Study design
The present study is an eight weeks double-blind, placebo-controlled clinical trial. Eligible subjects will be randomly assigned to 2 groups of placebo or intervention (2 soft gel containing 1 g of N. sativa extract oil each). The projected RCT will be conducted at the nutrition department of Tabriz medical university to evaluate the possible effect of Nigella sativa extract oil on patients with coronary artery disease. The Standard Protocol Items: Recommendations for Interventional Trials 2013 (SPIRIT) checklist is provided as an additional file.
Study population
Patients with coronary artery disease (CAD) will be recruited from Shahid Madani Heart center in Tabriz, Iran. Participants will be screened for the eligibility criteria by a cardiologist and based on their medical records. Then the included patients are informed by their physician about the trial protocol and routine appointments. After declaring their interest in the trial, they will be asked to sign a flyer with additional information about the study.
Inclusion criteria
Subjects will be recruited from Shahid Madani Heart center affiliated to Tabriz Medical Science, and those who meet the following criteria can participate in this project: a proof of 50% stenosis in at least one of their major coronary arteries in angiography, the ability and willingness to collaborate in the project, aged between 35-65 years, body mass index (BMI) of 18.5- 35.
Exclusion criteria
Exclusion criteria will include pregnancy or lactation, smoking cigarettes or opiates, cancer, acute or chronic renal failure, any hepatic or thyroid disorder, diabetes, any changes in medication, taking herbal supplementations, or any weight-loss drugs, having a history of food allergy.
Randomization and blinding
Eligible subjects will be randomly assigned in either control (taking placebo, n=30) or intervention (nigella sativa supplementation, n=30) group. A third party will use a computerize-generated random number to allocate the participant into the intervention and placebo groups. The company that produces the supplements will label the boxes with unique alphabetic codes based on the generated allocation sequence. Furthermore, the company will prepare two identical sealed boxes, each containing 30 soft gels inside. Every participant will receive four boxes during the supplementation. All of the investigators, participants, medical staff, and statisticians will be blind to the intervention, allocation, and study treatment until the final analysis. Any unintended unblinding will be reported to the main researcher. In such cases, applicant ID, date, and unblinding circumstances will be documented for inner control.
Sample size calculation
The sample size calculation was based on N. sativa supplementation on changes in total antioxidant capacity (TAC), which was conducted by Hadi et al. [37] it was computed by considering 95% confidence interval and 90% power. We also consider a 15% attrition rate, which at least 23 subjects are considered for each group.
Study procedure
At the baseline, a questionnaire, including past medical history, demographic information, drug history, and medication, will be filled. All the data will be confidential and accesable only by the investigators. Dietary intake will be assessed by 3-day questionnaires (two weekdays and one weekend) at the onset, internal, and the end of the study. Total calories, micro, and macro-nutrients intake will be calculated by NUT IV (the Hearst Corporation, San Bruno, CA, USA). Also, the physical activity level will be evaluated at the beginning and the end of the study by the international physical activity questionnaire (IPAQ). Table 1 shows the timetable for enrollment, involvement, and evaluation based on the Standard Protocol Items; Recommendations for Interventional Trials (SPIRIT). The anthropometric measures will be assessed with light clothes and without shoes. The height will be measured by stadiometer with the accuracy of 0.1-cm. Actual body weight, total fat mass, fat-free mass, visceral fat, and body muscle along with their percentage will be measured by the Tanita MC-780MA S body analyzer. Besides, waist/hip/neck circumferences and blood pressure will be assessed at each visit. Compliance will be assessed based on returned capsule count; those who miss more than ten percent of supplementation dosage will be excluded from the trial. At the start and end of the study, 10 milliliters of blood will be collected. Blood samples will be taken after 10-12 hours of fasting and will be stored at -80 C till the end of the interventions. Enzyme-linked immunosorbent assay (ELISA) kits will be used to evaluate the serum ICAM-1, VCAM-1, and insulin concentration. Other biochemical analyses such as high-density lipoprotein (HDL-C), triglyceride (TG), total cholesterol (TC), fasting blood sugar (FBS), superoxide dismutase (SOD), malondialdehyde (MDA), and total antioxidant capacity (TAC) will be measured. Fried Ewald equation will be used to estimate the low-density lipoprotein (LDL-C) concentration. Homeostasis model assessment – insulin resistance (HOMA-IR) will be calculated. Also, the atherogenic index of plasma will be assessed by the formula. Beck's Depression Inventory (BDI-II) and MacNew quality of life questionnaire related to heart disease will be used as the secondary outcomes.
Frequency of analyses
Only at the final analysis, outcome data will be analyzed, although statistical monitoring of safety data will be conducted throughout the study and reported at agreed intervals. Final analysis will take place eight weeks after the last patient is randomized.
Statistical analysis
We will use the SPSS program version 16.0 to analyze all the data (SPSS Inc., Chicago, IL), and P-value < 0.05 will be considered statistically significant. The efficacy of this trial will be assessed by both the per-protocol approach, and the intention-to-treat principle will be analyzed [38]. Missing or dropout data will be analyzed by modern imputation methods.
Subgroup analysis
Subgroup analysis are not planned yet.
Monitoring
Data monitoring
A clinical trial monitor occasionally supervises the study progress in each to ensure patient rights and well-being are safeguarded, that the protocol, ethical requirements, standards, and regulations are being followed, that the essential documentation is available and that collected data are accurate as there were recorded. Any change or amendments in the protocol will be shared.
Harm
There is no side effect reported after 2-g/day Nigella sativa supplementation. However, this clinical trial will be monitored by the data monitoring committee (DMC). Also, any possible side effects such as shortness of breath, allergy reaction, stomach disorders, muscular pain, and serious adverse events (SAEs) will be reported to the ethics committee of the Tabriz University of Medical Science.