The effect of grape products containing polyphenols on oxidative stress: a systematic review and meta-analysis of randomized clinical trials

Background The literature showed that Grape Products Containing Polyphenols (GPCP) had anti-oxidant activity. However, the effects of GPCP on different biomarkers of oxidative stress are still controversial. In this regard, this systematic review and meta-analysis aimed to evaluate the effect of Grape Products Containing Polyphenols (GPCP) intake on oxidative stress markers. Methods PubMed, Scopus, Web of Science, and Google Scholar data bases were searched up to August 20, 2020. A random-effects model, weighted mean difference (WMD), and 95% confidence interval (CI) were applied for data analysis. Meta-analysis was conducted over 17 eligible RCTs with a total of 633 participants. The study registration number is CRD42019116696. Results A significant increase was observed in Total Antioxidant Capacity (TAC) (weighted mean difference (WMD) = 1.524 mmol/L, 95% confidence interval (CI): 0.83, 2.21). Intake of GPCP enhanced Superoxide Dismutase (SOD) (WMD = 0.450 mmol/L, 95% CI: 0.23, 0.66), TAC (WMD = 2.829 mmol/L, 95% CI: 0.13, 5.52), and Oxygen Radical Absorbance Capacity (ORAC) (WMD = 0.524 μmol/L, 95% CI: 0.42, 0.62) among healthy participants. Higher GPCP doses increased SOD (WMD = 0.539 U/mgHb, 95% CI: 0.24, 0.82) and ORAC (WMD = 0.377 μmol/L, 95% CI: 0.08, 0.67), whereas longer intervention periods enhanced ORAC (WMD = 0.543 μmol/L, 95% CI: 0.43, 0.64). Conclusion GPCP intake may partly improve status of oxidative stress, but further well-designed trials are required to confirm these results. Supplementary Information The online version contains supplementary material available at 10.1186/s12937-021-00686-5.


Introduction
Under normal physiological conditions, various enzymatic systems, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) act as antioxidants and protect the cells against free radical damage, including reactive oxygen species (ROS) [1,2] Free radicals attack the main macromolecules and lead to cell damage and homeostasis. Increased number of free radicals reduce the detoxification capacity of tissues [3] and lead to oxidative stress. Oxidative stress occurs from the imbalance between production of ROS and protective effect of the antioxidant system, which is responsible for their neutralization and/or removal [4,5]. Oxidative stress can be caused by biological endogenous factors [6,7] or exogenous environmental factors [8].
High accumulation of free radicals overwhelms the antioxidant capacity of the body and leads to irreversible oxidative damage to nucleic acids, lipids, and proteins [9]. Oxidative damage lead to the cellular damage and cause changes in gene expression, cell proliferation, and apoptosis [9] Consequently, the aging process of the body is influenced and many chronic diseases develop, including cardiovascular disease, neural degeneration, cancer, and diabetes [2,10]. Endogenous antioxidant defense system involves a network of antioxidant enzymes and non-enzymatic molecules in cytoplasm of organs [2]. Antioxidant enzymes, such as SOD, catalase, and glutathione reductase, transform ROS into more stable molecules and maintain oxidative equilibrium [2]. In addition, reduced glutathione (GSH) is a tripeptide made of glutamine, cysteine, and glycine with protective function against oxidative stress [4]. Malondialdehyde (MDA), as a byproduct of polyunsaturated fatty acids peroxidation [11], can be toxic, potentially mutagenic, and atherogenic due to its reaction with biomolecules such as protein and nucleic acid [12]. It is also a biomarker of oxidative stress [13]. Oxygen radical absorbance capacity (ORAC) has been widely used for measuring the antioxidant activity [14]. Considering the difficulty of measuring plasma antioxidant capacity of each sample and regarding the interactions among different compounds [15], the total antioxidant capacity (TAC) was evaluated [16]. Generally, increased oxidative stress decreases TAC [17].
Although several clinical trials investigated the effect of GPCP intake on oxidative stress markers, no conclusive result exists on this issue. Furthermore, no systematic review and meta-analysis has ever been conducted in this area. Therefore, this systematic review and metaanalysis aimed to investigate the effect of GPCP on oxidative stress.

