Effect of bread fortification with pomegranate peel powder on metabolic profile in Type 2 Diabetes Mellitus patients: A randomized blind control trial

doi:10.21203/rs.3.rs-4976250/v1

Background

Among the leading causes of mortality in the world is type 2 diabetes (T2DM), and diet plays an important role in T2DM risk factors. A primary objective of this study was to explore the effects of pomegranate peel powder (PPP) fortified bread on glycemic control, lipid profile, and anthropometric indicators in patients with type 2 diabetes (T2DM).

Methods

The PPP was added to the bread by adding 0, 1.5, 2.5, 3.5, and 5 percent. We measured phenol content using Folin-Ciocalteu and antioxidant activity using DPPH (2, 2-diphenyl-1-picrylhydrazyl). Ninety participants were allocated to a randomized parallel controlled intervention study and the effect of consuming 100 g/day of bread with PPP (3.5%) on glycemic markers, lipid profile, and anthropometrics was assessed.

Results

No significant effects of bread with PPP intake compared with the control bread were observed on anthropometric, blood pressure, glycemic, and lipid markers after adjusting age, sex, and change in calorie.

Conclusions

Further investigations are warranted to elucidate the preventive roles of PPP on diabetes metabolic complications.

Trial Registration:

IRCT20191209045672N1 is the trial ID in the Iranian Registry of Clinical Trials. Date of registration 21/09/2020. https://en.irct.ir/trial/48132.

Pomegranate

Diabetes mellitus

Glycemic

Lipid profile

Pomegranates are a valuable source of phytochemicals.
functional foods provide some physiological benefits that direct the food industry.
Functional foods becoming ever more favorite in today’s world due to their safety.
The study's fortification strategy increased bread's total phenolic content and antioxidant capacity by adding Pomegranate Peel Powder.

The prevalence of type 2 diabetes mellitus (T2DM) is rising worldwide, estimated to reach 578 million (10.2%) in 2030 and 700 million (10.9%) in 2045.(1). During 2014–2020, the estimated overall prevalence of diabetes and pre-diabetes were 15% and 25.4% in Iran, respectively(2). In addition to dyslipidemia and hypertension, diabetes increases the risk of cardiovascular complications such as stroke, congestive heart failure, angina, and myocardial infarction. (3).

The cornerstones of chronic disease prevention remain lifestyle and dietary factors (4). A healthy diet can control plasma glucose levels and help prevent the development of T2DM (5).

Over 12,000 years ago, bread was a chief food item. A leavened food derived from the fermentation of wheat flour sugars derived from starch, involving chemical interactions between various ingredients (6). Since bread is a common diet ingredient, it may effectively deliver polysaccharides, fiber, and phenolic antioxidants. (6).

Foods containing high levels of dietary fiber and antioxidants are in high demand due to their health benefits, such as protecting against cancer, cardiovascular disease, and degenerative diseases. (7, 8).

Pomegranates (Punica granatum L., Punicaceae family) are a valuable source of phytochemicals. (9). Some Asian countries and other parts of the world have used it extensively in traditional medicine as a medicinal herb. (10).

Pomegranate peel powder (PPP) and other parts of the fruit contain almost 48 phenolic components (condensed tannins, flavonoids, and hydrolyzable tannins)(11, 12). Pomegranate peel extract improved HbA1c (Glycated Hemoglobin), lipid profile, and blood pressure in a randomized clinical trial by Grabez et al. (13). In another clinical study, pomegranate peel extract decreased lipid levels and blood pressure. (14).

It should be noted that food quality can be improved by fortifying cereals with plant-based and natural compounds, particularly PPP. Although previous studies have documented the benefits of PPP on T2DM, clinical trials of adding PPP to bread products with this dosage, using semi-bulky bread with sourdough have not been conducted. Therefore, the first objective was to formulate a bread fortified with PPP that contains a high level of dietary fiber and phytochemicals (phases 1 and 2). The second aim was to run a randomized, blinded, controlled clinical trial to investigate the effects of daily consumption of bread containing PPP on the lipid profile and glucose levels of patients with T2DM (phase 3).

Phase 1

2.1. Types of Breads:

Twenty-five patients with T2DM were selected to determine the suitable bread content Endocrine and Metabolism Research Center at the IUMS hosted the guests for breakfast over five days. Three types of semi-bulky bread and two flat breads were examined.Two days before baking, the flour was stored at the workshop temperature. The flatbread was cooked at 500 °C for three minutes. Water at a temperature of 20-22°C was used to prepare the dough. A uniform mixture of all dry ingredients was achieved by mixing them and then adding water after they were uniform. A two-hour fermentation was conducted in a closed chamber at a constant temperature and humidity. Water droplets entered the space as steam to adjust the humidity. A viscoelastic dough was obtained by mixing whole wheat flour, rye flour, water, salt, sourdough, sugar, and yeast in determined proportions. The dough was cut into loaves to complete the fermentation process and left in the hothouse for 120 minutes. We baked semi-bulky dough for 20 minutes at 210°C after leaving it for 2 hours at 39°C and 70% relative humidity. After air-cooling for 1 hour, they were packed in polyethylene bags until they reached a temperature of 20°C.

