Effect of Green Tea Supplementation on Antioxidant Status in Adults: A Systematic Review and Meta-Analysis of Randomized Clinical Trials

It is well-established that green tea supplementation has antioxidant properties. However, whether green tea supplementation leads to oxidative stress reduction remains unclear, as clinical investigations on this subject have yielded inconsistent outcomes. Consequently, we aimed to determine the effects of green tea supplementation on oxidative stress in adults. A systematic search of English language publications up to 21 August 2021 was carried out in PubMed, Scopus, Embase, and ISI Web of Science, utilizing pertinent keywords. These searches included randomized controlled trials (RCTs) evaluating the relationship between green tea supplementation, malondialdehyde (MDA), and total antioxidant capacity (TAC) in adults. A random-effects model was used to estimate the weighted mean difference (WMD) and 95% confidence intervals (95% CI). Meta-regression and non-linear dose-response analyses were performed to investigate the association between the dosage of green tea (mg/day) and the duration of the intervention (weeks) with pooled effect size. Sixteen RCTs with seventeen arms including 760 participants met the inclusion criteria. Our results indicated that green tea supplementation had significant effects on TAC (weighted mean difference [WMD]: 0.20 mmol/L; 95% CI: 0.09, 0.30, p < 0.001) and significant heterogeneity between studies (I2 = 98.6%, p < 0.001), which was largely related to gender and body mass index (BMI). Subgroup analysis in TAC identified a significant relationship except with low dose supplementation and obese individuals. No relationship between MDA and green tea supplementation was observed in any subgroups; however, meta-regression analysis revealed a linear inverse association between the dosage and significant change in MDA (r = −2117.18, p = 0.017). Our outcomes suggest that green tea supplementation improves TAC and affects MDA based on the dose of the intervention in adults. Future RCTs with longer durations are needed to expand our findings.


Introduction
All metabolic processes generate reactive oxygen species (ROS), which can induce oxidative damage to tissue and cell constituents such as lipids, proteins, and nucleic acids [1,2] when left unbalanced. Indeed, the overproduction and accumulation of large amounts of ROS lead to oxidative stress [3][4][5], which represents a major contributor to the risk, onset, and progression of chronic diseases [6][7][8][9][10][11][12]. A key component of oxidative stress implicated in adverse health effects is lipid peroxidation of polyunsaturated fatty acids and the subsequent production of malondialdehyde (MDA) (a major indicator of lipid oxidation) [9]. Therefore, interventions to intensify the body's antioxidant response to counteract oxidative stress and MDA production are desperately needed.
Natural dietary polyphenolic compounds are potent antioxidants that decrease oxidative stress and protect the body against several oxidative stress-associated diseases [13,14]. Among the common sources of natural dietary polyphenol in foods and beverages is green tea (originate from the leaves of Camellia sinensis), which has high antioxidant properties related to various compounds such as phenolics and flavonoids, and flavonols [15][16][17]. In fact, the major polyphenols with antioxidant properties in green tea are epicatechin (EC), epigallocatechin-3-gallate (EGCG), epicatechin-3-gallate (ECG), epigallocatechin (EGC), and gallocatechin gallate (GCG), which are also called catechins [18]. While some studies have reported that EGCG and other catechins in green tea increase the activity of antioxidant enzymes, decrease indices of oxidative damage and prevent and treat some diseases [19][20][21], there are equivocal effects on markers of oxidative stress in diabetes, metabolic syndrome, β-thalassemia major, and obesity [9,13,[22][23][24][25]. Methodological issues such as non-randomization, different outcome measures, short duration of treatment, low study quality, and population heterogeneity have precluded a comprehensive understanding of the extent to which green tea supplementation may exert a beneficial effect on oxidative stress. Further, the potential effectiveness of green tea supplementation in mitigating oxidative stress among adults with pre-existing inflammatory conditions has not been clarified. Thus, we performed a systematic review and meta-analysis of randomized controlled trials (RCTs) to evaluate the relationship between green tea supplementation and oxidative stress markers, as assessed by the MDA and total antioxidant capacity (TAC), in adults with and without inflammatory health conditions.

