Effects of Propolis Supplementation on The Severity of Disease In Irritable Bowel Syndrome Subjects: A Randomized, Double-Blind Clinical Trial

Abstract

The effects of propolis, a well-known functional food, on irritable bowel syndrome (IBS), a chronic gastrointestinal disorder, in humans have yet to be investigated. This study evaluated propolis effects in IBS subjects. In this clinical study, 56 patients with IBS diagnosed by Rome IV criteria were assigned for 6 weeks randomly to the study groups. At the baseline and endpoint phase, patients’ gastrointestinal symptoms, quality of life (QOL), anxiety state, dietary intakes, and anthropometric indices were assessed. Independent t-test, paired t-test, Mann-Whitney U test, Wilcoxon, Fisher's exact test, repeated measures analysis of variance and logistic regression test were used for analyzing the data. To adjust the effect of confounders, covariance analysis was used. The results of this study showed that after modulating the effect of potential confounders, propolis supplementation increased the chance of improving IBS severity by 6.22 (with a confidence interval of: 1.33 - 1.14 and P = 0.035). A significant abdominal pain improvement, anxiety state, and bowel habits dissatisfaction reduction was observed within- and between-group differences in propolis group compared to the placebo group (P = 0.040, P = 0.035, P = 0.029, retrospectively). The overall score of quality of life and its domains in the propolis group was statistically significant, but in comparison between the two groups, this difference was not significant. Also, regards to the food intakes and anthropometric indices, there were no significant differences between and within the two study groups. This study illustrated that propolis supplementation could be used as adjunctive therapy in IBS disease to reduce abdominal pain and anxiety state.

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Effects of Propolis Supplementation on The Severity of Disease In
Irritable Bowel Syndrome Subjects: A Randomized, Double-Blind
Clinical Trial
Mahsa Miryan
Tabriz University of Medical Sciences
Pejman Alavinejad
Ahvaz Jundishapur University of Medical Sciences
Mohammadreza Abbaspour
Mashhad University of Medical Sciences
Davood Soleimani
Kermanshah University of Medical Sciences
Alireza Ostadrahimi (  Ostadrahimi@tbzmed.ac.ir )
Tabriz University of Medical Sciences
Research Article
Keywords: Propolis, Irritable bowel syndrome, IBS-QOL, IBS-SSS, Dietary intake, Anxiety state
DOI: https://doi.org/10.21203/rs.3.rs-144646/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License

