Beneficial Effects of Pomegranate Peel Extract Treatment on Anthropometry and Body Composition of Overweight Patients With Diabetes Mellitus Type-2: a Randomised Clinical Trial

Department of Hygiene, Faculty of Medicine, University of Banja Luka, Banja Luka, the Republic of Srpska, Bosnia and Herzegovina. Department of Hygiene, Public Health Institute of Republic of Srpska, Banja Luka, the Republic of Srpska, Bosnia and Herzegovina. Department of Pharmacology, Toxicology and Clinical Pharmacology, Faculty of Medicine, University of Banja Luka, Banja Luka, the Republic of Srpska, Bosnia and Herzegovina. Institute for Medicinal Plant Research "Dr Josif Pančić", Belgrade, Serbia. Institute of Hygiene and Medical Ecology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia.


Introduction
patients are overweight or obese and obesity by itself is linked to insulin resistance and abnormalities in insulin secretion. 3,4 One of the most significant risk factors for metabolic disorders and its predisposition to DMT2 is abdominal distribution of fat. 5 Having in mind that DM is one of the leading causes of death worldwide and that DMT2 and obesity together increase the mortality, it is not surprising that the prevention and treatment of DM and obesity are important public health measures. 1,6 Diabetes can be managed by different approaches, including antidiabetic medication, nutrition, physical activity or herbal remedies. 7,8 There is an increasing interest in identifying herbal compounds that have lipid-lowering activities or properties to reduce obesity. 9 According to some studies, polyphenols exert very potent anti-inflammatory effects and can improve metabolic conditions. 8 Pomegranate is an excellent source of polyphenols (flavonoids, condensed tannins and hydrolysable tannins) with beneficial pharmacological properties and potential for treatment of various disorders including DM. [10][11][12][13] According to the literature data, pomegranate peel extract (PoPEx) can affect adipocyte differentiation. 9 Adipocytes, on the other hand, are very important in the development of metabolic disturbances that are related to obesity and DM. 9,14 A recent clinical study, performed in obese patients with DMT2, clearly showed that PoPEx, containing ellagitannins had a very potent hypolipaemic, hypoglycaemic and anti-hypertensive effects, but had no effect on body mass index (BMI), body weight, fat mass and fat-free mass. 15 However, the animal and cell culture studies suggest that dietary polyphenols may have a pronounced anti-inflammatory effect associated with a reduction of body weight and FM. 16,17 Therefore, this clinical trial, as an arm of the existing clinical investigation, was aimed to study the effects of PoPEx on anthropometry and body composition in overweight patients with DMT2.

Study population
This study was designed as a randomised, double-blind, placebo-controlled clinical trial in Figure 1: The study design and the participants flow diagram overweight patients with type 2 diabetes mellitus. Patients were recruited at the Department Endocrinology of the University Clinical Centre of the Republic of Srpska, Banja Luka. All participants (40 -65 years of age) were overweight (BMI ≥ 25 kg/m 2 ), had poor glycaemic control (glycosylated haemoglobin, HbA1C ≥ 6.5%) and were treated with metformin for the period of at least one year before being enrolled in the study. Patients not enrolled in the study were those with inflammatory diseases, with chronic kidney or liver disease or those on hormone replacement therapy and antioxidant supplements. Patients on insulin treatment were not considered for the study.

Ethical Considerations
All the subjects interested in participating in this study had to sign an informed consent. They were informed about the study purpose and protocol, as well as on the possible risks or benefits of treatment. This clinical study was approved by the Ethics Committee of the Faculty of Medicine, University of Banja Luka No 01-9-604-2/17 and the study was conducted according to the Declaration of Helsinki.

Study design and medication
After randomisation, sixty patients were allocated into two groups. The study group (n=30) received capsules (250 mg) containing PoPEx twice daily for 8-weeks period, and the placebo group (n=30) received capsules containing the same quantity of placebo. Participants had to follow the study protocol without changing their dietary habits, physical activities and medication regimens during the study period. They participants

Methods
Two patients (3.33 %), out of 60 enrolled in the study, did not complete the study. At baseline, there were no differences in age and DM and metformin therapy duration between the PoPEx group and the placebo group (57.9 ± 6.1 years were provided with a fixed number of capsules needed for the course of treatment. The participant flow diagram and study design are presented in Figure 1.

