Academic Editor: Peter A. McCullough
Background: Type 2 diabetes mellitus (T2DM) is commonly associated with
hyperglycemia, dyslipidemia, oxidative stress and inflammation which are well
known cardiovascular risk factors. Pomegranate peel polyphenols have a proven
hypolipemic, antioxidant and anti-inflammatory activity. However, there is a lack
of clinical studies that would confirm its antioxidant and anti-inflammatory
effects in diabetic patients. The potential of pomegranate peel extract (PoPEx)
to counteract inflammation and oxidative stress in T2DM patients was
investigated. For this purpose, a randomized, double-blind placebo-controlled
study involving adult T2DM patients treated with PoPEx or placebo for eight-weeks
was conducted. Methods: Patients were randomly divided into two groups:
the first group (n = 30) received capsules containing PoPEx 250 mg twice daily,
while the placebo group (n = 30) received placebo capsules twice daily. Plasma
concentration of inflammatory factors (interleukin 6 (IL-6), tumor necrosis
Type 2 diabetes mellitus (T2DM) makes 90–95% of all diabetes, affecting 6.28%
of the world population globally [1, 2]. The risk of T2DM increases with age, and
it occurs more frequently in obese individuals and individuals with a sedentary
lifestyle . Hyperglycemia, dyslipidemia, oxidative stress and inflammation are
involved in the pathogenesis of T2DM [3, 4, 5]. Thus, hyperglycemia can increase
production of free radicals, which potentiates lipid peroxidation and protein
nitration, activation of stress-sensitive pathways and increased DNA damage .
Exposure of pancreatic
Pomegranate (Punica granatum L.) is used in traditional medicine of different cultures. It is a rich source of polyphenols and other phytochemicals, including sterols, terpenoids, alkaloids and tocopherols, as compounds with important physiological actions [11, 12]. These phytochemicals extracted from different parts of the fruit possess significant anti-diabetic, antioxidant and anti-inflammatory effects, as well as lipid lowering and antimi-crobial activity [13, 14]. Pomegranate peel makes up to 40–50% weight of the fruit and due to its high polyphenol content possess strong antioxidant activity [15, 16]. The pomegranate extracts have been used as a functional food for the years [17, 18, 19]. The results of recently published studies have shown the beneficial effects of pomegranate peel extract (PoPEx) on body composition, systolic and diastolic blood pressure and lipid profile in overweight T2DM patients [20, 21]. In addition, PoPEx has demonstrated no effect on fasting blood glucose level, but decreased the level of glycolisated hemoglobin after of 8 weeks’ administration in diabetic patients. Although several biologically active polyphenols have been detected in the pomegranate peel, making it the richest source of tannins and flavonoids among all parts of the fruit, clinical studies are needed to confirm its anti-oxidant and anti-inflammatory effect in diabetic patients.
The aim of this study was to investigate the potential of PoPEx to attenuate the inflammation and oxidative stress in T2DM patients.
This randomized double-blind placebo-controlled study involved 60 overweight
patients with T2DM. The patients were recruited at the Endocrinology Department
of the University Clinical Centre of the Republic of Srpska, Banja Luka, Bosnia
and Herzegovina, from June to November 2018. The study inclusion criteria were:
40–65 years of age, BMI
After assessing the National Diabetes Register 84 patients who met the inclusion criteria were identified and 60 patients accepted to participate in the study. Patients were randomly divided into 2 groups, by an impartial person, using 1:1 block randomization: the first group (n = 30) received capsules containing PoPEx 250 mg, twice a day (PoPEx group), while the placebo group (n = 30) received visually identical capsules containing placebo (250 mg), twice a day. Both groups continued with regular diet during the 8 weeks’ intervention period. The study was double blinded, meaning that no patients nor investigators were aware of which intervention is being administrated.
Pomegranate (Punica granatum L.) was deposited in Botanical Garden
“Jevremovac” University of Belgrade (woutcher specimen No BEOU 17742).
Pomegranate fruits were picked and washed by hand in the east Herzegovina region
(south of Bosnia and Herzegovina). The peel was separated from fruit and dried
4–6 days and grounded in powder. For the preparation of peel extract, ethanol
was used as solvent. Phenolic compounds of PoPEx were quantified using high
performance liquid chromatography (HPLC) methodology . Quantification of
ellagic acid, gallic acids, punicalagin and punicalin was done using calibration
curves of authentic standards. The results are presented as milligrams per gram
of dry weight (mg/g DW). The obtained dry extract contained punicalagin 69.67
All parameters were measured at baseline and at the end of the study. Height was
measured using wall-mounted stadiometer to the nearest 0.1 cm. Body mass was
measured using Tanita body composition analyzer. Body mass index (BMI) was
calculated as weight (kg) divided by square of height (m
All the patients filled out 3 days’ prospective food records at baseline and at last 3 days of the intervention period to estimate energy intake and to follow potential changes during the study, that might affect the results. The energy intake was estimated according to the Serbian Food Composition Database, harmonized with EuroFIR standards end embedded in EuroFIR Food Platform and Balkan Food Platform .
