The Impact of Punica granatum Linn and Its Derivatives on Oxidative Stress, Inflammation, and Endothelial Function in Diabetes Mellitus: Evidence from Preclinical and Clinical Studies

Diabetes mellitus is recognized as the leading contributor to cardiovascular disease and associated mortality rates worldwide. Despite the use of pharmaceutical drugs to treat diabetes, its prevalence continues to rise alarmingly. Therefore, exploring remedies with a lower toxicity profile is crucial while remaining safe and effective in addressing this global public health crisis. Punica granatum Linn (pomegranate), known for its properties and safety profile, has been investigated in applied research and preclinical and clinical trials. However, conflicting reports still exist regarding its effects in diabetes. According to our knowledge, no systematic review has been conducted to critically analyze evidence from preclinical and clinical trials simultaneously, explicitly focusing on oxidative stress, inflammation, and endothelial function in diabetes. Therefore, in this systematic review, we searched for evidence on the impact of pomegranate in diabetes using databases such as PubMed, Scopus, and Google Scholar. Our inclusion criteria were limited to studies published in English. Of the 170 retrieved studies, 46 were deemed relevant and underwent critical analysis. The analyzed evidence suggests that pomegranate has the potential to alleviate oxidative stress, inflammation, and endothelial dysfunction in diabetes. Although a beneficial impact was noted in these markers, the endothelial function evidence still requires validation through further clinical trials with a powered sample size.


Introduction
Diabetes mellitus (DM) is a chronic metabolic condition characterized by hyperglycemia, which causes damage to the heart, blood vessels, eyes, kidneys, and nerves over time [1].A report by the International Diabetes Federation (IDF) indicated that about 537 million people were living with DM in 2021, which is anticipated to reach 783 million by 2045 [2].The IDF further revealed that about 90% of the DM population has type 2 diabetes globally (T2D) [2].Some of the mechanisms implicated in the pathophysiology of T2D include oxidative stress, which disrupts insulin signaling, damages pancreatic β-cells, and induces inflammation, thus promoting endothelial dysfunction [3,4].In oxidative stress, there is an imbalance between the rate of reactive oxygen species (ROS) production and the body's ability to eliminate them [5].This further predisposes the body to damage due to excessive ROS.Notably, endothelial dysfunction in T2D increases the risk of developing secondary complications and cardiovascular disease (CVD) (Figure 1). to excessive ROS.Notably, endothelial dysfunction in T2D increases the risk of developing secondary complications and cardiovascular disease (CVD) (Figure 1).The literature suggests that any therapeutic approach that can ameliorate oxidative stress may help control T2D and associated CVD.Several pharmaceutical drugs, including sodium-glucose cotransporter-2 (SGLT2) inhibitors, biguanides, glitazones, and α-glucosidase inhibitors, are widely used to control insulin sensitivity and reduce blood glucose in T2D patients [6,7].However, such drugs are associated with various adverse effects and related complications, ranging from megaloblastic anemia and neuropathy associated with vitamin B12 deficiency, increased low-density lipoproteins (LDL), hypoglycemia, water retention, acidosis, bone fractures, heart failure, weight gain, and gastrointestinal reactions [6][7][8][9].Due to all these secondary complications associated with pharmacological drugs, it would be ideal to evaluate the effect of functional foods and natural compounds with antioxidants on diabetes control and management.In fact, the World Health Organization (WHO) acknowledges the benefits of traditional, complementary, and alternative medicines (TCAMs), especially the use of plants that have been scientifically proven to be effective [10].This has prompted more research into natural remedies for diabetes.
In South Africa, traditional medicine has existed for a long time, with practitioners providing care to the public, although some of these medicinal plants are not properly verified by botanists for use in diabetes [11].More recently, Mokgalaboni et al. [12] reported a significant effect of Corchorus olitorius in animal models of diabetes on hyperglycemia, oxidative stress, and inflammation.Although the results showed potential benefits The literature suggests that any therapeutic approach that can ameliorate oxidative stress may help control T2D and associated CVD.Several pharmaceutical drugs, including sodium-glucose cotransporter-2 (SGLT2) inhibitors, biguanides, glitazones, and αglucosidase inhibitors, are widely used to control insulin sensitivity and reduce blood glucose in T2D patients [6,7].However, such drugs are associated with various adverse effects and related complications, ranging from megaloblastic anemia and neuropathy associated with vitamin B 12 deficiency, increased low-density lipoproteins (LDL), hypoglycemia, water retention, acidosis, bone fractures, heart failure, weight gain, and gastrointestinal reactions [6][7][8][9].Due to all these secondary complications associated with pharmacological drugs, it would be ideal to evaluate the effect of functional foods and natural compounds with antioxidants on diabetes control and management.In fact, the World Health Organization (WHO) acknowledges the benefits of traditional, complementary, and alternative medicines (TCAMs), especially the use of plants that have been scientifically proven to be effective [10].This has prompted more research into natural remedies for diabetes.
In South Africa, traditional medicine has existed for a long time, with practitioners providing care to the public, although some of these medicinal plants are not properly verified by botanists for use in diabetes [11].More recently, Mokgalaboni et al. [12] reported a significant effect of Corchorus olitorius in animal models of diabetes on hyperglycemia, oxidative stress, and inflammation.Although the results showed potential benefits in diabetes, this was only conducted in preclinical models, thus calling for more research in clinical trials.Although there are limitations associated with the translation of preclinical studies into clinical trials, the evidence from these studies may provide the basis for developing new alternative therapies to treat DM and prevent secondary complications.Pomegranate is another fruit that has gained research attention due to its antioxidant properties and, more recently, has been suggested to be a functional food due to its multiple health-promoting properties [13,14].This fruit is scientifically known as Punica granatum Linn and belongs to the Plantae kingdom and Punicaceae family [15].Several studies have investigated the effects of pomegranate on oxidative stress in diabetes.The evidence from preclinical studies has demonstrated the potential effect of pomegranate as an anti-oxidative agent, as revealed by its ability to reduce the levels of ROS in rodent models of diabetes [16][17][18][19][20][21][22][23][24].
Interestingly, the same results seem to be observable in clinical studies, as revealed by reduced markers of oxidative stress in T2D patients following treatment with pomegranate [25][26][27][28][29][30].However, there are still some inconsistencies in clinical trials on the impact of pomegranate, especially on different markers of oxidative stress and inflammation, and limited evidence on endothelial function in diabetes.More recently, a meta-analysis was conducted on pomegranate, and the researchers found no effect of pomegranate on oxidative stress and inflammation.One of the limitations is that their analysis focused on only total antioxidant capacity (TAC) and high-sensitive C-reactive protein (hs-CRP) as markers of oxidative stress and inflammation, respectively; hence, they might not independently be ideal predictors [27].In contrast, another meta-analysis showed a significant decrease in hs-CRP, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) without effects on CRP, soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular cell adhesion molecule-1 (sICAM-1), and malonaldehyde (MDA) [31].
Additionally, a meta-analysis by Morvaridzadeh et al. [32] on pomegranate showed no effect on TAC, glutathione peroxidase (GPx), paraoxonase-1 (PON1), and MDA.Furthermore, the analyzed studies encompassed a wide range of conditions, making it difficult to interpret and make conclusive remarks and recommendations to a wider diabetic population.The current review highlights the potential benefits of pomegranate extracts, focusing on various oxidative, inflammation, and endothelial markers in diabetes while elucidating its mode of action.

