The effects of pomegranate supplementation on biomarkers of inflammation and endothelial dysfunction: A meta-analysis and systematic review

Complementary Therapies in Medicine 49 (2020) 102358

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The effects of pomegranate supplementation on biomarkers of inflammation and endothelial dysfunction: A meta-analysis and systematic review

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Peng Wanga, Qiang Zhanga, Huijuan Houa, Zhiyong Liua, Li Wangb, Reyhaneh Rasekhmaghamc, Hamed Kord-Varkanehd, Heitor O. Santose, Guangtao Yaoa,* a

Department of Cardiovascular, Dezhou People's Hospital, No. 1166 Dongfanghong West Road, Decheng District, Dezhou City, Shandong Province, 253000, China Department of Gastroenterology, Yucheng People's Hospital, No. 753 Kaifa Road, Yucheng City, Shandong Province, 251200, China c Department of Nutrition, Faculty of Medical Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran d Student Research Committee, Department of Clinical Nutrition and Dietetics, Faculty of Nutrition and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran e School of Medicine, Federal University of Uberlandia (UFU), Uberlandia, Minas Gerais, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Meta-analysis Inflammation Endothelial function Pomegranate

Objects: Cardiovascular disease (CVD) is one of the leading causes of death worldwide. CVD is associated with increased levels of reactive oxygen species which are pro-inflammatory and can damage the endothelium. The pomegranate fruit is a rich source of phytochemicals with a high antioxidant and anti-inflammatory activity, possessing thus health benefits. This systematic review and meta-analysis aims to evaluate the effect of pomegranate juice on the biomarkers of inflammation and vascular dysfunction. Methods: Studies were identified using the PubMed/Medline and SCOPUS databases. Screening of relevant articles and references was carried out from inception until May 2019. This systematic review and meta-analysis was performed using the Preferred Items for Reporting of Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Results: Overall, 16 randomized controlled trials (RCTs) involving 572 subjects were included in this study. Combining effect sizes from 16 studies, we recorded that pomegranate supplementation significantly reduced hsCRP, IL-6 and TNF-α (Weighted Mean Diff ;erences (WMD): −6.57 mg/L, 95 % CI: −10.04 to −3.10, P = 0.000; WMD: −1.68 pg/mL, 95 % CI: −3.52, 0.157, P = 0.000; WMD: −2.37 pg/mL, 95 % CI: −3.67, −1.07, P = 0.00, respectively) levels, when compared to placebo. No significant reduction was found in CRP (WMD: 2.19 mg/dL, 95 % CI: −3.28, 7.67, P = 0.61), E-selectin (WMD: 8.42 ng/mL, 95 % CI: −22.9, 39.8, P = 0.599), ICAM (WMD= −17.38 ng/mL, 95 % CI: −53.43, 18.66, P = 0.107), VCAM (WMD: −69.32 ng/mL, 95 % CI: −229.26, 90.61, P = 0.396) or MDA (WMD: 0.031 μmol/L, 95 % CI: −1.56, 0.218, P = 0.746) comparing pomegranate supplementation to placebo. Conclusion: We found a significant effect of pomegranate supplementation on hs-CRP, IL-6 and TNF-α in adults. However, the effects of pomegranate supplementation on CRP, E-selectin, ICAM, VCAM or MDA were not significant in this meta-analysis.

1. Introduction

lead to cardiac ischemia, cardiomyopathy and acute coronary syndromes.6 CVD is associated with an increased production of reactive species of oxygen that are pro-inflammatory and damage the endothelium.7–9 Endothelial dysfunction increases the vascular permeability and reduces nitric oxide (NO) bioavailability, which in turn increases inflammation. On the other hand, cell adhesion molecules (CAMs) are highly expressed in the endothelium and play a key role in the development of atherosclerosis.6 Among lifestyle interventions, functional foods and nutritional supplements may attenuate the gene

Pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) are produced by macrophages, T lymphocytes, and adipocytes.1 TNF-α induces the expression of various inflammatory cytokines and translation factors.2 Inflammation has an important role in the cardiovascular disease (CVD).3,4 CVD is one of the leading causes of death in the world.5 CVD can present in many forms, starting from atherosclerosis, and over time can

⁎

Corresponding author. E-mail address: [email protected] (G. Yao).

https://doi.org/10.1016/j.ctim.2020.102358 Received 17 January 2020; Received in revised form 23 February 2020; Accepted 24 February 2020 Available online 26 February 2020 0965-2299/ © 2020 Elsevier Ltd. All rights reserved.