Blood samples were taken after 12–14 hours overnight fasting at beginning of
the study, and following the intervention period. Serum lipid profile including
triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol
(HDL-C), low-density lipoprotein cholesterol (LDL-C), fasting glucose,
glycosylated hemoglobin (HbA
Prooxidative parameters, index of lipid peroxidation, nitrites (NO
The nitrites (NO
The determination of superoxide anion radical is based on the reaction of
For measuerement of H
The homocysteine concentration was measured in serum by means of HPLC procedure with reverse-phase separation and fluorescence detection, as previously described. The fluorescence was determined spectrophotometrically at 390 nm, while emission was measured at 470 nm .
The TAC was assessed by commercially available Sigma-Aldrich total antioxidant assay kit.
Plasma cytokine concentrations were determined in triplicates using human
cytokine-specific ELISA assays (Human IL-6 DUOSET ELISA Development kit, R &
D Systems, USA; Human TNF-
The data were analyzed using the SPSS software (Version 20; IBM Chicago, IL,
USA). The distribution of variables, was assessed by Shapiro-Wilk’s test.
Baseline values were computed by Chi square test, Student’s t test and
Mann-Whitney U test depending on the data distribution. For analysis of
differences in the outcome variables at the end of study compared to the baseline
values, paired sample t-test or Wilcoxon Signed Rang test were
conducted. For assessing differences between the two groups we used mixed-model
analysis of variance (mixed ANOVA), and non-parametric alternative to mixed ANOVA
Bruner and Langer test. Correlations between delta values of inflammatory markers
and oxidative stress biomarkers were calculated using Spearman’s rank
correlation. Differences in p value of p
Sixty patients with T2DM were enrolled in the study, but two of them from the
placebo group were excluded from further analysis due to discontinuation of the
therapy. The baseline characteristics of the PoPEx and the placebo groups are
displayed in Table 1. No differences between the two groups in the age and gender
of study participants, serum level of HbA
|Parameters||PoPEX group (N = 30)||Placebo group (N = 30)||p value|
|Female N (%)||15 (50.00)||15 (50.00)||1.000|
|Age (years, mean
|Duration of DM (months, mean
|Age at onset of DM (years, mean
|Current smoking status N (%)||10 (33.33)||5 (17.86)||0.179|
|PoPEx, Pomegranate peel extract; BMI, Body mass index; HbA|
After 8 weeks’ of PoPEx supplementation we noticed significantly lowered serum
values of HbA
There were no differences in parameters of oxidative stress at baseline between
the two groups. However, after eight weeks of intervention, all oxidative stress
markers (TBARS, NO
|Oxidative stress parameters||PoPEx group||Placebo group||p value|
|Before treatment||After treatment||Before treatment||After treatment|
|Data are expressed as the mean |
No significant differences were found in the lipid profiles between the PoPEx group and the placebo group at the beginning of study. However, the eight weeks’ consumption of PoPEx capsules had a significant influence on lipid profile. As shown in Table 3, a significant decrease in the levels of TC, LDL-C and TG, associated with significant increase in HDL-C, were noticed in the PoPEx group, compared with baseline values. No significant changes were observed in the placebo group. Furthermore, analysis of between group changes showed significant differences in the levels of TG and HDL-C. A part of these results related to lipid dynamics have been already published .
|Lipid profile||PoPEx group||Placebo group||p value|
|Before treatment||After treatment||Before treatment||After treatment|
|Data are expressed as the mean |
The eight weeks of PoPEx supplementation resulted in significant decrease of the
inflammatory parameters, compared to baseline values (hsCRP: median,
Interquartile Range (IQR) 2.05 mg/L, 1.18–3.17 vs. 1.75 mg/L, 0.97–2.67; IL-6:
The effects of eight-week PoPEx consumption on the circulating
biomarkers of inflammation levels. (A–C) The high sensitivity c-reactive
protein (hsCRP), Interleukin 6 (IL-6), and tumor necrosis factor
Finally, the correlations between changes in oxidative stress and inflammatory
markers were tested. Significant inverse correlations were found between TAC and
all measured markers of inflammation:
|Group||ΔTBARS mmol/L||Δ O
||Δ TAC µmol/L|
|IL-6, Interleukin 6; TNF-|
A growing interest in polyphenol research was noticed in the last 20 years. Although the current evidence is mostly based on the results of observational and in vitro studies, which have elucidated the potential mechanisms of the protective effects of polyphenols. There are a meager number of clinical studies that clarify the effects of pomegranate . Within these, most studies have been conducted using pomegranate juice or whole fruit extracts. The doses, study design and the duration of these studies varied from study to study .
The interaction of oxidative stress and low-grade inflammation has been reported as a known pathway in the development and progression of diabetes mellitus and its complications, including CVD . Lipid metabolism and peroxidation are important for development of inflammation which is elevated in several diabetic complications . This randomized, placebo-controlled intervention study lasting eight weeks is, to the best of our knowledge, among the first studies that described the effects of pomegranate peel extract on the oxidative state and inflammation in T2DM patients. We found that consumption of a standardized PoPEx, used in this study, significantly influenced the markers of oxidative stress, inflammation parameters and lipid profile in diabetic patients.