Search Strategy
Evidence was retrieved by independent investigators (KM and SD) through online databases, including Scopus and PubMed, on 20 May 2023 and updated on 20 July 2023, according to the updated guidelines of the preferred reporting items for systematic review and meta-analysis (PRISMA) [33].The exact search strategy is presented in the Supplementary File (Tables S1-S6).The search comprised five key terms: "pomegranate", "oxidative stress", "inflammation", "endothelial dysfunction", and "diabetes mellitus".This was performed in a separate search with the use of the Boolean operator "AND".The search was restricted to evidence published in English, however, with no duration limitation.Additionally, two independent investigators verified the search before decisions could be made (WNP and PM).

Study Selection
Two independent researchers (KM and SD) selected the studies based on their title, abstract, keywords, and overall aims and findings.The third independent researcher read all of the studies that the prior researchers had disagreements on and made a conclusion.The studies that passed the initial screening were subjected to a full screening of their full text.All studies that passed the second phase of screening were included if they met the following criteria: (i) they used either pomegranate juice extract, powder, or its active compounds; (ii) they included a rodent model of diabetes or diabetic patients; and (iii) they reported any markers of oxidative stress, inflammation, or endothelial function.The exclusion criteria were (i) reviews, commentaries, book chapters, and letters to editors and (ii) in vitro studies.

Data Extraction
Two investigators (KM and SD) independently extracted data from all relevant studies.The data extracted from each clinical study included the first author's surname; publication year; the country where the study was conducted; the study design; the number of participants in both groups; the number and proportion of males; the mean age in years; the body mass index (BMI) of the group on pomegranate in kg/m 2 ; the duration of treatment with pomegranate; the dose; the form of pomegranate treatment; and the main findings.Similarly, for preclinical studies, the data extracted included the first author's surname, the experimental model, the form of pomegranate and the duration of treatment, the main findings, and the statistical data for effect measures.In cases of disparities between the two investigators, WNP made a decision by re-evaluating the study and items in question.