Pomegranate peel extract
PoPEx was provided by Institute for Medicinal Plant Research "Dr Josif Pančić", Belgrade, Serbia. Pomegranate fruits were obtained from Herzegovina, a southern region of Bosnia and Herzegovina. After being separated from the fruit, peels were dried at room temperature (4 -6 days). The dried peels were grounded with a laboratory mill to obtain the powder. Powdered pomegranate peel was extracted with 50% ethanol. After filtration, the extract was evaporated to dryness and put in capsules. Each capsule (250 mg) contained polyphenols (punicalagin, punicalin, ellagic acid, and gallic acid) in defined quantities. The detailed methods of preparation and quantification of phenolic compounds of PoPEx were described in detail in a recently published paper. 15 The chemical formulae of these polyphenols are presented in Figure 2.

Energy and nutrient intake
Using a 3-day food diary records, the dietary intake was assessed at the beginning and at the end of the treatment period. The energy and nutrition intake were estimated every day using the Serbi-an Food Composition Database, harmonised with the European Food Information Resource (Euro FIR) standards and integrated into the Euro FIR Food Platform and Balkan Food Platform. 18 The participants were advised not to change their usual daily diet.

Anthropometric measurements
The anthropometric measurements were performed at the beginning and the end of the study period by the same trained investigator. All measurements were taken in the morning hours on subjects wearing underwear only. Height was measured using a stadiometer (accuracy of 1 mm), waist circumference (WC) with a non-stretchable tape and the body mass by standard scale (accuracy of 100 g). Body composition (FM, fat-free mass, visceral fat level, and phase angle) was determined using a Tanita bioelectrical impedance analyser (Tanita Corporation, Tokyo, Japan). For skinfold thickness, Harpenden skinfold caliper (SF) was used. Triple measurements were taken on the right side of the body in four standard places (biceps, triceps, subscapular and suprailiac), and the average value was used. BMI was calculated using the following formula: [weight (kg)/height (m) 2 ].

