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Dietary flavonoids intake and the risk of stroke: A dose-

response meta-analysis of prospective cohort studies

Journal: BMJ Open

Manuscript ID: bmjopen-2015-008680

Article Type: Research

Date Submitted by the Author: 06-May-2015

Complete List of Authors: Tang, Zhenyu; The Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, People’s Republic of China, Department of Neurology Li, Min; The Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, People’s Republic of China, Department of Neurology Zhang, Xiaowei; The Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, People’s Republic of China,

Department of Neurology Hou, Wenshang; The Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, People’s Republic of China, Department of Neurology

<b>Primary Subject Heading</b>:

Evidence based practice

Secondary Subject Heading: Cardiovascular medicine, Evidence based practice, Neurology, Nutrition and metabolism, Public health

Keywords: Public health < INFECTIOUS DISEASES, Stroke < NEUROLOGY, International health services < HEALTH SERVICES ADMINISTRATION & MANAGEMENT

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Dietary flavonoids intake and the risk of stroke: A dose-response

meta-analysis of prospective cohort studies

Zhenyu Tang associate professora,1,*, Min Li master student

a,1, Xiaowei Zhang

master studenta, Wenshang Hou master student

a

a Department of Neurology, The Second Affiliated Hospital of Nanchang University, Nanchang

330006, Jiangxi Province, People’s Republic of China

* Correspondence to: Zhenyu Tang, Department of Neurology, The Second Affiliated Hospital of

Nanchang University, No. 1, Minde Road, Nanchang, Jiangxi 330006, People’s Republic of China

[email protected]

1 Zhenyu Tang and Min Li contributed equally to this work.

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Abstract

Objective To clarify and quantify the potential association between the intake of flavonoids and

risk of stroke.

Design Meta-analysis and systematic review of prospective cohort studies.

Data source Studies published before February 2015 identified through electronic searches using

PubMed, Embase and the Cochrane Library.

Eligibility criteria for selecting studies Prospective cohort studies with relative risks and 95%

confidence intervals for stroke according to the intake of flavonoids (assessed as dietary intake).

Results The meta-analysis yielded 10 prospective cohort studies involving 258,158 participants

and more than 4,581 stroke cases. The pooled estimate of multivariate relative risk of stroke for

the highest compared with the lowest dietary flavonoids intake was 0.90 (95% confidence interval,

0.82-0.98; p = 0.019). Dose-response analysis indicated that the summary relative risk of stroke

for an increase of 100 mg flavonoids consumed per day was 0.94 (95% confidence interval,

0.83-1.06) without heterogeneity among studies (I2 = 0%). Stratifying by duration, the relative risk

of stroke for flavonoids intake was 0.89 (95% confidence interval, 0.81-0.99) in longer than 10

years of follow-up studies.

Conclusions Results from this meta-analysis suggest that higher dietary flavonoids intake may

moderately lower the risk of stroke.

Article summary

Strengths and limitations of this study

To our knowledge, this is the largest meta-analysis on flavonoids intake and the risk of stroke. We

also investigated a dose-response relation between flavonoids consumption and risk of stroke.

The possibility of residual confounding or confounding by unmeasured factors, which cannot be

ruled out in any observational study, must be acknowledged. We cannot exclude the possibility of

recall bias in the assessments of diet based on the food frequency questionnaires.

1. Stroke is the second most common cause of death and the second highest cause of disability

worldwide.

2. Epidemiological studies suggest that the intake of flavonoids is beneficial in delaying or

preventing the development of stroke, though results from cohort studies are controversial.

3. Higher dietary flavonoids intake is associated with a significantly reduced risk of stroke.

4. Dose-response analyses indicated a 6% lower risk of stroke per 100 mg/day increment of

flavonoids.

5. Further evidence from preferably randomized controlled studies should explore what kind of

flavonoids can reduce the risk of stroke.

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Introduction

Stroke is the second most common cause of death, as well as the fourth leading cause of lost

productivity and the second highest cause of disability worldwide.1,2

The prevention of stroke is

thus clearly an important public health priority. In recent decades, concern has mounted regarding

the premature incidence and mortality associated with stroke, with growing interest in altering risk

factors and reversing this global epidemic. Among the known risk factors for stroke, dietary

factors, especially dietary flavonoids intake, have aroused particular attention. Clinical studies

have shown that intakes of flavonoids reduce cardiovascular disease (CVD) risk.3-5

Additionally,

experimental studies indicated that flavonoids have been shown to have both antioxidant and

antithrombotic properties.6,7

Over the last 2 decades, many prospective studies have assessed the association of dietary

flavonoids intake and risk of stroke.8-17

Although a recent meta-analysis that combined the results

from 8 cohort studies of flavonol intake and risk of stroke found a significant association of stroke

of 0.86 (95% CI, 0.75 to 0.99) for the highest versus lowest category of flavonol intake,18

the role

of flavonoids intake on stroke prevention is still controversial. In addition, flavonoids intake

differed substantially between studies, which makes it difficult to interpret the summary estimate

based on results from study populations with different ranges of flavonoids intake.19

To fill these gaps, we conducted a dose-response meta-analysis of the current evidence for the

association between flavonoids exposure, including cohort studies of dietary flavonoids, with risk

of stroke.

Methods

Literature search

The search strategy was conducted according to the recommendations of the Meta-analysis of

Observational Studies in Epidemiology (MOOSE).20

We performed a systematic search of

PubMed, Embase, and the Cochrane library through February, 2015. The following key words

were used in our search strategies: “flavonoids,” “polyphenols,” “phenolics,” “flavonols,”

“flavones,” “quercetin,” “kaempferol,” “myricetin,” “isorhamnetin,” “apigenin,” “luteolin,” and

“stroke,” “cerebrovascular disease,” “cerebrovascular disorders,” “cerebral infarct,” “ischemic

stroke,” “intracranial hemorrhage,” “intracranial artery disease,” “cardiovascular disease,”

“myocardial ischemia,” “myocardial infarct,” “ischemic heart disease,” “coronary heart disease,”

and “longitudinal studies,” “cohort studies,” “prospective studies,” “follow-up studies.” We

restricted the search to human studies. There were no language restrictions. In addition, we

reviewed the references lists of obtained articles to identify additional relevant studies. When the

same or similar patient cohort was included in several publications, only the most recent or

complete report was selected for analysis.

Study Selection

Studies were selected for the meta-analysis if they fulfilled the following entry criteria: (1) the

study of adult patients had a community-based or population-based, prospective cohort design; (2)

the exposure of interest was intake of dietary flavonoids (including: flavonols, flavones,

flavanones, flavan-3-ols, anthocyanidins, and isoflavones); (3) the outcome of interest was stroke,

including all types of stroke (fatal, nonfatal, ischemic, and hemorrhagic); (4) reported quantitative

estimates of the multivariate-adjusted relative risk (RR) and 95% confidence interval (CI) for

stroke incidence or mortality associated with flavonoids intake; and (5) longer than 1 year of

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follow-up. Studies were excluded if (1) the study design was cross-sectional, case-control; (2)

reported unadjusted or only age- or sex-adjusted RR; and (3) shorter than 1 year of follow-up.

Data Abstraction

All data were independently abstracted in duplicate by 2 investigators (ML, and XZ).

Discrepancies were resolved by consensus. When necessary, the original authors were contacted

for supplementary information. The following data were extracted from each study: first author’s

last name, publication year, country where the study was performed, location, number of

participants, participants’ age, follow-up years, assessment of dietary data and stroke, number of

cases, adjusted covariates and study quality.

Assessment of study quality

The Newcastle-Ottawa Scale (NOS) was used to assess the quality of studies.21

The quality of

cohort studies were evaluated in the following three major components: selection of the study

group (0-4 stars), quality of the adjustment for confounding (0-2 stars) and assessment of outcome

in the cohorts (0-3 stars). A higher score represents better methodological quality. The full score

was 9 stars. Studies were graded as the high-quality if they met ＞8 awarded stars.

Statistical Analysis

The relative risks (RRs) were used as the common measure of association between depression and

stroke, and the hazard ratios (HRs) were considered equivalent to RRs. Data analysis used

multivariate-adjusted outcome data. We converted these values in every study by using their

natural logarithms, and the SEs were calculated from these logarithmic numbers and their

corresponding 95% CIs. When the result on total stroke in our meta-analysis was not available, we

used data from ischemic stroke, nonfatal stroke, or fatal stroke as an equivalent to total stroke.22

We combined these estimates using a random-effects model, which takes into account both

within-study and between-study variabilities.23

In the dose-response analysis, the generalized least

square for trend estimation method described by Greenland and Longnecker24

and Orsini et al25

was used to calculate study-specific slopes (linear trends) and 95% confidence intervals. The

method requires the distributions of cases and person years for exposure categories, and

median/mean of flavonoids intake level for each comparison group. We assigned the midpoint of

the upper and lower boundaries of each comparison group to determine mean flavonoids intake

level if the median or mean intake was not provided. If the lower or upper boundary for the lowest

and highest category, respectively, was not reported, we assumed that the boundary had the same

amplitude as the closest category.19

Additionally, we first created restricted cubic splines with 3

knots at fixed percentiles 25%, 50%, and 75% of the distribution.26

A p value for nonlinearity was

calculated by testing the null hypothesis that the coefficient of the fractional polynomials

component is equal to zero. Heterogeneity among studies was evaluated using the chi-square test

based on Cochran’s Q test and I2 statistic at p＜0.10 level of significance,

23 which describes the

percentage of variability in the effect estimates that is due to heterogeneity rather than chance.27

We regard I2 of ＜40% as “heterogeneity might not be important” and ＞75% as “considerable

heterogeneity” based on the suggestion of Cochrane Handbook for Systemic Review of

Interventions.28

All available data were conducted in the primary analysis. Subsequent subgroup

analyses were conducted according to stroke outcomes (fatal/nonfatal versus ischemic), follow-up

duration (≤10 years versus ＞10 years), sex (male versus female versus combined), Geographical

area (United States versus Asian versus European), history of stroke (yes versus no), and study

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quality (high [score＞ 8] versus low [score ≤8]). To explore possible explanations for

heterogeneity and to test the robustness of the association, we conducted meta-regression,

sensitivity analyses and abovementioned stratified analysis. The possibility of publication bias was

evaluated using the Egger rank correlation test at p＜0.10 level of significance and visual

inspection of a funnel plot.29,30

In the case of publication bias, “nonparametric trim-and-fill”

method was used to compute risk estimates corrected for this bias.31

All the statistical analyses

were performed in STATA 12.0 (StataCorp, College Station, TX). p values were 2-sided and p＜

0.05 was considered statistically significant.

Results

Literature Search

Figure 1 shows the results of literature research and selection. We initially identified 181 citations.

After exclusion of duplicate records and studies that did not fulfill our inclusion criteria, 21 studies

remained, and we further evaluated the full texts of these 21 publications. Of these, we excluded

11 studies for the following reasons: no stroke outcomes (n=4), duplicate reports (n=3) and review

(n=4). Finally, 10 studies met the inclusion criteria and were included in the meta-analysis.8-17

Study Characteristics

The characteristics of the studies and of their participants are presented in Table 1 and

Supplemental table 1. A total of 10 studies involving 258,158 participants and more than 4,581

stroke cases were included in the meta-analysis.8-17

Among 10 studies, 3 were conducted primarily

in the United States,11,14,16

1 from an Asian country (Singapore),17

and 6 studies were from

European countries (including Finland, Netherlands).8-10,12,13,15

The number of participants ranged

from 755 in the study by Marniemi et al12

to 69,622 in the study by Cassidy et al.16

3 studies

included both men and women,10,12,17

3 studies included only men,8,9,15

and 4 studies only

women.11,13,14,16

The follow-up duration ranged from 6.1 to 28 years,8,10

with a median of 14.35

years. The dietary assessment of flavonoids intake varied across studies, in most of the studies,

intake of flavonoids was measured by food frequency questionnaires (FFQs) and dietary history

interview. In most of the studies, stroke was assessed by medical records or death certificates

based on ICD-8,9,10. All studies provided adjusted risk estimates (e.g., sex, body mass index,

smoking, education, et al), overall quality scores ranged for 7 to 9, and the median score was 8.

Flavonoids intake and Stroke Risk

The multivariable adjusted RRs of fatal or nonfatal stroke in relation to dietary flavonoids intake

from individual studies and the combined RR are presented in figure 2. For the 10 studies, only 1

showed that flavonoids intake was associated with decreased risk of stroke.10

Overall, participants

with the highest dietary flavonoids intake, compared with the lowest, experienced a significant

decreased risk for development of stroke (combined RR, 0.90; 95% CI, 0.82-0.98; Figure 2) after

adjustment for other risk factors. We saw no heterogeneity among studies (p = 0.733, I2

= 0%).

Among 10 studies, 4 studies were eligible for the dose-response analysis of flavonoids intake and

risk of stroke.9,14,16,17

Using a restricted cubic splines model, dose-response analysis found a

statistically nonsignificant inverse association with risk of stroke per 100-mg/day increment of

flavonoids intake (relative risk 0.94, 95% confidence interval 0.83 to 1.06, I2 = 0%; Figure 3 and

4). No publication bias was observed (p = 0.962).

Stratifying analysis

In most subgroups, dietary flavonoids intake was not associated with stroke. Stratifying by sex,

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the associations between dietary flavonoids intake and risk of stroke were similar between males

and females (table 2). The pooled RR of stroke were 0.88 (95% CI: 0.75-1.03) from studies

conducted in the United States, 0.88 (95% CI: 0.77-100) in European, and 0.97 (95% CI:

0.81-1.16) in Asian (table 2). Stratifying by duration, we observed the protective effect of dietary

flavonoids intake in longer than 10 years of follow-up studies. The associations were more evident

in several strata of study characteristics (table 2).

Sensitivity Analysis and Publication Bias

We tested the robustness of our results in sensitivity analysis, when a single study included in the

meta-analysis was deleted at a time, the results of meta-analysis remained largely unchanged,

indicating that the results of the present meta-analysis were stable (data not shown). Visual

inspection of the funnel plot identified asymmetry (Supplemental figure 1). There was no

statistical evidence of publication bias among studies for stroke risk by using Egger test (p =

0.222).

Meta Regression

In this meta-analysis, although no heterogeneity was observed among individual studies, a

multivariate regression was conducted to explore the potential sources of heterogeneity under

stroke. The results of regression suggested that stroke outcomes, follow-up duration, sex,

geographical area, history of stroke, and study quality were no significant source of heterogeneity.

Discussion

Our meta-analysis of 10 prospective studies indicates that dietary flavonoids intake may be

inversely associated with risk of stroke. In addition, dose-response analysis found a statistically

nonsignificant inverse association, with a RR of 0.94 (95% CI, 0.83-1.06) for each 100-mg/day

increment of flavonoids intake.

Potential benefits of flavonoids

Flavonoids, constitute a large class of polyphenols, are widely distributed in plants and are present

in considerable amounts in fruits, vegetables, tea and red wine. These bioactive polyphenols are

non-energetic, non-nutrient secondary metabolites present in plants and cannot be synthesizes by

humans.32

In recent decades, overwhelming evidence indicates that intake of flavonoids was

associated with well-known risk-factors for CVD. There was also some evidence that flavonoids

may have a direct role in the development of CVD (e.g., coronary heart disease, stroke, et al).33,34

In addition, although there are many putative biological mechanisms underlying a possible

cardioprotective role for flavonoids,35

including antioxidant,6 reductions in platelet aggregation,

36

anti-inflammatory,37

and the recovery of endothelial function properties of some of the compounds,

the effect of individual components or interactions between flavonoids is still largely unknown,

flavonols and isoflavones intake may explain some of this beneficial effect.