Results
A total of 95 records were obtained from Scopus, while 57 were retrieved from PubMed.Additionally, Google Scholar was searched, and about 18 records were found relevant.Prior to the screening process, it was determined that 34 records were duplicates found in both databases through these separate searches, and thus, they were excluded.As a result, 136 records were subjected to initial screening by their title, abstract, keywords, and objectives.Fifty-one records were considered irrelevant and excluded due to their title, abstract, and aims being outside the scope of our review.
Consequently, a total of 85 records underwent a thorough screening process.Among these 85 records, 39 were excluded based on the following criteria: 17 were review articles, 9 did not report on outcomes of interest, 4 did not focus on pomegranate as an intervention, 1 was a commentary, 1 was a book chapter, 5 were not related to diabetes or models, 1 was a cell culture study, and 1 was a study protocol.Hence, a total of 46 records (33 preclinical and 13 clinical trials) were deemed relevant as they reported on the effect measures of interest in diabetic models or patients (Figure 2).2).An overview of pomegranate, its bioactive compounds, and its bioavailability.Punica granatum L. (pomegranate) is a plant belonging to the Punicaceae family [15].It is widely cultivated in regions such as the Middle East, the Caucasus, northern and tropical Africa, Iran, the Indian subcontinent, Central Asia, Southeast Asia (in drier parts), and
Additionally, we found thirteen relevant clinical studies that investigated the impact of pomegranate on oxidative stress, inflammation, and endothelial function in individuals with diabetes.These studies were published in peer-reviewed journals between 2000 and 2022, thus providing a comprehensive range of evidence on the effects of pomegranate in diabetes.The research was conducted in various countries, including three studies in Israel [28,29,60], eight in Iran [25,26,30,[61][62][63][64][65], one in Turkey [66], and one in Bosnia and Herzegovina [67].A total of 468 participants with diabetes, with a mean age of 54.48 ± 1.64, were included in these studies, alongside 120 healthy control individuals.The mean body mass index for the diabetic group receiving pomegranate was 29.70 ± 2.42 kg/m 2 , and 145 participants were male.The pomegranate was administered orally as juice or a capsule for 6 weeks to 3 months (Table 2).Different study designs were included, with at least four being quasi-experimental; this study aims to evaluate interventions but does not use randomization.One was a case-control study and eight were randomized controlled trials (Table 2).
1 kilogram of pomegranates: the seeds were grounded to obtain juice.The concentration of the extracts was dissolved in distilled water.Diabetic rats were treated with PJ at (100 or 300 mg/kg) for four weeks.
Treatments significantly increased GSH, CAT, and SOD and decreased TBARS.
Fresh PJ was prepared by diluting 12.5 mL/L in 1 L of water to make 0.35 mmol.The diluted PJ was given to the mice orally in their drinking water for four months.
Ground powder of pomegranate flowers (PGFs) and powdered rat feed were mixed to make rat pellet feed.Diabetic rats were fed the mixed pellet as PGFs at 300, 400, and 500 mg/kg for eight weeks.
PGF treatment significantly decreased lipid peroxidation (LPO) and increased GSH levels.
Pomegranates were washed, crushed, and squeezed to make PJ.Concentrated PJ was diluted in water (20 mL of concentrated juice in 500 mL of distilled water) to make 2.5 mL diluted PJ.Diabetic rats were treated with PJ for ten weeks.
PJ treatment significantly decreased endothelial nitric oxide synthase (eNOS) expression and increased SOD without significant changes in GSH.
Pomegranate peel dried and ground to powder (250 mg/kg).Seeds were used to make fresh juice (5 mL/kg).Diabetic rats were treated orally fed 250 PP mg/kg PP mixed with diet and oral PJ at 5 mL/kg) daily for four weeks.
Treatment with both regimens significantly increased total antioxidant capacity (TAC).
Pomegranate seed extract (PSE), grounded to powder and dissolved in distilled water to form PSE (300 mg/kg/day), was given orally by gavage for four weeks.
PSE significantly decreased the pancreatic expression of nuclear factor kappa-beta (NF-κβ) and increased pancreatic GSH content.

Author, Country Experimental Model Intervention and Duration Main Findings
Aboonabi et al. [16] Malaysia STZ-nicotinamide (NAD)-induced diabetes in male Sprague Dawley rats.
The red pomegranate fruit was washed and peeled, and the arils were crushed and squeezed to make juice (1 mL of juice).
The pomegranate seeds (PSs) were freeze-dried and ground into powder.
The powder was dissolved into distilled water (100 mg of PSs + 1 mL DW).Pomegranate juice-seed (1 mL of PJ + 100 mg of PS) Diabetic rats were treated orally with pomegranate seeds and juice for 21 days.
Treatment with pomegranate significantly increased the enzymatic antioxidants, including CAT, SOD, and TAC, and decreased MDA in the plasma.
The leaf powder (100 g) was dissolved into methanol: water (70:30) for 72 h to obtain the hydroalcoholic extract.Diabetic rats were treated via oral route using an oral feeding needle once with 50, 100, and 200 mg/kg of ethyl acetate fraction of Punica granatum Linn.Leaves (EAPG) for 28 days.