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expression of pro-inflammatory cytokines,2 with polyphenols have been proposed to improve endothelial function.10 The Punica granatum L. (pomegranate) fruit is a rich source of phytochemicals with a high antioxidant and anti-inflammatory activity that deserves attention in many aspects of human health and diseases.11 Phytochemicals include flavonoids (anthocyanins, catechines, quercetin, rutin), other types of polyphenols, ellagitannins, and antioxidant vitamins.12–15 Natural polyphenols, such as anthocyanins, act as antiinflammatory agents by inhibiting the cyclooxygenase, nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) activity and phosphorylation of mitogen-activated protein kinase (MAPKs) proteins, while inducing NO expression.15–18 Studies have shown that pomegranate reduces lipid peroxidation, oxidative stress 19 and insulin resistance,20 improves the lipid profile and reduces obesity by affecting the peroxisome proliferator activated receptors (PPARs),21 and can be potentially useful in treating hypertension and improving the vascular function.22 To date, there are conflicting results regarding interventional studies. For instance, Asgary et al. (2013) noted that patients with hypertension receiving 150 mL of pomegranate juice, for two weeks, significantly decreased the serum levels of vascular cell adhesion protein 1 (VCAM-1) levels while increased E-selectin. In addition, pomegranate juice had no significant effect on serum interleukin-6 (IL-6), high-sensitivity C-reactive protein (hs-CRP) or intercellular adhesion molecule (ICAM) levels.6 Sohrab et al. (2014), in turn, supplemented patients with type 2 diabetes mellitus (T2DM) with 250 mL of pomegranate juice for 12 weeks, then observing a significant decrease of 32 % and 30 % in hs-CRP and IL-6 levels, respectively.23 Given the potential and controversies of pomegranate juice in cardiometabolic aspects, the aim of this study was to evaluate not only its ingestion, but also the effects of supplementing other processed pomegranate products (e.g. powder and oil), on biomarkers of inflammation and vascular dysfunction.

Fig. 1. Flow chart for study identification.

Studies were excluded if they: 1) were non-clinical trials studies; 2) were conducted on children, pregnant women or animals; 3) were not placebo-controlled trials; 4) lacked sufficient data for the outcomes of interest in the intervention or control group; and 5) examined the effect of pomegranate supplementation along with other interventions.

"Random Allocation"[Mesh] OR RCT[Title/Abstract] OR "Intervention Studies"[Title/Abstract] OR "intervention"[Title/Abstract] OR "controlled trial"[Title/Abstract] OR "randomized"[Title/Abstract] OR "randomised"[Title/Abstract] OR "random"[Title/Abstract] OR "randomly"[Title/Abstract] OR "placebo"[Title/Abstract] OR "assignment"[Title/Abstract])). The search strategy in SCOPUS was: ((TITLEABS-KEY (punicaceae) OR TITLE-ABS-KEY ("Punica*") OR TITLE-ABSKEY (pomegranate))) AND (((((TITLE-ABS-KEY ("Clinical Trials")) OR (TITLE-ABS-KEY ("Clinical Trial")) OR (TITLE-ABS-KEY ("Cross-Over Studies")) OR (TITLE-ABS-KEY ("Double-Blind Method")) OR (TITLEABS-KEY ("Single-Blind Method")) OR (TITLE-ABS-KEY ("Random Allocation")) OR (TITLE-ABS-KEY ("RCT"))) OR (TITLE-ABS-KEY ("random")) OR (TITLE-ABS-KEY ("randomly")) OR (TITLE-ABS-KEY ("placebo")) OR (TITLE-ABS-KEY ("assignment"))) OR ((TITLE-ABS-KEY ("Intervention Studies")) OR (TITLE-ABS-KEY ("intervention")) OR (TITLE-ABS-KEY ("controlled trial")) OR (TITLE-ABS-KEY ("randomized")) OR (TITLE-ABS-KEY ("Trial")) OR (TITLE-ABS-KEY ("randomised"))))). No restriction based on language or publication date was applied. To avoid missing any study, the reference lists of all the eligible articles, related reviews, and meta-analyses were also reviewed. Unpublished records, conference papers, theses, and patents were not included in this meta-analysis.