Meanwhile, the exact mechanism for the beneficial effect by which flavonoids intake may protect

against the development of stroke is still ambiguous. The first mechanism is the improved effect

of flavonoids on the amelioration of insulin resistance, flavonoids, especially quercetin, may be

beneficial in insulin resistance by inhibition of intestinal glucose transporters.38

Moreover,

flavonoids reduced mitochondrial lipid peroxidation and loss of mitochondrial transmembrane

electric potential caused by oxidative stress induced by ADP plus iron. Thus, it was mainly

attributed to antioxidant effects.39

The third mechanism is the protective action of flavonoids on

the anti-inflammatory properties in the brain, it may be attributable to raft disrupting and

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antioxidant effects.40

Because the flavonoids are very diverse in their bioavailability and

bioactivity, the rationale for assuming that intakes to all flavonoid classes might have effects on

stroke needs more consideration.

Results in relation to other studies

Over the past decades, despite extensive studies that investigated the role of flavonoids intake on

either cardiovascular diseases or cerebrovascular diseases, it remains unclear whether the

association between flavonoids intake and risk of stroke is causal. Some of the studies suggested

flavonoids associated with increased risk of stroke,10

the others failed to find the association.8,9

Importantly, the possibility of reverse causality should be addressed. Previous meta-analysis of

flavonol intake and risk of stroke showed that flavonol intake was inversely associated with stroke

incidence.34

That meta-analysis included 6 prospective cohorts studies, of which were from 3

different countries, 4 were conducted in Europe (3 in Finland and 1 in The Netherlands) and 2 in

the United States. The inverse associations between flavonol intake and stroke were observed in

two prospective cohort studies (Zutphen Elderly Study and Kuopio Study). Weak and

nonsignificant inverse associations were found in the Finnish Mobile Clinic Study and Women’s

Health Study, whereas no associations were noted in the Iowa Women’s Health Study and

Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. The results for flavonol were

consistent with our findings on flavonoids. But the data from studies included by previous

meta-analysis were limited to August 2009.34

Interestingly, since then many new studies involving

relationship between flavonoids intake and risk of stroke were published.16,17

To obtain a more

comprehensive estimate of the putative influence of the flavonoids on stroke, we conducted a

meta-analysis of prospective cohort studies. To our knowledge, this meta-analysis is the largest to

reveal the potential relationship between dietary flavonoids intake and risk of stroke. However,

most studies assess dietary flavonoids intake based on self-reported questionnaires using FFQs,

and medical records were not always available for stroke classification, the possibility that

misclassification of flavonoids intake and stroke was inevitable and likely to bias true association

among individual studies.

Strengths and limitations

Compared with the previous meta-analyses,18,34

our study has several more important strengths.

The present meta-analysis included 2 times more participants and 2 times more stroke cases, to

our knowledge, this is the largest meta-analysis on flavonoids intake and the risk of stroke. We

also explored possible source of heterogeneity using subgroup analyses and the meta-regression

method, all of them were no significant. Moreover, the presence of a dose-response relationship

further strengthened the association of dietary flavonoids intake with risk of stroke. Therefore, the

results should be more reliable.

In interpreting the results, some limitations of this meta-analysis should be acknowledged. First,

one limitation of any meta-analysis of observational studies is that residual confounding or

confounding by unmeasured factors (such as intake of other nutrients) may have affected the

significant association between flavonoids intake and stroke risk. Thus, these findings should be

treated with cautions. Second, we cannot exclude the possibility of recall bias in the assessments

of diet based on the FFQs. However, the prospective study design and exclusion of participants

with chronic diseases at baseline should minimize such bias. Third, the noticeable limitation of our

study was the potential for bias due to inevitable measurement error and misclassification,

especially for individual with lower consumption levels. We attempted to reduce measurement

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error in adjusting for energy or fiber intake and using of cumulatively averaged intake levels.

Fourth, we could not study the main sources of flavonoids because of the insufficient data.

However, it is most likely that the main source of flavonols, flavones and flavanones have been

the same.15

Finally, the possible limitation is due to language bias. We attempted to minimize this

bias by searching major electronic databases with no language restriction. However, several

articles published in non-English or unpublished reports might not appear in international journal

databases, and could be omitted by our searches.41

Conclusions

In summary, results from this meta-analysis suggest that higher dietary flavonoids intake may

moderately lower the risk of stroke after adjustment of established cardiovascular risk factors.

Further preferably randomized controlled studies are needed to evaluate the effects of flavonoids

intake on stroke risk.

Contributors: ML and ZT conceived and designed the study. ML and WH searched the databases

and checked them according to the eligible criteria and exclusion criteria. ZT helped develop

search strategies. XZ and WH extract quantitative data. XZ, and WH analyzed the data. ML wrote

the draft of the paper. All authors contributed to writing, reviewing, or revising the paper. ZT is the

guarantor.

Funding: This work was not funded by any foundation or program.

Competing interests: All authors have completed the ICMJE uniform disclosure form at

www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and

declare: no support from any organization for the submitted work; no financial relationships with

any organizations that might have an interest in the submitted work in the previous three years; no

other relationships or activities that could appear to have influenced the submitted work.

Ethical approval: Not required.

Data sharing: No additional data available.

Figures Information

Figure 1. Process of literature search and study selection.

(TIFF)

Figure 2. Random effects analysis of fully adjusted studies for highest versus lowest intake of

flavonoids and risk of stroke.

(TIFF)

Figure 3. Dose-response relationship between dietary flavonoids intake and stroke risk.

(TIFF)

Figure 4. Forest plot of flavonoids and risk of stroke.

(TIFF)

Appendix figure information

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Supplemental figure 1. Egger funnel plot for detection of publication bias for stroke risk.

(DOC)

Appendix table information

Supplemental table 1. Confounding factors and methods for adjustment.

(DOC)

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25. Orsini N, Li R, Wolk A, et al. Meta-analysis for linear and nonlinear dose-response relations:

examples, an evaluation of approximations, and software. Am J Epidemiol 2012;175:66-73.

26. Harrell FE Jr, Lee KL, Pollock BG. Regression models in clinical studies: determining

relationships between predictors and response. J Natl Cancer Inst 1988;80:1198-202.

27. Higgins JP, Thompson SG, Deeks JJ, et al. Measuring inconsistency in meta-analyses. BMJ

2003;327:557-60.

28. Higgins JPT, Green S. Cochrane Handbook for systematic Reviews of Interventions. Oxford,

UK: The Cochrane Collaboration; 2011.

29. Begg CB, Mazumdar M. Operating characteristics of a rank correlation test for publication

bias. Biometrics 1994;50:1088-101.

30. Sterne JA, Egger M. Funnel plots for detecting bias in meta-analysis: guidelines on choice of

axis. J Clin Epidemiol 2001;54:1046-55.

31. Duval S, Tweedie R. Trim and fill: A simple funnel-plot-based method of testing and adjusting

for publication bias in meta-analysis. Biometrics 2000;56:455-63.

32. Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance.

Nutr Rev 1998;56:317-33.

33. Huxley RR, Neil HA. The relation between dietary flavonol intake and coronary heart disease

mortality: a meta-analysis of prospective cohort studies. Eur J Clin Nutr 2003;57:904-8.

34. Hollman PC, Geelen A, Kromhout D. Dietary flavonol intake may lower stroke risk in men

and women. J Nutr 2010;140:600-4.

35. Geleijnse JM, PCh H. Flavonoids and cardiovascular health: which compounds, what

mechanisms. Am J Clin Nutr 2008;88:12-3.

36. Murphy KJ, Chronopoulos AK, Singh I, et al. Dietary flavanols and procyanidin oligomers

from cocoa (Theobroma cacao) inhibit platelet function. Am J Clin Nutr 2003;77:1466-73.

37. Landberg R, Sun Q, Rimm EB, et al. Selected dietary flavonoids are associated with markers

of inflammation and endothelial dysfunction in U.S. women. J Nutr 2011;141:618-25.

38. Li YQ, Zhou FC, Gao F, et al. Comparative evaluation of quercetin, isoquercetin and rutin as

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inhibitors of alpha-glucosidase. J Agric Food Chem 2009;57:11463-8.

39. Silva B, Oliveira PJ, Dias A, et al. Quercetin, kaempferol and biapigenin from Hypericum

perforatum are neuroprotective against excitotoxic insults. Neurotox Res 2008;13:265-79.

40. Perez-Vizcaino F, Duarte J. Flavonols and cardiovascular disease. Mol Aspects Med

2010;31:478-94.

41. Li M, Hou W, Zhang X, et al. Hyperuricemia and risk of stroke: a systematic review and

meta-analysis of prospective studies. Atherosclerosis 2014;232:265-70.

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Table 1. Characteristics of studies included in the meta-analysis

First author,

publication (yr) Country/Population

No. Participants (%

male)

Age rang or

mean (yr)

Follow-Up

Duration (yr)

Assessment of

dietary data

Stroke

Ascertainment

No. of stroke

cases

Pre-stroke

Excluded

Study

Quality

Hirvonen et al.8

2000

Finland/European

26497 (100)

50-69

6.1

A self-administered,

modified diet history

method

Any type of stroke

based on ICD-8, 9

codes

Ischemic: 736

Yes

8

Arts et al.9 2001

Netherlands/European

806 (100)

65-84

15

A cross-check

dietary history

method

Any type of stroke

based on ICD-9

codes

Fatal: 47,

Nonfatal: 88

No

7

Knekt et al.10

2002

Finland/European

9131 (NA)

30-69

28

A dietary history

interview

Any type of stroke

based on ICD-8

codes

Fatal or

nonfatal: 681

No

8

Sesso et al.11

2003

United States

38445 (0)

53.9

6.9

A food-frequency

questionnaire

Any type of stroke

based on clinical

diagnosis

Fatal or

nonfatal: NA

Yes

9

Marniemi et al.12

2005

Finland/European

755 (47.8)

65-99

10

A dietary history

interview

Any type of stroke

based on ICD-9

codes

Fatal: 45,

Nonfatal: 25

Yes

8

van der Schouw et

al.13

2005


16165 (0)

49-70

6.3

A validated

food-frequency

questionnaire

Any type of stroke

based on ICD-9

codes

Fatal or

nonfatal: 147

Yes

9

Mink et al.14

2007

United States

34489 (0)

55-69

16

A food-frequency

questionnaire

Any type of stroke

based on ICD-9

codes

Fatal: 469

Yes

9

Mursu et al.15

2008

Finland/European

1950 (100)

42-60

15.2

An instructed 4d

food recording by

Any type of stroke

based on ICD-9, 10

Ischemic: 102

Yes

9

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1

household measures codes

Cassidy et al.16

2012

United States

69622 (0)

30-55

14

A semiquantitative

food-frequency

questionnaires

interview

Any type of stroke

based on medical

records, autopsy

reports, and death

certificates

Ischemic: 943

Yes

8

Talaei et al.17

2014

Singapore/Asian

60298 (44.5)

45-74

14.7

A semiquantitative

food-frequency

questionnaires

interview

Any type of stroke

based on ICD-9

codes

Fatal: 1298

No

8

Abbreviations: ICD: international classification of diseases.

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2

Table 2. Stratified analyses of flavonoids intake and stroke risk

Group No. of studies RR (95% CI) Heterogeneity test

P value of pooled effect χχχχ2 P value I

2, %

Overall studies

Fatal/nonfatal stroke 10 0.90 (0.82-0.98) 2.35 0.733 0 0.019

Ischemic stroke 3 0.93 (0.80-1.07) 1.03 0.609 0 0.301

Geographical area

United States 3 0.88 (0.75-1.03) 1.59 0.511 0 0.112

Asian 1 0.97 (0.81-1.16) 0.33 - - 0.740

European 6 0.88 (0.77-1.00) 1.95 0.577 0 0.051

History of stroke

Yes 3 0.89 (0.78-1.02) 1.66 0.683 0 0.096

No 7 0.90 (0.80-1.02) 1.62 0.351 4.6 0.105

Sex

Male 3 0.95 (0.79-1.14) 0.55 0.651 0 0.580

Female 4 0.89 (0.77-1.04) 1.45 0.613 0 0.114

Combined 3 0.86 (0.72-1.04) 1.58 0.227 32.6 0.148

Mean follow-up, years

≤10 4 0.90 (0.74-1.09) 1.10 0.340 10.7 0.271

>10 6 0.89 (0.81-0.99) 2.09 0.750 0 0.036

Quality score

High, score＞8 4 0.87 (0.70-1.07) 1.34 0.534 0 0.181

Low, score≤8 6 0.91 (0.82-1.00) 1.97 0.589 0 0.048

Abbreviations: CI = confidence interval; RR = relative risk.

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152x101mm (300 x 300 DPI)

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152x101mm (300 x 300 DPI)

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152x101mm (300 x 300 DPI)

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152x101mm (300 x 300 DPI)

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Egger's publication bias plot

standardized effect

precision0 5 10

-2

-1

0

1

Supplemental figure 1 Egger funnel plot for detection of publication bias for stroke risk

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Supplemental table 1. Confounding factors and methods for adjustment

Reference Adjusted covariates Total stroke Nonfatal stroke Fatal Stroke Ischemic Stroke Hemorrhagic

stroke

Hirvonen et al.8 2000

Age, BMI, SBP, DBP, height, cholesterol, diabetes, history

CHD, smoking, alcohol, supplementation group and education.

equal to

ischemic stroke

results

0.98 (0.80-1.21)

Arts et al.9 2001

Age, BMI, smoking, alcohol, physical activity, coffee, diet,

energy, FA, prescribed diet, intakes of fish, coffee, cholesterol,

fiber, fish, vit C, vit E and β-carotene

0.92

(0.51-1.68)

Knekt et al.10

2002

Age, sex, geographic area, occupation, BMI, BP, cholesterol,

diabetes, region, SE and smoking

0.79

(0.64-0.98)

Sesso et al.11

2003

Age, exercise, aspirin, BMI, BP, postmenopausal hormone use,

cholesterol, diabetes, history of CHD, smoking, alcohol, F﹠V,

fiber, folate and vit E.

0.70

(0.46-1.07)

Marniemi et al.12

2005

Age, sex, smoking, functional capacity and weight adjusted

energy intake.

0.65

(0.34-1.23)

van der Schouw et

al.13

2005

Age, BMI, cholesterol, physical activity, diabetes,

hypertension, hypercholesterolemia, HRT, OC, MS, smoking,

alcohol, energy, F﹠A, fiber, protein, fruit, vegetable and

menopausal status.