STZ-induced diabetes in Sprague Dawley rats
Diabetic rats were treated orally with 1, 2, and 4 mL/200 g of PJ for four weeks.
Treatment with 2 mL/200 g of PJ significantly decreased MDA.
Leaf powder (100 g) was dissolved in methanol: water (70:30) for 72 h to obtain a hydroalcoholic extract.Diabetic rats were treated with a flavonoid-rich fraction of pomegranate leaves (PGFF) at 50, 100, and 200 mg/kg for 28 days.
Punicalagin (PU) powder dissolved in 0.2 mL saline solution and was intraperitoneally administered at 1 mg/kg daily for 15 days.
40 g of dried powder dissolved in 95% ethanol to make a hydroalcoholic extract.Leaf extract 100 and 200 mg/kg of pomegranate, fruit peel extract 100 mg/kg, and peel extract 200 mg/kg of pomegranate.
Treatment significantly increased SOD and CAT while decreasing TBARS.
Dried pomegranate peels were grounded into a fine powder, dissolved in distilled water (100 mg/1 mL), and given orally through the stomach tube to rats at a 200 mg/kg dose for 20 days.
Pomegranate peel powder significantly increased SOD and TAC, while MDA and nitric oxide (NO) decreased.
Diabetic rats were treated with 400 mg/kg of PPE via oral gavage for four weeks.
Treatment significantly increased anti-oxidative activity, GSH, and TAC.

Author, Country Experimental Model Intervention and Duration Main Findings
Mollazadeh et al. [23] Iran STZ-induced diabetes in male Wistar rats.
Diabetic rats were orally treated daily with pomegranate seed oil (PSO) at 0.4 and 0.8 mg/kg for 28 days.
PSO at both concentrations significantly increased total thiol content and decreased MDA levels in the heart and kidneys.

Onal et al. [19] Turkey
STZ-induced diabetes in male Sprague Dawley rats.Diabetic rats were treated with PJ at 100 mg kg for ten weeks.
Treatment with PJ significantly decreased MDA levels without significant changes in inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) protein.
PSO dissolved in dimethyl sulfoxide, and the rats were treated orally with PSO at 0.4 and 0.8 mL/kg for three weeks.
PSO treatment significantly increased CAT, SOD, and GPx activity and decreased oxidative stress index values in tissue and mitochondrial fractions.
The rats were orally treated with 1 mL of PJ or 100 mg of pomegranate seed powder (PS) in 1 mL distilled water for 21 days.

Gabr et al. [37] Egypt
Alloxan-induced diabetes in male albino rats.PJ or peel extract at 500 mg/kg orally for four weeks.
Treatment with either juice or seed extract increased CAT and decreased MDA levels.
PJ of 20 mL concentrated juice in 500 mL of distilled water to make 100 µL.PJ treatment was administered at 100 µL through gastric gavage for ten weeks.
Treatment significantly increased GSH and GPx and decreased MDA without changes in SOD.