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Abstract
The effects of propolis, a well-known functional food, on irritable bowel syndrome (IBS), a chronic gastrointestinal disorder, in
humans have yet to be investigated. This study evaluated propolis effects in IBS subjects.In this clinical study, 56 patients with IBS
diagnosed by Rome IV criteria were assigned for 6 weeks randomly to the study groups. At the baseline and endpoint phase, patients’
gastrointestinal symptoms, quality of life (QOL), anxiety state, dietary intakes, and anthropometric indices were assessed.
Independent t-test, paired t-test, Mann-Whitney U test, Wilcoxon, Fisher's exact test, repeated measures analysis of variance and
logistic regression test were used for analyzing the data. To adjust the effect of confounders, covariance analysis was used. The
results of this study showed that after modulating the effect of potential confounders, propolis supplementation increased the
chance of improving IBS severity by 6.22 (with a condence interval of: 1.33 - 1.14 and P = 0.035). A signicant abdominal pain
improvement, anxiety state, and bowel habits dissatisfaction reduction was observed within- and between-group differences in
propolis group compared to the placebo group (P = 0.040, P = 0.035, P = 0.029, retrospectively). The overall score of quality of life
and its domains in the propolis group was statistically signicant, but in comparison between the two groups, this difference was not
signicant. Also, regards to the food intakes and anthropometric indices, there were no signicant differences between and within the
two study groups. This study illustrated that propolis supplementation could be used as adjunctive therapy in IBS disease to reduce
abdominal pain and anxiety state.
Introduction
Irritable bowel syndrome (IBS) is a common functional gastrointestinal (GI) disease that is manifested by recurrent abdominal pain
and altered bowel addiction (1). No specic markers or laboratory parameters are available yet to diagnose the disease. Recently in
clinical practice, the Rome IV criteria have been proposed as the latest diagnostic tool for IBS, based on the GI symptoms (2). IBS is
estimated to affect women approximately three times more than men, with an overall prevalence of 10% (3, 4).
The pathophysiology of IBS is not well known, but several factors have been attributed to an individual’s susceptibility to IBS
including the alterations in gut microbiota, brain-gut interaction, motility or/and permeability, and intestinal immune system function;
GI microscopic inammation; psychological stress; chronic infections; specic nutrients and foods; and genetic factors (5). Recent
investigations also reveal the role of inammatory and oxidative stress factors in increasing nervous system sensitivity and
perception of abdominal pain in IBS subjects (3).
Various strategies are recommended to improve or even treat IBS symptoms but, unfortunately, often with little success so far (6).
The initial strategy would be to base the prohibition of consuming gas-producing foods by following a diet low in FODMAPs. But if
this is not helpful, the consumption of these special foods should not be avoided for a long time (7). Emerging evidence has shown
an important role of the modulating GI immune system and gut microbiota using prebiotic and/or probiotic supplements in
ameliorating the symptoms of IBS, which has been benecial for many patients (8). Dietary polyphenols and their secondary
metabolites also have a crucial role in maintaining the balance of the GI microbiome by altering bacterial metabolites that can raise
mucin gene expression that resulted in an increase in the thickness of the GI mucosal layer and also reduction in GI inammation (9).
Propolis, a popular traditional medicine, is a resinous hive product collected by honeybees from varied petals and plant buds sources
(10). With the advent of new methods such as high-performance liquid chromatography (HPLC), more than 300 types of
phytochemicals have been identied in this hive product, mainly from the family of polyphenols. They are secondary plant
metabolites with well-known antioxidant properties (11). Recent studies have been shown that propolis, due to the unique diversity of
its components (especially polyphenols), not only has antioxidant effects but also can modulate the inammatory pathways,
immune system function, gut microbiota, and GI permeability (12-15).
Considering the wide range of probable causes and symptoms in IBS patients which propolis may modify; we aimed to evaluate the
ecacy of propolis supplementation on the severity of gastrointestinal symptoms, dietary intakes, anxiety state, and quality of life in
patients with IBS.
Materials And Methods
Study design and subjects

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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 identied as an IBS patient if he/she had at least two of the following
criteria: 
I. Improvement with defecation
II. Onset related to a change in stool frequency
III. Onset related to exchange in stool form (appearance)
Enrolled participants
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 ll 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.
Excluded participants
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, benets, 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 stratication 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.
Supplementation
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 avonoids), 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 microora, a period of six weeks is adequate to boost intestinal microora 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 ll 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.
Primary Outcome

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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 lled
out by patients pre- and post-intervention. The IBSSS questionnaire included ve 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).
Secondary outcomes
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classied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
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 signicance 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 signicant. 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).