2.3. Search strategy

2.4. Data extraction

This systematic review and meta-analysis was conducted based on the Preferred Reporting Items for Systematic Reviews and MetaAnalyses (PRISMA) (Fig. 1). A total of 1061 relevant articles published up to May 2019 were detected after searching the PubMed/Medline and SCOPUS databases. The MESH and non-MESH keywords used in PubMed/Medline were: ((((pomegranate[tiab]) OR "Punicaceae"[Mesh]) OR "Punicaceae"[tiab])) AND (("Cross-Over Studies"[Mesh] OR "Double-Blind Method"[Mesh] OR "Single-Blind Method"[Mesh] OR

Data extraction was performed by two independent investigators. The following data were extracted from the included studies: first author’s name; year of publication; study location; study duration; mean age and gender of participants; study design; health status of the study population; number of participants in each groups; type and dose of pomegranate supplements; and mean ± standard deviation (SD) of the biomarkers levels of inflammation and endothelial dysfunction at baseline and the end of the study. Any probable controversies were

2. Methods 2.1. Eligibility criteria Studies were selected if they met the following criteria based on using the PICO items: description of the Population (P), Intervention (I), Comparison (C), and Outcomes (O): i) population was subjects s ≥18 years old; ii) pomegranate administration was prescribed, iii) examined the effect of pomegranate administration compared with the control group, iv) reported CRP or/and hs-CRP or/and TNF-α or/and IL-6 or/ and malondialdehyde (MDA) or/and E-Selectin or/and ICAMs or/and VCAMs before and after the intervention in both placebo and the treatment group, and v) full-text manuscript written in English. 2.2. Exclusion criteria

2

44 28

19 54

250 mL /juice

240 mL /juice

500 mg /extract

1000 mg /extract

500 mL /juice

220 mL /juice

1000 mg /extract

500 mg /extract

750 mg /juice

400 mg/ oil

100 ml/juice

50 mL/ juice

538.5 mg /extract

Yes Endothelial

Yes Endothelial

Yes Endothelial

Yes Endothelial

Yes Endothelial

Yes Endothelial

3

Yes Endothelial

Yes Endothelial

Yes Endothelial

Yes Endothelial

Yes Endothelial

Yes Endothelial

Yes Endothelial

101

45

18

55

27

50

30

42

38

21

150 mL /juice

Yes Endothelial

Inflammation Markers: Function: Yes Inflammation Markers: Function: Yes Inflammation Markers: Function: No Inflammation Markers: Function: No Inflammation Markers: Function: No Inflammation Markers: Function: No Inflammation Markers: Function: No Inflammation Markers: Function: No Inflammation Markers: Function: No Inflammation Markers: Function : No Inflammation Markers: Function: No Inflammation Markers: Function: No Inflammation Markers: Function: No Inflammation Markers: Function: No

Sample size

Intervention Dosage (type)

Outcomes (Yes/No)

Table 1 Characteristics of the eligible studies.

30

21

66

53

21

48.5

52.5

51.5

49.5

19

55

59

Mean age

8

8

48 hours

4

48 hours

8

24

5 days

1

4

8

2

12

2

Duration (week)

Both

Men

Men

Both

Men

Both

Men

Both

Both

Women

Men

Both

Both

Gender

Adults with no symptomatic disease

Well-trained rowers

Hemodialysis patients

Dyslipidemic patients

Elite weightlifters

Rheumatoid arthritis patients

Obese women with dyslipidemia Overweight and obese individuals Patients with metabolic syndrome Patients with ischemic heart disease Hemodialysis patients

Patients with type 2 diabetes mellitus Young healthy males

Hypertensive subjects

Target population

Randomised, double blind placebocontrolled trial Randomised, double blind placebocontrolled trial Randomised placebo controlled doubleblind trial Randomised, double blind placebocontrolled trial Randomised double-blinded placebocontrolled clinical trial

Randomized double blind clinical trial/ parallel Randomized double blind clinical trial/ parallel Randomised, double blind placebocontrolled trial Randomized, placebo controlled, crossover study Randomized double blind clinical trial/ parallel Randomized double blind clinical trial/ parallel Randomized double blind clinical trial

Randomised, placebo-controlled trial

Single blind

Study design

A. Stockton et al.

A. Urbaniak et al.

Lilach Shema-Didi et al.

GOLALEH ASGHARI et al.

Achraf Ammar et al.

M Ghavipour et al.

Pei-Tzu Wu et al.

Zahra Razani et al.

Hossein Moazzen et al.

Mahdiyeh Khadem Haghighian et al. Banafshe Hoseini et al.

Mohammad Mazani et al.

Golbon Sohrab et al.

Sedigheh Asgary et al.

