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 [1]. 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 [6]. Exposure of pancreatic $\beta$-cells to the oxidative stress may reduce insulin gene expression through the inhibition of promoter activity and insulin mRNA expression [7]. Furthermore, oxidative stress induced by hyperglycemia is believed to increase the level of pro-inflammatory proteins and cytokines [8]. The increased secretion of tumor necrosis factor $\alpha$ (TNF-$\alpha$) is linked with obesity-associated insulin resistance [9]. Adipose tissue, mostly visceral, represent the main source of pro-inflammatory mediators in T2DM such as TNF-$\alpha$, IL-1, IL-6, IL-10, leptin, adiponectin and other bioactive substances collectively called adipokines [10]. TNF-$\alpha$ and IL-6 can directly impair pancreatic $\beta$-cell function but also indirectly, by producing free fatty acids. These mechanisms promote the development of T2DM confirming the link between insulin resistance and inflammation. Previous research reports showed that inflammation and oxidative stress are associated with the risk of developing cardiovascular complication in T2DM patients [4]. Understanding the role of pro-inflammatory cytokines and inflammatory processes in the development of T2DM suggests that therapeutic approaches that can inhibit inflammation could be useful in the prevention and control of T2DM, as well as cardiovascular compliations in diabetic patients [4]. Considering a large number of people affected by obesity and obesity-related metabolic disorders, including T2DM worldwide, these therapeutic approaches can have a huge economic and public health impact.

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.

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${}_1$C, duration of T2DM and metformin therapy were found. The results of 3 days’ food record showed no significant changes between groups in mean energy and micronutrient intake during the study [20]. In both study groups, no adverse effects were reported during the follow-up period.

After 8 weeks’ of PoPEx supplementation we noticed significantly lowered serum values of HbA${}_1$C in PoPEx group (7.55 $\pm$ 1.22 vs. 7.32 $\pm$ 1.02%, p 0.001), while the values remained unchanged in the placebo group.

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${}^{-}$, O${}_2$${}^{-}$ and homocysteine) except H${}_2$O${}_2$ decreased significantly in the PoPEx group, while the TAC significantly increased. At the same time, no differences were detected in the placebo group except the TAC, which also increased. Thus, markedly lower values of oxidative stress markers (TBARS, NO${}^{-}$, O${}_2$${}^{-}$) were found in the PoPEx group than in the placebo group at the end of the study. The results were similar after conducted mixed ANOVA, except for the TAC value (Table 2).

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 [21].

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: 139.50 $\mu$mol/L, 94.04–199.95 vs. 123.50 $\mu$mol/L, 77.29–168.75, and TNF$\alpha$ 172.80 pg/L 140.30–233.10 vs. 139.00 pg/L, 104.91–189.83) (Fig. 1A–C). At the same time, serum levels of hsCRP, IL-6 and TNF-$\alpha$ were higher at the end of he study in placebo group. The differences between the basal and final values (dellta values) of inflammatory parameters were significantly different between the two groups after the intervention period (Fig. 1D–F).

Fig. 1.

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 $\alpha$ (TNF-$\alpha$) levels, baseline and after eight-weeks intervention period in the Diabetes mellitus type 2 patients. Wilcoxon signed rank test was done and asterisk (*) indicate significant differences compared with baseline value within the groups *p 0.05, **p 0.01. (D–F) The delta values of circulating inflammation biomarkers levels (hsCRP, IL-6, and TNF-$\alpha$) in the PoPEx group and the Placebo group. Mixed ANOVA was done and sign (†) indicate significant differences between the groups after controlling for the baseline values †p 0.05, ††p 0.01.

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: $\mathrm{\Delta }$ IL-6, $\mathrm{\Delta }$ TNF-$\alpha$, and $\mathrm{\Delta }$ hsCRP. In addition, $\mathrm{\Delta }$ IL-6 inversely correlated with $\mathrm{\Delta }$ H${}_2$O${}_2$ and positively with $\mathrm{\Delta }$ NO${}^{-}$ (Table 4).

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 [30]. 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 [13].

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 [4]. Lipid metabolism and peroxidation are important for development of inflammation which is elevated in several diabetic complications [31]. 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${}_1$C in PoPEx group, while fasting blood glucose was unaffected [21]. Several studies show that the inadequate glycemic control indicated higher risk of diabetes-related complication and death [32]. Better glucoregulation and, in the same time antioxidant and anti-inflammatory activities of PoPEx contribute to better diabetes management [33].

Homocysteine, a sulphur containing amino acid is an independent risk factor for cardiovascular disorders [29]. It may induce mitochondrial dysfunction trough an increase in reactive oxygen species (ROS) production [34] and promote oxidative injury to vascular cells [35]. A previous study suggested that synergistic effects of moderately elevated levels of homocysteine and oxidative stress markers in diabetes promote the development of atherosclerosis [35]. However, homocysteine levels in diabetic patients are controversial. Numerous studies reported elevated level of homocysteine [36, 37], but some other studies found unchanged [38] and even reduced homocysteine level in these patients [39]. 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 $\mu$mol/L is associated with a decrease of 16% in risk of ischemic heart disease and 24% in risk of stroke [40].