2.2. Blood Sugar Measurement

The patients consumed five types of bread for five consecutive days. The fasting and 2h post-prandial blood sugar levels were measured with a glucometer (Glucocheck, Taiwan) before and after breakfast.

Phase 2

2.3. Preparation of pomegranate peel powder

Iranian pomegranates were obtained from Fars Province. The type of pomegranate was Robab. The pomegranates were manually peeled and dried. The peels were ground and then sifted through a 12-mesh sieve using a grinding machine to achieve a fine powder. For further use, dried products were stored in plastic bags at -20°C.

2.4.Organoleptic Evaluation of Baked Bread

According to the World Health Organization monograph, the recommended oral daily dosage is 2.5 to 4.5 g (15). Twenty-five untrained persons with previous sensory evaluation experience and people with or without diabetes consumed bread containing various levels of PPP (0 %, 1.5, 2.5, 3.5, 5% w/w) for organoleptic analysis. The test was evaluated on a five-point hedonic scale from one (extremely poor quality) to five (remarkably good quality). After testing each sample, they were asked to drink some water to cleanse their mouths. A score sheet was used to record and rate the characteristics of aroma, color, texture, mouth feel, and taste. (16).

2.5. Determination of total phenolic content (TPC)

Folin-Ciocalteu reagent was used to determine TPC in bread with and without PPP. The measurement was performed using a spectrophotometer at 760 nm. The results were expressed in mg gallic acid/g of bread, and all determinations were performed in triplicate. To dry the dough and bread, they were placed in an oven at 40-50 °C. After centrifuging with methanol for 10 minutes, they were placed in a shaker incubator at 200 RPM for 2 hours. After recentrifuging for 10 minutes at 1000 rpm, Whatman paper was used to filter the samples. Following this, 100 µliters of water were mixed with 300 µliters of Folin-Ciocalteu reagent. The absorbance at 760 nm was measured against a blank after 900 µliters of Na2CO3 solution (20 g/100mL) was added and stored in the dark for 30 minutes. Based on the calibration curve of gallic acid, total phenolic contents were estimated and expressed as gallic acid equivalents (GAE), in milligrams per gram (Table 1) (17).

2.6. The antioxidant activity of the samples

In this study, the DPPH technique was used to measure antioxidant activity. Ten milliliters of methanol were used to extract one gram of bread and dough powder. Analyses were conducted on the supernatant. The amount of 1 mL of the supernatant was added to 3.9 mL of ethanol solution of DPPH (6× 10-9 mol/lit) and the mixture was then kept at room temperature. Spectrophotometry was used to measure the absorbance after 30 minutes.

We used the following equation to calculate the DPPH radical scavenging capacity (Table 1).

(eq1): DPPH = [1 – (Absorbance of the sample/absorbance of the control)] × 100

(17-19).

2.7. The chemical composition of breads

Different samples were analyzed for their chemical composition. AOAC methods were used to analyze ash, moisture, protein, fat, and crude fiber (20) (Table 2), while carbohydrate content was determined according to FAO as follows:

(eq2): Carbohydrate % =100 – (crude fiber + protein % + ash % + fat %+ moisture %)

Kjedahl's method was used to measure protein (21). ISIRI (Iranian Institute of Standards and Industrial Research) was used to measure moisture and ash.

Phase 3:

2.8. Study population

Several centers were selected for patient referral for the study. The University Endocrine Metabolic Research Center and the Health Center provided the patients. From May 2021 to January 2023, 90 type 2 diabetic patients of both sexes participated in a blind, randomized clinical trial. Primary outcomes included glycemic indices and lipid profiles. Patients were informed of the purpose and protocol of the investigation, the possible risks and advantages related to the study, the proposed testing, and the study timeframe during the enrollment phase. At the beginning of the study, all participants completed the demographic and general questionnaires.

Those with T2DM who met the following inclusion criteria were eligible to participate:

a) Patients with T2DM diagnosed within the last five years

b) The HbA1c levels of patients have remained at 8.0% over the past three months

c) The participants between the ages of 40 and 60 year

d) Body mass index (BMI) of 30 kg/m²and more

People with liver disease (cirrhosis and hepatitis) or chronic kidney disease, taking hormone replacement therapy, antioxidant supplements, or insulin were excluded.

Those who had changed their physical activity or diet within the past six months, smoking and/or alcohol or drug abuse history, weight loss of over 10%, pregnancy/lactation, use of sulfonylureas, participation in another clinical study, history of malignancy, being on a special diet (e.g., vegetarian or ketogenic), heart disease, other factors that could affect compliance with research procedures, and non-compliance with bread consumption were also excluded from the study. The trial was terminated if patients showed serious problems during the intervention. An endocrinologist involved in the study (MK) discussed any medical conditions or abnormalities with the participants during the intervention. We registered the protocol in the Iranian Registry of Clinical Trials (ID: IRCT20191209045672N1). https://en.irct.ir/trial/48132. Additionally, the current study was described according to the Consolidated Standards of Reporting Trials (CONSORT) statement (22).