Author

P. Wang, et al.

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the basis of title and abstract; in next step, 32 eligible studies selected for a careful full-text assessment. We excluded 18 articles based on following reasons: studies were not published in English (n = 3), studies which lacked a control group (n = 7), those which examined the effect of pomegranate in combination with another intervention (n = 1), and publications which lacked the outcomes of interest (n = 4). In addition, we excluded studies which evaluated the same dataset (n = 3). Overall, 14 eligible RCTs with 16 treatment arms were included in this meta-analyses as shown in Fig. 1. Among them, 3 articles reported the effect of pomegranate supplementation on the concentrations of CRP, 7 articles on hs-CRP, 4 articles on TNF-α, 5 articles on IL-6, 6 articles on MDA, and 2 articles on biomarkers of endothelial function/ dysfunction (including E-Selectin, ICAMs and VCAMs).

resolved by the third reviewer (AM). With regards to studies in which data was provided at different time points, data from the latest measurement was obtained. When an individual study reported the effect of pomegranate supplementation in different doses, each dose was analyzed as a separate study. In addition, when relevant data was missing from the studies, we contacted the publications’ authors to retrieve it. 2.5. Quality assessment of studies In this study, the risk of bias was assessed using the methods depicted in the Cochrane Handbook.24 The assessment took into consideration the following six items: random sequence generation; allocation concealment; blinding of participants and personnel; blinding of outcome assessment; incomplete outcome data; selective reporting and other sources of bias. Each item was categorized as “high risk” (at least one item has a high risk of bias), “low risk” (all items have a low risk of bias) or “unclear” (at least one item has an unclear risk of bias). Other biases included profit bias and the sample calculation. We ranked each study as having poor, fair, or good quality based on the Agency for Healthcare Research and Quality (AHRQ) standards. Articles were judged to be of poor quality if the allocation concealment, random sequence generation, and blinding demonstrate unclear or high risk of bias.

3.2. Study characteristics The basic characteristics of the included studies are shown in Table 1. Our study included 572 participants, 305 in the intervention group and 267 in the control group, with a mean age of 37.5 years. The studies were published between 2011 and 2018. Of these studies, 11 were conducted in Iran 5,6,23,27–34 and five in: the United States,35 Tunisia,36 Israel,37 Poland,38 and the United Kingdom.39 In 8 studies, the intervention group received pomegranate juice in a dose of 50−750 mL5,23,28,30,36–38; in 5 studies, the intervention group received pomegranate extract in a dose of 500−1000 mg 29,31,33,35,39; in one study, the intervention group received 480 mg/day of pomegranate oil 32 for a period of 48 h to 12 weeks. Five studies were conducted on men, 28,35–38 one study on women 33 and 7 studies on both sexes,6,23,30–32.34,39 The studies recruited healthy young people,28,39 bodybuilders,36 canoe athletes, 38 hypertensive patients,6 T2DM patients,23 obese and overweight subjects,29,33 patients with ischemic heart disease,30 metabolic syndrome 34 or dyslipidemia,32,33 patients undergoing dialysis,35,37 and patients diagnosed with rheumatoid arthritis.31 In 4 studies, hs-CRP did not decrease significantly in the intervention group.23,28,30,32 In 7 studies, no significant changes in CRP, TNF-α, IL-6 or MDA were recorded in the intervention and control groups,6,30–32.35,37,39 ICAMs and VCAMs showed a significant decrease in the intervention group of Sohrab’s 27 and Agary’s 6 reports at the end of the intervention. The quality assessment and the risk of bias assessment of the included studies is provided in Supplementary Table 1. Five arms have fair quality,31,36–38,40 three has poor quality 6,28,30, and six have good quality.27,29,32,35,39,41 Most trials revealed adequate quality for key factors.