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Results
3.1. General characteristics of the trial
Between September 2019 and January 2020, a total of 168 patients were enrolled in the trial and were screened, of whom 56 patients
met eligibility criteria and underwent randomization (28 patients to the propolis group and 28 patients to the placebo group). A total
of51 patients (26 patients in the propolis group and 25 patients in the placebo group) completed this trial while 5 patients (3 patients
in the placebo group and 2 patients in the propolis group) discontinued the study for a reason unrelated to the trial treatment and 1
patient in the propolis group discontinued the study because of a drug-related adverse event (abdominal distention). The trial
owchart is shown in Figure 1. There was no signicant differences in terms of compliance rates between the propolis and placebo
groups at the end of the trial (93% for propolis vs. 90% for placebo; P: 0.73).
3.2. Demographic characteristics
The baseline demographic characteristics of the participants in both groups are shown in Table 1. Prior to the intervention, there
were no statistically signicant differences between the two groups in terms of gender, marital status, education levels, occupational
status, physical activity levels (metabolic equivalents), IBS subtypes, duration of IBS symptoms, and anxiety state.
Dietary intakes,anthropometric indices, and physical activity
As observed in Table 2, caffeine and lactose intake had a signicant change throughout the study in both groups, whereas other
nutrients did not signicantly change. However, these changes were related to the effect of time and were not the effect of the
intervention. The intakes of energy, macronutrients, lactose, and caffeine had no signicant changes from baseline to the end of the
trial in both groups (P >0.05). None of the participants in the present study reported alcohol consumption at the beginning and during
the study. Also, there were no signicant changes in terms of weight, BMI, WC, and physical activity (METs) in the both group as
observed in Table 3.
3.3. Severity of IBS
As shown in Table 5, overall scores of IBS symptoms severity and all its components scores signicantly reduced in the propolis
group at the end of the trial (P <0.05). In the placebo group, the scores of severity of abdominal distention decreased signicantly
while other components did not change at the end of the trial (P <0.05). There were signicant between-group differences in the
severity of abdominal pain and dissatisfaction with bowel habits after adjusting the potential confounders based on model 2.
The changes in the grade of IBS are illustrated in Figure 2. At the end of the trial, the grade of IBS decreased in 21 patients (80.7%)
and had no change in 5 (19.3%) who received propolis supplements. The improvement in the grade of IBS was statistically
signicant in the propolis group (P=0.001). In the placebo group, the grade of IBS decreased in 13 (52%) patients; had no changes in
11 (44%) patients, and increased in 1 (4%) patient. The improvement in the grade of IBS was not signicant in the placebo group
(P=0.501). Mann-Whitney U test showed that the proportion of patients experienced reduction in the IBS severity by at least one grade
was signicantly higher in the propolis group than the placebo group (80.7% vs. 52% P=0.015). In addition, the adjusted odds of
improvement of IBS was 6.22 (95% CI: 1.14 to 33.9; P=0.035) with propolis treatment as compared with placebo.
3.4. Quality of life
As shown in Table 4, there were no signicant between-group changes for the total QOL-IBS and its components scores after
adjusting the potential confounders based on model 1 throughout the study, except for body image. In model 2 after adjusting the
potential confounders no signicant between-group changes were observed. The signicant within-group reduction in the QOL-IBS
and its components scores were observed in the propolis group, compared to the placebo group post-intervention (P<0.05).
3.5. Anxiety
Propolis supplementation decreased the frequency of severe anxiety in IBS patients from 23.07% to 7.69%. Additionally, it raised the
frequency of minimum anxiety from 23.07% to 34. 61%, and mild anxiety from 30.76% to 46.15% in the propolis group. Also, another
nding of this study was that there was a signicant reduction in patients’ anxiety in the propolis group versus the placebo group