2.9. Study Design:

Of the 11,675 cases assessed, only 520 met the inclusion criteria for diabetic patients. Then,112 patients agreed to participate in the study. As previously reported, the original protocol included a three-month intervention period (23). Ninety patients were randomly assigned to two groups after randomization. Forty-five participants were given 100 grams of wheat bread with 3.5% PPP for 12 weeks, and 45 control participants were given free PPP wheat bread for 12 weeks. Based on previous studies, the dosage was chosen (24). During the study, participants did not change their medication regimens, physical activity, or dietary habits. They were instructed to freeze the remaining bread for later use. At the beginning of the study, all participants completed a demographic questionnaire. Weekly phone interviews and routine visits were to monitor compliance with bread intake. Patients needed to consume three bread baguettes (32-35 g) per day for 90 consecutive days. The packaging and size of both types of bread were similar. Consumed bread was replaced with a new amount of bread.

2.10. Anthropometric variables

Standard methods were used to collect anthropometric data. Participants were measured in underwear in the morning by trained personnel. Using a stadiometer (Seca, Hamburg, Germany), the height was measured to the nearest 0.1 cm without footwear. A Seca Beam Balance (Seca, Germany) was used to measure weight to the nearest 0.1 kg with light clothing. Weight/height² (kg/m²) was used to calculate BMI. Using an anthropometric tape, the waist circumference (WC) was measured midway between the lowest rib and the iliac crest.

2.11. Run-in period

The run-in period was conducted for 14 days before the study began. During the run-in period, investigators verified the patients' diets and identified their concerns. Patients consumed wheat bread without PPP during the run-in period. As a part of the run-in period, the volunteers were also required to demonstrate their motivation to comply with the intervention. The compliance of the patient was assessed using diaries since there is no biomarker to measure bread intake.

2.12. Randomization, Allocation Concealment, and Blinding

Computer-generated random numbers were used to generate sequentially numbered envelopes containing balanced assignments to either control or intervention groups at the end of the run-in period. A blind study was designed by labeling control and treatment bread according to the randomization sequence output by an independent investigator not involved in the study design or analysis.

2.13. Dietary intake and physical activity evaluation

At baseline and after the intervention period, participants' physical activity, energy, and nutrient intake were assessed. The quantity and nature of food and drinks consumed by participants were recorded. The questionnaires on physical activity and food records were explained by a trained nutritionist (MZ). Patients were asked to write down their daily beverage and food intake for three days to evaluate their dietary consumption using food records for three non-sequential days (two weekdays and a weekend). For better accuracy, a food album was also used by the researcher (MZ). We excluded patients who consumed less than 1200 calories or more than 4000 calories daily. A customized version of Nutritionist IV software (First Data Bank; Hearst Corp, San Bruno, CA, USA) was used to analyze energy, micro- and macronutrient intakes.

Each item was coded after all measurements were converted to grams (25).

Pre- and post-intervention, participants reported their daily physical activity.

2.14. Biochemical assays

To do laboratory tests, patients who met the inclusion criteria were asked to attend The University Endocrine and Metabolism Research Center between 8:00 a.m. and 10:00 a.m. after 12 hours of fasting. At the beginning and at the end of the study, 10 milliliters of fasting blood were collected. For 15 minutes, the collected samples were centrifuged at 3500 rpm and 25 °C. Until further analysis, the isolated serums were transferred to six sterile microtubes and stored at -80°C. Before and after each intervention period, fasting blood glucose (FBG), insulin, and HbA1c (glycated hemoglobin) were measured. The glucose oxidase technique (Pars Azmun, Tehran, Iran) was used to determine FBG levels. Intra- and inter-assay coefficients of variation were ≤2% for the glucose test. A turbidimetric immunoassay was used to measure HbA1c levels using the BT1500 device (Made in Spain) (26). The serum insulin levels were measured using the ELISA technique (Monobind Inc, Costa Mesa, CA, USA) (27). Homeostatic model evaluation of insulin resistance (HOMA-IR) and beta-cell function (HOMA-B) were calculated using Matthews' equation: HOMA-IR = [(glucose-insulin)/405; Where glucose is fasting glucose (mg/dL) and insulin is fasting insulin (mu/mL)(28). HOMA-B; calculated as [20 × fasting insulin (29)/FG {mmol/L} − 3.5](30). The quantitative insulin sensitivity check index (QUICKI) was used to calculate insulin sensitivity (31): QUICKI = 1/ [log fasting serum insulin (μU/ml) + log fasting plasma glucose (mg/dL)]. We also measured plasma lipids (triglycerides [TG], total cholesterol [TC], LDL-, and HDL-cholesterol) using a photometric assay kit (Pars Azmoun, Tehran, Iran). Intra- and inter-assay coefficients of variation were ≤2% for both TG and TC. The intervention group assignments were blinded to all laboratory skilled workers.

2.15. Blood pressure

Before measurement, all patients were asked not to drink coffee/tea or engage in heavy physical activity for approximately 30 minutes. A sphygmomanometer (Omron Intell Sense, HEM-907XL, Omron Company, Kyoto, Japan) was used to measure blood pressure (BP) on the right arm in the sitting position. We measured the mean values of two consecutive measurements taken at 5-minute intervals.