2.6. Statistical analysis Mean differences and standard deviations (SD) of the following outcomes comparing pomegranate supplementation and controls were used to calculate the overall effect size: (1) CRP (mg/dL) (2) hs-CRP (mg/L) (3) TNF-α (pg/mL) (4) IL-6 (pg/mL) (5) MDA (μmol/L) (6) Eselectin (ng/mL) (7) ICAMs (ng/mL) (8) VCAMs (ng/mL). A randomeffects model was employed to estimate the overall effect size. When the SDs for mean diff ;erences were not reported, we calculated them by the following formula: SD change=√[(SD baseline 2+SDfinal 2) −(2×R × SD baseline × SD final)]. Based on previous studies in which mean diff ;erences were reported, a correlation coefficient of 0.8 was assumed as the R-value in the aforementioned formula. In addition, the standard error of mean (SEM) was converted to SD using the following formula: SD = SEM×√n (n = number of participants in each group). Moreover, we used the GetData Graph Digitizer 2.26.0.20 to estimate mean differences and SDs in studies that reported the outcomes of interest as graphs only. The inter-study heterogeneity was examined using the Cochrane’s Q test (significance point at P < 0.1) and I2 statistic. In order to find the potential sources of heterogeneity, we performed subgroup analysis using fixed-effects models. Sensitivity analysis was employed to verify the impact of each trial on the pooled effect size using the one-study- removed (leave-one-out) method. Random-effects meta-regression analysis by an unlimited maximum likelihood method was used to evaluate the relationship between effect size and potential moderator variables including dose and duration of supplementation. In addition, publication bias was assessed by the visual inspection of funnel plots and Begg's rank correlation, and Egger's weighted regression statistics.25 When any publication bias was discovered, it was tested by the trim and fill approach.26. All statistical analyses were carried out using the Stata software version 14 (Stata Corp. College Station, Texas, USA). P < 0.05 was considered statistically significant.

3.3. Effect of pomegranate supplementation on hs-CRP levels Combining the effect sizes from seven studies, we recorded that pomegranate supplementation significantly reduced hs-CRP (weighted mean differences (WMD): −6.57 mg/L, 95 % CI: −10.04 to -3.10, P = 0.000) levels compared to controls, with a considerable between-study heterogeneity (I2 = 98.4 %, p < 0.000) (Supplementary Fig. 1). Between-study heterogeneity disappeared after subgroup analysis by intervention dose (I2 = 98.4 %, P = 0.000), intervention duration (I2 = 98.4 %, P = 0.000) and participants’ gender (I2 = 98.4 %, P = 0.000). However, the effect of pomegranate supplementation on hs-CRP levels was significant in studies with a duration < 8 weeks (WMD: −1.17 mg/L, 95 % CI: −1.58, -0.75, P = 0.002) and studies conducted on females (WMD: −8.90 mg/L, 95 % CI: −11.23, -6.56, P = 0.000). Studies involving a dose of pomegranate > 500 mL (WMD: −1.43 mg/ L, 95 % CI: −2.03, −0.83, P = 0.075) and studies on males (WMD: −2 mg/L, 95 % CI: −5.11, 1.11, P = 0.209) did not provide significant results.

3. Results 3.1. Study selection The initial search resulted in 1061 relevant records (262 articles in PubMed/Medline, 799 in SCOPUS). In addition, by hand-searching the references of the relevant articles, we identified 6 additional studies. After removing duplicates (n = 375), the 686 records were reviewed on

3.4. Effect of pomegranate supplementation on CRP levels Combining 4

findings

from

three

studies,

pomegranate

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individual trial on the pooled effect sizes.

supplementation did not significantly decrease CRP (WMD: 2.19 mg/ dL, 95 % CI: −3.28, 7.67, P = 0.61) versus placebo, with no significant between-study heterogeneity (I2 = 0.0 %, P = 0.43) (Supplementary Fig. 1). Subgroup analysis based on study duration (I2 = 0.0 %, P = 0.614) and participants’ gender (males: I2 = 0.0 %, P = 0.73) were performed, but did not yield significant results. The effect of pomegranate supplementation on CRP was not significant in trials that were performed in males (WMD: 1.10 mg/dL, 95 % CI: −5.24, 7.44, P = 0.73).

3.9. Publication Bias Evaluation of publication bias by visual inspection of funnel plot demonstrated no evidence of publication bias in the meta-analysis of pomegranate supplementation on biomarkers of inflammation and endothelial dysfunction (hs-CRP: Begge's(p = 0.102), and Egger's(p = 0.210), CRP: Begge's(p = 0.402), and Egger's(p = 0.399), TNF-α: Begge's(p = 0.386), and Egger's(p = 0.288), IL-6: Begge's(p = 0.625), and Egger's(p = 0.586), MDA: Begge's(p = 0.09), and Egger's(p = 0.102)) (Supplementary Fig. 2).

3.5. Effect of pomegranate supplementation on TNF-α levels Using pooled effect sizes from four studies, pomegranate supplementation significantly reduced TNF-α compared to the control group (WMD: −2.37 pg/mL, 95 % CI: -3.67, -1.07, P = 0.00). No significant heterogeneity was seen between the included studies (I2 = 7.8 %, P = 0.35) (Supplementary Fig. 1). Subgroup analysis based on the gender of the participants (I2 = 7.8 %, P = 0.354) and the duration of the supplementation (I2 = 7.8 %, P = 0.66) was performed, but no significant decrease based on gender or duration of the supplementation was observed.