Search strategy
This systematic review and meta-analysis was conducted according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses Guidelines) [47].
The protocol of this study was also registered on PROS-PERO, an International Prospective Register of Systematic Reviews (http://www.crd.york.ac.uk/PROSPERO) with the registration no of CRD42019116696.
We searched PubMed, Scopus, Web of Science, and Scholar up to August 20, 2020 using Medical Subject Heading terms (MeSH) and non-MeSH terms to evaluate the effect of GPCP supplementation on oxidative stress biomarkers. The following keywords were used in the search: (grape* OR "grape polyphenol" OR "grape seed extract" OR "grape seed" OR "Grape Seed Proanthocyanidins" OR raisin* OR polyphenol* OR "Vitis vinifera" OR raisin* OR "grape extract" OR wine* OR "grape polyphenols" OR "grape powder" OR "concord grape juice" OR "grape juice") AND (" oxidative stress " OR " Superoxide Dismutase" OR " Superoxide Peroxidase " OR "oxidative stress indices" OR "Glutathione Peroxidase" OR " oxidative stress markers" OR "oxidative mediators" OR "oxidative biomarker" OR "F 2isoprostanes" OR " isoprostanes " OR malondialdehyde* OR MDA OR "Catalase" OR CAT OR " total antioxidant capacity " OR " total antioxidant status" OR "total oxidant status " OR TAS OR TOS OR Glutathione* OR TBARS OR " Reduced Glutathione" OR " Thiobarbituric Acid Reactive Substances " OR ORAC OR "Oxygen Radical Absorbance Capacity " OR TAC OR GPX OR SOD OR GSH) AND (trial* OR "randomized controlled trials" OR RCT OR " Clinical Trials as Topic " OR "clinical trials" OR "randomized controlled clinical trial" OR "randomized clinical trials" OR "controlled clinical trials" OR intervention OR Intervention OR randomized OR randomised OR random OR randomly OR placebo OR assignment OR cross-over OR parallel) NOT (animal* OR rat OR rats OR rabbit* OR rattus OR monkey* OR mice* OR mouse* OR hen* OR chicken* OR duck* OR pig* OR cow* OR "cell line" OR nonhuman OR "In-vitro" OR "In-vivo"). Furthermore, Reference lists of the related original and review articles were also carefully checked to obtain other eligible studies.

Selection criteria
The inclusion criteria for the studies were: 1) having an RCT design; 2) evaluating the effect of GPCP on oxidative stress biomarkers versus placebo or other intervention, such as water or usual wine; 3) reporting the dose of GPCP; 4) having participants with 18 years of age or older, and 5) being in English.

Study selection
Two researchers separately performed the initial screening on the basis of the titles and abstracts of the articles. In the next step, the full texts of all related articles were investigated by reviewers to find studies over the effect of GPCP on oxidative stress. Eventually, any disagreements were discussed and resolved by consensus with third researcher (Fig. 1).

Data extraction
Two independent researchers summarized the data on the studies' author's family name, publication year, sample size and rate of sample loss, dose and type of intervention, study duration, cross-over or parallel study design, gender, age and healthy status of participants, as well as mean and SD of oxidative stress biomarkers levels at the baseline and end of trial. The collected information was double-checked by a third researcher.

Quality assessment
At this stage, two reviewers independently evaluated the methodological quality of the eligible studies through Cochrane Collaboration's tool including six domains of: 1) random sequence generation (selection bias); 2) allocation concealment (selection bias); 3) blinding of participants and personnel (performance bias); 4) blinding of outcome assessment (detection bias); 5) incomplete outcome data (attrition bias); and 6) selective reporting (reporting bias). Each domain was classified to three categories: low risk of bias, high risk of bias and unclear risk of bias. According to the mentioned domains, the overall quality of each study was considered as good (low risk for more than 2 item), fair (low risk for 2 item), or weak (low risk for less than 2 item) [48].