2.16. Sample size calculation

According to the differences in level TC obtained from the same study, we determined the sample size(14) . We finally reached 45 subjects per group, and 90 participants with T2DM were included in the trial, assuming a type 1 error of 5% and a type 2 error of 20%. (14).

2.17. Statistical analysis

In the clinical trial phase, SPSS software (Version 20; SPSS Inc., Chicago, IL, USA) was used for all statistical analyses. Based on the per-protocol approach, the analysis was carried out. Participant characteristic data as well as mean energy and nutrient intake were recorded at the baseline and presented as means ± SD while categorical variables were presented as frequency (percentage).

Quantitative variables were examined for normality using Skewness and Kolmogorov-Smirnov tests. Descriptive statistics (means ± SDs) were determined for quantitative variables and differences the beginning between the two groups were analyzed by independent t-test. Using paired t-tests, we calculated the mean difference between baseline and end-point outcomes for each group. Multivariate covariance (MANCOVA) test adjusted for sex, age, and calorie intake detected differences between the two groups after 12 weeks of intervention. Pearson's correlation was used to determine the relationship between TPC and antioxidant activity. For further analysis, Tukey's post hoc comparison test was conducted. Differences at p-value ˂0. 05 were considered significant.

2.18. Main outcome

Primary outcomes were glycemic status (FBG, HbA1C, and insulin) and lipid profile (TG, TC, LDL-chol, and HDL-chol). BP levels and anthropometric indices were secondary endpoints.

3.1. Analysis of bread characteristics

Among the 5 types of bread, there were no significant differences in FBG and 2-hour post-prandial levels. Using 3.5% PPP concentration in semi-bulky bread resulted in overall acceptable quality. Bread with PPP 5% reduced bread's taste, texture, color, and overall acceptability. The darker color and bitter taste of PPP may be due to the soluble tannins content.

Table 1 shows the antioxidant activity and total phenol of breads. It is shown that the antioxidant activity of the samples widely varies, between 13.90 ±0.51% and 93.71 ±0.35 %. The TPC in samples was between 27.97±2.3 (mg gallic acid/g) extract and 78.33±3.2 (mg gallic acid/g) extract. The chemical composition of the breads tested is shown in Table 2.

3.2. Analysis of participants characteristics:

77 participants (39 in the treatment group and 38 in the control group) completed the 12-week intervention. Six individuals in the intervention group (due to gastrointestinal discomfort [n=3], COVID-19 strike [n=1] and non-compliance [n=2] and seven individuals in the control group (due to business mission [n=1], COVID-19 strike[n=1], non- compliance[n=2], gastrointestinal discomfort[n=1] and surgery[n=2] were lost to follow up (figure 1).

Group baseline characteristics and medication details are provided in Table 3. In terms of current medications, weight, BMI, WC, and gender, there were no significant differences between the groups. There were no significant differences in dietary intakes of energy, macronutrients, and micronutrients between the two groups based on the 3-day dietary records. (Table 4).

3.3. Anthropometrics and biochemical variables

Anthropometric parameters such as BMI, weight, WC, and waist-to-hip ratio, did not change significantly following 12-week treatment. Compared with the control group, bread with PPP did not alter plasma levels of TG, TC, LDL-C, and HDL-C. In terms of serum insulin concentrations, insulin sensitivity, HOMA-IR, HOMA-B, QUICKI, FBG, and HbA1C, (Table 5). After controlling for confounding factors such as sex, age, and calorie intake, the results did not statistically change (Table 5).

Nowadays, functional foods provide some physiological benefits that direct the food industry to focus its research on products of this nature(32). Functional food ingredients becoming ever more favorite in today’s world due to their safety and validity as well as having fewer side effects than chemical medicines(33). The PPP, a fruit product contains bioactive compounds such as flavonoids, phenolic compounds, polyphenols (including hydrolyzable tannins), dietary fiber, minerals, and vitamins(34). According to the present study, phenolic compounds significantly contribute to the antioxidant capacity of the investigated bread with PPP. The fortification strategy in the current study resulted in added PPP to bread which significantly increased its TPC and antioxidant capacity.

After baking and freezing, antioxidant activity and TPC decreased slightly for all samples. The antioxidant activity also decreased after fermentation, possibly due to the oxidative reactions that occurred during fermentation(35). The difference in results between our study and that of Akkuş et al. in TPC and antioxidant activity may be attributed to differences in the dosage of PPP, the variety used, and the incorporation of sourdough.(15).

Our knowledge of the beneficial effects of bread fortified with PPP in T2DM patients is limited. It is a continuous challenge for diabetics to control their blood glucose levels. T2DM participants with poor glycemic control suffer from macro- and micro-vascular complications over the long term(36). Previous clinical trials have investigated the significant positive effects of pomegranate fractions (peels, flowers, and seeds) and some of their active components on metabolic and biochemical variables in patients with type 2 diabetes (37). However, no beneficial effects of bread with PPP on glycemic markers including HbA1c, FBG, serum insulin concentrations, and insulin sensitivity were documented in our study. To date, there was one publication assessing the effects of bread with PPP on metabolic measurements(15). Akkuş et al. showed that bread with PPP did not significantly affect blood glucose and insulin (15). Also, an 80% methanol extract of pomegranate peel did not affect blood glucose levels(38). Moreover, previous trials on pomegranate juice with similar polyphenols to PPP failed to show the beneficial effects of the intervention on glycemic indices (39, 40). It should be noted that applying PPP as a pure supplement and extract had promising results in diabetes management(41-44).

