Study design and subjects
This randomized, double-blind, parallel-design, placebo-controlled clinical trial was conducted on subjects who their IBS has been determined by a gastroenterologist according to Rome IV criteria. Patients were recruited from the Soroush Special Clinic of Ahvaz, Iran, between September 2019 and January 2020. Based on the Rome IV criteria, patients who had recurrent abdominal pain or discomfort (at least 1 day/week in the last 3 months) were identified as an IBS patient if he/she had at least two of the following criteria:
- Improvement with defecation
- Onset related to a change in stool frequency
- Onset related to exchange in stool form (appearance)
The inclusion criteria in this trial included patients aged 18-65 years who had a constipation subtype of IBS (IBS-C) or a mixed subtype of IBS (IBS-M) based on the Bristol stool form scale (BSFS); have no allergy to bee products; and fill out a written consent form. The exclusion criteria of the study were pregnancy or breastfeeding; patients with malignancy or other chronic GI diseases; regular use of drugs that modify GI movements (such as Metoclopramide, Cisapride, Narcotics, Diphenoxylate, and etc.); regular use of laxatives and/or antibiotics; the history of major surgery in the digestive system (such as Billroth's operation, having an ostomy and any resection of any part of the digestive tract); being on diet; regular use of prebiotic and/or probiotic compounds; and use of psychotherapy drugs.
Patients with taking less than 80% of their supplements, unwilling to continue collaboration in the study, experiencing severe physical and mental trauma, or changing their diet plan or physical activity during the study were withdrawn from follow-up.
The trial protocol, available at Iranian Registry of Clinical Trials (https://en.irct.ir/trial/40983, registration date: 26/12/2019, Registration number: IRCT20190708044154N1), was approved by the Ethics Committee of Tabriz University of Medical Sciences. This trial was performed in accordance with the Declaration of Helsinki. All patients were provided verbally with information on the objectives, benefits, and possible health risks of the trial at the time of enrollment and then provided written informed consent.
Randomization and intervention
Eligible patients were randomly allocated in a 1:1 ratio to receive propolis or placebo tablets. In this study, a random-number table was used to generate randomization sequences with a block size of 4 and stratification according to IBS subtypes and sex. For proper blinding, the propolis and placebo were prepared in precisely the same color, size, odor, and packaging. Also, numbered drug containers were used to conceal random allocation. No one was aware of treatment assignments, except the pharmacist.
The supplements were prepared by Mashhad School of Pharmacy, Mashhad University of Medical Sciences, Iran; under the supervision of a clinical pharmacist. Propolis tablets consist of 450 mg of propolis extract (containing 90 mg of the polyphenols and 67 mg flavonoids), whereas the placebo tablets contain microcrystalline cellulose (a powder that had no taste, calories, smell, or nutrients) and various edible colors (16). The tablets were similar in color, shape, and packaging; and were administrated before lunch and dinner for six weeks. The optimal dosage of propolis (900mg/day) was extracted from animal studies which its method is completely described in the published protocol article of this study (17). Due to the same mechanism of propolis and pre-and probiotics for intestinal microflora, a period of six weeks is adequate to boost intestinal microflora and/or GI symptoms in patients based on former studies (18, 19). One of the researchers was responsible for follow-up patients by phone calls, weekly. She was asked each patient to report any adverse effect they may be experienced during the study, and fill out the supplement checklist on which patients recorded supplements consumed. In each visit, compliance was assessed by the supplement checklists and by counting the return of uneaten supplements.