The effect of pomegranate and its derivatives on markers of inflammation in animal models of diabetes.
Evidence from preclinical studies has revealed, to some extent, the potential of pomegranate as an anti-inflammatory remedy.For instance, El-Deeb et al. [41] used Sprague Dawley STZ-NAD-induced diabetic rats to explore the benefits of 200 mg/kg of ethanolic extract of pomegranate for four weeks.This study demonstrated a significantly lower level of TNF-α (43.2 ± 1.51 pg/mL) compared to untreated diabetic rats (73.0 ± 0.87 pg/mL), p < 0.05.The same researcher further reported significantly decreased IL-6 (46.1 ± 1.31 pg/mL) compared to the untreated group (66.9 ± 0.99 pg/mL), p < 0.05.Moreover, these findings were confirmed by a previous study that used the same model [54] with a dose of 1 mL of PJ or 100 mg of pomegranate seed powder for 21 days.Interestingly, they reported a significant decrease in markers of inflammation, including TNF-α, IL-6, and NF-κβ.For instance, the mean of TNF-α was 2944.02pg/mL, p > 0.05, and 2844.92pg/mL, p < 0.05, at 1 mL of PJ and 100 mg of PS, respectively, compared to the untreated group, with 3074.37 pg/mL.Additionally, there was a decrease in the levels of IL-6 in the respective dosages: 296.42 pg/mL, p < 0.01, and 316.51 pg/mL, p < 0.01, compared to 355.86 pg/mL.Furthermore, the same study showed reduced NF-κβ in PJ, 2228.89pg/mL, p < 0.05, and 2138.68 pg/mL in the PS group compared to the untreated group, with 2337.14 pg/mL, p < 0.01.Similar results are partly corroborated by Shaker et al. [44], who also used the same model of diabetes and a slightly higher dosage (300 mg/kg) of pomegranate seed extract for four weeks.Consistently, they demonstrated a significant decrease in the pancreatic expression of NF-κβ, mean, SD, 765.6 ± 9.9 pg/gm, compared to the untreated group, with 992.5 ± 8.46 pg/gm, p < 0.01.Furthermore, transforming growth factor beta (TGF-β) was significantly decreased in the treated group, 477.5 ± 4.24 pg/gm, compared to the untreated group, 879.16 ± 6.91 pg/gm, p < 0.01.In another investigation, diabetic Sprague Dawley rats induced by STZ were subjected to an oral pomegranate regimen of 150 mg/kg daily for 18 days [56].In this experiment, the researchers made notable observations, specifically a substantial reduction in embryonic IL-1β, 2.78 ± 0.48 pg/mL, compared to 4.12 ± 0.57 pg/mL and IL-6, 1.30 ± 0.15 pg/mL compared to the untreated group, 2.18 ± 0.29 pg/mL.Moreover, in the diabetic mother rats, the same results were shown.For instance, there was a decrease in IL-6: 3.74 ± 1.08 pg/mL and IL-1β, 5.12 ± 1.24 pg/mL compared to the untreated diabetic groups: IL-6, 6.44 ± 1.98 pg/mL, p < 0.05 and IL-1β, 7.99 ± 1.23 pg/mL, p < 0.05.These results are further supported by Abo-Saif et al. [43], who demonstrated a significant decrease in IL-1β following eight weeks of PPE at a 150 mg/kg dose in diabetic Wistar rats.El-Missiry et al. [42] used the same model with an intraperitoneal punicalagin dose of 1 mg/kg for 15 days.Consistently, the treatment resulted in a significant decrease in IL-6 (45.9 ± 2.27 pg/mL compared to 72.7 ± 1.98 pg/mL, p < 0.001); TNF-α, (37.3 ± 0.42 pg/mL compared to 63.8 ± 2.75 pg/mL, p < 0.001); and IL-1β (75.7 ± 2.09 pg/mL compared to 124.9 ± 4.44 pg/mL, p < 0.001).In the most recent study by Abdulhadi et al. [53], STZinduced diabetic Wistar rats were intraperitoneally injected with punicalagin at a dose of 1 mg/kg for 15 days; interestingly, this was associated with a significant decrease in CRP (p < 0.001) and MCP-1 (p < 0.001) levels compared to the untreated diabetic group.On the contrary, a different study found no significant effect of pomegranate oil on CRP (p < 0.05) in HFD-fed mice [59].
The effects of pomegranate and its derivatives on markers of endothelial function in rodent models of diabetes.
Endothelial dysfunction in diabetes increases the risk of developing CVD complications such as atherosclerosis due to the formation of foam cells in the blood vessels [82].Thus, to curb these secondary complications in diabetes, it is important to control endothelial dysfunction in diabetes.Therefore, pomegranate may improve endothelial function due to its antioxidant potential, especially in diabetes.For example, in Sprague Dawley rats with STZ-NAD-induced diabetes, a four-week administration of 200 mg/kg of ethanolic extract of pomegranate demonstrated a significant decrease in NO bioavailability: 34.4 ± 0.60 µmol/L, p < 0.05 compared to 56.3 ± 1.30 µmol/L [41].A study by Çukurova et al. [58], also using the same animal model, revealed that 2.5 mL of PJ treatment significantly decreased endothelial nitric oxide synthase (eNOS) expression (p < 0.05).Additionally, in Wistar rats with STZ-NAD-induced diabetes, lower levels of NADPH oxidase (NOx) were observed following oral administration of PAJ at 100 (13 ± 1.5 µmol/g) or 300 mg (11.6 ± 1.0 5 µmol/g) compared to 31.7 ± 1.45 µmol/g, p < 0.001 [36].However, Onal et al. [19], after using 100 mg/kg pomegranate for ten weeks in Sprague Dawley STZ-induced diabetic rats, observed no significant difference in iNOS (p > 0.05) and eNOS (p > 0.05) protein levels.Most recently, Abdulhadi et al. [53] investigated 1 mg/kg intraperitoneal injection of punicalagin in STZ-induced diabetic Wistar rats for 15 days.Interestingly, this study reported significantly lower levels of ICAM-1 (p < 0.001), VCAM-1 (p < 0.001), Eselectin (p < 0.001), and endothelin-1 (ET-1) (p < 0.001) compared to the untreated diabetic group.The aforementioned results were corroborated by El-Mansi et al. [56], who used the same model coupled with an oral methanolic extract of pomegranate at a 150 mg/kg dose for 18 days.This study demonstrated a significant decrease in the level of ET-1 in both the embryo (3.59 ± 0.27 pg/mL) and its mother (6.87 ± 0.41 pg/mL) compared to the untreated diabetic embryo (5.16 ± 0.37 pg/mL) and its mother (8.87 ± 0.68 pg/mL).Increased ET-1 stimulates the production of ROS [83,84].Its reduction in diabetes following pomegranate treatment shows its potential to ameliorate endothelial dysfunction and associated CVD complications.