4. Discussion This current meta-analysis was conducted to determine the effect of pomegranate supplementation on biomarkers of inflammation and endothelial dysfunction in humans. By evaluating 16 RCTs, we showed that pomegranate supplementation was associated with a significant reduction in hs-CRP, TNF-α, IL-6, E-selectin and VCAM levels. The positive results can be observed due to the high concentrations of polyphenols in pomegranate, which contain potent tannins and anthocyanins.42,43 Nevertheless, pomegranate supplementation did not lead to a significant reduction in MDA, CRP or ICAM levels. Concerning MDA concentrations, the pomegranate supplementation showed a significant lowering-effect, compared to placebo, through subgroup analysis by which the amount of pomegranate juice was > 500 mL and duration of intervention > 8 weeks. Such a result may be important biochemically, as the MDA is considered a biomarker of lipid peroxidation, a process which is involved in the development of several disorders 44; however, the long-term adherence to this posology may be unviable under real-world conditions. Although pomegranate supplementation significantly reduced hsCRP levels, the effect occurred when duration of treatment was < 8 weeks and for female subjects. Flavonoid content of pomegranate is primarily a possible factor that contributed to the reduction of hs-CRP levels and, most importantly, possessing certain benefits by modulating vascular wall,45,46 as the improvement in endothelial function.47,48 In contrast, it should be noted that many studies reported that hs-CRP levels did not decrease significantly in the intervention group following pomegranate supplementation23,28,30,32 and, therefore, a recommendation of ingesting pomegranate products for reducing the levels of hs-CRP may fail to accompany their normalization. Given that hs-CRP and CRP are global biomarkers in routine of both inpatients and outpatients, it is crucial to indicate their differences and clinical applicability. Albeit they are related to the measurement of the same protein, hs-CRP comprises assays that detect lower levels whereby cardiovascular events can be predicted, while CRP is associated with bacterial and viral infections.49 Along these lines, as a surrogate biomarker of future cardiovascular events, when hs-CRP levels range < 1, 1–3, and > 3 mg/l (< 0.10, 0.10 to 0.30, and > 0.30 mg/dl), patients are at low-, moderate-, and high-risk.50,51 Hence, an attention upon effects of nutritional strategies on the status of hs-CRP is more reasonable as a means of accompanying low-grade inflammation presented in cardiometabolic dysregulations, for which pomegranate products may be a candidate as reported herein. In keeping with this stand, the WMD of −6.57 mg/L for hs-CRP values reported by this current metaanalysis indeed is a pivotal variation toward clinical scenario. Importantly, there are long-term studies that investigated the effects of pomegranate juice supplementation on arterial parameters, in conjunction with cardiometabolic biomarkers, of patients with serious cardiovascular problems. Davidson et al. supplemented subjects at moderate coronary heart disease risk with 240 ml/d of pomegranate juice, thereby noting slowed carotid intima–media thickness (CIMT) progression specifically in subjects with increased oxidative stress and higher ratio of triglycerides/high-density lipoprotein levels.52 Indeed,