The main outcome of the trial was the percentage of patients with an improvement of at least one stage of IBS disease from baseline to the sixth week of intervention. To assess IBS severity, the IBS symptom severity scale (IBS-SSS) was used. It was filled out by patients pre- and post-intervention. The IBSSS questionnaire included five clinically applicable items over a 10-day period include: Ι) the abdominal pain intensity, ΙΙ) the frequency of abdominal pain, ΙΙΙ) the abdominal distension intensity, IV) dissatisfaction with bowel movements, and V) potential impact of IBS on the patient’s daily life. The mean score of each scale is a maximum of 100 and the questionnaire total score reaches a maximum of 500, eventually. Scores of <75, 75-175, 175-300, and ≥ 300 displayed mild, moderate, and severe stages of the IBS disease, respectively (20).
The secondary outcomes of the trial were change in IBS-quality of life (IBS-QoL), anxiety state, body mass index (BMI), and waist circumference (WC) to the sixth week of intervention. Patients’ quality of life (QoL) was assessed using the 34-item IBS-QoL questionnaire which consists of 8 subscales including (a) food avoidance, (b) dysphoria, (c) body image, (d) interference with activity, (e) health worry, (f) sexual, social reaction, and (g) relationships.(21) Participant responses to all the 34 items were summed and then transformed to a 0-to-100 scale. The Beck anxiety inventory (BAI) was used to assess patients’ anxiety status. BAI is a 21-item scale validated as an anxiety screening questionnaire based on Fydrich et al (22). Each item expresses one of the symptoms of anxiety commonly experienced by patients who are clinically anxious or in anxious conditions. The questionnaire scores ranged from 0 to 63. The anxiety state was classified as minimal (scores range from 0 to 7), mild (scores range from 8 to 15), moderate (scores range from 16 to 25), and severe (scores range from 30 to 63) (23). Due to the anxiety is a potential confounder for the study it was assessed before and after the intervention and its effect was adjusted at the end of the trial by the statistical analysis. Weight was measured close to 0.1 kg by a calibrated scale (Seca, Hamburg, Germany) with light clothes and no shoes. Height was measured close to 0.1 cm by an audiometer (Seca, Hamburg, Germany). Then, BMI was calculated as weight (kg)/height2 (m). WC was measured as the smallest circumference between the costal and iliac crests using a non-stretchable measuring tape to the nearest 0.1 cm.
Confounding factors assessment:
Dietary intake was appraised by a three-day food record (two nonconsecutive weekdays, and one weekend) before and after the intervention. Dietary intakes were assessed by the Nutritionist IV software. A validated international physical activity questionnaire-short form (IPAQ-SF) was used for evaluating the physical activity of the patients at baseline and the endpoint. Responses were converted to Metabolic Equivalent Task minutes per week (MET-min/ week). It consisted of 7 questions that will collect all types of physical activity as part of daily life (24).
Statistical analysis was conducted using IBM SPSS Statistics software, version 16 (SPSS Inc., and Chicago, IL, USA). The sample size was 28 patients in each group by assuming a between-group difference of 25% points in the main outcome (19) on the basis of a two-sided significance level of 5%, a power of 80%, and a withdrawal rate of 30% with the use of A'Hern's single-stage phase II methodology (25).
According to the patterns of missing data, a suitable multiple imputation approach followed for completing missing data. The authors checked the data entry double times.
Data were presented as mean (SD) for numerical data, frequency (percentage) for categorical variables, and median (25th, 75th) for values with skewed distribution. For evaluating the differences between the 2 groups at baseline, independent samples t-test or Mann-Whitney U test were used for values with normal and non-normal distribution, respectively. Paired samples t-test and Wilcoxon signed-rank test were used for assessing within-group changes, as appropriate. To judge between-and within-group differences of qualitative variables, Fisher's exact test and Sign test were applied, respectively. For adjusting the confounding factors the analysis of covariance (ANCOVA) test was used. In this study two separate models were used to achieve the goal. Model 1 included baseline values, and model 2 included the model 1, and changes in physical activity, and energy intake. P values under 0.05 were observed as statistically significant. The binary logistic regression was used to calculate the odds of achieving the main outcome with propolis supplementation in both crude and adjusted models. Further details of the study method are presented in the protocol article of this study (17).