The administration of bread with PPP did not result in significant effects in reducing hyperlipidemia. We observed similar findings in this randomized controlled trial as those reported in another study. (39). In contrast, a study conducted in Turkey found a significant effect on blood TC and TG(15).Two studies conducted in IRAN concluded that pomegranate peel extract improved lipid profile in obese women and another study found that pomegranate peel extract in doses between 50-300 mg/kg/day decreased serum lipids in rats (14, 45). Cupcakes containing 20% PPP for 6 weeks could improve lipid profiles in rats (46). The lipid profile of rats was improved in 10 weeks by a mixture of beans and pomegranate seeds and peeled pomegranate (44). The addition of PPP and pomegranate peel extract to the diet for four weeks significantly reduced serum TC, TG, LDL-Chol, and lipid peroxidation in hypercholesterolemic rats (47). In rats fed a high-fat diet, pomegranate ellagic acid reduced plasma TC and TG levels dose-dependently(48).

The consumption of bread with PPP did not affect body weight, BMI, and BP. In the Akkuş study, bread with PPP showed a significant change in weight, WC, and BP(15). A clinical study carried out on obese participants with T2DM patients clearly showed that PoPEx, containing ellagitannins had potent anti-hypertensive effects with no effects on BMI, body weight, fat mass, and fat-free mass (13).

Differences in results could be attributed to different doses and varieties of pomegranates and using pomegranate peel extract and supplement instead of PPP. It is also important to note that various factors such as individual differences, genotype, dietary factors, patient's health status, medications, and study duration could impact the results. Moreover, the reductions observed in the Akkuş study could have been partially caused by the hypocaloric diet. So, their results should be interpreted with more caution. (15). Baking and freezing may reduce the amounts of absorbed phenols by the body. It is important to note that the combined effects of different dietary components (synergic and anti-synergic) and the healthy nature of applied intervention for the control group (bread with sourdough and yeast) could influence the outcome. Furthermore, most studies have been conducted on rats. In comparison with PPP supplements, adding PPP to bread may reduce the effects of pure PPP, requiring further study comparing the effects of PPP alone and in combination with bread. Moreover, the participants of the current survey had already established glycemic control, and this might attenuate the potential net effect of PPP on outcomes. However, some clinical trials confirmed the worthwhile effects of pomegranate on the metabolic features of T2DM patients.

Plausible mechanisms support the role of PPP in managing T2DM. Administration of PoPEx has been suggested to have hypoglycemic effects by delaying the activity of α-glycosidase and α-amylase (49, 50). Pomegranate polyphenols like ellagic acid and punicalagin appear to have inhibitory effects on the activity of α-amylase, α-glucosidase, and dipeptidyl peptidase-4 (DPP-4) in free-cell systems and adipocyte cell lines(51). The polyphenols act as α-amylase inhibitors like acarbose. These two polyphenols, along with urolithin A (a metabolite produced during intestinal microbiota metabolism of pomegranate ellagitannins), also significantly decreased triglyceride accumulation and gene expression associated with adipocyte formation(51). Overall, the study demonstrated that punicalagin, ellagic acid, and urolithin A are capable of inhibiting enzymes involved in carbohydrate-triglyceride-related metabolisms, such as GLUT4 (glucose transport4), DPP-4, and lipase(51). Researchers have studied flavonoids and plant-derived flavonoid-rich extracts for their ability to modulate glucose transport and carbohydrate digestion in the gastrointestinal tract. Both are somewhat predicated on polyphenols' ability to interact non-specifically with macromolecules such as protein and starch (52, 53). The flavonoids may also inhibit glucose translocation from the lumen into enterocytes by interacting directly with GLUT2 (glucose transporters2) and Sodium/glucose cotransporter 1(SGLT1) (54). As a result, both gluconeogenesis and glucose transport from absorbed foods may be reduced. Fiber is abundant in PPP, and it can also improve hypercholesterolemia and atherosclerosis (47). It was suggested that pomegranate punicalagin inhibits α-amylase, causing sugars to be released more gradually, thus preventing spikes in sugar levels(55). Pomegranate punicalagin, sitosterol, and phytosterol act downstream of the mevalonate metabolic point and inhibit cellular cholesterol synthesis. Inhibition of pancreatic lipase activity is another possible mechanism of action of pomegranate polyphenols (56).

Several studies have shown that pomegranate peel polyphenols lower cholesterol in a dose-dependent manner. The molecular mechanism may contain activation of peroxisome proliferator-activated receptor γ (PPARγ) and strengthen cholesterol metabolism in L-02 cells (57). The PPP can prevent fat accumulation and body weight gain by controlling obesity-related hormones (leptin-adiponectin route). The presence of tannin, mainly at a high level, may decrease the food intake and performance of rats through their negative effect on palatability. Liu et al. (2016) reported that pomegranate peel extracts were capable of promoting cholesterol catabolism by increasing total bile acid content(48).