Data synthesis and analysis
Statistical analyses were conducted using STATA software, version 11.2 (STATA Corp, College Station, TX). The random effects model which takes the between-study heterogeneity into account was used to calculate the weighted mean difference (WMD) and its 95% confidence intervals (CIs) [49]. To incorporate between-study variation, a random effects model was also applied to combine effect sizes. This model takes between-study heterogeneity into account. To evaluate heterogeneity among studies, I 2 and Q statistic were used. If I 2 > 50% and p-value of Q statistic < 0.05, statistically significant heterogeneity was recognized [50]. Subgroup analysis was conducted to explore the possible source of heterogeneity among the studies for all of oxidative biomarkers were addressed in our meta-analysis. Subgroup analysis included the following: duration, grape polyphenol doses, study design (parallel and crossover), study quality (weak, fair, good), and health status of study population (healthy individuals: people with no clinical condition versus unhealthy individuals: overweight; obese; chronic obstructive pulmonary disease; chronic kidney disease; coronary artery disease or ≥ 1 cardiac risk factor; type 2 diabetes). Publication bias was assessed by examination of the funnel plot and formal testing for "funnel plot" asymmetry using Begg's test and Egger's test [51]. Sensitivity analysis was performed to identify whether a specific study or a particular group of studies affected the outcomes [51]. P values of less than 0.05 were considered significant.

Meta-regression
Meta-regression was conducted to evaluate the association of estimated effect size with dose and duration of the GPCP intake.

Effect of GPCP on ORAC levels
Meta-analysis of five RCTs [37-39, 116, 117] demonstrated no significant effect of GPCP on ORAC levels (WMD = 0.210 μmol/L; 95% CI: − 0.15, 0.57; P = 0.25) (Fig. 4). This result did not change after the sensitivity analysis) Supplementary Fig. 7). A significant heterogeneity was seen among the studies (P < 0.001, I 2 = 96.02). No significant publication bias was found with regard to the related studies based on the funnel plots ( Supplementary  Fig. 8