Materials and Methods
Findings from this systematic review and meta-analysis were reported based on the Preferred Reporting Items of Systematic Reviews and Meta-Analysis (PRISMA) guideline [26]. The PICOS model was used to prepare the question: population (all individuals), intervention (green tea), comparison (studies which had control group), outcome (studies that reported two markers of oxidative stress including MDA and TAC), and study design (RCT, Registration code: CRD42021240026) [27].

Search Strategy
We conducted database searches of PubMed, Scopus, Embase, and ISI web of science from inception to 21 August 2021. All relevant studies written in the English language were included using. Medical subject headings (MeSH) and text words. Search words included: "green tea" OR "green tea extract" OR "green tea extract AR25" OR "catechin" OR "catechins" OR "EGCG" OR "Camellia sinensis" OR "tea polyphenols" OR "catechinic acid" OR "acid catechinic" OR "sinensis Camellia" OR "Thea sinensis" OR "sinensis Thea" OR "tea polyphenols" AND "oxidative stress" OR malondialdehyde OR MDA OR glutathione OR GSH OR "total antioxidant capacity" OR TAC OR "total antioxidant status" OR TAS AND.
intervention OR "intervention study" OR "intervention studies" OR "controlled trial" OR randomized OR randomized OR random OR randomly OR placebo OR assignment OR "clinical trial" OR trial OR assignment OR "randomized controlled trial" OR "randomized clinical trial" OR RCT OR blinded OR "double blind" OR "double blinded" OR trial OR "clinical trial" OR trials OR "pragmatic clinical trial" OR "cross-over studies" OR "cross-over" OR "cross-over study" OR parallel OR "parallel study" OR "parallel trial". Reference lists of the included studies were manually checked to identify additional articles. Unpublished documents and grey literature such as thesis, patents, and conference papers were not included.

Eligibility Criteria
Studies were included if they met the following criteria: RCT design, population aged >18 years, and reported MDA and/or TAC in both the intervention and placebo groups. Studies with duplicated data, non-RCT design, animal studies, studies that assess the effects of green tea supplementation and other interventions, and those with insufficient data for the outcomes of interest or did not meet inclusion criteria of our meta-analysis were excluded.

Data Extraction
Two independent researchers extracted data and evaluated any disagreement was resolved through open discussion with a third independent investigator (KhM). First author's name, year of publication, study location, duration and design, gender, mean age and mean body mass index (BMI) of population, the health status of participants, number of participants in each group, type and dose of green tea, and results (means and standard deviations for MDA and TAC before and after intervention) were extracted from the full text of included studies.

Statistical Analysis
This meta-analysis was performed using STATA software, version 14.0 (Stata Corp. LP, College Station, TX, USA). The effect of green tea supplementation on oxidative stress (MDA and TAC) was estimated by pooling mean and standard deviation (SD) values of the baseline and final of the studies in both intervention and placebo groups. Missing SDs for changes were calculated utilizing the following formula: SD change = square root [(SD baseline 2 + SD final 2 ) − (2 × R × SD baseline × SD final)], if the studies did not report mean and SD. We considered a correlation coefficient of 0.9 as the R-value of the mentioned formula [38]. We used the following formula to calculate standard errors (SE) from SD: SE √ n. If a study provided medians (interquartile ranges), we converted them to means (SD) as described by Hozo and colleagues [39]. If the study results were reported graphically, we used GetData Graph Digitizer software to estimate mean (SD). At first, a fixed-effect model was performed to pool the data. A random-effects model was used to estimate the weighted mean difference (WMD) and 95% confidence (95% CI). Heterogeneity among studies was assessed with the Q test and the I 2 index statistic. If p < 0.1 and I 2 > 50%, it was considered that heterogeneity existed among studies. If p > 0.1 and I 2 < 50%, fixed-effect models would be applied. Sensitive analysis and subgroup analysis were performed to evaluate the source of heterogeneity and verified the stability of results. In the sensitivity analysis, one study was omitted at each turn to evaluate the influence of each study on the results. Subgroup analysis was performed by stratifying study type, region, study quality score, and maximum category. Contour-enhanced funnel plots with an Egger linear regression test and a Begg rank correlation test were used to evaluate the potential publication bias. Meta-regression and non-linear dose-response analyses were performed to investigate the association between the dosage of green tea (mg/day) and the duration of the intervention (weeks) with pooled effect size. Because all the data used for analyses were extracted from the published studies, ethical approval and informed consent were not necessary.

