The trial was a single center, prospective, randomized, controlled trial that conducted in critically ill patient with primary sepsis between December 2018 and October 2019 in the Intensive Care Units, Imamreza Hospital. The study protocol and consent forms were approved by the Research Ethics Committee of Mashhad University of Medical Sciences (registration code: IR.MUMS.MEDICAL.REC.1397.290) and was registered in the Iranian Registry of Clinical Trials (registration code: IRCT20181025041460N1) (26). Written informed consent was obtained from all patients or their legal representatives before inclusion in the study.
Patients and interventions
Fifty five patients were randomly assigned to four groups of the study using block randomization to propolis (n=14), propolis plus melatonin (n=14), melatonin (n=14) or control groups (n=13). Patients who had the eligibility criteria include age 18 to 75 years, admission to the intensive care unit with the criteria for systemic inflammatory response syndrome (primary sepsis), providing written, informed consent form by the patient or first-degree relatives (legal representatives) of the patient and the Glasgow coma scale (GCS) is equal to 7 or higher entered to this study. Exclusion criteria were; pregnancy and lactation, patients who are not able to start nutritional support in the first 24-48 hours, autoimmune disorders, cancer, severe and advanced sepsis, chemotherapy and radiotherapy in one last month, received positive inotropic drugs (including dopamine, dobutamine, and epinephrine), severe liver failure, severe and active bleeding, human immunodeficiency virus (HIV), known food allergies, and morbid obesity (body mass index (BMI)> 40). In addition all selected patients were treated based on the sepsis guideline (27), and at any time of the study, participants were excluded from the study if they were reluctant to continue the study, any of the criteria for non-entry, or sensitivity to melatonin or propolis supplements.
The intervention groups in addition to the usual treatments, received 1000 mg/day propolis alone, 1000 mg/day propolis plus 20 mg/day melatonin, or 20 mg/day melatonin alone, respectively for the 10-day, however, in the control group, only routine treatments were provided.
The combination of aqueous and ethanolic extract propolis was purchased as syrup from Soren Tech Toos Company (Soren tech-Toos Co, Mashhad-Iran, Batch Number; STT5.006) and melatonin was purchased from Amin Pharmaceutical Company (Aminpharma Co, Isfahan, Iran). The melatonin-receiving groups received 20 mg of melatonin daily (in two divided doses of 10 mg at noon and 3 hours before bedtime) and groups receiving propolis daily received 1000 mg of propolis (in two divided doses in the morning and evening) for 10 days.
Data collection and measurements
A checklist was designed by the research team in which information including age, gender, underlying disease, medical history; drug history, etc. were collected and recorded. In the present study inflammatory markers and clinical outcomes assessed as primary outcomes and also oxidative stress markers, infection indices, gavage intakes, and 28-day survival assessed as secondary outcomes.
Clinical and nutritional outcomes measurements
Anthropometric measurements including estimated height based on ulna length and patient age (28), weight (using the patient's bedside scale (Seca-Germany)), body mass index (BMI) were calculated using the estimated height and weight of the patients (Kg/m2 where Kg is a patient's weight in kilograms and m2 is their height in meters squared), and also mid arm circumference (MAC) using non-elastic tape meters for each patient was measured at the beginning of the study.
Nutritional support of patients using enteral nutrition with hospital base formula that has a certain amount of calorie, macro and micronutrients was used (testing by TESTA Quality Control Laboratory, Mashhad-Iran), Enteral nutrition through the nasopharyngeal tube was started using a hospital gavage at a rate of 25 ml/hour and increased at the same rate every 6 hours according to the patient's tolerance to achieve the desired calories and the volume of gastric residue was not significant (300 ml>). The calorie requirement of each patient was calculated by hospital nutrition experts and the research team based on 25 kcal/kg actual body weight and the required protein content was considered to be 1.5 g/kg ideal body weight.
The NUTrition Risk in the Critically ill (NUTRIC score) Questionnaire was used at the beginning and end of the study to assess nutritional status and estimate malnutrition of the patients (29).
To estimate the risk of mortality and severity classification of the disease, the Acute Physiology and Chronic Health Evaluation II (APACHE II) questionnaire was used at the beginning and after the intervention (30), and Sequential Organ Failure Assessment (SOFA) questionnaire was also used to evaluate the function of the organs of the body during the study on days 1, 5, and 10 of the study (31). Patients were also followed up from the beginning of the study to 28 days after the intervention to assess mortality rate.
Hematological and Inflammatory markers measurements
The white blood cell count was measured by Sysmex K-1000 Hematology Analyzer-Japan and then the differential counts were performed under a microscope to accurately measure neutrophil values before and after the intervention. The serum CRP level was measured using the immunomodibrometry method and the quantitative CRP detection kit (Bionik, Iran) with the BT3500 (Biotecnica Instruments SpA -Italy) auto-analyzer and also, the measurement of interleukin 6 by ELISA method using kit (Demeditec-Germany) was performed in the beginning and end of the study.
Oxidative stress markers measurements
In the beginning and end of the study to assess the pro-oxidant-antioxidant balance (PAB), Hamidi Alamdari et al.'s method was used (32) and also malondialdehyde levels using kit (Zell Bio-Germany) was measured.
Sample size and statistical analysis
Sample size calculation was made based on 80% power and an alpha error of 5% to detect the inflammation effect based on changes in IL-6 by propolis supplementation using the method of Zhao and colleagues study using the below formula (20).
Based on the above formula, the sample size was at least 9 patients in each group and due to the high probability of dropout in the intensive care unit, at least 13 patients entered in each group.
All analyses were performed on an intention-to-treat (ITT) basis using the statistical package for social sciences (SPSS, Inc., Chicago, IL, USA) software version18. The normal distribution of variables was evaluated using the Shapiro-Wilk test and based on the results of this test, all data were normally distributed. In this study, chi-square test was used to compare the qualitative variables, we used paired samples t-test to assess the effects of propolis and melatonin supplements on primary and secondary outcomes measurements within groups, and the ANOVA test to compare between groups changes and, if necessary, post hoc tests followed by the Tukey comparison test was used. Also we adjusted the variables based on their baseline values by using Analysis of Covariance (ANCOVA). p < 0.05 is considered statistically significant.