1.05

(0.64-1.70)

Mink et al.14

2007

Age, BMI, BP, diabetes, HRT, MS, education, smoking,

activity, estrogen use, WHR and energy

equal to fatal

stroke results

0.94 (0.69-1.29)

Mursu et al.15

2008

Age, examination years, BMI, SBP, HM, cholesterol, TAG,

maximal oxygen uptake, smoking, history of CVD, diabetes,

alcohol, energy-adjusted intake of folate and vit E.

equal to

ischemic stroke

results

0.71 (0.37-1.37)

Cassidy et al.16

2012 Age, physical activity, smoking, HRT, BMI, aspirin use, equal to 0.90 (0.73-1.11)

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diabetes, hypercholesterolemia, history of CHD, alcohol,

menopausal status, energy, use of multivitamins and history of

hypertension

ischemic stroke

results

Talaei et al.17

2014

Age, sex, dialect, year of interview, educational level, BMI,

physical activity, smoking duration, alcohol, diabetes,

hypertension, CHD, stroke, energy and fiber

equal to fatal

stroke results

0.97 (0.81-1.16)

Abbreviations: BMI, body mass index; CVD, cardiovascular disease; CHD, coronary heart disease; BP, blood pressure; SBP: systolic blood pressures; DBP: diastolic blood

pressures; vit C: vitamin C; vit E: vitamin E; FA: fatty acids; F﹠V: fruit and vegetable intake; MS: menopausal status; HRT: hormonal replacement therapy; OC: oral

contraceptives; WHR: waist-to-hip ratio; HM: hypertension medication。

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MOOSE Checklist



Min Li master student1, Xiaowei Zhang master student

1, Wenshang Hou master

student1, Zhenyu Tang associate professor

1

1Department of Neurology, The Second Affiliated Hospital of Nanchang University,

Nanchang 330006, Jiangxi Province, People’s Republic of China

Correspondence to: Zhenyu Tang, Department of Neurology, The Second Affiliated

Hospital of Nanchang University, No. 1, Minde Road, Nanchang, Jiangxi 330006,

People’s Republic of China

Email: [email protected]

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Criteria Brief description of how the criteria were handled in

the meta-analysis

Reporting of background should

include

√ Problem definition Stroke is the second most common cause of death, as well

as the fourth leading cause of lost productivity and the

second highest cause of disability worldwide. The

prevention of stroke is thus clearly an important public

health priority. In recent decades, concern has mounted

regarding the premature incidence and mortality

associated with stroke, with growing interest in altering

risk factors and reversing this global epidemic. Among

the known risk factors for stroke, dietary factors,

especially dietary flavonoids intake, have aroused

particular attention. Clinical studies have shown that

intakes of flavonoids reduce cardiovascular disease

(CVD) risk. Additionally, experimental studies indicated

that flavonoids have been shown to have both antioxidant

and antithrombotic properties.

√ Hypothesis statement Flavonoids intake decrease risk of stroke.

√ Description of study outcomes Stroke.

√ Type of exposure or

intervention used

Flavonoids

√ Type of study designs used We included (1) original studies (eg, not review articles,

meeting abstracts, editorials, or commentaries); (2)

prospective cohort design (eg, not cross sectional design,

case-control design).

√ Study population We placed no restriction.

Reporting of search strategy

should include

√ Qualifications of searchers The credentials of the two investigators WH and ML are

indicated in the author list.

√ Search strategy, including time

period included in the

synthesis and keywords

PubMed from 1965 –February 2015

Embase from 1974 –February 2015

Cochrane library from 1990- February 2015

Keywords: (“flavonoids,” “polyphenols,” “phenolics,”

“flavonols,” “flavones,” “quercetin,” “kaempferol,”

“myricetin,” “isorhamnetin,” “apigenin,” “luteolin,” and

“stroke,” “cerebrovascular disease,” “cerebrovascular

disorders,” “cerebral infarct,” “ischemic stroke,”

“intracranial hemorrhage,” “intracranial artery disease,”

“cardiovascular disease,” “myocardial ischemia,”

“myocardial infarct,” “ischemic heart disease,” “coronary

heart disease,”) AND (“longitudinal studies,” “cohort

studies,” “prospective studies,” “follow-up studies.”).

√ Databases and registries

searched

PubMed, Embase, and the Cochrane library

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√ Search software used, name

and version, including special

features

We did not employ a search software. EndNote was used

to merge retrieved citations and eliminate duplications

√ Use of hand searching We hand-searched bibliographies of retrieved papers for

additional references,

√ List of citations located and

those excluded, including

justifications

Details of the literature search process are outlined in the

process of literature search and study selection. The

citation list is available upon request

√ Method of addressing articles

published in languages other

than English

We placed no restrictions on language; local scientists

fluent in the original language of the article were

contacted for translation

√ Method of handling abstracts

and unpublished studies

We had contacted a few authors for unpublished studies

on the association.

√ Description of any contact with

authors

We contacted authors who had conducted multivariate

analysis with coronary heart disease as a covariate, but

the exposure of interest was not intake of dietary

flavonoids.

Reporting of methods should

include

√ Description of relevance or

appropriateness of studies

assembled for assessing the

hypothesis to be tested

Detailed inclusion and exclusion criteria were described

in the methods section.

√ Rationale for the selection and

coding of data

Data extracted from each of the studies were relevant to

the population characteristics, study design, exposure,

outcome, and possible effect modifiers of the association.

√ Assessment of confounding Restricted the analysis to age- or sex-adjusted estimates

only. Conducted sensitivity analyses by eliminating

studies that had not adjusted for possible confounders.

√ Assessment of study quality,

including blinding of quality

assessors; stratification or

regression on possible

predictors of study results

The Newcastle-Ottawa Scale (NOS) was used to assess

the quality of studies. The quality of cohort studies were

evaluated in the following three major components:

selection of the study group (0-4 stars), quality of the

adjustment for confounding (0-2 stars) and assessment of

outcome in the cohorts (0-3 stars). A higher score

represents better methodological quality. The full score

was 9 stars. Studies were graded as the high-quality if

they met ＞8 awarded stars.

√ Assessment of heterogeneity Heterogeneity of the studies were explored within two

types of study designs using Cochrane’s Q test of

heterogeneity and I2 statistic that provides the relative

amount of variance of the summary effect due to the

between-study heterogeneity.

√ Description of statistical

methods in sufficient detail to

be replicated

Description of methods of meta-analyses, sensitivity

analyses, subgroup analyses, meta regression and

assessment of publication bias are detailed in the

methods.

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√ Provision of appropriate tables

and graphics

We included 1 flow chart, several summary tables and

figures.

Reporting of results should

include

√ Graph summarizing individual

study estimates and overall

estimate

Figure 2, 3 and 4

√ Table giving descriptive

information for each study

included

Table 1 and Supplemental table 1

√ Results of sensitivity testing

Table 2

√ Indication of statistical

uncertainty of findings

95% confidence intervals were presented with all

summary estimates, I2 values and results of sensitivity

analyses

Reporting of discussion should

include

√ Quantitative assessment of bias Subgroup analyses indicate heterogeneity in strengths of

the association due to most common biases in cohort

studies.

√ Justification for exclusion We excluded studies that had not adjusted for or were

standardized by age or sex, a potential confounder, and

used different exposure or outcome assessment for the

comparison groups.

√ Assessment of quality of

included studies

We discussed the results of the subgroup analyses, and

potential reasons for the observed heterogeneity.

Reporting of conclusions should

include

√ Consideration of alternative

explanations for observed

results

We discussed that potential unmeasured confounders such

as other chronic diseases may have caused residual

confounding, but the measured factors that are correlated

with such confounders would have mitigated the bias.

We noted that the variations in the strengths of

association may be due to true population differences, or

to differences in quality of studies.

√ Generalization of the

conclusions

Our meta-analysis suggests that dietary flavonoids intake

may be inversely associated with risk of stroke. In

addition, dose-response analysis found a statistically

nonsignificant inverse association, with a relative risk of

0.94 (95% confidence intervals, 0.83-1.06) for each 100-

mg/day increment of flavonoids intake.

√ Guidelines for future research We recommend future preferably randomized controlled

studies should explore what kind of flavonoids can reduce

the risk of stroke.

√ Disclosure of funding source No separate funding was necessary for the undertaking of

this systematic review.

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Dietary flavonoid intake and the risk of stroke: A dose-

response


Journal: BMJ Open

Manuscript ID bmjopen-2015-008680.R1


Date Submitted by the Author: 26-Nov-2015

Complete List of Authors: Tang, Zhenyu; The Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, People’s Republic of China, Department of Neurology

Li, Min; The Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, People’s Republic of China, Department of Neurology Zhang, Xiaowei; The Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, People’s Republic of China, Department of Neurology Hou, Wenshang; The Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, People’s Republic of China, Department of Neurology



Secondary Subject Heading: Cardiovascular medicine, Evidence based practice, Neurology, Nutrition and

metabolism, Public health

Keywords: Stroke < NEUROLOGY, NUTRITION & DIETETICS, Epidemiology < INFECTIOUS DISEASES


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a,1, Xiaowei Zhang


a





[email protected]


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1

Abstract


risk of stroke.

Design Meta-analysis and systematic review of prospective cohort studies.







the highest compared with the lowest dietary flavonoids intake was 0.90 (95% confidence interval,




of stroke for flavonoids intake was 0.89 (95% confidence interval, 0.81-0.99) in longer than 10


Conclusions Results from this meta-analysis suggest that higher dietary flavonoids intake may


Article summary

Strengths and limitations of this study

To our knowledge, this is the largest meta-analysis on flavonoids intake and the risk of stroke. We

also investigated a dose-response relation between flavonoids consumption and risk of stroke.

The possibility of residual confounding or confounding by unmeasured factors, which cannot be

ruled out in any observational study, must be acknowledged. We cannot exclude the possibility of

recall bias in the assessments of diet based on the food frequency questionnaires.

1. Stroke is the second most common cause of death and the second highest cause of disability

worldwide.

2. Epidemiological studies suggest that the intake of flavonoids is beneficial in delaying or

preventing the development of stroke, though results from cohort studies are controversial.

3. Higher dietary flavonoids intake is associated with a significantly reduced risk of stroke.


flavonoids.

5. Further evidence from preferably randomized controlled studies should explore what kind of

flavonoids can reduce the risk of stroke.

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2

Introduction









Additionally,








the role

of flavonoids intake on stroke prevention is still controversial. In addition, flavonoids intake

differed substantially between studies, which makes it difficult to interpret the summary estimate

based on results from study populations with different ranges of flavonoids intake.19



of stroke.

Methods

Literature search












reviewed the references lists of obtained articles to identify additional relevant studies. When the

same or similar patient cohort was included in several publications, only the most recent or

complete report was selected for analysis.

Study Selection








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3



Data Abstraction









The quality of






The relative risks (RRs) were used as the common measure of association between depression and

stroke, and the hazard ratios (HRs) were considered equivalent to RRs. Data analysis used









and Orsini et al25



median/mean of flavonoids intake level for each comparison group. We assigned the midpoint of

the upper and lower boundaries of each comparison group to determine mean flavonoids intake








component is equal to zero. Heterogeneity among studies was evaluated using the chi-square test

based on Cochran’s Q test and I2 statistic at p＜0.10 level of significance,

23 which describes the

percentage of variability in the effect estimates that is due to heterogeneity rather than chance.27

We regard I2 of ＜40% as “heterogeneity might not be important” and ＞75% as “considerable

heterogeneity” based on the suggestion of Cochrane Handbook for Systemic Review of

Interventions.28

All available data were conducted in the primary analysis. Subsequent subgroup

analyses were conducted according to stroke outcomes (fatal/nonfatal versus ischemic), follow-up

duration (≤10 years versus ＞10 years), sex (male versus female versus combined), Geographical

area (United States versus Asian versus European), history of stroke (yes versus no), and study

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4

quality (high [score＞ 8] versus low [score ≤8]). To explore possible explanations for

heterogeneity and to test the robustness of the association, we conducted meta-regression,

sensitivity analyses and abovementioned stratified analysis. The possibility of publication bias was

evaluated using the Egger rank correlation test at p＜0.10 level of significance and visual

inspection of a funnel plot.29,30

In the case of publication bias, “nonparametric trim-and-fill”

method was used to compute risk estimates corrected for this bias.31

All the statistical analyses

were performed in STATA 12.0 (StataCorp, College Station, TX). p values were 2-sided and p＜

0.05 was considered statistically significant.

Results

Literature Search








Supplemental table 1. A total of 10 studies involving 258,158 participants and more than 4,581

stroke cases were included in the meta-analysis.8-17

Among 10 studies, 3 were conducted primarily

in the United States,11,14,16







3 studies

included both men and women,10,12,17


and 4 studies only

women.11,13,14,16



years. The dietary assessment of flavonoids intake varied across studies, in most of the studies,








showed that flavonoids intake was associated with decreased risk of stroke.10


with the highest dietary flavonoids intake, compared with the lowest, experienced a significant



= 0%).

Among 10 studies, 4 studies were eligible for the dose-response analysis of flavonoids intake and




flavonoids intake (relative risk 0.94, 95% confidence interval 0.83 to 1.06, I2 = 0%; Figure 3 and

4). No publication bias was observed (p = 0.962).


In most subgroups, dietary flavonoids intake was not associated with stroke. Stratifying by sex,

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the associations between dietary flavonoids intake and risk of stroke were similar between males

and females (table 2). The pooled RR of stroke were 0.88 (95% CI: 0.75-1.03) from studies

conducted in the United States, 0.88 (95% CI: 0.77-100) in European, and 0.97 (95% CI:

0.81-1.16) in Asian (table 2). Stratifying by duration, we observed the protective effect of dietary

flavonoids intake in longer than 10 years of follow-up studies. The associations were more evident

in several strata of study characteristics (table 2).







0.222).

Meta Regression





Discussion

Our meta-analysis of 10 prospective studies indicates that dietary flavonoids intake may be

inversely associated with risk of stroke. In addition, dose-response analysis found a statistically

nonsignificant inverse association, with a RR of 0.94 (95% CI, 0.83-1.06) for each 100-mg/day

increment of flavonoids intake.





humans.32







36



the effect of individual components or interactions between flavonoids is still largely unknown,


Meanwhile, the exact mechanism for the beneficial effect by which flavonoids intake may protect

against the development of stroke is still ambiguous. The first mechanism is the improved effect

of flavonoids on the amelioration of insulin resistance, flavonoids, especially quercetin, may be

beneficial in insulin resistance by inhibition of intestinal glucose transporters.38

Moreover,

flavonoids reduced mitochondrial lipid peroxidation and loss of mitochondrial transmembrane

electric potential caused by oxidative stress induced by ADP plus iron. Thus, it was mainly




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bioactivity, the rationale for assuming that intakes to all flavonoid classes might have effects on



Over the past decades, despite extensive studies that investigated the role of flavonoids intake on


association between flavonoids intake and risk of stroke is causal. Some of the studies suggested

flavonoids associated with increased risk of stroke,10


Importantly, the possibility of reverse causality should be addressed. Previous meta-analysis of


incidence.34






Health Study, whereas no associations were noted in the Iowa Women’s Health Study and

Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. The results for flavonol were

consistent with our findings on flavonoids. But the data from studies included by previous

meta-analysis were limited to August 2009.34

Interestingly, since then many new studies involving

relationship between flavonoids intake and risk of stroke were published.16,17

To obtain a more

comprehensive estimate of the putative influence of the flavonoids on stroke, we conducted a

meta-analysis of prospective cohort studies. To our knowledge, this meta-analysis is the largest to

reveal the potential relationship between dietary flavonoids intake and risk of stroke. However,

most studies assess dietary flavonoids intake based on self-reported questionnaires using FFQs,

and medical records were not always available for stroke classification, the possibility that

misclassification of flavonoids intake and stroke was inevitable and likely to bias true association

among individual studies.


Compared with the previous meta-analyses,18,34

our study has several more important strengths.

The present meta-analysis included 2 times more participants and 2 times more stroke cases, to

our knowledge, this is the largest meta-analysis on flavonoids intake and the risk of stroke. We

also explored possible source of heterogeneity using subgroup analyses and the meta-regression

method, all of them were no significant. Moreover, the presence of a dose-response relationship

further strengthened the association of dietary flavonoids intake with risk of stroke. Therefore, the

results should be more reliable.




significant association between flavonoids intake and stroke risk. Thus, these findings should be

treated with cautions. Second, we cannot exclude the possibility of recall bias in the assessments

of diet based on the FFQs. However, the prospective study design and exclusion of participants

with chronic diseases at baseline should minimize such bias. Third, the noticeable limitation of our



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Fourth, we could not study the main sources of flavonoids because of the insufficient data.