As one of the strengths of this study, we investigated the effects of PPP-fortified bread on participants with type 2 diabetes glycemia and lipid profile. To determine which type of bread is best for diabetics, we tested 5 types of bread. In addition, measuring the antioxidant activity and TPC in the dough after freezing is a positive aspect. In this research, sourdough was used in bread which affects the secretion of incretins that are effective in improving blood sugar. It is suggested to investigate bread with and without sourdough in the future studies. Non-significant differences between groups in serum blood glucose and lipid profile levels may have become statistically significant with long-term follow-up. Bread with PPP can have a role in changing lifestyles and has received attention. There are some limitations to our study that need to be addressed. As a result of the COVID-19 strike. Some people were not able to participate in the intervention. Patients with BMIs below 30 were also difficult to select. Our study does not support the notion that bread containing PPP might improve glycemia or lipid profile. These results should, however, be interpreted cautiously. Further research is needed to clarify the impact of bread with PPP on blood sugar levels and lipids, using a specific design for non-inferiority trials. Moreover, a previous study has shown that a longer fermentation time (about 2.5 hours) would have a remarkable effect on total antioxidant activity(58) . Accordingly, future studies should increase the fermentation time and assess its potential impact on metabolic characteristics. This work can be regarded as a new step toward future research on PPP and the findings of the current study could be used as a pilot for other studies to improve glycemia, and lipid profile disorders through consuming PPP-fortified foods.

Beta-cell function: (HOMA-B)

Body mass index: (BMI)

Dipeptidyl peptidase-4: (DPP-4)

DPPH: (2, 2-diphenyl-1-picrylhydrazyl)

Fasting blood glucose: (FBG)

GLUT4: (glucose transport4)

GLUT2 :(glucose transporters2)

HbA1c: (glycated hemoglobin)

ISIRI: (Iranian Institute of Standards and Industrial Research)

Insulin resistance: (HOMA-IR)

Multivariate covariance: (MANCOVA)

Pomegranate peel powder: (PPP)

Peroxisome proliferator-activated receptor γ: (PPARγ)

Quantitative insulin sensitivity check index: (QUICKI)

Sodium/glucose cotransporter 1:(SGLT1)

Type 2 diabetes :(T2DM)

Total phenolic content: (TPC)

Triglycerides: (TG)

Total cholesterol: (TC)

waist circumference:(WC)

Acknowledgments

The present work is part of the Ph.D. thesis approved by the Isfahan University of Medical Sciences (approval no: (IR.MUI. RESEARCH.REC.1399.087)). We would like to thank all the patients who kindly cooperated with us. We are also grateful to the Simin Nan factory, Isfahan, Iran that helped with preparing the bread. The authors also wish to thank the staff at the Endocrine and Metabolism Research Center for their kind cooperation, especially Mr. Torbatian, Mrs. Nowrozi, Mrs. Najafi, and Mr. Naderian. The authors are especially grateful to Dr. Sahar Saraf-Bank, Dr. Sanaz Mehrabani, Mahsa Rezazadegan, and Azam Khani.

Funding

A grant by Isfahan University of Medical Science under project number 398978 supported this study.

Conflict of interest

No competing financial interests or personal relationships affected the work, according to the authors.

Availability of data

The data supporting this study's findings are available from the corresponding author upon reasonable request.

Ethics Declarations

Ethics approval and consent to participate

The study adheres to the principles outlined in the Declaration of Helsinki. All participants provided written informed consent before taking part in this study. The Medical Ethics Committee of Isfahan University of Medical Sciences approved the study protocol (approval no.IR.MUI.RESEARCH.REC.1399.087), and all personal information about participants will be kept secure in a database.

CRediT authorship contribution statement:

Maryam Zare: Project administration, Investigation, Methodology, writing the original draft. Atefe Rezaei: Investigation, writing – reviewing and editing the draft. Mozhgan Karimifar: clinical assistance, Investigation, and editing draft. Mohammad Javad Tarrahi: developed the statistical design and analysis, reviewing and editing the draft. Sayed

Sahar Saraf-Bank: Investigation, writing – reviewing and editing the draft.

Amir Hossein Goli: Food Science. Methodology, reviewing, and editing the draft.

Reza Amani: Supervision of study, conceptualization, writing - reviewing & editing. All of the authors have seen and approved the last version of the manuscript.

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Table 1: Antioxidant activity, total phenolic content and chemical composition in bread with and without pomegranate peel powder (PPP)

Type of bread and dough	Antioxidant activity (%)	Total phenolic content (mg gallic acid/g)
Dough without PPP	14.50 ±0.42^a	29.34±1.2^a
Baked bread without PPP	14.20 ± 0.45^a	28.75±3.5^a
Freeze bread without PPP	13.90 ±0.51^a	27.97±2.3^a
Dough with PPP	93.71 ±0.35 ^d	78.33±3.2^d
Baked bread with PPP	81.30 ±0.33^c	74.60±2.5^c
Froze bread with PPP	78.87 ± 0.25^b	61.85±1.5^b

The results are presented as mean± SD and different low letters in the columns antioxidant and polyphenol are significantly different (p˂ 0.05)