The effect of pomegranate and its derivatives on markers of oxidative stress in a rodent model of diabetes.
The effect of pomegranate on markers of endothelial function in diabetic patients.
Endothelial dysfunction contributes to the development of CVD.This study focused on sICAM-1 and sVCAM-1 from clinical trials.Our evidence in clinical trials showed conflicting findings on endothelial function following treatment with 250 mL of pomegranate oil in diabetes [65].For instance, we observed a significant decrease in sICAM-1 levels from baseline (151 ± 17 ng/mL) to post treatment (138 ± 12 ng/mL, p < 0.001) and a decrease in sE-selectin levels from 19 ± 7 ng/mL to 13 ± 6 ng/mL post treatment (p < 0.001).However, there was no significant difference in sVCAM-1 levels: 27 ± 11 ng/mL compared to 31 ± 19 ng/mL (p > 0.05).
The effect of pomegranate and its derivatives on markers of inflammation in diabetic patients.
Inflammation is implicated in the development and progression of diabetes, especially T2D.Diabetes and inflammation have a complex relationship that involves several cellular and molecular pathways.Some implicated mechanisms include inflammatory pathways such as the activation of nuclear factor-kappa-beta (NF-κβ) [85].Various inflammatory markers, such as TNF-α, IL-6, CRP, and NF-κβ, are evaluated to assess inflammation and serve as targets for potential therapies [85,86].In the current review, evidence from clinical studies showed a significant decrease in the circulating levels of CRP (p < 0.05), IL-6 (p < 0.01), and TNF-α (p < 0.01) in diabetic patients on 8-week treatment with 500 mg of pomegranate peel extract [67].Similar results were reported by a study conducted in Iran, where 12 weeks of 250 mL of PJ significantly decreased plasma hs-CRP (baseline: 3243 ± 2935 ng/mL compared to post treatment: 1791 ± 1657 ng/mL, p < 0.05) and IL-6 (10.9 ± 4.4 ng/L compared to 7.1 ± 5.6 ng/L, p < 0.05) [62].Furthermore, this study showed a significant decrease in TNF-α between the baseline (37.0 ± 19.3 ng/L) and posttreatment (30.4 ± 17.5 ng/L) levels, p < 0.01.Consistently, PSO at a dose of 3g for eight weeks significantly reduced IL-6 (before treatment: 5.2 ± 2.2 pmol/mL compared to after treatment: 4.5 ± 1.9 pmol/mL, p = 0003) and TNF-α (before treatment: 9.2 ± 4.1 pmol/mL compared to after: 7.7 ± 2.4 pmol/mL, p = 0.028) in T2D patients.However, no significant differences were observed in the serum levels of hs-CRP (before: 1.4 ± 1.8 pg/mL compared to after: 0.9 ± 0.6 pg/mL, p = 0.11) [63].Similar findings were acknowledged in a quasiexperimental design [61] that used concentrated PJ (50 g) for four weeks and observed a significant decrease in IL-6 levels from baseline (31.12 ± 3.12 pg/mL) to post treatment (23.40 ± 2.27 pg/mL, p = 0.04).Surprisingly, no statistically significant differences were observed in the levels of TNF-α (baseline: 18.72 ± 0.95 pg/mL compared to post treatment: 17.66 ± 1.41 pg/mL, p = 0.42) and CRP (2.37 ± 0.24 ng/ML compared to 2.44 ± 0.23 ng/ML, p = 0.74).Likewise, administering 250 mL of PJ for 12 weeks in diabetic patients showed no significant effect on NF-κβ [65].Although these findings revealed some contradictory reports, the gathered evidence suggests that pomegranate and its derivatives may have the potential to modulate inflammation by reducing markers associated with inflammation in diabetic patients.
The effect of pomegranate and its derivatives on markers of oxidative stress in diabetic patients.
Oxidative stress plays an important role in the development and progression of both T1D and T2D due to the accumulation of ROS [5].The accumulation of ROS results in cell, tissue, and organ damage, thus predisposing them to oxidative stress [5].Several treatment strategies aim to target oxidative stress to minimize associated complications.In this review, we focused on the effect of pomegranate on various markers of oxidative stress, including MDA, SOD, CAT, and AOC, as presented in Table 2.The overall evidence gathered from clinical studies revealed that pomegranate exerts an antioxidant effect in patients with diabetes (Table 2).For instance, a study by Rosenblat et al. [29] showed that 50 mL of pomegranate juice administered for three months significantly decreased serum oxidative stress.Consistently, this was associated with lower lipid peroxide (p < 0.01) and TBARS levels (p < 0.05) and an increase in PON1 arylesterase (p < 0.05) activity.Furthermore, the study reported an increase in GSH levels (p < 0.05), concomitant with a decrease in ox-LDL uptake (p < 0.05), following pomegranate juice treatment.
The gathered evidence suggests the potential effects of pomegranate and its derivatives in reducing oxidative stress in diabetic patients.These effects include reduced lipid peroxidation, increased antioxidant enzyme activity, improved glutathione levels, and enhanced TAC.To completely understand the underlying mechanisms and identify the ideal dosage and duration of pomegranate supplementation for efficient therapy for the management of oxidative stress, further research is required.