However, it is most likely that the main source of flavonols, flavones and flavanones have been

the same.15

Finally, the possible limitation is due to language bias. We attempted to minimize this

bias by searching major electronic databases with no language restriction. However, several

articles published in non-English or unpublished reports might not appear in international journal

databases, and could be omitted by our searches.41

Conclusions

In summary, results from this meta-analysis suggest that higher dietary flavonoids intake may


Further preferably randomized controlled studies are needed to evaluate the effects of flavonoids

intake on stroke risk.



search strategies. XZ and WH extract quantitative data. XZ, and WH analyzed the data. ML wrote

the draft of the paper. All authors contributed to writing, reviewing, or revising the paper. ZT is the

guarantor.









Figures Information


(TIFF)



(TIFF)

Figure 3. Dose-response relationship between dietary flavonoids intake and stroke risk.

(TIFF)

Figure 4. Forest plot of flavonoids and risk of stroke.

(TIFF)


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(DOC)



(DOC)

REFERENCES



















Nutr 2001;74:227-32.


J Clin Nutr 2002;76:560-8.





2005;15:188-97.



2005;111:465-71.





Study. Br J Nutr 2008;100:890-5.


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9

Stroke 2012;43:946-51.






















2003;327:557-60.










Nutr Rev 1998;56:317-33.




and women. J Nutr 2010;140:600-4.








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10





2010;31:478-94.



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First author,


No. Participants (%

male)

Age rang or

mean (yr)

Follow-Up

Duration (yr)

Assessment of

dietary data

Stroke

Ascertainment

No. of stroke

cases

Pre-stroke

Excluded

Study

Quality

Hirvonen et al.8

2000

Finland/European

26497 (100)

50-69

6.1



method

Any type of stroke

based on ICD-8, 9

codes

Ischemic: 736

Yes

8

Arts et al.9 2001


806 (100)

65-84

15

A cross-check

dietary history

method

Any type of stroke

based on ICD-9

codes

Fatal: 47,

Nonfatal: 88

No

7

Knekt et al.10

2002

Finland/European

9131 (NA)

30-69

28

A dietary history

interview

Any type of stroke

based on ICD-8

codes

Fatal or

nonfatal: 681

No

8

Sesso et al.11

2003

United States

38445 (0)

53.9

6.9

A food-frequency

questionnaire

Any type of stroke

based on clinical

diagnosis

Fatal or

nonfatal: NA

Yes

9

Marniemi et al.12

2005

Finland/European

755 (47.8)

65-99

10

A dietary history

interview

Any type of stroke

based on ICD-9

codes

Fatal: 45,

Nonfatal: 25

Yes

8

van der Schouw et

al.13

2005


16165 (0)

49-70

6.3

A validated

food-frequency

questionnaire

Any type of stroke

based on ICD-9

codes

Fatal or

nonfatal: 147

Yes

9

Mink et al.14

2007

United States

34489 (0)

55-69

16

A food-frequency

questionnaire

Any type of stroke

based on ICD-9

codes

Fatal: 469

Yes

9

Mursu et al.15

2008

Finland/European

1950 (100)

42-60

15.2

An instructed 4d

food recording by

Any type of stroke

based on ICD-9, 10

Ischemic: 102

Yes

9

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1


Cassidy et al.16

2012

United States

69622 (0)

30-55

14

A semiquantitative

food-frequency

questionnaires

interview

Any type of stroke

based on medical

records, autopsy

reports, and death

certificates

Ischemic: 943

Yes

8

Talaei et al.17

2014

Singapore/Asian

60298 (44.5)

45-74

14.7

A semiquantitative

food-frequency

questionnaires

interview

Any type of stroke

based on ICD-9

codes

Fatal: 1298

No

8


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2




2, %

Overall studies


Ischemic stroke 3 0.93 (0.80-1.07) 1.03 0.609 0 0.301

Geographical area

United States 3 0.88 (0.75-1.03) 1.59 0.511 0 0.112

Asian 1 0.97 (0.81-1.16) 0.33 - - 0.740

European 6 0.88 (0.77-1.00) 1.95 0.577 0 0.051

History of stroke

Yes 3 0.89 (0.78-1.02) 1.66 0.683 0 0.096

No 7 0.90 (0.80-1.02) 1.62 0.351 4.6 0.105

Sex

Male 3 0.95 (0.79-1.14) 0.55 0.651 0 0.580

Female 4 0.89 (0.77-1.04) 1.45 0.613 0 0.114

Combined 3 0.86 (0.72-1.04) 1.58 0.227 32.6 0.148


≤10 4 0.90 (0.74-1.09) 1.10 0.340 10.7 0.271

>10 6 0.89 (0.81-0.99) 2.09 0.750 0 0.036

Quality score

High, score＞8 4 0.87 (0.70-1.07) 1.34 0.534 0 0.181

Low, score≤8 6 0.91 (0.82-1.00) 1.97 0.589 0 0.048


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152x101mm (300 x 300 DPI)

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152x101mm (300 x 300 DPI)

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152x101mm (300 x 300 DPI)

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sta

nd

ard

ize

d e

ffect

precision0 5 10

-2

-1

0

1


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stroke




equal to

ischemic stroke

results

0.98 (0.80-1.21)

Arts et al.9 2001




0.92

(0.51-1.68)

Knekt et al.10

2002



0.79

(0.64-0.98)

Sesso et al.11

2003




0.70

(0.46-1.07)

Marniemi et al.12

2005


energy intake.

0.65

(0.34-1.23)

van der Schouw et

al.13

2005




menopausal status.

1.05

(0.64-1.70)

Mink et al.14

2007



equal to fatal

stroke results

0.94 (0.69-1.29)

Mursu et al.15

2008




equal to

ischemic stroke

results

0.71 (0.37-1.37)

Cassidy et al.16

2012 Age, physical activity, smoking, HRT, BMI, aspirin use, equal to 0.90 (0.73-1.11)

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hypertension

ischemic stroke

results

Talaei et al.17

2014




equal to fatal

stroke results

0.97 (0.81-1.16)



contraceptives; WHR: waist-to-hip ratio; HM: hypertension medication。

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Supplemental table 2. Flavonoid subclasses and compounds for each study.

Reference Adjusted covariates

Hirvonen et al.8 2000 Quercetin, kaempherol, myricetin, luteolin, and apigenin..

Arts et al.9 2001 Quercetin, kaempferol, myricetin, luteolin, and apigenin..

Knekt et al.10 2002

4 flavonols (kaempferol, quercetin, myricetin, and

isorhamnetin), 2 flavones (apigenin and luteolin), and 3

flavanones (hesperetin, naringenin, and eriodictyol).

Sesso et al.11 2003 Quercetin, kaempferol, myricetin, luteolin, and apigenin..

Marniemi et al.12 2005 Quercetin, kaempferol, myricetin, luteolin, and apigenin..

van der Schouw et al.13

2005

Daidzein, Genistein, Formononetin, Biochanin A.

Mink et al.14 2007




Mursu et al.15 2008

Flavonols, flavones, flavanones, flavan-3-ols and

anthocyanidins.

Cassidy et al.16 2012


isorhamnetin), 2 flavones (apigenin and luteolin), 6

anthocyanins (cyaniding, delphinindin, malvidin, pelargonidin,

petunidin, peonidin), 2 flavan-3-ols (catechins, epicatachins), 3

polymers (proanthocyanidins, theaflavins, thearubigins), and 3


Talaei et al.17 2014 Genistein, daidzein, and glycitein.

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MOOSE Checklist






1







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the meta-analysis


include










especially dietary flavonoids intake, have aroused






√ Hypothesis statement Flavonoids intake decrease risk of stroke.



intervention used

Flavonoids







should include






PubMed from 1965 –February 2015

Embase from 1974 –February 2015

Cochrane library from 1990- February 2015



“myricetin,” “isorhamnetin,” “apigenin,” “luteolin,” and

“stroke,” “cerebrovascular disease,” “cerebrovascular

disorders,” “cerebral infarct,” “ischemic stroke,”

“intracranial hemorrhage,” “intracranial artery disease,”



heart disease,”) AND (“longitudinal studies,” “cohort



searched


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features







justifications






than English







on the association.


authors




flavonoids.


include








coding of data




























be replicated



assessment of publication bias are detailed in the

methods.

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and graphics


figures.


include



estimate

Figure 2, 3 and 4



included

Table 1 and Supplemental table 1

√ Results of sensitivity testing

Table 2





analyses


include



studies.




comparison groups.


included studies




include



results









conclusions

Our meta-analysis suggests that dietary flavonoids intake





mg/day increment of flavonoids intake.



the risk of stroke.



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response


Journal: BMJ Open



Date Submitted by the Author: 08-Feb-2016









BMJ Open on O


http://bmjopen.bm

j.com/

BM


jopen-2015-008680 on 8 June 2016. Dow

nloaded from



Dietary flavonoid intake and the risk of stroke: A dose-response



a,1, Xiaowei Zhang


a





[email protected]


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1

Abstract


risk of stroke.

Design Meta-analysis of prospective cohort studies.







the highest compared with the lowest dietary flavonoid intake was 0.90 (95% confidence interval,




of stroke for flavonoid intake was 0.89 (95% confidence interval, 0.81-0.99) in longer than 10


Conclusions Results from this meta-analysis suggest that higher dietary flavonoid intake may


Article summary

Strengths of this study

1. This is the largest meta-analysis on flavonoid intake and the risk of stroke.

2. Higher dietary flavonoid intake is associated with a significantly reduced risk of stroke.


flavonoids.

Limitations of this study

4. The possibility of residual confounding or confounding by unmeasured factors, which cannot be

ruled out in any observational study, must be acknowledged.

5. We cannot exclude the possibility of recall bias in the assessments of diet based on the food

frequency questionnaires.

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2

Introduction









Additionally,








the role

of flavonoid intake on stroke prevention is still controversial. In addition, flavonoid intake differed

substantially between studies, which makes it difficult to interpret the summary estimate based on

results from study populations with different flavonoid intakes.19



of stroke.

Methods

Literature search












reviewed the reference lists of obtained articles and contacted authors to identify additional

relevant studies and information. When the same or similar patient cohort was included in several

publications, only the most recent or complete report was selected for analysis.

Study Selection








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Data Abstraction









The quality of






The relative risks (RRs) were used as the common measure of association between flavonoid

intake and stroke, and the hazard ratios (HRs) were considered equivalent to RRs. Data analysis

used multivariate-adjusted outcome data. We converted these values in every study by using their








and Orsini et al25



median/mean of flavonoid intake level for each comparison group. We assigned the midpoint of

the upper and lower boundaries of each comparison group to determine mean flavonoid intake








component is equal to zero. When the result on total flavonoid intake in this dose-response

meta-analysis was not available, we used data from flavonols, flavones, flavanones, flavan-3-ols,

and isoflavones (in the sequential order) as an equivalent to total flavonoid. Heterogeneity among

studies was evaluated using the chi-square test based on Cochran’s Q test and I2 statistic at p＜

0.10 level of significance,23

which describes the percentage of variability in the effect estimates

that is due to heterogeneity rather than chance.27

We regard I2 of ＜40% as “heterogeneity might

not be important” and ＞75% as “considerable heterogeneity” based on the suggestion of

Cochrane Handbook for Systemic Review of Interventions.28

All available data was conducted in

the primary analysis. Subsequent subgroup analyses were conducted according to stroke outcomes

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4

(fatal/nonfatal versus ischemic), follow-up duration (≤10 years versus ＞10 years), sex (male

versus female versus combined), Geographical area (United States versus Asian versus European),

history of stroke (yes versus no), and study quality (high [score＞8] versus low [score ≤8]). To

explore possible explanations for heterogeneity and to test the robustness of the association, we

conducted meta-regression, sensitivity analyses and above mentioned stratified analysis. The

possibility of publication bias was evaluated using the Egger rank correlation test at p＜0.10 level

of significance and visual inspection of a funnel plot.29,30

In the case of publication bias,

“nonparametric trim-and-fill” method was used to compute risk estimates corrected for this bias.31

All the statistical analyses were performed in STATA 12.0 (StataCorp, College Station, TX). p

values were 2-sided and p＜0.05 was considered statistically significant.

Results

Literature Search








Supplemental tables 1 and 2. A total of 10 studies involving 258,158 participants and more than

4,581 stroke cases were included in the meta-analysis.8-17

Among 10 studies, 3 were conducted

primarily in the United States,11,14,16


and 6 studies were

from European countries (including Finland, Netherlands).8-10,12,13,15

The number of participants

ranged from 755 in the study by Marniemi et al12


3

studies included both men and women,10,12,17


and 4 studies only

women.11,13,14,16



years. The dietary assessment of flavonoid intake varied across studies, in most of the studies,








showed that flavonoid intake was associated with decreased risk of stroke.10


with the highest dietary flavonoid intake, compared with the lowest, experienced a significant



= 0%).

Among 10 studies, 4 studies were eligible for the dose-response analysis of flavonoid intake and




flavonoid intake (relative risk 0.94, 95% confidence interval 0.83 to 1.06, I2 = 0%, p = 0.313;

Figure 3 and 4). No publication bias was observed (p = 0.962).

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In most subgroups, dietary flavonoid intake was not associated with stroke. Stratifying by sex, the

associations between dietary flavonoid intake and risk of stroke were similar between males and

females (table 2). The pooled RR of stroke were 0.88 (95% CI: 0.75-1.03) from studies conducted

in the United States, 0.88 (95% CI: 0.77-1.00) in European, and 0.97 (95% CI: 0.81-1.16) in Asian

(table 2). Stratifying by duration, we observed the protective effect of dietary flavonoid intake in

longer than 10 years of follow-up studies. The associations were more evident in several strata of

study characteristics (table 2).







0.222).

Meta Regression





Discussion

Our meta-analysis of 10 prospective studies indicates that dietary flavonoid intake may be

inversely associated with risk of stroke. In addition, dose-response analysis indicated that a

100-mg/day increment of flavonoids intake was associated with a 6% lower risk of stroke (RR

0.94, 95% CI 0.83 to 1.06). However, this association was not statistical significance.





humans.32







36



the effect of individual compounds or interactions between flavonoids is still largely unknown,


Although, the exact mechanism for the beneficial effect by which flavonoid intake may protect

against the development of stroke is still ambiguous, a possible mechanism is the amelioration of

insulin resistance, especially by quercetin, due to inhibition of intestinal glucose transporters.38

Moreover, flavonoids reduced mitochondrial lipid peroxidation and loss of mitochondrial

transmembrane electric potential caused by oxidative stress induced by ADP plus iron, mainly



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bioactivity, the rationale for assuming that intakes of all flavonoid classes might have effects on



Over the past decades, despite extensive studies that investigated the role of flavonoid intake on


association between flavonoid intake and risk of stroke is causal. One study suggested flavonoids

are associated with increased risk of stroke,10


More,

importantly, the possibility of reverse causality should be addressed. Previous meta-analysis of


incidence.34






Health Study, this result was consistent with a detailed overview by Vogiatzoglou et al,41

whereas

no associations were noted in the Iowa Women’s Health Study and Alpha-Tocopherol,

Beta-Carotene Cancer Prevention Study. The results for flavonol were consistent with our findings

on flavonoids. But the data from studies included by previous meta-analysis were limited to

August 2009.34

Interestingly, since then new studies involving relationship between flavonoids

intake and risk of stroke were published.16,17

To obtain a more comprehensive estimate of the

putative influence of the flavonoids on stroke, we conducted a meta-analysis of prospective cohort

studies. To our knowledge, this meta-analysis is the largest to reveal the potential relationship

between dietary flavonoid intake and risk of stroke. However, most studies assess dietary

flavonoid intake based on self-reported questionnaires using FFQs, and medical records were not

always available for stroke classification, the possibility that misclassification of flavonoid intake

and stroke was inevitable and likely to bias true association among individual studies.