Discussion
Findings showed that GPCP intake had a significant effect on increasing the TAC levels; however, it had no significant impact on other oxidative stress biomarkers.
In the sub-group analysis, GPCP significantly increased SOD, TAC, and ORAC levels in healthy participants. Furthermore, higher GPCP doses increased ORAC and SOD levels significantly. Longer intervention periods also increased the ORAC levels. Finally, cross-over study design enhanced the TAC and SOD levels.
The most notable bioactivity of GPCP is their antioxidant properties [133]. Grape products containing polyphenols use various mechanisms such as sweeping reactive oxygen and nitrogen species [134,135], chelating metals and minerals, modulating transcription factors, inhibiting and inducing pro-oxidant and antioxidant enzymes, and exerting synergistic effects on other antioxidants to exert their antioxidant activities [136,137].
In fact, nuclear factor-erythroid-2-related factor-2 (Nrf2) signaling pathway, as an essential pathway for protection against oxidative stress, regulates the expression of many intracellular antioxidant genes [138]. Thus, this pathway is one of the most important therapeutic targets for the prevention and treatment of oxidative stress and its related diseases [139]. The GPCP including grape seed extract proanthocyanidin induce and activate Nrf2 [46,138]. Grape products containing polyphenols also inhibit lipid peroxidase by increasing paraoxonase activity plasma [140], which consequently inhibits LDL oxidation [141].
Our subgroup analyses showed a significant increase in the ORAC levels in longer-term studies; this finding is confirmed by other studies [32,44,119]. Metaregression analysis also indicated a significant relationship between duration of GPCP consumption and TAC, ORAC, and MDA levels. The literature indicated that supplementation duration affected the efficiency of the dietary polyphenols. Furthermore, the supplementation duration should be sufficiently long to influence the oxidative stress markers. In order to demonstrate the doseresponse relationship, long-term studies are needed to exert significant effects on the antioxidant biomarkers [31,32,142]. The impact of grape seed extract on blood pressure was also more pronounced in longer study [83].
Our results indicated a significant elevation in SOD and ORAC levels in higher doses of grape polyphenols. The meta-regression showed that the changes in SOD, TAC, ORAC, and GPX levels were dependent on grape polyphenols supplementation dose. Similar to our results, a study indicated that different GPCP supplementation doses had different effects. For example, the flowmediated dilation (FMD) improved better at a higher dose of red grape powder, while the diastolic blood pressure decreased better at a lower dosage of this product [123]. Contrary to our results, a meta-analysis showed that systolic and diastolic blood pressure decreased significantly at lower doses of GSE [83]. Moreover, 25 and 50% (v / v) concentrations of GPCP showed similar antioxidant activity. These concentrations were better than the 10% (v / v) concentration. These results show that the antioxidant capacity of the phenols is probably in the saturated concentration range and does not increase with higher concentrations [143].
The significant increase in SOD levels in studies with cross over designs might be due to the fact that these trials (range: 0.4-300 g, mean: 150.22 g) had significantly higher GPCP doses than the parallel trials (range: 0.1-0.35 g, mean: 0.8 g).
Subgroup analysis also indicated a significant effect of GPCP on TAC, SOD, and ORAC in healthy individuals. Similar to our results, other studies showed that the antioxidant capacity of the studied population may vary with their health status [1]. A meta-analysis found that the effect of grape seed extract on blood pressure depended on the individuals' initial blood pressure level, although the confounding effect of the patient's medications should be considered in unhealthy individuals [83]. Another metaanalysis reported that the elevated levels of FMD were different between the individuals with cardiovascular risk factors and the healthy participants [56]. In addition, the dose-response mechanism of the grape products may vary based on the individuals' health status [144]. According to a previous study, supplementation with GPCP had different effects on smokers since they need higher levels of antioxidants; consequently higher doses of GPCP are required for clearer results [43]. In health status, a balance exists between production of free radicals and the antioxidant defense system that prevents the disease. In disease conditions, the balance shifts towards producing free radicals and increasing oxidative stress [145]; consequently, unhealthy people are expected to have higher levels of oxidative stress [1] and require higher doses of GPCP to improve antioxidant macros.
The present research has some strength. This is the first study over the effect of GPCP on oxidative stress. Subgroup analyses were also conducted on the study type, duration, and quality, the products' dosage, and the participants' health status. However, this meta-analysis had several limitations. Few oxidative biomarkers evaluated in most RCTs also, oxidative markers had rapid mechanism of oxidation, future studies should evaluate all of related biomarkers especially TAC, therefore, the results will be more accurate. The RCTs included in the present study had limited follow-up periods. Moreover, the investigated articles were heterogeneous considering their populations' characteristics as well as the administered type and doses of GPCP. So, further clinical trials are needed over the effect of grape polyphenol on the oxidative biomarkers as primary outcome using different doses and type of GPCP. Polyphenol contents in grape products are varied widely because many factors influence their contents, such as grape cultivars, season, processing, storage condition, and duration. Future researchers are suggested to report the amount of grape polyphenol in their test products and serum levels of polyphenols in participants. Since most studies did not consider the effects of confounders, including lifestyle, diet, physical activity, smoking, health/disease, age and medications, we were unable to evaluate these effects.

Conclusions
In conclusion, the results of this study demonstrated that supplementation with GPCP had a significant effect on increasing the TAC levels, but it had no effect on other oxidative stress biomarkers. The effect of GPCP on SOD, GPX, ORAC, and TAC levels depended on the administered dosage. In the same regard, the supplementation duration affected MDA, TAC, and ORAC levels. However, further well-designed RCTs with larger sample sizes and longer-durations are required in this area.