Discussion
Our systematic review is composed of 33 preclinical and 13 clinical studies.Indeed, more evidence supports using pomegranate as an antioxidant and anti-inflammatory agent, showing the benefits of improving endothelial function.Although there was no uniform evaluation of the same markers for all these parameters, the evidence gathered in this study suggests that pomegranate can ameliorate oxidative stress, inflammation, and endothelial dysfunction in diabetes.However, its mechanisms of action are still not well documented.When significant focus was placed on the effect of pomegranate on oxidative stress in rodent models of diabetes, a few markers that were considered included MDA, SOD, CAT, TBARS, GSH, GPx, TAC/AOC, ROS, PON1, FFA, and lipid peroxidation.Due to a physiological imbalance or condition, a decline in the activity of antioxidant enzymes such as GSH, SOD, CAT, and GPx enhances the vulnerability to oxidative stress.
MDA levels were reduced in several preclinical studies [16,19,20,23,34,[36][37][38][39][41][42][43][45][46][47]51,56].Interestingly, similar findings were observed in clinical studies, as shown by a decrease in MDA levels [26,30,53,66].Another biomarker of oxidative stress that was evaluated included TBARS.This takes into account the level of LPO degradation products in cells and tissues.In fact, diabetes is associated with increased TBARS, thus indicating increased LPO and further oxidative stress [88].Therefore, it is imperative that LPO be inhibited in diabetes in order to alleviate oxidative stress.In the current review, TBARS levels were decreased in preclinical and clinical studies following pomegranate treatment in diabetes [24,29,47,48,60,67].This shows the potential of pomegranate as an anti-oxidative agent in the state of diabetes.
Moreover, pomegranate treatment has been shown to alleviate oxidative stress by neutralizing ROS [21] and preventing LPO [43,53].This beneficial effect can be explained, at least in part, by the high content of anthocyanins and ellagic acid in pomegranate (Figure 4).Likewise, pomegranate inhibits LPO, thus reducing MDA production and mitigating oxidative damage [21].We found that in rodent models of diabetes, various markers of inflammation were assessed.After treatment with pomegranate, these markers were remarkably decreased.Some of these markers included TNF-α [35,41,42,54], IL-6 [41,42,56], IL-1β [35,42,43,54,56], NF-κβ [44,54], MCP-1 [35], and CRP [53].Similarly, in clinical studies, we found that the administration of pomegranate decreased TNF-α [62,63,67], IL-6 [61][62][63]67], and CRP [62,67].Although the results from preclinical have been replicated in clinical trials, there are still some inconsistencies in inflammation, as reported by other researchers.For instance, no significant effect of pomegranate was observed on TNF-α [61,62], CRP [61], hsCRP [63], and NF-κβ [65] in clinical studies.This points out the limitation of this fruit as an anti-inflammatory agent; however, some reasons may be due to the preparation of the plant extract, part of fruit used, dosage, and the stage of diabetes.Some of the modes All of these findings support the use of pomegranate as an anti-oxidative agent, as evidenced by its ameliorative effect on the various markers of oxidative stress.The antioxidant compounds in pomegranate include anthocyanins and ellagic acid [89][90][91], which ameliorate oxidative stress in diabetes.A few studies suggest that anthocyanins, ellagic acid, and PU mediate pomegranate's anti-oxidative stress effects by degrading and scavenging free radicals [42,[92][93][94][95][96].
We found that in rodent models of diabetes, various markers of inflammation were assessed.After treatment with pomegranate, these markers were remarkably decreased.Some of these markers included TNF-α [35,41,42,54], IL-6 [41,42,56], IL-1β [35,42,43,54,56], NF-κβ [44,54], MCP-1 [35], and CRP [53].Similarly, in clinical studies, we found that the administration of pomegranate decreased TNF-α [62,63,67], IL-6 [61][62][63]67], and CRP [62,67].Although the results from preclinical have been replicated in clinical trials, there are still some inconsistencies in inflammation, as reported by other researchers.For instance, no significant effect of pomegranate was observed on TNF-α [61,62], CRP [61], hsCRP [63], and NF-κβ [65] in clinical studies.This points out the limitation of this fruit as an antiinflammatory agent; however, some reasons may be due to the preparation of the plant extract, part of fruit used, dosage, and the stage of diabetes.Some of the modes of action of pomegranate in the amelioration of inflammation are mediated by anthocyanins, which inhibit the cyclooxygenase (COX), NF-κβ activity, and phosphorylation of mitogen-activated protein kinase (MAPK) proteins while inducing nitric oxide (NO) expression [97][98][99].Regarding endothelial function, a few preclinical studies have shown reduced levels of the following endothelial markers: VCAM-1 [53], ICAM-1 [53], E-selectin [53], ET-1 [56], eNOS [58], and NO bioavailability [36,39,41].Similarly, sICAM-1 and sE-selectin were also reduced without any effect on sVCAM-1 [65].Reduced markers of endothelial function following pomegranate treatment in diabetes support its use as an agent to improve endothelial function in diabetes.One mechanism by which pomegranates enhance endothelial function is by reducing oxLDL levels [25].This is partly because the accumulation of ox-LDL is associated with endothelial dysfunction and the development of atherosclerosis [100].Therefore, our results show that pomegranate may preserve healthy endothelial function by preventing LDL oxidation.In clinical studies, there have been contradictory findings on oxLDL following pomegranate treatment.On the other hand, in a study, pomegranate showed no effect on eitheriNOS and eNOS [19].For example, a report by Rosenblat [29] indicated an increase in oxLDL, whereas Sohrab [25] reported a decrease in oxLDL.Oxidized LDL impairs endothelial function and vascular health, forming foam cells and atherosclerotic plaques.This in turn may lead individuals to develop secondary complications.Due to these controversies, further research is required to better understand the effects of pomegranate treatment in diabetes on endothelial functions.
Although the current review has shown some of the potential benefits of pomegranate in diabetes, it is important to consider some of the following limitations.Firstly, most of the included studies were conducted in Asia, which comprises countries known to be major pomegranate-producing countries.Secondly, the diabetic rodent models presented in this study were induced through the administration of STZ or alloxan, causing T1D, unlike T2D observed in humans (as shown in Table 2).As a result, the physiological pathways leading to oxidative stress, endothelial function, and inflammation may differ between the two conditions, affecting how pomegranate regulates them.Additionally, the method of administration of pomegranate, especially in rodents, varied from different studies, with some using powder in their diet, such as pomegranate juice, while others prepared extracts from fruit peel and seeds.These differences in administration could contribute to conflicting findings across the studies.
Moreover, the human studies also employed different methods and doses, which led to contradicting results.For instance, some studies used pomegranate juice, while others used powdered pomegranate at varying doses.These variations in the approaches used further complicate the interpretation of the results.Amongst the strengths of the current review is the inclusion of evidence from preclinical studies and clinical trials.Evaluation of various treatment regimens also allows us to find a possible effective and safe dose.The stringent eligibility criteria, selection, and multiple database searches strengthened our review.