We has several important strengths compared with the previous meta-analyses.18,34

The present

meta-analysis included 2 times more participants and 2 times more stroke cases. To our knowledge,

this is the largest meta-analysis on flavonoid intake and the risk of stroke. We also explored

possible source of heterogeneity using subgroup analyses and the meta-regression method, all of

them were not significant. Moreover, the presence of a dose-response relationship further

strengthened the association of dietary flavonoid intake with risk of stroke. Therefore, the results

should be more reliable.




strength of association between flavonoid intake and stroke risk. Additionally, all studies did not

include complete data on total flavonoid intake in this dose-response analysis, the intake of

flavonoid may have been underestimated in these original studies. Thus, these findings should be

treated with caution. Second, we cannot exclude the possibility of recall bias in the assessments of

diet based on the FFQs. However, the prospective study design and exclusion of participants with

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chronic diseases at baseline should minimize such bias. Third, the noticeable limitation of our




Fourth, we tried to study the main sources of flavonoids. But, we failed it because of the

insufficient data. Different sub-classes for flavonoid were used in the primary studies included in

this meta-analysis. Among 10 studies, 4 studies included the same sub-classes (including quercetin,

kaempherol, myricetin, luteolin, and apigenin),8,9,11,12

another 4 studies included the similar

sub-classes (including flavonols, flavones, flavanones, flavan-3-ols, and so on),10,14-16

and 2

studies included the similar compounds (including daidzein, genistein, gormononetin, biochanin A,

and glycitein),13,17

as summarized in supplemental table 2. Thus, we evaluated the effects of total

flavonoid intake on stroke risk rather than the wide-range of flavonoid sub-classes. On the one

hand, in order to balance the risk of a type I error (the true effect is zero but we reject the null) and

a type II error (the true effect is not zero but we fail to reject the null), we combined these

estimates using a random-effects model, which takes into account both within-study and

between-study variabilities.23

Thus, these results should be treated with caution. Finally, the

possible limitation is due to language bias. We attempted to minimize this bias by searching major

electronic databases with no language restriction. However, several articles published in

non-English or unpublished reports might not appear in international journal databases, and could

be omitted by our searches.42

Conclusions

In summary, results from this meta-analysis suggest that higher dietary flavonoid intake may


Randomized controlled studies are needed to evaluate the effects of flavonoid intake on stroke

risk.



search strategies. XZ and WH extracted quantitative data. XZ, and WH analyzed the data. ML

wrote the draft of the paper. All authors contributed to writing, reviewing, or revising the paper.

ZT is the guarantor.









Figures Information


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8

(TIFF)



(TIFF)

Figure 3. Dose-response relationship between dietary flavonoids intake and stroke risk. The

solid line represents point estimates of the association between flavonoid intake and stroke

risk, and the dotted lines are 95% CIs. The horizontal line is the reference line.

(TIFF)

Figure 4. Forest plot of flavonoid and risk of stroke.

(TIFF)



(DOC)



(DOC)


(DOC)

REFERENCES


















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9


Nutr 2001;74:227-32.


J Clin Nutr 2002;76:560-8.





2005;15:188-97.



2005;111:465-71.





Study. Br J Nutr 2008;100:890-5.


Stroke 2012;43:946-51.






















2003;327:557-60.




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10







Nutr Rev 1998;56:317-33.




and women. J Nutr 2010;140:600-4.












2010;31:478-94.

41. Vogiatzoglou A, Mulligan AA, Lentjes MA, et al. Flavonoid intake in European adults (18 to

64 years). PLoS One 2015;10:e0128132.



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First author,


No. Participants

(% male)

Age range or

mean (yr)

Follow-Up

Duration (yr)

Assessment of

dietary data

Stroke

Ascertainment

No. of stroke

cases

Pre-stroke

Excluded

Study

Quality

Hirvonen et al.8

2000

Finland/European

26497 (100)

50-69

6.1



method

Any type of stroke

based on ICD-8, 9

codes

Ischemic: 736

Yes

8

Arts et al.9 2001


806 (100)

65-84

15

A cross-check

dietary history

method

Any type of stroke

based on ICD-9

codes

Fatal: 47,

Nonfatal: 88

No

7

Knekt et al.10

2002

Finland/European

9131 (NA)

30-69

28

A dietary history

interview

Any type of stroke

based on ICD-8

codes

Fatal or

nonfatal: 681

No

8

Sesso et al.11

2003

United States

38445 (0)

53.9

6.9

A food-frequency

questionnaire

Any type of stroke

based on clinical

diagnosis

Fatal or

nonfatal: NA

Yes

9

Marniemi et al.12

2005

Finland/European

755 (47.8)

65-99

10

A dietary history

interview

Any type of stroke

based on ICD-9

codes

Fatal: 45,

Nonfatal: 25

Yes

8

van der Schouw et

al.13

2005


16165 (0)

49-70

6.3

A validated

food-frequency

questionnaire

Any type of stroke

based on ICD-9

codes

Fatal or

nonfatal: 147

Yes

9

Mink et al.14

2007

United States

34489 (0)

55-69

16

A food-frequency

questionnaire

Any type of stroke

based on ICD-9

codes

Fatal: 469

Yes

9

Mursu et al.15

2008

Finland/European

1950 (100)

42-60

15.2

An instructed 4d

food recording by

Any type of stroke

based on ICD-9, 10

Ischemic: 102

Yes

9

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1


Cassidy et al.16

2012

United States

69622 (0)

30-55

14

A semiquantitative

food-frequency

questionnaires

interview

Any type of stroke

based on medical

records, autopsy

reports, and death

certificates

Ischemic: 943

Yes

8

Talaei et al.17

2014

Singapore/Asian

60298 (44.5)

45-74

14.7

A semiquantitative

food-frequency

questionnaires

interview

Any type of stroke

based on ICD-9

codes

Fatal: 1298

No

8


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2




2, %

Overall studies


Ischemic stroke 3 0.93 (0.80-1.07) 1.03 0.609 0 0.301

Geographical area

United States 3 0.88 (0.75-1.03) 1.59 0.511 0 0.112

Asian 1 0.97 (0.81-1.16) 0.33 - - 0.740

European 6 0.88 (0.77-1.00) 1.95 0.577 0 0.051

History of stroke

Yes 3 0.89 (0.78-1.02) 1.66 0.683 0 0.096

No 7 0.90 (0.80-1.02) 1.62 0.351 4.6 0.105

Sex

Male 3 0.95 (0.79-1.14) 0.55 0.651 0 0.580

Female 4 0.89 (0.77-1.04) 1.45 0.613 0 0.114

Combined 3 0.86 (0.72-1.04) 1.58 0.227 32.6 0.148


≤10 4 0.90 (0.74-1.09) 1.10 0.340 10.7 0.271

>10 6 0.89 (0.81-0.99) 2.09 0.750 0 0.036

Quality score

High, score＞8 4 0.87 (0.70-1.07) 1.34 0.534 0 0.181

Low, score≤8 6 0.91 (0.82-1.00) 1.97 0.589 0 0.048


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152x101mm (300 x 300 DPI)

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sta

nd

ard

ize

d e

ffect

precision0 5 10

-2

-1

0

1


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stroke




equal to

ischemic stroke

results

0.98 (0.80-1.21)

Arts et al.9 2001




0.92

(0.51-1.68)

Knekt et al.10 2002



0.79

(0.64-0.98)

Sesso et al.11 2003




0.70

(0.46-1.07)

Marniemi et al.12

2005


energy intake.

0.65

(0.34-1.23)

van der Schouw et

al.13 2005




menopausal status.

1.05

(0.64-1.70)

Mink et al.14 2007



equal to fatal

stroke results

0.94 (0.69-1.29)

Mursu et al.15 2008




equal to

ischemic stroke

results

0.71 (0.37-1.37)

Cassidy et al.16 2012 Age, physical activity, smoking, HRT, BMI, aspirin use, equal to 0.90 (0.73-1.11)

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hypertension

ischemic stroke

results

Talaei et al.17 2014




equal to fatal

stroke results

0.97 (0.81-1.16)



contraceptives; WHR: waist-to-hip ratio; HM: hypertension medication; MI: myocardial infarction.

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Reference Adjusted covariates



Knekt et al.10 2002







2005


Mink et al.14 2007


isorhamnetin), 2 flavones (apigenin and luteolin), 3 flavanones

(hesperetin, naringenin, and eriodictyol).

Mursu et al.15 2008


anthocyanidins.





petunidin, peonidin), 2 flavan-3-ols (catechins, epicatechins), 3

polymers (proanthocyanidins, theaflavins, thearubigins), and 3



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MOOSE Checklist






1







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the meta-analysis


include










especially dietary flavonoid intake, have aroused






√ Hypothesis statement Flavonoid intake decreases risk of stroke.



intervention used

Flavonoids







should include






PubMed from 1965 –January 2016

Embase from 1974 –January 2016

Cochrane library from 1990- January 2016



“myricetin,” “isorhamnetin,” “apigenin,” “luteolin,”

“proanthocyanidins,” “anthocyanins,” “anthocyanidins,”

“flavan-3-ols,” “isoflavones,” and “stroke,”

“cerebrovascular disease,” “cerebrovascular disorders,”

“cerebral infarct,” “ischemic stroke,” “intracranial

hemorrhage,” “intracranial artery disease,”



heart disease,” and “longitudinal studies,” “cohort


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searched




features







justifications






than English







on the association.


authors




flavonoids.


include








coding of data






























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be replicated assessment of publication bias are detailed in the

methods.


and graphics


figures.


include



estimate

Figure 2, 3 and 4



included

Table 1 and Supplemental tables 1 and 2

√ Results of sensitivity testing Table 2





analyses


include



studies.




comparison groups.


included studies




include



results









conclusions

Our meta-analysis suggests that dietary flavonoid intake





mg/day increment of flavonoid intake.



the risk of stroke.



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response


Journal: BMJ Open



Date Submitted by the Author: 29-Mar-2016









BMJ Open on O


http://bmjopen.bm

j.com/

BM


jopen-2015-008680 on 8 June 2016. Dow

nloaded from






a,1, Xiaowei Zhang


a





[email protected]


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1

Abstract


risk of stroke.


Data source Studies published before January 2016 identified through electronic searches using














Article summary





flavonoids.






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2

Introduction







factors, especially dietary flavonoid intake, have aroused particular attention. Clinical studies have

shown that intakes of flavonoids reduce cardiovascular disease (CVD) risk.3-5

Additionally,




flavonoid intake and risk of stroke.8-18




the role





association between flavonoid exposure, including cohort studies of dietary flavonoids, with risk

of stroke.

Methods

Literature search






“flavones,” “quercetin,” “kaempferol,” “myricetin,” “isorhamnetin,” “apigenin,” “luteolin,”

“proanthocyanidins,” “anthocyanins,” “anthocyanidins,” “flavan-3-ols,” “isoflavones,” and








publications, only the most recent or complete report was selected for analysis. This search

strategy was updated on January 14, 2016.

Study Selection




flavanones, flavan-3-ols, anthocyanidins, proanthocyanidins, and isoflavones); (3) the outcome of

interest was stroke, including all types of stroke (fatal, nonfatal, ischemic, and hemorrhagic); (4)

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3

reported quantitative estimates of the multivariate-adjusted relative risk (RR) and 95% confidence

interval (CI) for stroke incidence or mortality associated with flavonoids intake; and (5) longer

than 1 year of follow-up. Studies were excluded if (1) the study design was cross-sectional,

case-control; (2) reported unadjusted or only age- or sex-adjusted RR; and (3) shorter than 1 year

of follow-up.

Data Abstraction









The quality of






The relative risks (RRs) were used as the common measure of association between flavonoid

intake and stroke, and the hazard ratios (HRs) were considered equivalent to RRs. Data analysis

used multivariate-adjusted outcome data. We converted these values in every study by using their


corresponding 95% CIs. When data on total stroke was not available, we used data from ischemic

stroke, nonfatal stroke, or fatal stroke as an equivalent to total stroke.23

We combined these



In the dose-response analysis, the generalized least square for trend

estimation method described by Greenland and Longnecker25

and Orsini et al26

was used to

calculate study-specific slopes (linear trends) and 95% confidence intervals. The method requires

the distributions of cases and person years for exposure categories, and median/mean of flavonoid

intake level for each comparison group. We assigned the midpoint of the upper and lower

boundaries of each comparison group to determine mean flavonoid intake level if the median or

mean intake was not provided. If the lower or upper boundary for the lowest and highest category,

respectively, was not reported, we assumed that the boundary had the same amplitude as the

closest category.20

Additionally, we first created restricted cubic splines with 3 knots at fixed

percentiles 25%, 50%, and 75% of the distribution.27

A p value for nonlinearity was calculated by

testing the null hypothesis that the coefficient of the fractional polynomials component is equal to

zero. When the result on total flavonoid intake in this dose-response meta-analysis was not

available, we used data from flavonols, flavones, flavanones, flavan-3-ols, and isoflavones (in the

sequential order) as an equivalent to total flavonoid. Heterogeneity among studies was evaluated

using the chi-square test based on Cochran’s Q test and I2 statistic at p＜0.10 level of

significance,24

which describes the percentage of variability in the effect estimates that is due to

heterogeneity rather than chance.28

We regard I2 of ＜40% as “heterogeneity might not be

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important” and ＞75% as “considerable heterogeneity” based on the suggestion of Cochrane

Handbook for Systemic Review of Interventions.29

All available data was utilized in the primary

analysis. Subsequent subgroup analyses were conducted according to stroke outcomes












Results

Literature Search




17 studies for the following reasons: no stroke outcomes (n=8), duplicate reports (n=4) and

reviews (n=5). Finally, 11 studies met the inclusion criteria and were included in the

meta-analysis.8-18





Among 11 studies, 4 were conducted in

the United States,11,14,16,17






to 98,469 in the study by McCullough et al.17

4 studies

included both men and women,10,12,17,18


and 4 studies only

women.11,13,14,16


with a median of 14 years.

The dietary assessment of flavonoid intake varied across studies, in most of the studies, intake of

flavonoids was measured by food frequency questionnaires (FFQs) and dietary history interview.

In most of the studies, stroke was assessed by medical records or death certificates based on

ICD-8,9,10. All studies provided adjusted risk estimates (e.g., sex, body mass index, smoking,

education, et al), overall quality scores ranged for 7 to 9, and the median score was 8.




showed that flavonoid intake was significantly associated with decreased risk of stroke.10

Overall,

participants with the highest dietary flavonoid intake, compared with the lowest, experienced a

significant decreased risk for development of stroke (combined RR, 0.89; 95% CI, 0.82-0.97;

Figure 2) after adjustment for other risk factors. We saw no heterogeneity among studies (p =

0.774, I2

= 0%). Among 11 studies, 4 studies were eligible for the dose-response analysis of

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flavonoid intake and risk of stroke.9,14,16,18

Using a restricted cubic splines model, dose-response

analysis found a statistically nonsignificant inverse association with risk of stroke per 100-mg/day

increment of flavonoid intake (relative risk 0.94, 95% confidence interval 0.83 to 1.06, I2 = 0%, p

= 0.313; Figure 3 and 4). No heterogeneity was observed (p = 0.962).















0.238).

Meta Regression




geographical area, history of stroke, and study quality were no significant sources of

heterogeneity.