Additionally, as we previously reported, consumption of PoPEx significantly
lowered serum values of HbA
Homocysteine, a sulphur containing amino acid is an independent risk factor for
cardiovascular disorders . It may induce mitochondrial dysfunction trough an
increase in reactive oxygen species (ROS) production  and promote oxidative
injury to vascular cells . A previous study suggested that synergistic
effects of moderately elevated levels of homocysteine and oxidative stress
markers in diabetes promote the development of atherosclerosis . However,
homocysteine levels in diabetic patients are controversial. Numerous studies
reported elevated level of homocysteine [36, 37], but some other studies found
unchanged  and even reduced homocysteine level in these patients . At the
beginning of the present study the level of homocysteine was within the normal
range, but after eight weeks of PoPEx treatment the serum level of homocysteine
was significant decreased. Lowering homocysteine level by 3
The results of this study demonstrated that PoPEx supplementation significantly
reduced oxidative stress markers such as TBARS, NO
Our findings confirmed previously published results that an eight-week
consumption of PoPEx induces statistically significant lipid profile improvement
in patients with T2DM, compared to the placebo group . We have confirmed a
significant reduction in the plasma levels of TG, TC and LDL-C, and an increase
in HDL-C in PoPEx group (Table 3). The study conducted by Hosseini et
al. , reported that a four-week-long administration of the concentrated
extract of whole pomegranate induced a significant improvement in lipid profiles
in obese people. Also, these outcomes are in accordance with previous studies
[55, 56, 57, 58, 59], which used pomegranate juice or pomegranate seed oil supplementation.
On the other hand, some authors reported that administration of pomegranate
polyphenols did not resulted in any improvement in lipid profile [60, 61]. The
inconsistency of these results can be explained by several reasons: small sample
size, differences in the applied doses, supplementation periods, and form of
pomegranate products . Previously, in vivo and in vitro
studies suggested that pomegranate extracts had regulatory effects on lipid
metabolism in human and animal adipose tissue. The eight-week administration of
the hydromethanol peel extract in diabetic rats showed hypolipemic activities
that was attributed to the powerful reactive oxygen scavenging properties of
these compounds . The inhibition of lipid synthesis and the inhibition of
lipolysis are the mechanisms by which some intervention prevents ectopic lipid
storage and insulin resistance. The molecular mechanism may involve the
activation of peroxisome proliferator-activated receptor
Elevated TyG index as a reliable marker of insulin resistance, and it is associated with progression of coronary artery calcification reflecting cardiovascular risk . Also,TyG index was strongly associated with cardiovascular events in patients with T2DM . As it can be seen in the Table 3, PoPEx significantly reduced this index, suggesting the potential effect of POPEx in reducing risk for cardiovascular complications in T2DM patients.
The increased levels of inflammatory biomarkers such as IL-6, TNF-
In the present study, a reduction of plasma concentration of inflammatory
parameters IL-6, TNF-
The proposed mechanism of anti-inflammatory action of pomegranate is a decrease
of the expression of cyclooxygenase-2 (COX-2) via inhibition of the
The strenght of this study is in the use of standardized PoPEx polyphenols in T2DM patients with strong exclusion/inclusion criteria. The major limitations are that this is a small-scale study that is performed in one clinic, and that the study lacked the assessment of dietary polyphenol intake. Further larger studies should be conducted in order to document solid conclusions on whether a dietary component can improve health parameters in T2DM patients.
The study demonstrated that eight-week-long PoPEx administration had favorable effects on inflammatory status and oxidative stress biomarkers in diabetic patients. At the same time, the lipid profile improved significantly. Consumption of PoPEx could be advocated to be used as a functional food ingredient, considered as a dietary and natural remedy with pharmacological properties for prevention and treatment of diabetic patients.
MG, RŠ and MPS—contributed to the conception and design of the study; MG and VRG—collected data; MG, VV, VJ, DMD—analysed the data; MG, RŠ, MPS, VV, VRG,VJ, DMD, RS, KŠ, DB, NV—wrote and revised the manuscript. All authors have read and agreed to the published version of the manuscript.
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the Faculty of Medicine University of Banja Luka (No 01-9-604-2/17), the Republic of Srpska, Bosnia and Herzegovina, and by Ethics Committee o the University Clinical Centre of the Republic of Srpska (No18/4.37/17), Bosmia and Herzegovina. The study protocol was explained and written informed consent was obtained from all the participants involved in the study.
The authors would like to thank all the participants in the present study. The authors express appreciation to the Institute for Medicinal Plant Research “Dr Josif Pančić”, Belgrade, Serbia for preparing the pomegranate peel extract.
This research was supported by the Ministry for Scientific-Technological Development, Higher Education and Information Society (No 19/6-020/961-81/18) Government of the Republic of Srpska.
The authors declare no conflict of interest. Vladimir Jakovljević and Dragan M Djuric are serving as Guest editors of this journal. We declare that Vladimir Jakovljević and Dragan M Djuric had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Peter A. McCullough.