3.6. Effect of pomegranate supplementation on IL-6 levels Combining findings from six studies, a significant reduction in IL-6 levels (WMD: −1.68 pg/mL, 95 % CI: −3.52, 0.157, P = 0.000) was detected after pomegranate supplementation versus placebo, with a significant between-study heterogeneity (I2 = 77.4 %, P = 0.000) (Supplementary Fig. 1). Subgroup analysis based on the study duration (I2 = 0.0 %, P = 0.512), participants’ gender (males: I2 = 47.3 %, P = 0.169), intervention dose (dose < 500 mL: I2 = 0.0 % P = 0.87; dose > 500 mL: I2 = 0.0 % P = 0.543) was performed, but no significant decrease based on gender, duration of the supplementation or dosage was seen. 3.7. Effect of pomegranate supplementation on E-selectin, ICAM and VCAM levels Combining findings from 2 studies, no significant reduction in Eselectin or VCAM levels (WMD: 8.42 ng/mL, 95 % CI: −22.9, 39.8, P = 0.599, WMD: −69.32 ng/mL, 95 % CI: −229.26, 90.61, P = 0.396, respectively) was detected after pomegranate supplementation versus placebo, with a significant between-study heterogeneity (I2 = 87.2 %, P = 0.005, I2 = 91.4 %, P = 0.001, respectively) (Supplementary Fig. 1). Moreover, pomegranate supplementation did not significantly decrease ICAM levels compared to controls (WMD= −17.38 ng/mL, 95 % CI: −53.43, 18.66, P = 0.107). 3.8. Effect of pomegranate supplementation on MDA levels Combining findings from 8 studies, MDA did not decrease significantly (WMD: 0.031 μmol/L, 95 % CI: −1.56, 0.218, P = 0.746) following pomegranate consumption versus placebo, with a significant between-study heterogeneity (I2 = 64.2 %, P = 0.007) (Supplementary Fig. 1). Subgroup analysis based on the study duration (> 8 weeks, I2 = 72.6 %, P = 0.026), participants’ gender (males: I2 = 0.0 %, P = 0.709, both genders: I2 = 59.0 %, P = 0.062) and intervention dose (< 500 mL: I2 = 20.6 % P = 0.286; dose > 500 mL I2 = 79.2 % P = 0.002) was performed, yielding a significant reduction in MDA levels when the dose of pomegranate supplementation exceeded 500 mL or when the duration of the supplementation surpassed 8 weeks. 3.8.1. Sensitivity analysis To evaluate the influence of each single trial on the pooled results we removed each study from the examination, stage by stage and accounted for their uniqueness. We did not note significant impact for any 5

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Davidson et al.’ study provides a valuable finding, once the researchers employed a randomized, double-blind, parallel trial of 146 and 143 patients receiving pomegranate juice and control beverage, respectively, for up to 18 months. In an another study working a considerable length of intervention,53 ten atherosclerotic patients with carotid artery stenosis (CAS) were submitted to consume 50 ml/d of pomegranate juice for one year. After this period, serum low-density lipoprotein (LDL) oxidative state significantly reduced by 90 %, whereas serum levels of total antioxidant status (TAS) increased by 130 % and serum paraoxonase 1 (PON 1) activity was increased by 83 %, when compared to baseline values. The small sample of this study is a limitation, but in addition to serum biomarkers, endartherectomy was performed to collect arterial lesions then detecting reduced levels of cholesterol, lipid peroxides and LDL oxidation after the intervention with pomegranate juice and compared to controls, having increased the levels of reduced glutathione (GSH) in this circumstance as well. Moreover, in a randomized placebo controlled double-blind trial in which 101 chronic hemodialysis patients were enrolled to receive 100 ml of pomegranate juice during each dialysis, or matching placebo, three times a week for one year, only 5% of supplemented group presented progression in the atherosclerotic process, while more than 50 % of placebo group had the progression.54 These long-term studies complement the present meta-analysis by relevant cardiovascular endpoints, as displayed in Fig. 2, where we joined them to our findings.52–54 While these long-term studies support the effects of only pomegranate juice,52–54 this meta-analysis encompassed other forms of supplementation. Herein, the majority of studies used pomegranate juice or extract. Regarding the posology, the daily dose of pomegranate juice varied from 50−750 mL,5,23,28,30,36–38 pomegranate extract from 500−1000 mg,29,31,33,35,39 and one study applied 480 mg of pomegranate oil 32; overall, the period of treatment ranged from 48 h to 12 weeks. To the best of our knowledge, there are

no long-term clinical studies that addressed the effects of pomegranate extract and oil, hence their potential should not be overlooked. Due to the heterogeneity among the populations, there is no evidence to recommend a specific dosage of pomegranate products according to the diseases. As such, the studies recruited patients with diseases and metabolic dysregulations (hypertensive patients,6 T2DM patients,23 obese and overweight subjects,29,33 patients with ischemic heart disease, 30 metabolic syndrome 34 or dyslipidemia,32,33 patients undergoing dialysis,35,37 and patients diagnosed with rheumatoid arthritis31), athletes (bodybuilders 36 and canoe athletes 38), and healthy subjects.28,39 Finally, at least pomegranate juice may be considered an attractive foodstuff for the general public and, therefore, it would be practicable its implementation among the practice of dietitians and physicians to optimize the antioxidant status of patients who have a poor diet and/or metabolic dysregulations. Since that juice intake have evolved in the layperson as an unhealthy habit through anecdotal evidence and speculation by some healthcare professionals, this study support that pomegranate juice is a healthy food item with adjuvant potential in modulating cardiometabolic parameters. Unlike being a “villain”, some fruit juices may be “friends” as a means of providing antioxidants with therapeutic potentials. Recently, orange and cherry juice have gained substantial attention as dietary tools able to attenuate the cardiometabolic markers (e.g. blood pressure levels and metabolic biomarkers such as lipid and glycemic indices), even being a source of carbohydrates.55,56 Likewise, we corroborate that pomegranate juice is another option employing against biomarkers of low-grade inflammation related to the cardiometabolic aspects. Of course, it is important avoid the addition of sugar and very high intake of the juice in order to not dysregulate the daily ingestion of carbohydrates and calories.