Study Selection
We found 3204 publications in Scopus, PubMed, Embase, and ISI web of science in the initial search. Of these, eight hundred seventy-four articles were duplicated. Thus, a total of 2330 articles were assessed for the title and abstract screening. After screening of title and abstract, 2288 unrelated studies were discarded due to primary evaluation of inclusion criteria: unrelated title (n = 1729), review (n = 123), and animal study (436). Consequently, 42 studies were retrieved for full-text review, 26 of which were excluded due to: lack of sufficient data and the use of two or more interventions at the same time [2,21,22,24,30,34,. Finally, sixteen RCTs with one double-arm study [4,5,9,10,13,22,24,29,37] were eligible for this systematic review and meta-analysis. The flow chart of the literature search is shown in Figure 1.

Study Characteristics
Sixteen RCTs with one double-arm study assessing the effects of green tea supplementation on oxidative stress were identified. Included studies carried out in various countries such as Iran [5,9, [10]. Publication dates ranged from 2009 and 2020. The follow-up period ranged from 2 weeks to 36 weeks. The sample size of the included studies ranged from eight to forty-eight participants. Some studies enrolled only females [35] or males

Publication Bias and Sensitivity Analysis
The sensitivity analysis indicated that removing any of the studies would not substantially change the effect of green tea supplementation on oxidative stress (Figure 2). We used Egger's weighted regression tests to explore the publication bias. There was no evidence of publication bias for studies examining the effect of green tea supplementation on MDA (p = 0.335, Egger's test) (p = 0.711 Begg test), and TAC (p = 0.093, Egger's test) (p = 0.893 Begg test) ( Figure 3A,B).  Table 3).

Publication Bias and Sensitivity Analysis
The sensitivity analysis indicated that removing any of the studies would not substantially change the effect of green tea supplementation on oxidative stress (Figure 2). We used Egger's weighted regression tests to explore the publication bias. There was no evidence of publication bias for studies examining the effect of green tea supplementation on MDA (p = 0.335, Egger's test) (p = 0.711 Begg test), and TAC (p = 0.093, Egger's test) (p = 0.893 Begg test) ( Figure 3A,B).

Non-Linear Dose-Response between Dose and Duration of Green Tea Supplementation and Antioxidant Status
Non-linear dose-response analysis was conducted between the dose and duration of green tea supplementation for TAC and MDA. Dose-response analysis indicated that green tea supplementation did not affect TAC and MDA concentrations ( Figures 4A,B and 5A,B).

Non-Linear Dose-Response between Dose and Duration of Green Tea supplementation and Antioxidant Status
Non-linear dose-response analysis was conducted between the dose and duration of green tea supplementation for TAC and MDA. Dose-response analysis indicated that green tea supplementation did not affect TAC and MDA concentrations ( Figures 4A,B and  5A,B).

Meta-Regression Analysis
We performed meta-regression analysis to evaluate the association between the dose of green tea (mg/day) and the duration of the intervention (weeks) with antioxidant status. The results of the analysis revealed that green tea supplementation significantly changed MDA (r = −2117.18, p = 0.017) based on the dose of the intervention ( Figure 6) and did not

Meta-Regression Analysis
We performed meta-regression analysis to evaluate the association between the dose of green tea (mg/day) and the duration of the intervention (weeks) with antioxidant status. The results of the analysis revealed that green tea supplementation significantly changed MDA (r = −2117.18, p = 0.017) based on the dose of the intervention ( Figure 6) and did not show a linear relationship between dose and duration with significant changes in TAC (Figures 7 and 8A,B). show a linear relationship between dose and duration with significant changes in TAC (Figures 7 and 8A,B).   show a linear relationship between dose and duration with significant changes in TAC (Figures 7 and 8A,B).