The results of this study demonstrated that PoPEx supplementation significantly reduced oxidative stress markers such as TBARS, NO${}^{-}$ and O${}_2$${}^{-}$ and increase the TAC in T2DM patients. Previous studies reported that flavonoids from pomegranate juice a responsible for the improvement in antioxidant capacity and also the beneficial effect in limiting oxidative damage [41, 42]. Furthermore, these findings are supported by the results in women with polycystic ovarian syndrome and diabetic patients [43, 44]. Studies also reported an increase of NO serum level in T2DM patients [45, 46]. Hyperglycemia as a common feature in T2DM, and duration of disease may enhance the NO production [45]. The higher level of NO combined with increased production of O${}_2$${}^{-}$ in formation of peroxynitrate extremely reactive form of ROS, which damages proteins, lipids, and nucleic acids and promote LDL-oxidation and atherosclerotic plaque development, which further lead to cardiovascular complications [8, 47]. The levels of these harmful compounds are significantly reduced after PoPEx intervention (Table 2). Possible mechanisms for improving oxidative stress following eight weeks of PoPEx supplementation include free radical scavenging and supporting antioxidant system [14, 48]. Although several studies failed to show alleviation of cardiovascular complication using antioxidant supplementation (Vitamin E) [49, 50] positive effects of polyphenols are more prominent [14, 51]. However, there is a great variability and seasonal variations in polyphenols consumption between countries. The largest antioxidant contribution achieved through food consumption, was obtained from vegetables and fruits intake [52]. In population living in the four season zones the higher consumption of fruits and vegetables was observed in spring and summer [53]. This dietary intake pattern could explain the increased TAC noticed in the placebo group.

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 [21]. 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. [54], 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 [62]. 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 [19]. 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 $\gamma$ (PPAR$\gamma$) and the increased metabolism of cholesterol in LO2 cells [63], or decreased expression of sterol regulatory binding protein-1c (SREBP-1c) [64]. The main components of PoPEx, punicalagin and ellagic acid, have lipid-lowering effect in a dose-dependent manner. Disturbances of lipid metabolism in T2DM patients may lead to overproduction of lipid peroxidation products which may cause elevation of oxidative stress [65].

Elevated TyG index as a reliable marker of insulin resistance, and it is associated with progression of coronary artery calcification reflecting cardiovascular risk [66]. Also,TyG index was strongly associated with cardiovascular events in patients with T2DM [67]. 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-$\alpha$ and hsCRP observed in T2DM have been widely reported [68]. It is known that raised levels of IL-6, and TNF-$\alpha$ can promote insulin resistance and contribute to the development of T2DM [69, 70]. Furthermore, hsCRP as a mediator of atherosclerotic diseases is strongly associated with the cardiovascular disease risk in T2DM patients [71].

In the present study, a reduction of plasma concentration of inflammatory parameters IL-6, TNF-$\alpha$ and hsCRP (21%, 12% and 20%, respectively) following the eight-week consumption of PoPEx in diabetic patients was observed. In line with our results, the anti-inflammatory effects of pomegranate or its derivatives had been previously shown in several studies. Significant decreases in IL-6 and hsCRP concentrations (by 30% and 32%, respectively) were observed after twelve-week administration of 250 mL/day pomegranate juice in diabetic patients [72]. Boldaji and coworkers demonstrated beneficial effects of eight-week consumption of 100 mL pomegranate juice in hemodialysis patients by reduction of CRP and IL-6 [73]. In an experimental models of rheumatoid arthritis administration 200 mg/kg of pomegranate rind extract significantly ameliorate inflammation parameters such as TNF-$\alpha$, IL-6 and nuclear factor kappa B (NF-$\kappa$B) suggesting that inhibition of NF-$\kappa$B could be responsible for its anti-inflammatory effect [74]. Mastrogiovanni et al. [75] suggested that punicalagin as bioactive component could be related to anti-inflammatory activity of PoPEx trough inhibition of prostaglandin production. These results are supported by the results of a recent study that showed that punicalagin and ellagic acid, as main components of pomegranate peel, possess a potent antioxidative and anti-inflammatory effect [76]. However, biological effects of polyphenols in vivo depend on their metabolism and this aspect will be addressed in a separate study.

The proposed mechanism of anti-inflammatory action of pomegranate is a decrease of the expression of cyclooxygenase-2 (COX-2) via inhibition of the NF-$\kappa$B and mitogen activated protein kinase (MAPK) pathways reducing the generation of pro-inflammatory prostaglandins [77, 78]. This is in accordance with our recent results on the fatty acid composition of plasma lipids of T2DM patients that the consumption of PoPEx inhibited conversion of arachidonic acid to pro-inflammatory eicosanoids and this could be one of possible anti-inflammatory mechanism of PoPEx activity [21].

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.