Discussion




0.94, 95% CI 0.83 to 1.06). However, this association was not statistically significant.




non-energetic, non-nutrient secondary metabolites present in plants and cannot be synthesized by

humans.33

In recent decades, overwhelming evidence indicates that intake of flavonoid was

associated with well-known risk-factors for CVD. There is also some evidence that flavonoids

may have a role in preventing the development of CVD (e.g., coronary heart disease, stroke, et

al).34,35



including anti-inflammatory,37

antioxidant,6 recovery of

endothelial function properties of some of the compounds, and reductions in platelet

aggregation,38

and the effect of individual compounds or interactions between flavonoids is still

largely unknown, flavonol and isoflavone intake may explain some of this beneficial effect.19,39

Although, the exact mechanism for the beneficial effect by which flavonoid intake may protect

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against the development of stroke is still ambiguous, a possible mechanism is the regulation of

blood pressure, especially by anthocyanins, due to increase endothelial-derived nitric oxide

(NO).40

Moreover, flavonoids reduced mitochondrial lipid peroxidation and loss of mitochondrial

transmembrane electric potential caused by oxidative stress induced by ADP plus iron, mainly


A third mechanism is the protective action of flavonoids on the

anti-inflammatory responses in the brain, that may be attributable to raft disrupting and



bioactivity, the rationale for assuming that intakes of all flavonoid classes might have effects on








More,

importantly, the possibility of reverse causality should be addressed. A previous meta-analysis of


incidence.35

That meta-analysis included 6 prospective cohorts studies from 3 different countries,

4 were conducted in Europe (3 in Finland and 1 in The Netherlands) and 2 in the United States.

The inverse associations between flavonol intake and stroke were observed in two prospective

cohort studies (Zutphen Elderly Study and Kuopio Study). Weak and nonsignificant inverse

associations were found in the Finnish Mobile Clinic Study and Women’s Health Study, this result

was consistent with a detailed overview by Vogiatzoglou et al,43

whereas no associations were

noted in the Iowa Women’s Health Study and Alpha-Tocopherol, Beta-Carotene Cancer

Prevention Study. The results for flavonol were consistent with our findings on flavonoids. But the

data from studies included by previous meta-analysis were limited to August 2009.35

Interestingly,

since then 3 new studies involving relationship between flavonoids intake and risk of stroke were

published.16-18

To obtain a more comprehensive estimate of the putative influence of the

flavonoids on stroke, we conducted a meta-analysis of prospective cohort studies. To our

knowledge, this meta-analysis is the largest to reveal a potential relationship between dietary

flavonoid intake and risk of stroke. However, most studies assess dietary flavonoid intake based

on self-reported questionnaires using FFQs, and medical records were not always available for

stroke classification; the possibility that misclassification of flavonoid intake and stroke was

inevitable and likely to bias true association among individual studies.


We have several important strengths compared with previous meta-analyses.19,35

The present



possible sources of heterogeneity using subgroup analyses and the meta-regression method, all of







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strength of association between flavonoid intake and stroke risk. Additionally, all the studies used

in this dose-response analysis did not include complete data on total flavonoid intake; the

flavonoid intake may have been underestimated in these original studies. Thus, these findings

should be treated with caution. Second, we cannot exclude the possibility of recall bias in the

dietary assessment based on the FFQs. However, the prospective study design and exclusion of

participants with chronic diseases at baseline should minimize such bias. Third, a noticeable

limitation of our study was the potential for bias due to inevitable measurement error and

misclassification, especially for individuals with lower consumption levels. We attempted to

reduce measurement error by adjusting for energy or fiber intake and using cumulatively averaged

intake levels. Fourth, we tried to study the main sources of flavonoids. But, we failed because of



kaempherol, myricetin, luteolin, and apigenin),8,9,11,12



and 2

studies included the similar compounds (including daidzein, genistein, gormononetin, biochanin A,


as summarized in supplemental table 2. Among 11 studies, only 4 studies

were eligible for the dose-response analysis, and the compounds of flavonoid

subclasses are different among these studies. Of these, 2 studies included flavanones, 2

studies included flavones, 2 studies included flavonols, 2 studies included flavan-3-ols, and 2

studies included isoflavones. Due to the limited number of studies that met dose-response analyses

and the insufficient statistical power, we did not conduct dose-response analysis separately on

each class. Thus, we evaluated the effects of total flavonoid intake on stroke risk rather than the

wide-range of flavonoid sub-classes. On the one hand, in order to balance the risk of a type I error

(the true effect is zero but we reject the null) and a type II error (the true effect is not zero but we

fail to reject the null), we combined these estimates using a random-effects model, which takes

into account both within-study and between-study variabilities.24

Thus, these results should be

treated with caution. Finally, a possible limitation is due to language bias. We attempted to

minimize this bias by searching major electronic databases with no language restriction. However,

several articles published in non-English or unpublished reports might not appear in international

journal databases, and could be omitted by our searches.44

Conclusions




risk.








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8







Figures Information


(TIFF)



(TIFF)




(TIFF)

Figure 4. Forest plot of flavonoid and risk of stroke. 4 cohort studies were eligible for the

dose-response analysis of flavonoid intake and risk of stroke. Relative risk of less than 1.0

favours exposure to greater intake of flavonoid. CI, Confidence interval.

(TIFF)



(DOC)



(DOC)


(DOC)

REFERENCES

1. Feigin VL, Forouzanfar MH, Krishnamurthi R, et al. Global and regional burden of stroke

during 1990-2010: findings from the Global Burden of Disease Study 2010. Lancet

2014;383:245-54.

2. Mozaffarian D, Benjamin EJ, Go AS, et al. Heart Disease and Stroke Statistics-2016 Update:

A Report From the American Heart Association. Circulation 2015.


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9














Nutr 2001;74:227-32.


J Clin Nutr 2002;76:560-8.





2005;15:188-97.



2005;111:465-71.





Study. Br J Nutr 2008;100:890-5.


Stroke 2012;43:946-51.

17. McCullough ML, Peterson JJ, Patel R, et al. Flavonoid intake and cardiovascular disease

mortality in a prospective cohort of US adults. Am J Clin Nutr 2012;95:454-64.












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10











2003;327:557-60.










Nutr Rev 1998;56:317-33.




and women. J Nutr 2010;140:600-4.







39. Sagara M, Kanda T, NJelekera M, et al. Effects of dietary intake of soy protein and

isoflavones on cardiovascular disease risk factors in high risk, middle-aged men in Scotland. J

Am Coll Nutr 2004;23:85-91.

40. Steffen Y, Gruber C, Schewe T, et al. Mono-O-methylated flavanols and other flavonoids as

inhibitors of endothelial NADPH oxidase. Arch Biochem Biophys 2008;469:209-19.




2010;31:478-94.


64 years). PLoS One 2015;10:e0128132.



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First author,


No. Participants

(% male)

Age range or

mean (yr)

Follow-Up

Duration (yr)

Assessment of

dietary data

Stroke

Ascertainment

No. of stroke

cases

Pre-stroke

Excluded

Study

Quality

Hirvonen et al.8

2000

Finland/European

26497 (100)

50-69

6.1



method

Any type of stroke

based on ICD-8, 9

codes

Ischemic: 736

Yes

8

Arts et al.9 2001


806 (100)

65-84

15

A cross-check

dietary history

method

Any type of stroke

based on ICD-9

codes

Fatal: 47,

Nonfatal: 88

No

7

Knekt et al.10

2002

Finland/European

9131 (NA)

30-69

28

A dietary history

interview

Any type of stroke

based on ICD-8

codes

Fatal or

nonfatal: 681

No

8

Sesso et al.11

2003

United States

38445 (0)

53.9

6.9

A food-frequency

questionnaire

Any type of stroke

based on clinical

diagnosis

Fatal or

nonfatal: NA

Yes

9

Marniemi et al.12

2005

Finland/European

755 (47.8)

65-99

10

A dietary history

interview

Any type of stroke

based on ICD-9

codes

Fatal: 45,

Nonfatal: 25

Yes

8

van der Schouw et

al.13

2005


16165 (0)

49-70

6.3

A validated

food-frequency

questionnaire

Any type of stroke

based on ICD-9

codes

Fatal or

nonfatal: 147

Yes

9

Mink et al.14

2007

United States

34489 (0)

55-69

16

A food-frequency

questionnaire

Any type of stroke

based on ICD-9

codes

Fatal: 469

Yes

9

Mursu et al.15

2008

Finland/European

1950 (100)

42-60

15.2

An instructed 4d

food recording by

Any type of stroke

based on ICD-9, 10

Ischemic: 102

Yes

9

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1


Cassidy et al.16

2012

United States

69622 (0)

30-55

14

A semiquantitative

food-frequency

questionnaires

interview

Any type of stroke

based on medical

records, autopsy

reports, and death

certificates

Ischemic: 943

Yes

8

McCullough et

al.17

2012

United States

98469 (38.8)

70

7

A semiquantitative

food-frequency

questionnaires

interview

Any type of stroke

based on ICD-9, 10

codes

Fatal: 573

Yes

9

Talaei et al.18

2014

Singapore/Asian

60298 (44.5)

45-74

14.7

A semiquantitative

food-frequency

questionnaires

interview

Any type of stroke

based on ICD-9

codes

Fatal: 1298

No

8


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2




2, %

Overall studies


Ischemic stroke 3 0.93 (0.80-1.07) 1.03 0.609 0 0.301

Geographical area

United States 4 0.86 (0.76-0.98) 1.50 0.683 0 0.026

Asian 1 0.97 (0.81-1.16) 0 - - 0.740

European 6 0.88 (0.77-1.00) 3.81 0.577 0 0.051

History of stroke

Yes 3 0.89 (0.78-1.02) 2.10 0.351 4.6 0.105

No 8 0.89 (0.80-0.99) 4.38 0.735 0 0.027

Sex

Male 4 0.82 (0.64-1.06) 4.90 0.179 38.8 0.130

Female 5 0.92 (0.80-1.05) 2.39 0.664 0 0.216

Combined 3 0.86 (0.72-1.04) 2.97 0.227 32.6 0.114


≤10 5 0.88 (0.77-1.01) 3.78 0.437 0 0.067

>10 6 0.89 (0.81-0.99) 2.68 0.750 0 0.036

Quality score

High, score＞8 5 0.85 (0.73-0.99) 2.26 0.688 0 0.038

Low, score≤8 6 0.91 (0.82-1.00) 3.73 0.589 0 0.048


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Process of literature search and study selection.

152x101mm (300 x 300 DPI)

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Random effects analysis of fully adjusted studies for highest versus lowest intake of flavonoids and risk of stroke.

152x101mm (300 x 300 DPI)

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Dose-response relationship between dietary flavonoids intake and stroke risk. The solid line represents point

estimates of the association between flavonoid intake and stroke risk, and the dotted lines are 95% CIs. The

horizontal line is the reference line.

152x101mm (300 x 300 DPI)

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Forest plot of flavonoid and risk of stroke. 4 cohort studies were eligible for the dose-response analysis of flavonoid intake and risk of stroke. Relative risk of less than 1.0 favours exposure to greater intake of

flavonoid. CI, Confidence interval. 152x101mm (300 x 300 DPI)

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152x101mm (300 x 300 DPI)

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stroke




equal to

ischemic stroke

results

0.98 (0.80-1.21)

Arts et al.9 2001




0.92

(0.51-1.68)

Knekt et al.10 2002



0.79

(0.64-0.98)

Sesso et al.11 2003




0.70

(0.46-1.07)

Marniemi et al.12

2005


energy intake.

0.65

(0.34-1.23)

van der Schouw et

al.13 2005




menopausal status.

1.05

(0.64-1.70)

Mink et al.14 2007



equal to fatal

stroke results

0.94 (0.69-1.29)

Mursu et al.15 2008




equal to

ischemic stroke

results

0.71 (0.37-1.37)


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hypertension

ischemic stroke

results

McCullough et al.17

2012

Age, smoking, beer and liquor intake, history of hypertension,

history of cholesterol, family history of MI, BMI, physical

activity, energy intake, aspirin use, HRT, and sex

equal to fatal

stroke results

0.83 (0.66-1.04)

Talaei et al.18 2014




equal to fatal

stroke results

0.97 (0.81-1.16)



contraceptives; WHR: waist-to-hip ratio; HM: hypertension medication; MI: myocardial infarction.

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Reference Flavonoid subclasses



Knekt et al.10 2002







2005


Mink et al.14 2007


isorhamnetin), 2 flavones (apigenin and luteolin), 3 flavanones

(hesperetin, naringenin, and eriodictyol), flavan-3-ols (apples,

red wine, green tea, black tea) and anthocyanidin (bluederries,

raspberries, red wine).

Mursu et al.15 2008


anthocyanidins.





petunidin, peonidin), flavan-3-ols (catechins, epicatechins), 3

polymer classes (proanthocyanidins, theaflavins, thearubigins),

and 3 flavanones (hesperetin, naringenin, and eriodictyol).

McCullough et al.17 2012

Anthocyanidin, flavan-3-ols, flavanones, flavones, flavonols,

proanthocyanidins, isoflavones.


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1

MOOSE Checklist






1







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2


the meta-analysis


include



















intervention used

Flavonoids







should include





















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3


searched




features







justifications






than English







on the association.


authors




flavonoids.


include








coding of data






























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4


methods.


and graphics


figures.


include



estimate

Figure 2, 3 and 4



included







analyses


include



studies.




comparison groups.


included studies




include



results









conclusions









the risk of stroke.



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5

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response


Journal: BMJ Open



Date Submitted by the Author: 23-Apr-2016







Keywords: STROKE MEDICINE, NEUROLOGY, PUBLIC HEALTH


BMJ Open on O


http://bmjopen.bm

j.com/

BM


jopen-2015-008680 on 8 June 2016. Dow

nloaded from






a,1, Xiaowei Zhang


a





[email protected]


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1

Abstract


risk of stroke.
















Article summary





flavonoids.






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2

Introduction





the premature incidence and mortality associated with stroke, and there is growing interest in

altering risk factors and reversing this global epidemic. Among the known risk factors for stroke,

dietary factors, especially dietary flavonoid intake, have aroused particular attention. Clinical

studies have shown that intakes of flavonoids reduce cardiovascular disease (CVD) risk.3-5

Additionally, experimental studies indicated that flavonoids have been shown to have both

antioxidant and antithrombotic properties.6,7






the role






of stroke.

Methods

Literature search







“proanthocyanidins,” “anthocyanins,” “anthocyanidins,” “flavan-3-ols,” “isoflavones,” and








publications, only the most recent or complete report was selected for analysis. This search

strategy was updated on January 14, 2016.

Study Selection






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3



than 1 year of follow-up. Studies were excluded if (1) the study design was cross-sectional; or


of follow-up.

Data Abstraction









The quality of




was 9 stars. Studies were graded as the high-quality if they were awarded ＞8 stars.


Relative risks (RRs) were used as the common measure of association between flavonoid intake

and stroke, and hazard ratios (HRs) were considered equivalent to RRs. Data analysis used





We combined these





and Orsini et al26

was used to







closest category.20





zero. When data on total flavonoid intake included in this dose-response meta-analysis was not




significance,24



We regard I2 of ＜40% as “heterogeneity that might not be

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4
















Results

Literature Search






meta-analysis.8-18













4 studies



and 4 studies only

women.11,13,14,16






ICD-8,9,10.33-35

All studies provided adjusted risk estimates (e.g., sex, body mass index, smoking,






Overall,




0.774, I2


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5

flavonoid intake and risk of stroke.9,14,16



















0.238).

Meta Regression





heterogeneity.