Conclusions
Based on a thorough examination of the various studies in this review, a comprehensive body of research consisting of 33 preclinical and 13 clinical studies involving 468 patients with T2D suggests that pomegranate shows promising results as a potential agent for improving oxidative stress, inflammation, and endothelial dysfunction in diabetes.Although these benefits have been acknowledged in both preclinical and clinical studies, there is limited evidence regarding endothelial function, particularly in clinical studies, which necessitates further investigation, particularly in diabetes.Additionally, it is worth noting that the clinical evidence presented in this study was based on small sample sizes ranging from 10 to 60 T2D patients, indicating that these trials may have been insufficiently powered.Therefore, based on this observation, we recommend conducting future clinical trials with larger sample sizes to gain a better understanding of the underlying mechanisms through which pomegranate exerts its effects and to determine the optimal dosage and duration of pomegranate treatment that can be used to achieve optimal anti-inflammatory and anti-oxidative benefits.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antiox12081566/s1. Table S1: Search on Scopus for oxidative stress; Table S2: Search on Scopus for endothelial function; Table S3: Search on Scopus for inflammation; Table S4: Search on PubMed for oxidative stress; Table S5: Seach on PubMed for inflammation; Table S6: Search on PubMed for endothelial function.
Author Contributions: Conceptualization, K.M. and S.L.L.; methodology, K.M., S.D., W.N.P. and P.M; software, K.M.; validation, K.M., S.D., W.N.P., P.M. and S.L.L.; formal analysis, K.M.; investigation, K.M.; resources, K.M. and S.L.L.; data curation, K.M., S.D. and W.N.P.; funding, K.M.; writingoriginal draft preparation, K.M.; writing-review and editing, K.M., S.D., W.N.P., S.L.L. and P.M.; supervision, S.L.L.All authors have read and agreed to the published version of the manuscript.Acknowledgments: All figures were generated by KM using Biorender.W.N.P is currently funded by the University of South Africa Women in Research (WiR of 2023), which forms part of wider research funding under "the use and functional properties of African indigenous fruits and vegetables in alleviating household food and nutrition insecurity for local communities".The content herein is the sole responsibility of the authors and does not necessarily represent the official views of the funders.

Conflicts of Interest:
The authors declare no conflict of interest.

Antioxidants 2023 ,
12, x FOR PEER REVIEW 5 of 26 being quasi-experimental; this study aims to evaluate interventions but does not use randomization.One was a case-control study and eight were randomized controlled trials (Table

Figure 2 .
Figure 2. PRISMA flow chart depicting the study selection, screening, and inclusion.

Figure 2 .
Figure 2. PRISMA flow chart depicting the study selection, screening, and inclusion.

Figure 3 .
Figure 3.The active compounds found in pomegranate.

Figure 3 .
Figure 3.The active compounds found in pomegranate.

Funding:
This research was partially funded by Research Development Grants for nGAP Scholars (reference number NGAP23022780506) and the Research Excellence Award for Next Generation Researchers: NONF230515106418.The content herein is the sole responsibility of the authors and does not necessarily represent the official views of the National Research Foundation.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.

Table 1 .
General overview of the effect of pomegranate extract and its active compounds on oxidative stress in rodent models of diabetes.

Table 2 .
Overview of the effect of pomegranate extract and its active compounds on oxidative stress and inflammation in diabetic patients.