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throughout the study (16.88 to 11.19 versus 17.68 to 16.44; P=0.040). It was also found that the anxiety score of patients’ within-
group signicantly decreased in the propolis group contrary to the placebo group (P=0.002, P=0.462, retrospectively).
Discussion
The current trial revealed that supplementation with 900 mg/day of propolis for 6 weeks signicantly improved anxiety state, some
clinical symptoms of IBS in patients with IBS (abdominal pain, dissatisfaction with bowel habits). However, it had no signicant
effects on dietary intakes, anthropometric indices, quality of life, and physical activity levels. The current trial is the rst study
evaluating the effects of propolis administration in patients with IBS.
Our results showed that propolis had no signicant effect on energy intakes and energy-adjusted dietary intakes. In line with our
results, Soleimani et al. reported that the administration of 900 mg/day of propolis for 4 months in patients with non-alcoholic fatty
liver disease (NAFLD) had no signicant effect on energy and dietary intakes (16). In the study of Samadi et al., the administration of
900 mg/day propolis for 3 months did not affect the dietary intakes in the propolis group in type 2 diabetic patients (26). In another
study, consumption of 900 mg/day propolis for 18 months did not affect energy and nutrients intakes in patients with type 2
diabetes mellitus (T2DM).(27) Furthermore, another study showed the consumption of a high dose of propolis (1500 mg/day)
through 8 weeks did not affect energy and nutrients intakes in patients with T2DM (28).
Our results also showed that propolis had no signicant effect on weight, BMI, and WC after adjusting the potential confounders
such as changes in energy intakes and physical activity levels. In line with our results, the study of Soleimani et al. showed no
signicant effect of propolis administration on weight, BMI, body fat mass, and fat free mass in patients with NAFLD (16). Likewise,
Mujica et al. reported that daily consumption of 30 drops of 3% propolis extract for 3 months did not affect weight, BMI, and WC (29).
Another research showed that a daily supplement of 1500 mg of propolis for 8 months did not affect body weight and BMI (30).
Conversely, Samadi et al. reported that propolis administration (900 mg/day for 3 months) in T2DM reduced weight and BMI, while it
did not affect WC (26). This inconsistency can be effect of confounders such as changes in dietary intakes and physical activity
levels throughout the study of Samadi et al (26).
Our results also showed that propolis had no signicant effect on IBS quality of life in patients with IBS after adjusting the potential
confounders. Contrary to our ndings, Pessolato et al. reported receive 5% propolis ointment administered daily for mices with grade
2 burns on the burn sites for 21 days improved their QOL (31). Also, the study of Samet et al. showed that 500 mg/day of propolis for
6 months can improve the QOL of patients with recurrent aphthous stomatitis in the oral cavity (32).
Our results showed that propolis administration improved anxiety state in patients with IBS after adjusting the potential confounders.
The anxiolytic effects observed in propolis might be related to its terpenoids compounds, which can reduce adrenocorticotropic
hormone (ACTH) levels and subsequently reduce cortisol levels and increase the activity of the body's own antioxidant defense
system, which ultimately strengthens the antioxidant system in Brain tissue is through the hypothalamic-pituitary-adrenal axis (33).
Our results showed that propolis administration improved the IBS (score and grade) in patients with IBS after adjusting the potential
confounders. Recent systematic review of pre-clinical studies concluded that propolis intake might have benecial effects on many
aspects of clinical, macroscopic, and histological features of colitis (34). Nonetheless, limited studies examined the effects of
propolis on the severity of inammation-based diseases in humans. A human study showed propolis decreased the severity of pain
related to oral mucositis (35). Propolis by its anti-inammatory effects could reduce the inammation related to IBS disease, and
based on this study ndings it can reduce the severity of abnormal pain and dissatisfaction with bowel habits. Non-specic propolis
extract is involved in the immune response by activating macrophages, which does this by releasing hydrogen peroxide and
inhibiting the production of nitric oxide (dose-dependent effect), which can be affected by its effect on Inhibition of inducible nitric
oxide synthase (iNOS) gene expression and iNOS catalytic activity is justied (36, 37). Laboratory studies have shown the inhibitory
effect of propolis on free radicals (38, 39). Some of the specic effects shown by the aqueous form of propolis include an inhibitory
effect on platelet aggregation, an inhibitory effect on the synthesis of prostaglandins in vitro, and inhibition of 5-lipoxygenase (5-
LOX) (40-42). Studies have also shown that alcoholic propolis extract inhibits transcription of the iNOS gene through its effect on
Nuclear factor kappa B (NF-κB) sites in the NF-κB promoter, which is dose-dependent (43). Also, alcoholic extract of propolis can
interfere with inammatory response mechanisms, which has a very important effect on controlling cellular epithelial function (44).