Statistical analyses
For statistical analyses, the IBM SPSS 20 software was used (Chicago, IL, USA). All results were expressed as mean ± standard deviation and p < 0.05 was considered significant. For comparisons between the groups, Student   low-up period. The baseline characteristics of the PoPEx and the placebo groups are presented in Table 1. There were no significant differences in anthropometric variables between the PoPEx and the placebo group at baseline.
Mean energy intake at baseline was similar in both groups (2333.6 ± 307.9 kcal and 2265.6 ± 343.4 kcal, respectively). The 3-day diary food intake showed no significant changes in the energy and macronutrient intake during the study period. The mean fat energy intake was 920.2 ± 99.3 kcal in the PoPEx group and 881.1 ± 83.8 kcal in the placebo group, accounting for nearly 40% of fat energy. The average daily intakes of saturated fat the energy were 289.6 ± 31.5 kcal and 296.7 ± 30.6 kcal in the PoPEx group and the placebo group, respectively ( Table 2).
After the intervention period, significant increase in BMI (0.18 ± 0.30 kg/m 2 ) and body mass (0.48 ± 0.93 kg) were noticed in the PoPEx group; z = -2.646, p = 0.008 (r = 0.34) and z = -2.391, p = 0.016 (r =0.30), respectively. However, at the same time, the intake of PoPEx produced a significantly decreased WC, z = -4.613, p < 0.001 (r = 0.60), indicating a large effect size using Cohen's test and a non-significant decrease in the  level of visceral fat. In the placebo group, there were no statistical differences in the anthropometric characteristics at the end of the intervention period (Table 3). The relation between the bioelectrical impedance analysis (BIA) and skinfold thicknesses (SF) caliper measurements are illustrated in Figure  3. The correlation factor was high in the total sample (r = 0.887; p < 0.001), but it was lower This clinical study was performed to examine the effects of the 8-week treatment with PoPEx on body composition and anthropometric parameters in overweight subjects with DMT2.
The results indicated that PoPEx had a beneficial effect on WC, but the effects on body mass and anthropometric characteristics were not consistent. Compared to the placebo group, a significant effect on BMI increase and WC decrease was noticed in the PoPEx group. However, the percentage of FM was decreased (-0.58 %) but the change was statistically not significant. Moreover, the recent results of an additional arm of this study undoubtedly demonstrated hypolipaemic activity of PoPEx treatment with a beneficial effect on fatty acid composition indicating a strong influence on lipid metabolism. 15 Life-style management, including balanced nutrition and physical activity, are very important keys for improving glucose control in the context of DM self-management. 19 Having that in mind, at the beginning of this study, all participants completed a 3-day food diary and all participants were asked not to change the nutritional pattern. The results showed that diabetic patients had a similar proportion of macronutrients in the diet as the rest of the population which what is in accordance with the 2019 Consensus Report. 20 Nearly 40 % of calories taken originated from fat and saturated fat energy and it was was higher than recommended. Epidemiological studies suggested a positive relationship between the saturated fat intake and plasma cholesterol levels. 21 Furthermore, a meta-analysis of randomised controlled clinical trials on modification of dietary fats on cardiovascular disease (CVD) risk suggested that saturated fat energy reduction might reduce cardiovascular events by 14% in patients with DMT2. 22 In diabetic patients replacing 2% saturated fat energy with polyunsaturated fatty acids (PUFA) ener-gy was associated with a 12 %-decrease in CVD mortality rate. 23 The results of this study clearly showed that at the end of the intervention period, the energy intake and energy macronutrient proportion remained the same in both study groups.
The 8-weeks consumption of PoPEx induced a significant reduction in WC in the treatment group. WC is an independent predictive factor of chronic disease including DMT2. 24 According to Lou et al, 25 26,27 have indicated that one of the possible mechanisms by which leaf extract affects the body mass is similar to the mechanism of drug orlistat (Xenical) causing the decrease in the activity of intestinal lipase, fat absorption, and increase of fat excretion. The administration of the whole pomegranate extract significantly decreased the body mass in overweight people, while there were no changes in the placebo group. 28 Another study noted that intake of 120 mL of pomegranate juice during the 30 days led to a decrease in body mass and total body fat percentage.29 Discrepancies in outcomes of these studies could be explained by the differences in study protocols and treatment regimens (different study designs, different duration of the interventional period, variations among subjects, dosages and forms of pomegranate used). 30 The phase angle value has been reported previously as a biomarker of fat-free mass and changes of phase angle value and fat free-mass in the PoPEX group confirm that assumption. 31,32 Concerning the body composition changes in the PoPEx group, average weight gain was 0.48 ± 0.9 kg, but the percent of FM was decreased by 0.58 ± 2.2%. For the assessment of body composition in this study, two measurement tools were used, including BIA and SF caliper. The results showed a highly significant correlation between BIA and SF caliper (p< 0.001, r =0.887).

Discussion
for women compared with men (r = 0.93). The results showed a non-significant reduction in fat mass percentage in the PoPEx group (-0.58 ± 2.21 %, p = 0.159) compared with the placebo group (0.14 ± 1.24 %, p = 0.546). Besides, changes in phase angle value and fat-free mass were non-significant (0.78 ± 0.24 and 0.61 ± 0.25).
The results of this study showed that eightweek supplementation with PoPEx had a beneficial effect on anthropometry and body composition of overweight diabetic patients. Further studies are needed to explore the potential effect of PoPEx on adipokines and adipocyte functions in obese patients.
In DMT2 patients, atherosclerotic CVD is the major cause of morbidity and mortality. Although multiple factors play key roles in the development of CVD in DMT2, obesity is one of the factors that can be modified by dietary interventions. 4 Previous studies investigating the effects of the pomegranate consumption on BMI and body composition are inconsistent. According to Ghefalti et al, 33 supplementation with pomegranate extract showed a tendency to exert a beneficial effect on weight and the percentage of FM. Some in vivo and in vitro studies suggested that pomegranate had regulatory effects on dyslipidaemia and adipose tissue metabolism in human and animal adipose tissue. 34 Polyphenols as functional food components exert a potential anti-obesity effect through an impact on white adipocyte browning and activation of the brown adipose tissue. Induction of the beige adipocytes may be mediated via the adrenergic membrane receptors, resulting in the stimulation of lipolysis and thermogenesis. 35 Conclusion