Discussion






Flavonoids, a large class of polyphenols, are widely distributed in plants and are present in

considerable amounts in fruits, vegetables, tea and red wine. These bioactive polyphenols are

non-energetic, non-nutrient secondary metabolites present in plants and cannot be synthesized by

humans.36

In recent decades, overwhelming evidence indicates that flavonoid intake is associated

with well-known risk-factors for CVD. There is also some evidence that flavonoids may have a

role in preventing the development of CVD (e.g., coronary heart disease, stroke, et al).37,38

In

addition, although there are many putative biological mechanisms underlying a possible




endothelial function by some compounds, and reductions in platelet aggregation,41

and the effect

of individual compounds or interactions between flavonoids is still largely unknown, flavonol and

isoflavone intake may explain some of this beneficial effect.19,42

Although, the exact mechanism by which flavonoid intake may protect against the development of

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6

stroke is still ambiguous, a possible mechanism is the regulation of blood pressure, especially by

anthocyanins, due to an increase in endothelial-derived nitric oxide (NO).43

Moreover, mainly

attributed to antioxidant mechanisms, flavonoids reduced mitochondrial lipid peroxidation and

loss of mitochondrial transmembrane electric potential caused by oxidative stress induced by ADP

plus iron.44

A third mechanism is the protective action of flavonoids on the anti-inflammatory

responses in the brain, that may be attributable to raft disrupting and antioxidant effects.45

Because

the flavonoids are very diverse in their bioavailability and bioactivity, the rationale for assuming

that intakes of all flavonoid classes might have effects on stroke needs more consideration.







More,

importantly, the possibility of reverse causality should be addressed. A previous meta-analysis of


incidence.38

That meta-analysis included 6 prospective cohorts studies from 3 different countries,

4 were conducted in Europe (3 in Finland and 1 in The Netherlands) and 2 in the United States.

The inverse associations between flavonol intake and stroke were observed in two prospective

cohort studies (Zutphen Elderly Study and Kuopio Study). Weak and nonsignificant inverse

associations were found in the Finnish Mobile Clinic Study and Women’s Health Study, this result

was consistent with a detailed overview by Vogiatzoglou et al,46

whereas no associations were

noted in the Iowa Women’s Health Study and Alpha-Tocopherol, Beta-Carotene Cancer

Prevention Study. The results for flavonols were consistent with our findings on flavonoids. But

the data from studies included by previous meta-analysis were limited to August 2009.38

Interestingly, since then 3 new studies on the relationship between flavonoids intake and risk of

stroke were published.16-18

To obtain a more comprehensive estimate of the putative influence of

the flavonoids on stroke, we conducted a meta-analysis of prospective cohort studies. To our

knowledge, this meta-analysis is the largest to reveal a potential relationship between dietary

flavonoid intake and risk of stroke. However, most studies assess dietary flavonoid intake based

on self-reported questionnaires using FFQs, and medical records were not always available for

stroke classification; the possibility that misclassification of flavonoid intake and stroke was




The present










strength of the association between flavonoid intake and stroke risk. Additionally, all the studies

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7

used in this dose-response analysis did not include complete data on total flavonoid intake; the








intake levels. Fourth, we tried to study the main sources of flavonoids. But, we failed because of



kaempferol, myricetin, luteolin, and apigenin),8,9,11,12



and 2

studies included the similar compounds (including daidzein, genistein, formononetin, biochanin A,


as summarized in supplemental table 2. Among 11 studies, only 3 studies

were eligible for the dose-response analysis, and the compounds of flavonoid

subclasses are different among these studies. Of these, 2 studies included flavanones, 2

studies included flavones, 2 studies included flavonols, 2 studies included flavan-3-ols, and 2

studies included isoflavones. Due to the limited number of studies that met dose-response analyses

and the insufficient statistical power, we did not conduct dose-response analysis separately on

each class. Thus, we evaluated the effects of total flavonoid intake on stroke risk rather than the

wide-range of flavonoid sub-classes. On the one hand, in order to balance the risk of a type I error

(the true effect is zero but we reject the null) and a type II error (the true effect is not zero but we

fail to reject the null), we combined these estimates using a random-effects model, which takes

into account both within-study and between-study variabilities.24

Thus, these results should be

treated with caution. Finally, a possible limitation is due to language bias. We attempted to

minimize this bias by searching major electronic databases with no language restriction. However,

several articles published in non-English or unpublished reports might not appear in international

journal databases, and could be omitted by our searches.47

Conclusions




risk.









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8






Figures Information


(TIFF)



(TIFF)




(TIFF)




(TIFF)



(DOC)



(DOC)


(DOC)

REFERENCES



2014;383:245-54.


A Report From the American Heart Association. Circulation 2015.



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9













Nutr 2001;74:227-32.


J Clin Nutr 2002;76:560-8.





2005;15:188-97.



2005;111:465-71.





Study. Br J Nutr 2008;100:890-5.


Stroke 2012;43:946-51.















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10










2003;327:557-60.









33. Moriyama IM. The eighth revision of the International Classification of Diseases. Am J Public

Health Nations Health 1966;56:1277-1280.

34. Kupka K. International classification of diseases: ninth revision. WHO Chron

1978;32:219-225.

35. Bramer GR. International statistical classification of diseases and related health problems.

Tenth revision. World Health Stat Q 1988;41:32-36.

36. Guillermo R, Alejandro Z, Miguel Z, et al. Chrysoeriol and other polyphenols from Tecoma

stans with lipase inhibitory activity. J Ethnopharmacol 2016.




and women. J Nutr 2010;140:600-4.









Am Coll Nutr 2004;23:85-91.






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11

2010;31:478-94.


64 years). PLoS One 2015;10:e0128132.



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First author,


No. Participants

(% male)

Age range or

mean (yr)

Follow-Up

Duration (yr)

Assessment of

dietary data

Stroke

Ascertainment

No. of stroke

cases

Pre-stroke

Excluded

Study

Quality

Hirvonen et al.8

2000

Finland/European

26497 (100)

50-69

6.1



method

Any type of stroke

based on ICD-8, 9

codes

Ischemic: 736

Yes

8

Arts et al.9 2001


806 (100)

65-84

15

A cross-check

dietary history

method

Any type of stroke

based on ICD-9

codes

Fatal: 47,

Nonfatal: 88

No

7

Knekt et al.10

2002

Finland/European

9131 (NA)

30-69

28

A dietary history

interview

Any type of stroke

based on ICD-8

codes

Fatal or

nonfatal: 681

No

8

Sesso et al.11

2003

United States

38445 (0)

53.9

6.9

A food-frequency

questionnaire

Any type of stroke

based on clinical

diagnosis

Fatal or

nonfatal: NA

Yes

9

Marniemi et al.12

2005

Finland/European

755 (47.8)

65-99

10

A dietary history

interview

Any type of stroke

based on ICD-9

codes

Fatal: 45,

Nonfatal: 25

Yes

8

van der Schouw et

al.13

2005


16165 (0)

49-70

6.3

A validated

food-frequency

questionnaire

Any type of stroke

based on ICD-9

codes

Fatal or

nonfatal: 147

Yes

9

Mink et al.14

2007

United States

34489 (0)

55-69

16

A food-frequency

questionnaire

Any type of stroke

based on ICD-9

codes

Fatal: 469

Yes

9

Mursu et al.15

2008

Finland/European

1950 (100)

42-60

15.2

An instructed 4d

food recording by

Any type of stroke

based on ICD-9, 10

Ischemic: 102

Yes

9

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1


Cassidy et al.16

2012

United States

69622 (0)

30-55

14

A semiquantitative

food-frequency

questionnaires

interview

Any type of stroke

based on medical

records, autopsy

reports, and death

certificates

Ischemic: 943

Yes

8

McCullough et

al.17

2012

United States

98469 (38.8)

70

7

A semiquantitative

food-frequency

questionnaires

interview

Any type of stroke

based on ICD-9, 10

codes

Fatal: 573

Yes

9

Talaei et al.18

2014

Singapore/Asian

60298 (44.5)

45-74

14.7

A semiquantitative

food-frequency

questionnaires

interview

Any type of stroke

based on ICD-9

codes

Fatal: 1298

No

8


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2




2, %

Overall studies


Ischemic stroke 3 0.93 (0.80-1.07) 1.03 0.609 0 0.301

Geographical area

United States 4 0.86 (0.76-0.98) 1.50 0.683 0 0.026

Asian 1 0.97 (0.81-1.16) 0 - - 0.740

European 6 0.88 (0.77-1.00) 3.81 0.577 0 0.051

History of stroke

Yes 3 0.89 (0.78-1.02) 2.10 0.351 4.6 0.105

No 8 0.89 (0.80-0.99) 4.38 0.735 0 0.027

Sex

Male 4 0.82 (0.64-1.06) 4.90 0.179 38.8 0.130

Female 5 0.92 (0.80-1.05) 2.39 0.664 0 0.216

Combined 3 0.86 (0.72-1.04) 2.97 0.227 32.6 0.114


≤10 5 0.88 (0.77-1.01) 3.78 0.437 0 0.067

>10 6 0.89 (0.81-0.99) 2.68 0.750 0 0.036

Quality score

High, score＞8 5 0.85 (0.73-0.99) 2.26 0.688 0 0.038

Low, score≤8 6 0.91 (0.82-1.00) 3.73 0.589 0 0.048


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152x101mm (300 x 300 DPI)

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Supplemental table 1. Confounding factors and methods for adjustmentReference Adjusted covariates Total stroke Nonfatal stroke Fatal Stroke Ischemic Stroke Hemorrhagic

strokeHirvonen et al.8 2000 Age, BMI, SBP, DBP, height, cholesterol, diabetes, history

CHD, smoking, alcohol, supplementation group and education.equal toischemic strokeresults

0.98 (0.80-1.21)

Arts et al.9 2001Age, BMI, smoking, alcohol, physical activity, coffee, diet,energy, FA, prescribed diet, intakes of fish, coffee, cholesterol,fiber, fish, vit C, vit E and β-carotene

0.92(0.51-1.68)

Knekt et al.10 2002 Age, sex, geographic area, occupation, BMI, BP, cholesterol,diabetes, region, SE and smoking

0.79(0.64-0.98)

Sesso et al.11 2003 Age, exercise, aspirin, BMI, BP, postmenopausal hormone use,cholesterol, diabetes, history of CHD, smoking, alcohol, F﹠V,fiber, folate and vit E.

0.70(0.46-1.07)

Marniemi et al.12

2005Age, sex, smoking, functional capacity and weight adjustedenergy intake.

0.65(0.34-1.23)

van der Schouw etal.13 2005

Age, BMI, cholesterol, physical activity, diabetes,hypertension, hypercholesterolemia, HRT, OC, MS, smoking,alcohol, energy, F﹠A, fiber, protein, fruit, vegetable andmenopausal status.

1.05(0.64-1.70)

Mink et al.14 2007 Age, BMI, BP, diabetes, HRT, MS, education, smoking,activity, estrogen use, WHR and energy

equal to fatalstroke results

0.94 (0.69-1.29)

Mursu et al.15 2008 Age, examination years, BMI, SBP, HM, cholesterol, TAG,maximal oxygen uptake, smoking, history of CVD, diabetes,alcohol, energy-adjusted intake of folate and vit E.

equal toischemic strokeresults

0.71 (0.37-1.37)


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diabetes, hypercholesterolemia, history of CHD, alcohol,menopausal status, energy, use of multivitamins and history ofhypertension

ischemic strokeresults

McCullough et al.17

2012Age, smoking, beer and liquor intake, history of hypertension,history of cholesterol, family history of MI, BMI, physicalactivity, energy intake, aspirin use, HRT, and sex


0.83 (0.66-1.04)

Talaei et al.18 2014 Age, sex, dialect, year of interview, educational level, BMI,physical activity, smoking duration, alcohol, diabetes,hypertension, CHD, stroke, energy and fiber


0.97 (0.81-1.16)

Abbreviations: BMI, body mass index; CVD, cardiovascular disease; CHD, coronary heart disease; BP, blood pressure; SBP: systolic blood pressures; DBP: diastolic bloodpressures; vit C: vitamin C; vit E: vitamin E; FA: fatty acids; F﹠V: fruit and vegetable intake; MS: menopausal status; HRT: hormonal replacement therapy; OC: oralcontraceptives; WHR: waist-to-hip ratio; HM: hypertension medication; MI: myocardial infarction.

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Supplemental table 2. Flavonoid subclasses and compounds for each study.Reference Flavonoid subclassesHirvonen et al.8 2000 Quercetin, kaempherol, myricetin, luteolin, and apigenin..Arts et al.9 2001 Quercetin, kaempferol, myricetin, luteolin, and apigenin..Knekt et al.10 2002 4 flavonols (kaempferol, quercetin, myricetin, and

isorhamnetin), 2 flavones (apigenin and luteolin), and 3flavanones (hesperetin, naringenin, and eriodictyol).

Sesso et al.11 2003 Quercetin, kaempferol, myricetin, luteolin, and apigenin..Marniemi et al.12 2005 Quercetin, kaempferol, myricetin, luteolin, and apigenin..van der Schouw et al.13

2005Daidzein, Genistein, Formononetin, Biochanin A.

Mink et al.14 2007 4 flavonols (kaempferol, quercetin, myricetin, andisorhamnetin), 2 flavones (apigenin and luteolin), 3 flavanones(hesperetin, naringenin, and eriodictyol), flavan-3-ols,anthocyanidins, isoflavones, proanthocyanidins.

Mursu et al.15 2008 Flavonols, flavones, flavanones, flavan-3-ols andanthocyanidins.

Cassidy et al.16 2012 4 flavonols (kaempferol, quercetin, myricetin, andisorhamnetin), 2 flavones (apigenin and luteolin), 6anthocyanins (cyaniding, delphinindin, malvidin, pelargonidin,petunidin, peonidin), flavan-3-ols (catechins, epicatechins), and3 flavanones (hesperetin, naringenin, and eriodictyol).

McCullough et al.17 2012 Anthocyanidins, flavan-3-ols, flavanones, flavones, flavonols,proanthocyanidins, isoflavones.


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1

MOOSE Checklist






1







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the meta-analysis


include



















intervention used

Flavonoids







should include





















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3


searched




features







justifications






than English







on the association.


authors




flavonoids.


include








coding of data






























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methods.


and graphics


figures.


include



estimate

Figure 2, 3 and 4



included







analyses


include



studies.




comparison groups.


included studies




include



results









conclusions









the risk of stroke.



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response


Journal: BMJ Open



Date Submitted by the Author: 16-May-2016







Keywords: STROKE MEDICINE, NEUROLOGY, PUBLIC HEALTH


BMJ Open on O


http://bmjopen.bm

j.com/

BM


jopen-2015-008680 on 8 June 2016. Dow

nloaded from






a,1, Xiaowei Zhang


a





[email protected]


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1

Abstract


risk of stroke.
















Article summary





flavonoids.






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2

Introduction





the premature incidence and mortality associated with stroke, and there is growing interest in

altering risk factors and reversing this global epidemic. Among the known risk factors for stroke,

dietary factors, especially dietary flavonoid intake, have aroused particular attention. Clinical

studies have shown that intakes of flavonoids reduce cardiovascular disease (CVD) risk.3-5

Additionally, experimental studies indicated that flavonoids have been shown to have both

antioxidant and antithrombotic properties.6,7






the role






of stroke.

Methods

Literature search







“proanthocyanidins,” “anthocyanins,” “anthocyanidins,” “flavan-3-ols,” “isoflavones,”

“flavanones,” “catechins” and “stroke,” “cerebrovascular disease,” “cerebrovascular disorders,”

“cerebral infarct,” “ischemic stroke,” “intracranial hemorrhage,” “intracranial artery disease,”

“cardiovascular disease,” “myocardial ischemia,” “myocardial infarct,” “ischemic heart disease,”

“coronary heart disease,” and “longitudinal studies,” “cohort studies,” “prospective studies,”

“follow-up studies.” We restricted the search to human studies. There were no language

restrictions. In addition, we reviewed the reference lists of obtained articles and contacted authors

to identify additional relevant studies and information. When the same or similar patient cohort

was included in several publications, only the most recent or complete report was selected for

analysis. This search strategy was updated on January 14, 2016.

Study Selection






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3



than 1 year of follow-up. Studies were excluded if (1) the study design was cross-sectional; or


of follow-up.

Data Abstraction









The quality of




was 9 stars. Studies were graded as the high-quality if they were awarded ＞8 stars.