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Propolis could also reduce the inammatory cytokines (45). Propolis compounds such as caffeine phenyl ester inhibit NF-κB activity
(Figure 3) (46).
The current trial had some strength. One of the most important strengths of this study was that it conducted on the IBS patients
diagnosed by Rome IV criteria which is the newest tool for IBS diagnosis; also using stratied block randomization with a block size
of 4 (based on IBS subtypes and sex) led to the distribution of features between the study groups and the other strengths of this
study were the high compliance rateofpatientsto the treatment in each group.However, this trial had a few limitations including
self-reporting of physical activity and dietary intakes.
Conclusions
The present trail, for the rst time, revealed that supplementation with 900 mg/day propolis for 6 weeks could signicantly improve
abdominal pain, decrease dissatisfaction with bowel habits as well as the reduction in the anxiety state. Although, propolis
supplementation had no effect on IBS-QOL, dietary intakes, and anthropometric indices. This trial suggested that propolis could be
used as adjunctive therapy in IBS disease to reduce abdominal pain and the anxiety of IBS patients. Further RCT studies on the effect
of propolis supplementation on gut microbiome of IBS patients are suggested to achieve valid data in the management of IBS.
Abbreviations
ANCOVA: Analysis of covariance; BAI: Beck anxiety inventory; BMI: Body mass index; BSFS: Bristol stool form scale; GI:
Gastrointestinal; HPLC: High-performance liquid chromatography; IPAQ-SF: International physical activity questionnaire-short form;
IBS: Irritable bowel syndrome; IBS-C: Constipation subtype of IBS; IBS-M: Mixed subtype of IBS; IBS-QOL: Irritable bowel syndrome
quality of life; IBS-SSS: Irritable bowel syndrome symptom severity scale; iNOS; Inducible nitric oxide synthase; MCC: Microcrystalline
cellulose; METs: Metabolic equivalents; NF-kB: Nuclear factorkappa-light-chain-enhancer of activated B cells; OM: Oral mucositis;
QOL: Quality of life; WC: Waist circumference.
Declarations
Authorship
MM, AO, PA, DS, and MA designed research and contributed to the conception of the project, development of the overall research
plan, and study oversight. MM drafted the manuscript and analyzed and interpreted the data. MM and DS were the statistic
counselors and PA diagnosed the patients with Rome IV criteria. MM was involved in the sampling and data collection. All authors
have given nal approval of the version to be published.
Declaration of competing for interest
All authors declare that there is no conflict of interest.
Acknowledgment
We sincerely thank the patients who participated in the present study.
Funding
The study was nancially supported by the Nutrition Research Center of Tabriz University of Medical Sciences. The funder is not
involved in the study design, collection, management, analysis, and interpretation, writing of the manuscript and the decision to
submit the report for publication, including whether they will have ultimate authority over any of these activities. This is based on the
data obtained from an MSc dissertation of rst author (grant number: 63292) submitted to Tabriz University of Medical Sciences.
Ethics and Approval for human experiments:
This project was found to be in accordance with the ethical principles and the national norms and standards for conducting Medical
Research in Iran. Evaluated by Tabriz University of Medical Sciences. Approval ID: IR.TBZMED.REC.1398.473. In the study, we used
the Rome IV questionnaires for IBS disease (the Rome IV diagnostic questionnaire for IBS disease, Persian versions of IBS-SSS, and

Page 8/15
IBS-QOL questionnaires) after obtaining correspondence and authorization from Rome Foundation. The questionnaires were
provided to the researchers under a contract.
The present trial was approved by the Ethics Committee of Tabriz University of Medical Sciences with ethics code:
IR.TBZMED.REC.1398.473 and was registered in the Iranian registry of clinical trials (https://en.irct.ir/trial/40983, registration date:
26/12/2019, registration number: IRCT20190708044154N1).
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Tables