Table 2: Chemical composition of tested breads

bread	Fat(g)	Carbohydrate(g)	Protein(g)	Moister (%)	Ash(g)
With PPP	0.1	56.5	9.9	32.2	1.3
Without PPP	˂0.1	55.1	10.6	32.8	1.4

Pomegranate peel powder (PPP)

Table 3: Baseline characteristics of study subjects (n = 90)

Parameter	Intervention	Control	p-value^*
Weight (Kg)	72.51 ± 8.41	69.29 ± 9.67	0.09
BMI(Kg/m²⁾	27.23 ± 2.38	27.17 ± 2.53	0.91
WC (cm)	94.24 ± 5.68	91.76 ± 7.76	0.08
University graduated (%)	77.8	80	0.79
MET (hrs/day)	32.33 ± 4.48	32.55 ± 4.91	0.84
Sex (male/female) (%)	46.7 /53.3	31.1 / 68.9	0.13
Age	51.88 ± 5.10	55.02 ± 4.97	0.004
Current medication in use (%)
Anti-diabetic meds (%)	88.9	77.8	0.16
Metformin (%)	84.4	77.8	0.42
Anti-hyperlipidemia meds (%)
Statins (%)	66.7	60	0.51
Anti-hypertensive (%)	33.3	40	0.51
Anti-depressants (%)	13.3	4.4	0.26
COVID-19 history (%)	17.8	13.6	0.59
Hypothyroidism (%)	20	8.9	0.13

Note: Data are presented as Mean ± SD. Abbreviations: BMI, body mass index; WC. Waist circumference; MET hrs/day, metabolic equivalent of task hours per day.

Values are reported as mean ± SD and frequency n (%) for quantitative and qualitative variables, respectively.

P^* resulted from a Chi-squared test for qualitative variables and an independent t-test for quantitative variables.

Table 4: Nutritional consumption of the subjects from baseline to the end of the study.

Nutrients	Intervention				Control				P**
	Pre	Post	Change	P*	Pre	Post	Change	P*
Energy (kcal/day)	1998.98 ± 468.98	1937.80 ± 455.17	-61.17 ± 383.92	0.39	1979.65 ±444.51	1862.66 ±384.50	-116.98 ± 318.76	0.08	0.57
Protein(g/d)	69.39 ± 19.51	67.27 ± 16.32	-2.11 ± 21.70	0.60	70.82 ±14.97	67.53 ±17.40	-3.28 ± 16.73	0.34	0.82
Carbohydrate(g/d)	300.16 ± 79.44	289.57 ± 77.35	-10.59 ± 63.03	0.37	306.15 ± 78.60	284.99 ± 68.38	-21.15 ± 49.56	0.04	0.50
Fat(g/d)	62.26 ± 20.67	60.39 ± 19.02	-1.87 ± 21.50	0.64	55.83 ±19.48	53.83 ± 17.11	-2.00 ± 21.67	0.65	0.98
Omega-3 (g/day)	0.66 ± 0.34	0.6 ± 0.2	-0.05 ± 0.39	0.45	0.66 ± 0.35	0.55 ± 0.31	-0.10 ± 0.42	0.25	0.68
Fiber(g/d)	17.26 ± 5.30	17.15 ± 6.08	-0.11 ± 5.17	0.90	17.45 ± 6.08	17.45 ± 6.32	0.00 ± 4.57	0.99	0.93
Vit E(mg/d)	20.97 ± 8.40	20.39 ± 7.17	-0.58 ± 11.53	0.78	20.59 ± 11.66	19.39 ± 9.11	-1.20 ± 13.78	0.67	0.85
Vit C(mg/d)	109.50 ±55.32	119.37 ±76.47	9.86 ± 84.54	0.53	117.82 ± 65.58	116.73 ± 58.37	-1.08 ± 70.23	0.94	0.61
Vit A(mcg/d)	983.58 ± 784.04	925.37 ± 1087.60	-58.20 ±1169.33	0.79	798.69 ± 454.00	588.24 ± 758.38	-210.45 ± 920.71	0.27	0.60
Beta caroten(mcg/d)	397.35 ± 716.74	479.55 ±644.78	82.19 ± 648.73	0.50	275.25 ± 311.68	214.02 ± 278.45	-61.23 ±362.13	0.41	0.33
Selenium(mcg/d)	0.08 ± 0.03	0.09 ± 0.04	0.00 ± 0.05	0.67	0.08 ± 0.03	0.08 ± 0.03	-0.00 ± 0.04	0.75	0.60
Zinc(mg/d)	7.2 ± 2.3	7.45 ± 2.15	0.24 ± 2.93	0.65	6.98 ± 1.96	7.10 ± 2.16	0.11 ± 2.79	0.84	0.87
Chromium(mcg/d)	0.03 ± 0.02	0.03 ± 0.01	0.00 ± 0.02	0.94	0.03 ± 0.01	0.03 ± 0.01	0.00 ± 0.01	0.78	0.90

Variables are expressed as mean ± SD. P* values by (Paired sample-t test). P** values by independent t-test.