Fig. 2. Completed pathway based on the positive findings of this meta-analysis and long-term studies. The supplementation of pomegranate products decreases the levels of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) then hindering the generation of CRP—particularly reducing hs-CRP levels—, whose protein stimulates many complication factors associated with atherosclerosis. In addition, supplementing with pomegranate products lead to increased concentrations of reduced glutathione (GSH), total antioxidant status (TAS), and augmented activity of paraoxonase 1 (PON-1), which collectively display a crucial role by hindering the oxidation of low-density lipoprotein (LDL) and being proposed to mitigate the progression of intima-media thickness. IL-6, interleukin-6; GSH, glutathione; hsCRP, high-sensitivity C-reactive protein; Ox-LDL, oxidized low-density lipoprotein; PON-1, paraoxonase 1; TAS, total antioxidant status; TNF-α, tumor necrosis factor alpha; VCAM-1, vascular cell adhesion protein 1. 6

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5. Limitations

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Our meta-analysis has limitations that should be pointed. In terms of the sample size, the studied population was considered relatively small. Nevertheless, the pooled sizes for this study allowed deriving a statistically significant result. Additionally, confounded results arising from heterogeneity may still be possible, although subgroup analyses were performed to address potential confounders. Taking into account the length of intervention, the nature of the studies’ designs did not cover a long-term follow-up to observe major adverse cardiovascular events (total death, myocardial infarction, coronary revascularization, stroke, and hospitalization because of heart failure). Therefore, it would be required interventional studies working on long-term fashion to verify whether or not the positive effects of processed pomegranate products on vascular function and inflammatory biomarkers translate into clinical practice of CVD prevention. Worthy of note, we discussed the existing long-term interventions to expand the body of literature in this regard. 6. Conclusion This systematic review and meta-analysis provide evidence regarding the effects of supplementing processed pomegranate products on biomarkers of inflammation and endothelial dysfunction. Based on these results, pomegranate supplementation may be considered an adjuvant agent to mitigate the vascular dysfunction and low-grade inflammation, as reflected by the significant reduction in hs-CRP, TNF-α and IL-6 levels. Further well-controlled clinical studies are needed to assess the potential clinical benefits of ingesting pomegranate products on the vascular dysfunction, as well as their anti-inflammatory properties, in a long-term fashion. Funding No funding was received for this study. Transparency document The Transparency document associated with this article can be found in the online version. Declaration of Competing Interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.ctim.2020.102358. References 1. Bastard JPMM, Lagathu C, Kim MJ, Caron M, Vidal H, et al. Recent advances in the relationship between obesity, inflammation, and insulin resistance. Eur Cytokine Netw. 2006;17:4–12. 2. Zhang HPY, Wu J, Chen X, Lee S, Yang J, et al. Role of TNF-alpha in vascular dysfunction. Clin Sci. 2009;116:219–230. 3. Saghizadeh MOJ, Garvey WT, Henry RR, Kern PA. The expression of TNF alpha by human muscle. Relationship to insulin resistance. J Clin Invest. 1996;97:1111–1116. 4. Gaman M-A, Dobrica E-C, Pascu EG, et al. Cardio metabolic risk factors for atrial fibrillation in type 2 diabetes mellitus: focus on hypertension, metabolic syndrome and obesity. J Mind Med Sci. 2019;6(1):157–161. 5. Sedigheh Asgary MK, Sahebkar Amirhossein, Hashemi Mohamad. Mahmoud Rafieian-Kopaei Clinical investigation of the acute effects of pomegranate juice on blood pressure and endothelial function in hypertensive individuals. ARYA Atheroscler. 2013;9(6):326–331. 6. Sedigheh Asgary MK, Sahebkar A, Rafieian-Kopaei M, Afshani MR, Haghjooyjavanmard S. Clinical evaluation of blood pressure lowering, endothelial function improving, hypolipidemic and anti-inflammatory effects of Pomegranate Juice in hypertensive subjects. Phytother Res. 2013.

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