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Table1. Baseline characteristics of the study participants in the both groups.
Variables Propolis group
(N = 26)
Placebo group
(N = 25)
P-value
Age; years 38.92 ± 12.65 44.92 ± 12.10 0.090†
Females; n (%) 13 (50%) 14 (56%) 0.781‡
Married; n (%) 19 (73.07%) 23 (92%) 0.076‡
Academic Education; n (%) 10 (38.46%) 11 (44%) 0.885‡
Employee; n (%)  20 (77%) 20 (80%) 0.761‡
METs; minutes/week 691.50 [196.00 - 1629.00] 360.00 [173.25 - 1768.00] 0.883†
IBS Duration; years 12.32 ± 10.5 8.31 ± 7.36 0.366†
IBS Type IBS-C; n (%)  19 (73.07%) (68%) 17 0.764‡
IBS-M; n (%)  7 (26.92%) (32%) 8
Anxiety Normal; n (%)  6 (23.1%) 6 (24%) 0.952‡
Mild; n (%)  8 (30.8%) 6 (24%)
Moderate; n (%)  6 (23.1%) 6 (24%)
Severe; n (%)  6 (23.1%) 7 (28%)
Abbreviations. IBS, Irritable bowel syndrome; IBS-C, Constipation subtype of IBS; IBS-M, Mixed subtype of IBS; METs, Metabolic
equivalents
Physical activity level are presented as median [25th, 75th]. Age and duration of IBS are presented as mean±SD; other variables
are presented as number (%).
† Values were obtained from independent samples t-test.
‡ Values were obtained from Chi-square test.
            

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Table 2. Energy-adjusted nutrients intake across study follow-up
Variables Propolis group  Control group  Effect
week0week3week6Paweek0week3week6PaPbPcPd
Energy (Kcal) 1200
(389.01) 1263
(391.47) 1287
(454.7) 0.665 1373
(475.28) 1251
(533.31) 1175
(530.36) 0.366 0.851 0.81 0.240
Protein (g) 52.54
(18.44) 54.72
(17.41) 55.19
(17.19) 0.298 60.17
(21.38) 54
(23.47) 51.06
(20.39) 0.801 0.801 0.667 0.251
Fat (g) 22.25
(9.97) 25.34
(5.59) 26.14
(7.04) 0.233 26.60
(10.75) 25.11
(7.54) 24.19
(7.34) 0.580 0.742 0.945 0.164
Carbohydrate
(g) 119.95
(61.65) 210.04
(63.55) 210.97
(81.53) 0.744 228.49
(77.99) 207.99
(87.78) 191.24
(96.77) 0.305 0.871 0.405 0.238
Soluble Fiber
(g) 270
(130) 290 (10) 320
(110) 0.166 430
(170) 290 (30) 290
(110) 0.178 0.624 0.423 0.134
Insoluble
ber (g) 1560
(1440) 1590
(50) 1770
(690) 0.65 2430
(2050) 1590
(90) 1550
(705) 0.052 0.258 0.149 0.051
Fructose
(mg) 6090
(1950) 6860
(590) 7520
(1610) 0.223 6870
(2250) 6950
(1045) 7125
(1920) 0.863 0.625 0.980 0.101
Lactose (mg) 3250
(840) 2970
(520) 3920
(1680) 0.004 3680
(1030) 2760
(590) 3510
(1810) 0.023 0.974 0.001 0.172
Caffeine
(mg) 570
(340) 1090
(850) 180 (1) 0.001 870
(520) 1020
(1090) 180 (30) 0.003 0.66 0.001 0.46
Values are expressed as mean (SD).
a Paired sample
t
-test.
b P-intervention for comparing within each group trend, Repeated-measure analysis of variance test.
c P-time, Repeated-measure analysis of variance test.
d P-interaction, Repeated-measure analysis of variance test.
Table 3. Adjusted mean changes in anthropometric parameters in the both groups.
Variables Group Before After P-value†Changes‡P-value‡
Weight; kg Propolis 72.10 ± 13.65 72.28 ± 13.84 0.717 0.114 ± 0.094 0.677
Placebo 75.64 ± 15.08 75.61 ± 15.08 0.939 0.063 ± 0.102
BMI; kg/m2Propolis 25.61 ± 4.00 25.58 ± 3.96 0.711 -0.088 ± 0.028 0.775
Placebo 27.75 ± 5.85 27.73 ± 5.81 0.877 0.023 ± 0.031
WC; cm Propolis 85.94 ± 15.77 87.28 ± 2.45 0.711 1.34 ± 0.245 0.593
Placebo 95.20 ± 4.00 94.96 ± 2.74 0.784 -0.23 ± 0.268
Abbreviations. WC: Waist circumference; BMI: Body mass index
Dataare presented as mean ± standard deviation.
† Values were obtained from Paired- sample t-test.
‡ Values were obtained from ANCOVA test with baseline values and changes in physical activity and energy intake as covariates.