Table 5: The effects of bread with and without PPP on anthropometric and glycemic indices in type 2 diabetic patients (n_{1, 2}=45)

Variables

Intervention

Control

P**

p***

Pre

Post

Change

P*

Pre

Post

Change

P*

Weight(kg)

73.31 ± 8.53

73.44 ± 8.30

0.13 ±1.91

0.67

68.94 ± 9.88

68.78 ± 9.78

-0.15 ± 1.87

0.61

0.51

0.31

BMI (kg/m²)

27.24 ± 2.45

27.29 ± 2.40

0.05 ± 0.72

0.65

27.05 ± 2.45

26.99 ± 2.46

-0.05 ±0.72

0.61

0.49

0.31

Waist(cm)

94.12 ± 5.96

93.92 ± 5.74

-0.20 ± 2.41

0.59

91.03 ± 7.49

91.11 ± 7.73

0.07 ± 3.24

0.88

0.66

0.39

Waist/hip

0.92 ± 0.04

0.91 ± 0.04

-0.00 ± 0.02

0.08

0.89 ± 0.06

0.88 ± 0.06

-0.00 ± 0.02

0.06

0.85

0.25

SBP (mmHg)

113.56 ± 14.94

116.02 ± 15.31

2.45 ± 12.04

0.93

120.00 ± 14.65

119.82 ± 13.54

-0.17 ± 12.78

0.22

0.37

0.42

DBP (mmHg)

76.37 ± 10.50

75.37 ± 11.85

-1.00 ± 9.68

0.87

77.88 ± 11.58

77.60 ± 8.92

-0.28 ± 10.51

0.53

0.76

0.81

FBS (mg/dl)

112.53 ± 23.89

126.28 ± 28.03

13.74 ± 16.07

0.00

114.34 ± 29.16

127.15 ± 38.27

12.81 ± 25.52

0.004

0.84

0.23

Cholesterol (mg/dl)

170.56 ± 45.52

174.97 ± 39.14

4.41 ± 37.28

0.46

177.97 ± 32.36

170.36 ± 33.90

-7.60 ± 36.14

0.20

0.15

0.96

TG (mg/dl)

148.74± 53.38

151.12± 72.22

2.38± 65.43

0.82

170.21± 95.76

160.71 ± 79.48

-10.13± 92.13

0.48

0.84

0.55

HDL (mg/dl)

40.46 ± 10.18

41.07 ± 8.91

0.61 ± 6.61

0.56

45.00 ± 10.79

44.63 ± 9.31

-0.36 ± 9.55

0.81

0.60

0.86

TG/HDL

3.88 ± 1.62

3.81 ± 1.70

-0.06 ± 1.21

0.73

4.02 ± 2.49

3.83 ± 2.14

-0.08 ± 2.06

0.55

0.96

0.47

LDL (mg/dl)

100.35 ± 39.71

103.67 ± 35.10

3.31 ± 31.15

0.51

97.38 ± 38.31

92.29 ± 30.72

-5.08 ± 33.74

0.14

0.13

0.87

LDL/HDL

2.54 ± 0.91

2.63 ± 0.95

-0.08 ± 0.72

0.47

2.25 ± 0.82

2.11 ± 0.70

-0.14 ± 0.61

0.17

0.15

0.50

Insulin(mMoL/L)

7.46 ± 5.13

7.81 ± 5.65

0.35 ± 4.98

0.66

7.98 ± 4.66

8.15 ± 5.17

0.16 ± 3.69

0.78

0.86

0.56

Insulin sensitivity

0.73 ± 0.42

0.62 ± 0.42

-0.10 ± 0.48

0.18

0.63 ± 0.44

0.60 ± 0.42

-0.03 ± 0.34

0.54

0.46

0.26

Homa-IR

2.03 ± 1.39

2.39 ± 1.62

0.36 ± 1.43

0.12

2.29 ± 1.41

2.62 ± 1.92

0.32 ± 1.41

0.17

0.90

0.36

Homa-b

58.16± 40.71

49.92± 37.21

-9.65 ± 31.21

0.06

58.97 ± 40.25

45.08 ± 32.42

-13.88± 20.49

0.00

0.63

0.82

quiki

0.35 ± 0.03

0.34 ± 0.03

0.00 ± 0.03

0.12

0.35± 0.03

0.34 ± 0.03

0.00 ± 0.02

0.37

0.51

0.25

HbA1c (%)

6.51 ± 0.96

6.74 ± 1.18

0.32 ± 0.73

0.009

6.49 ± 0.97

6.53 ± 0.94

-0.08 ± 0.92

0.60

0.03

0.14

Note. Data were presented as mean (SD).

* Calculated by paired-sample T-test.

** Calculated by independent sample t-test

*** Obtained from multivariable analysis of covariance (MANCOVA), adjusted for age, sex, and calorie intake.

HOMO‐IR: homeostatic model assessment insulin resistance; QUICKI: quantitative insulin sensitivity check index.

SBP; Systolic Blood Pressure; DBP; Diastolic Blood Pressure; LDL; low-density lipoprotein, HDL; high-density lipoprotein, HbA1c; glycated hemoglobin.

No competing interests reported.

Effect of bread fortification with pomegranate peel powder on metabolic profile in Type 2 Diabetes Mellitus patients: A randomized blind control trial

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1. Background

2. Material and Methods

3. Results

4. Discussion

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Ethics approval and consent to participate

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