get a similar smell. The absorbed Bunium Persicum volatile oil in the placebo capsules was very low to change health parameters. According to a previous study, the dose of supplements was determined 2 grams/day as 2 capsules per meal with food (26). However, the intake and absorption of the PB either with or without food should be investigated further. The supplements were distributed monthly basis and checking compliance status was also done monthly through face to face and weekly by telephone. The duration of the intervention period was 8 weeks.
The PB voucher number was Bunium persicum (Boiss) B. Fedtch, Family: Apiaceae, KF1141-1. The analysis of the whole PB was performed by the Herbarium center of the Kerman Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran. The contents of Bunium persicum essential oil according to the gas chromatography-mass spectrometry (GC-MS) were 46.1 % γ-Terpinene and 15.5 % cumin aldehyde.
-Assessments and Measurements:
General Characteristics, Dietary Intakes, and Physical Activity:
The T2DM patients were identified, the eligibility criteria checked, the study details were clarified, and informed consent was obtained by the main investigator (BSC student). The questionnaires including the general questionnaire, 24-hour food recall (at the beginning and end), and short-form IPAQ (SF-IPAQ) questionnaire (at the beginning and end) were administered through interviewing.
The dietary status was determined using gram per day of values from 24-hour food recall (valid in Iran (27)) by the Nutritionist 4 software (27, 28).
The IPAQ questionnaire provides information on physical activity that people do as part of their everyday lives. The questions are about the time when the person has been active during the last 7 days. This questionnaire addresses the activities in the workplace or as part of the homework and the garden, place to place movement, exercises, and leisure activities. It also considers all the intense activities over the past 7 days. Intense activities require a great deal of physical power and more intense breathing. The IPAQ addresses only continuous activities for at least 10 minutes. Its short form has 7 classified questions determining the three activity levels (1-3 or low-to-high levels). This questionnaire had been validated in Iran (29, 30).
Weight (at the beginning and end), height (at the beginning) and waist circumference (at the beginning and end) were determined by using a digital scale and stadiometer (Seca® Germany, Model: 755 1021994). They were measured as such: Weight without shoes, with minimal clothing, and with 100-gram accuracy; height without shoes, standing, heels sticking to the wall, flat and forward head, and with 0.5-centimeter accuracy; and waist circumference with minimal clothing, at the middle of the last rib and the iliac crest. Body mass index (BMI) was calculated via dividing weight in kilograms by squared height in meters.
Blood biomarkers measurement:
At the beginning and the end of the study, 10 ml of blood (at the beginning and end) was taken from the peripheral vein after 12-hour fasting during the night and centrifuged for 20 min (3000 g). Serum glucose was determined on the same day of blood withdrawal. The remaining serums were frozen and stored at -80°C up to the analysis.
Serum nesfatin was determined using the sandwich ELISA and kit as Shanghai Crystal Day Biotech Co. Ltd®; Intra-assay CV<8 %, Inter-assay CV<10 % by an automatic device (Elisys Uno Human®). Similarly, the ELISA kit for FBI was DiaMetra® Co of Italy, DCM076-8; Intra-assay CV≤5 %, Inter-assay CV≤10 %. Serum glucose was measured according to the glucose oxidase method using the Hitachi analyzer device (q17®) and the specific kit as Bionik®, Liquid Stable, Glucose oxidase GOD-POD, Mono-reagent; Intra-assay CV≤2.10 %, Inter-assay CV≤3.09 %. Also, the serum levels of TC, TG, LDL-C, and HDL-C were measured using Hitachi analyzer device (q17®) and the specific kits as 1-Bionik®, Liquid Stable, Enzymatic Colorimetric CHOD-POD, 2-Bionik®, Liquid Stable, GPO-POD, Mono-reagent, 3-Bionik®, Liquid Stable, Direct. Enzymatic Colorimetric, and 4-Bionik®, Liquid Stable, Direct. Enzymatic Colorimetric, respectively. The intra- and inter-assay coefficients of variation for TC, TG, LDL-C, and HDL-C were ≤1.216% and ≤6.906%, ≤1.573% and ≤7.704%, ≤1.76% and ≤0.65 %, and ≤0.7% and ≤1.5%, respectively. HOMA-IR and QUICKI indices were calculated by the following formulas:
QUICKI = 1 / (log fasting insulin [µIU/ml]) + log fasting glucose [mg/dl])
HOMA-IR = FBI [µIU/ml] × FBS [mg/dl] / 405
as regards, no article was found similar to our study, the sample size determined by considering the sample size of the similar studies in obese and overweight patients (31, 32) and using the extract of anti-obesity factors (25), The sample size determined from 8 to 44 in each group. The sample size for this study according to study limitations (control of various factors such as powder consumption, diet control, physical activity, nutritional counseling, cost) in each group 27 patients are considered but accounting to 10% missing during the study, the number of patients is 30 in each group.
-Data Analysis and Accessibility:
Data management including entry, security, coding, and storage was performed at this stage. The missing data of the follow-up stage and baseline of one patient were estimated by modified-intention to treat (m-ITT) analysis and regression imputation method. The Kolmogorov-Smirnov, Chi-square, Fisher Exact, and t or Mann-Whitney tests assessed normality of continuous variables as well as categorical and continuous baseline characteristics, respectively. Two-way repeated measures analysis of variance (TWRM-ANOVA) was used to determine time effects and time by treatment interaction effects on all dependent variables. Moreover, TWRM-ANOVA was adjusted for dietary intake of vitamin B12. Also, 95% confidence interval (CI) and a P-value<0.05 were considered for reporting the measurements. Data analysis was conducted using SPSS16 (statistical package for the social sciences) and STATA11SE (general-purpose statistical software package by Stata Corp) software. The main investigator had access to the final dataset and the results were presented by the publication.