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Table 4. Quality of life of IBS participants throughout the study.
Variables  Propolis (n = 26) Placebo (n = 25) Mean Difference (95 % CI), P
Dysphoria

Baseline 59.12 (31.03) 59.61 (28.37) -0.049 (-16.23,17.21), 0.953b
6weeks 72.75 (22.52) 64.54 (25.04) 8.21 (-21.62,5.21), 0.225b, 0.813c,
0.404d
MD (95 %
CI),Pa
 13.6 (-22.11, -5.13),
0.003 1.69 (-14.66, 4.80),
0.307 
Interference with
activity Baseline 55.85 (25.20) 59.34 (21.40) -3.49 (-9.65, 16,62), 0.597b
6weeks 67.42 (24.55) 54.31 (18.95) 13.11(-25.43, -0.79), 0.037b, 0.424c,
0.932d 
MD (95 %
CI),Pa
11.57 (-19.9, -4.05),
0.004 -5.02 (-0.42, 10.48),
0.069 
Body image

Baseline 70.75 (28.28) 73.63 (13.10) -2.88 (-9.44, 15.21), 0.640b
6weeks 80.89 (19.49) 69.71 (13.62) 11.18 (-20,60, -1.74), 0.021b, 0.039c,
0.193d
MD (95 %
CI),Pa
10.14 (-15.81, -4.46),
0.001 3.91 (-1.24, 9.07),
0.130- 
Health worry Baseline 45.66 (29.96) 47.75 (25.77) -2.09 (-13.61, 17.79), 0.790b
6weeks 63.33 (27.42) 48.39 (23.69) 14.94 (-30.73, -3.14), 0.017b,0.243c,
0.824d
MD (95 %
CI),Pa
17.66 (-30.33, -9.26),
0.001 0.64 (-9.21, 7.68),
0.853 
Food avoidance Baseline 53.33 (30.80) 53.83 (21.38) -0.5 (-14.89, 15.91), 0.947b
6weeks 64 (22.29) 57.37 (24.41) 6.63 (-21.18, 7.93), 0.365b, 0.876c,
0.645d
MD (95 %
CI),Pa
10.66 (-20.32, -1.01),
0.032 3.52 (-12.95, 5.90),
0.449 
Social reaction Baseline 70.01 (21.77) 73.55 (21.38) -3.54 (-8.59, 15.69), 0.560b
6weeks 82.26 (14.40) 74.17 (13.54) 8.09 (-15.95, -0.22), 0.044b, 0.415c,
0.423d
MD (95 %
CI),Pa
12.25 (-17.73, -6.76),
0.0001 0.61 (-7.23, 6,00),
0.850 
Sexual Baseline 72.50 (31.04) 75 (29.36) -2.5 (-14.49, 19,49), 0.769b
6weeks 77 (22.44) 69.23 (31.07) 7.77 (-23.07, 7.53), 0.313b, 0.679c,
0.705d
MD (95 %
CI),Pa
4.5 (-14.13, 5.13), 0.345 5.76 (-6.11, 17.65),
0.327- 
Relationships Baseline 56 (29.41) 58.65 (22.54) -2.65 (-12.05, 17,36), 0.179b
6weeks 66 (23.80) 60.89 (21.57) 5.11 (-17.87, 7.67), 0.426b, 0.803c,
0.217d

