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

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 condence interval of: 1.33 - 1.14 and P = 0.035). A signicant 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 signicant, but in comparison between the two groups, this difference was not signicant. Also, regards to the food intakes and anthropometric indices, there were no signicant 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. 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 inammation; psychological stress; chronic infections; specic nutrients and foods; and genetic factors (5). Recent investigations also reveal the role of inammatory and oxidative stress factors in increasing nervous system sensitivity and perception of abdominal pain in IBS subjects (3). confounder for the study it was assessed and after the intervention and its effect was at the end of the trial the statistical was measured close to 0.1 kg by a calibrated scale (Seca, Hamburg, with light clothes and no shoes. Height was 0.1 by an audiometer (Seca, BMI was calculated as weight (kg)/height 2 (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. NF-κB activations, whereas avone, isorhamnetin, naringenin, and pelargonidin inhibit only NF-κB activation along with their inhibitory effect on iNOS expression and NO production in activated macrophages.


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)(13)(14)(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.

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 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
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 21item 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)/height 2 (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 questionnaireshort 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).

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).

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.

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.

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).

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 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' withingroup 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 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)(41)(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).
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.

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 IBS-QOL questionnaires) after obtaining correspondence and authorization from Rome Foundation. The questionnaires were provided to the researchers under a contract.      Figure 1 Study ow of enrolment, allocation, intervention, and assessment.

Figure 2
The changes in the severity of IBS from baseline to 6-week intervention in the propolis and placebo group. * Within-group comparisons with the use of a Wilcoxon rank-sum test showed a signi cant improvement in the degree of IBS in the propolis group from baseline to 6-week intervention. Mann-Whitney U test showed that the improvement in the degree of IBS in the propolis group was signi cantly higher than the placebo group (P value=0.015).