Discussion
To our knowledge, this is the first meta-analysis to evaluate the effects of green tea or green tea extract supplementation on oxidative stress (assessed TAC and circulating MDA concentrations). We identified 16 RCTs and 17 arms with 760 participants. These studies suggest that green tea and green tea extract supplementation were positively associated with TAC, but no association with MDA was observed in all subgroups. The association was independent of sex, BMI, duration, and green tea dosage.
Increased oxidative stress elevates the risk of chronic diseases, such as cardiovascular and end-stage renal disease, as it is related to the onset and progression of these adverse

Discussion
To our knowledge, this is the first meta-analysis to evaluate the effects of green tea or green tea extract supplementation on oxidative stress (assessed TAC and circulating MDA concentrations). We identified 16 RCTs and 17 arms with 760 participants. These studies suggest that green tea and green tea extract supplementation were positively associated with TAC, but no association with MDA was observed in all subgroups. The association was independent of sex, BMI, duration, and green tea dosage.
Increased oxidative stress elevates the risk of chronic diseases, such as cardiovascular and end-stage renal disease, as it is related to the onset and progression of these adverse conditions [11,12,62,63]. A greater antioxidant intake has been previously related to a lower risk of disease mortality [62]. In general, Camellia sinensis and its tea are known as one of the significant sources of antioxidants such as flavonoids. It has also been shown that green tea is especially rich in a very strong antioxidant called epigallocatechin gallate [63,64]. For the first time, our research demonstrated a significant beneficial effect of green tea supplementation on TAC via a systematic review and meta-analysis, indicating the decreasing impact of this compound on oxidative stress development. The existence of catechins such as EGCG and ECG in green tea, which have numerous free hydroxyl groups, have significant beneficial effects on free radical scavenging activity, and therefore, can lessen the product of lipid peroxidation [65]. A growing body of evidence has suggested the possible role of green tea supplementation in enhancing the antioxidant capacity [30,[66][67][68][69]. Additionally, catechin improves antioxidant capacity and may result in a significant decrease in lipid peroxidation production, which has been supported by the study of Lin et al. [66].
Several possible mechanisms are responsible for green tea polyphenols on increased plasma TAC. The vigorous antioxidative function of catechins is related to the dihydroxyl or trihydroxyl groups on the B-ring and the meta-5,7-dihydroxyl groups on the A ring. This function can also be heightened by the trihydroxyl structure in the D-ring (gallate) in EGCG and epicatechin gallate. Based on the polyphenolic structure, free radicals can be neutralized by Electron delocalization [67]. Moreover, reactive oxygen and nitric radicals can be excluded by tea catechins [68,69].
The concentration of lipid peroxidation products representing oxidative stress across health and disease has not been adequately referenced [17][18][19][20]. In this present research, the MDA was proposed as an indicator of lipid peroxidation productions [70]. Regardless of the antioxidant activity of catechin, there are no consistent results on its effects on MDA. For instance, Hadi et al. in 2017 suggested that MDA concentrations were substantially subsided in athletes who consumed green tea extract [5]. This lowering effect of green tea on MDA was also confirmed by Noronha et al. 2019, which explains our findings [25]. Although we found that catechin has no significant effect on MDA concentrations in all subgroups, our meta-regression analysis demonstrated that green tea supplementation reduced MDA concentrations based on the dose of the intervention (mg/day).
On the contrary, several studies have shown no significant association between catechin and the value of MDA. The beneficial effects of green tea catechins in the elimination of ROS have been shown in vitro, although Heijnen et al. supported the idea that it may not be effective in vivo [71]. It has been observed that even with the highest dosage, the number of flavonoids and polyphenol in serum and intracellular are remarkably less than other types of antioxidant concentrations. It is also worth mentioning that some polyphenols, such as catechins, are the result of primitive polyphenols, and thus have poorer antioxidants levels than the parental polyphenols. It seems likely that dietary flavonoid actions as an antioxidant in vivo are not noticeable [72].
The present systematic review and meta-analysis is not without limitations. Most analyses had high levels of heterogeneity, but this was expected because included studies had different types of participants, doses, and intervention durations. In addition, there were not enough studies to evaluate all components of oxidative stress (MDA and TAC) in certain diseases. Moreover, our search was limited to RTCs published in English. Our meta-analytic work has several strengths, including RCTs and diverse participants (both healthy and diseased) from three different continents. Further well-designed RCTs are warranted to corroborate and expand on our outcomes, particularly in regions where green tea supplementation is not popular and in subjects with different diseases that may benefit from a decline in oxidative stress, such as those with cancer.

Conclusions
In conclusion, combined data from interventional studies showed green tea supplementation has a beneficial effect on TAC. Additionally, a linear inverse association between the dose of green tea supplementation and a significant change in MDA was reported.
Green tea and green tea extract can be proposed as suitable drinks to increase antioxidant status in adults, which may decrease the risk and progression of chronic diseases related to higher oxidative stress levels.