Page 14/15
MD (95 %
CI),Pa
10 (-17.75, -2.24), 0.014 2.24 (-9.40, 4.91),
0.524 
Overall score Baseline 59.90 (24.77) 62.10 (18.31) -2.2 (-10.02, 14.42), 0.719b
6weeks 71.96 (19.24) 62.08 (17.63) 9.88 (-10.13, 14.53), 0.062b, 0.565c,
0.708d
MD (95 %
CI),Pa
12.05 (-18.35, -5.75),
0.001 0.26(-5.87, 5.83),
0.993 
IBS, Irritable bowel syndrome.
Mean (SD) and Mean Difference (95 % CI) are presented for data.
a Paired samples
t
-test.
b Independent samples
t
-test.
c ANCOVA test, adjusted for baseline values (Model 1).
d ANCOVA test, adjusted for baseline values, changes in physical activity, and energy intake (Model 2).

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Table 5. IBS symptoms severity score of participants throughout of study.
Variables  Propolis (n = 26) Placebo (n = 25) Mean Difference (95 % CI), P
Severity of abdominal
pain Baseline 55.76 ± 28.16 55.60 ± 35.55 0.16 (-17.23, 17.57); 0.984b
6weeks 33.07 ± 26.94 53.60 ± 29.98 - 20.53 (-36.55, -4.49); 0.013b,
0.020c, 0.040d
MD (95%
CI); Pa
-22.69 (-36.18, -9.19);
0.002 -2.00 (-7.38, 11.38);
0.664 
Frequency of abdominal
pain Baseline 5.15 ± 3.35 5.08 ± 3.39 0.07 (-1.82, 1.97); 0.938b
6weeks 3.07 ± 3.22 4.80 ± 2.76 -1.73 (-3.41, -0.02); 0.046b, 0.150c,
0.153d
MD (95%
CI); Pa
-2.70 (-3.80, -0.34);
0.021 -0.28 (-1.23, 0.67);
0.552 
Severity of abdominal
distention Baseline 64.61 ± 32.02 63.60 ± 22.70 1.01(-14.66, 16.69); 0.897b
6weeks 42.69 ± 25.22 48.80 ± 19.64 - 6.11(-18.86, 6.65); 0.341b, 0.327c,
0.305d
MD (95%
CI), Pa
- 21.92 (-32.82, -11.02);
0.001 -14.80 (-25.79, 3.80);
0.010 
Dissatisfaction with
bowel habits Baseline 60.00 ± 26.83 64.40 ± 26.93 -4.40 (-19.53, 10.73), 0.562b
6weeks 42.69 ± 23.24 64.00 ± 24.15 -21.31 (-34.64, -7.96), 0.002b,
0.006c, 0.035d
MD (95 %
CI), Pa
-17.30 (-27.56, -7.05),
0.002 -0.40 (-12.34, 13.14),
0.949 
Interference with quality
of life

Baseline 46.92 ± 35.52 55.60 ± 29.45 -8.68 (-27.07, 9.72), 0.348b
6weeks 34.23 ± 28.16 53.60 ± 24.81 -19.37 (-34.32, -4.40), 0.012b,
0.023c, 0.207d
MD (95 %
CI), Pa
-12.69 (-24,59, -0.78),
0.038 -2.00 (-18.25, 14.25),
0.802 
Overall score Baseline 288.84 ± 111.40 290.00 ± 108.97 -1.16 (-73.19, 50.88), 0.719b
6weeks 183.46 ± 106.46 268.00 ± 81.54 -84.54 (-138.06, -31.01), 0.003b,
0.008c, 0.036d
MD (95 %
CI), Pa
-95.38 (-139.89,
-50.87), 0.0001 -22.00 (-60.74,
16.74), 0.253 
Abbreviation. IBS, Irritable bowel syndrome
Mean (SD) and Mean Difference (95 % CI) are presented for data.
a Paired samples
t
-test.
b Independent samples
t
-test.
c ANCOVA test, adjusted for baseline values (Model 1).
d ANCOVA test, adjusted for baseline values, changes in physical activity and energy intake (Model 2).