Relative risks (RRs) were used as the common measure of association between flavonoid intake

and stroke, and hazard ratios (HRs) were considered equivalent to RRs. Data analysis used





We combined these





and Orsini et al26

was used to







closest category.20





zero. When data on total flavonoid intake included in this dose-response meta-analysis was not




significance,24



We regard I2 of ＜40% as “heterogeneity that might not be

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4
















Results

Literature Search






meta-analysis.8-18









European countries (Finland and the Netherlands).8-10,12,13,15




4 studies



and 4 studies only

women.11,13,14,16






ICD-8,9,10.33-35

All studies provided adjusted risk estimates (e.g., sex, body mass index, smoking,






Overall,




0.774, I2


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5

flavonoid intake and risk of stroke.9,14,16









in the United States, 0.88 (95% CI: 0.77-1.00) in Europe, and 0.97 (95% CI: 0.81-1.16) in Asian










0.238).

Meta Regression





heterogeneity.

Discussion






Flavonoids, a large class of polyphenols, are widely distributed in plants and are present in

considerable amounts in fruits, vegetables, tea and red wine. These bioactive polyphenols are

non-energy, non-nutrient secondary metabolites present in plants and cannot be synthesized by

humans.36

In recent decades, overwhelming evidence indicates that flavonoid intake is associated

with well-known risk-factors for CVD. There is also some evidence that flavonoids may have a

role in preventing the development of CVD (e.g., coronary heart disease, stroke, et al).37,38

In

addition, although there are many putative biological mechanisms underlying a possible




endothelial function by some compounds,41

and reductions in platelet aggregation,42

and the effect

of individual compounds or interactions between flavonoids is still largely unknown, flavonol and

isoflavone intake may explain some of this beneficial effect.19,43

Although, the exact mechanism by which flavonoid intake may protect against the development of

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6

stroke is still ambiguous, a possible mechanism is the regulation of blood pressure, especially by

anthocyanins, due to an increase in endothelial-derived nitric oxide (NO).44

Moreover, mainly

attributed to antioxidant mechanisms, flavonoids reduced mitochondrial lipid peroxidation and

loss of mitochondrial transmembrane electric potential caused by oxidative stress induced by ADP

plus iron.45

A third mechanism is the protective action of flavonoids on the anti-inflammatory

responses in the brain, that may be attributable to raft disrupting and antioxidant effects.46

Because

the flavonoids are very diverse in their bioavailability and bioactivity, the rationale for assuming

that intakes of all flavonoid classes might have effects on stroke needs more consideration.




association between flavonoid intake and risk of stroke is causal. One study suggested that persons

with higher flavonoids intakes had lower risk from stroke,10

the others failed to find the

association.8,9

More, importantly, the possibility of reverse causality should be addressed. A

previous meta-analysis of flavonol intake and risk of stroke showed that flavonol intake was

inversely associated with stroke incidence.38

That meta-analysis included 6 prospective cohorts

studies from 3 different countries, 4 were conducted in Europe (3 in Finland and 1 in The

Netherlands) and 2 in the United States. The inverse associations between flavonol intake and

stroke were observed in two prospective cohort studies (Zutphen Elderly Study and Kuopio Study).

Significant inverse associations were found in the Finnish Mobile Clinic Study and Women’s

Health Study, this result was consistent with a detailed review by Wang et al,5 whereas no

associations were noted in the Iowa Women’s Health Study and Alpha-Tocopherol, Beta-Carotene

Cancer Prevention Study. The results for flavonols were consistent with our findings on flavonoids.

But the data from studies included in the previous meta-analysis were limited to articles published

before August 2009.38

Since then, 3 new studies on the relationship between flavonoids intake and

risk of stroke were published.16-18

To obtain a more comprehensive estimate of the putative

influence of the flavonoids on stroke, we conducted a meta-analysis of prospective cohort studies.

To our knowledge, this meta-analysis is the largest to reveal a potential relationship between

dietary flavonoid intake and risk of stroke. However, most studies assess dietary flavonoid intake

based on self-reported questionnaires using FFQs, and medical records were not always available

for stroke classification; the possibility that misclassification of flavonoid intake or stroke was




The present










strength of the association between flavonoid intake and stroke risk. Additionally, all the studies

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used in this dose-response analysis did not include complete data on total flavonoid intake; the








intake levels. Fourth, we tried to study the main sources of flavonoids. But, we failed due to



kaempferol, myricetin, luteolin, and apigenin),8,9,11,12



and 3

studies included isoflavones,13,17,18

as summarized in supplemental table 2. Among 11 studies,

only 3 studies were eligible for the dose-response analysis, and the compounds of

flavonoid subclasses are different among these studies. Of these, 2 studies included

flavanones, 3 studies included flavones, 3 studies included flavonols, 2 studies included

flavan-3-ols, and 1 study included isoflavones. Due to the limited number of studies that met

dose-response analyses and the insufficient statistical power, we did not conduct dose-response

analysis separately on each class. Thus, we evaluated the effects of total flavonoid intake on stroke

risk rather than the wide-range of flavonoid sub-classes. On the one hand, in order to balance the

risk of a type I error (the true effect is zero but we reject the null) and a type II error (the true

effect is not zero but we fail to reject the null), we combined these estimates using a

random-effects model, which takes into account both within-study and between-study

variabilities.24

Thus, these results should be treated with caution. Finally, a possible limitation is

due to language bias. We attempted to minimize this bias by searching major electronic databases

with no language restriction. However, several articles published in non-English or unpublished

reports might not appear in international journal databases, and could be omitted by our

searches.47

Conclusions




risk.









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8






Figures Information


(TIFF)



(TIFF)




(TIFF)




(TIFF)



(DOC)



(DOC)


(DOC)

REFERENCES



2014;383:245-54.


A Report From the American Heart Association. Circulation 2016;133:e38-e360.



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9













Nutr 2001;74:227-32.


J Clin Nutr 2002;76:560-8.





2005;15:188-97.



2005;111:465-71.





Study. Br J Nutr 2008;100:890-5.


Stroke 2012;43:946-51.







20. Peterson JJ, Dwyer JT, Jacques PF, et al. Associations between flavonoids and cardiovascular

disease incidence or mortality in European and US populations. Nutr Rev 2012;70:491-508.







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10










2003;327:557-60.


UK: The Cochrane Collaboration; 2011:261-6.







33. Moriyama IM. The eighth revision of the International Classification of Diseases. Am J Public

Health Nations Health 1966;56:1277-1280.

34. Kupka K. International classification of diseases: ninth revision. WHO Chron

1978;32:219-225.

35. Bramer GR. International statistical classification of diseases and related health problems.

Tenth revision. World Health Stat Q 1988;41:32-36.

36. Goya L, Martin MA, Sarria B, et al. Effect of Cocoa and Its Flavonoids on Biomarkers of

Inflammation: Studies of Cell Culture, Animals and Humans. Nutrients 2016;8. Doi:

10.3390/nu8040212.




and women. J Nutr 2010;140:600-4.





41. Boesten DM, von USN, den Hartog GJ, et al. Protective Pleiotropic Effect of Flavonoids on

NAD(+) Levels in Endothelial Cells Exposed to High Glucose. Oxid Med Cell Longev

2015;2015:894597.





Am Coll Nutr 2004;23:85-91.


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11





2010;31:478-94.



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First author,


No. Participants

(% male)

Age range or

mean (yr)

Follow-Up

Duration (yr)

Assessment of

dietary data

Stroke

Ascertainment

No. of stroke

cases

Pre-stroke

Excluded

Study

Quality

Hirvonen et al.8

2000

Finland/European

26497 (100)

50-69

6.1



method

Any type of stroke

based on ICD-8, 9

codes

Ischemic: 736

Yes

8

Arts et al.9 2001


806 (100)

65-84

15

A cross-check

dietary history

method

Any type of stroke

based on ICD-9

codes

Fatal: 47,

Nonfatal: 88

No

7

Knekt et al.10

2002

Finland/European

9131 (NA)

30-69

28

A dietary history

interview

Any type of stroke

based on ICD-8

codes

Fatal or

nonfatal: 681

No

8

Sesso et al.11

2003

United States

38445 (0)

53.9

6.9

A food-frequency

questionnaire

Any type of stroke

based on clinical

diagnosis

Fatal or

nonfatal: NA

Yes

9

Marniemi et al.12

2005

Finland/European

755 (47.8)

65-99

10

A dietary history

interview

Any type of stroke

based on ICD-9

codes

Fatal: 45,

Nonfatal: 25

Yes

8

van der Schouw et

al.13

2005


16165 (0)

49-70

6.3

A validated

food-frequency

questionnaire

Any type of stroke

based on ICD-9

codes

Fatal or

nonfatal: 147

Yes

9

Mink et al.14

2007

United States

34489 (0)

55-69

16

A food-frequency

questionnaire

Any type of stroke

based on ICD-9

codes

Fatal: 469

Yes

9

Mursu et al.15

2008

Finland/European

1950 (100)

42-60

15.2

An instructed 4d

food recording by

Any type of stroke

based on ICD-9, 10

Ischemic: 102

Yes

9

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1


Cassidy et al.16

2012

United States

69622 (0)

30-55

14

A semiquantitative

food-frequency

questionnaires

interview

Any type of stroke

based on medical

records, autopsy

reports, and death

certificates

Ischemic: 943

Yes

8

McCullough et

al.17

2012

United States

98469 (38.8)

70

7

A semiquantitative

food-frequency

questionnaires

interview

Any type of stroke

based on ICD-9, 10

codes

Fatal: 573

Yes

9

Talaei et al.18

2014

Singapore/Asian

60298 (44.5)

45-74

14.7

A semiquantitative

food-frequency

questionnaires

interview

Any type of stroke

based on ICD-9

codes

Fatal: 1298

No

8

Abbreviations: ICD: international classification of diseases; NA: not available.

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2




2, %

Overall studies


Ischemic stroke 3 0.93 (0.80-1.07) 1.03 0.609 0 0.301

Geographical area

United States 4 0.86 (0.76-0.98) 1.50 0.683 0 0.026

Asian 1 0.97 (0.81-1.16) 0 - - 0.740

European 6 0.88 (0.77-1.00) 3.81 0.577 0 0.051

History of stroke

Yes 3 0.89 (0.78-1.02) 2.10 0.351 4.6 0.105

No 8 0.89 (0.80-0.99) 4.38 0.735 0 0.027

Sex

Male 4 0.82 (0.64-1.06) 4.90 0.179 38.8 0.130

Female 5 0.92 (0.80-1.05) 2.39 0.664 0 0.216

Combined 3 0.86 (0.72-1.04) 2.97 0.227 32.6 0.114


≤10 5 0.88 (0.77-1.01) 3.78 0.437 0 0.067

>10 6 0.89 (0.81-0.99) 2.68 0.750 0 0.036

Quality score

High, score＞8 5 0.85 (0.73-0.99) 2.26 0.688 0 0.038

Low, score≤8 6 0.91 (0.82-1.00) 3.73 0.589 0 0.048


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152x101mm (300 x 300 DPI)

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Supplemental table 1. Confounding factors and methods for adjustmentReference Adjusted covariates Total stroke Nonfatal stroke Fatal Stroke Ischemic Stroke Hemorrhagic

strokeHirvonen et al.8 2000 Age, BMI, SBP, DBP, height, cholesterol, diabetes, history

CHD, smoking, alcohol, supplementation group and education.equal toischemic strokeresults

0.98 (0.80-1.21)

Arts et al.9 2001Age, BMI, smoking, alcohol, physical activity, coffee, diet,energy, FA, prescribed diet, intakes of fish, coffee, cholesterol,fiber, fish, vit C, vit E and β-carotene

0.92(0.51-1.68)

Knekt et al.10 2002 Age, sex, geographic area, occupation, BMI, BP, cholesterol,diabetes, region, SE and smoking

0.79(0.64-0.98)

Sesso et al.11 2003 Age, exercise, aspirin, BMI, BP, postmenopausal hormone use,cholesterol, diabetes, history of CHD, smoking, alcohol, F﹠V,fiber, folate and vit E.

0.70(0.46-1.07)

Marniemi et al.12

2005Age, sex, smoking, functional capacity and weight adjustedenergy intake.

0.65(0.34-1.23)

van der Schouw etal.13 2005

Age, BMI, cholesterol, physical activity, diabetes,hypertension, hypercholesterolemia, HRT, OC, MS, smoking,alcohol, energy, F﹠A, fiber, protein, fruit, vegetable andmenopausal status.

1.05(0.64-1.70)

Mink et al.14 2007 Age, BMI, BP, diabetes, HRT, MS, education, smoking,activity, estrogen use, WHR and energy


0.94 (0.69-1.29)

Mursu et al.15 2008 Age, examination years, BMI, SBP, HM, cholesterol, TAG,maximal oxygen uptake, smoking, history of CVD, diabetes,alcohol, energy-adjusted intake of folate and vit E.

equal toischemic strokeresults

0.71 (0.37-1.37)


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diabetes, hypercholesterolemia, history of CHD, alcohol,menopausal status, energy, use of multivitamins and history ofhypertension

ischemic strokeresults

McCullough et al.17

2012Age, smoking, beer and liquor intake, history of hypertension,history of cholesterol, family history of MI, BMI, physicalactivity, energy intake, aspirin use, HRT, and sex


0.83 (0.66-1.04)

Talaei et al.18 2014 Age, sex, dialect, year of interview, educational level, BMI,physical activity, smoking duration, alcohol, diabetes,hypertension, CHD, stroke, energy and fiber


0.97 (0.81-1.16)

Abbreviations: BMI, body mass index; CVD, cardiovascular disease; CHD, coronary heart disease; BP, blood pressure; SBP: systolic blood pressures; DBP: diastolic bloodpressures; vit C: vitamin C; vit E: vitamin E; FA: fatty acids; F﹠V: fruit and vegetable intake; MS: menopausal status; HRT: hormonal replacement therapy; OC: oralcontraceptives; WHR: waist-to-hip ratio; HM: hypertension medication; MI: myocardial infarction.

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Supplemental table 2. Flavonoid subclasses and compounds for each study.Reference Flavonoid subclassesHirvonen et al.8 2000 Quercetin, kaempherol, myricetin, luteolin, and apigenin..Arts et al.9 2001 Quercetin, kaempferol, myricetin, luteolin, and apigenin..Knekt et al.10 2002 4 flavonols (kaempferol, quercetin, myricetin, and

isorhamnetin), 2 flavones (apigenin and luteolin), and 3flavanones (hesperetin, naringenin, and eriodictyol).

Sesso et al.11 2003 Quercetin, kaempferol, myricetin, luteolin, and apigenin..Marniemi et al.12 2005 Quercetin, kaempferol, myricetin, luteolin, and apigenin..van der Schouw et al.13

2005Daidzein, Genistein, Formononetin, Biochanin A.

Mink et al.14 2007 4 flavonols (kaempferol, quercetin, myricetin, andisorhamnetin), 2 flavones (apigenin and luteolin), 3 flavanones(hesperetin, naringenin, and eriodictyol), flavan-3-ols,anthocyanidins, isoflavones, proanthocyanidins.

Mursu et al.15 2008 Flavonols, flavones, flavanones, flavan-3-ols andanthocyanidins.

Cassidy et al.16 2012 4 flavonols (kaempferol, quercetin, myricetin, andisorhamnetin), 2 flavones (apigenin and luteolin), 6anthocyanins (cyaniding, delphinindin, malvidin, pelargonidin,petunidin, peonidin), flavan-3-ols (catechins, epicatechins), and3 flavanones (hesperetin, naringenin, and eriodictyol).

McCullough et al.17 2012 Anthocyanidins, flavan-3-ols, flavanones, flavones, flavonols,proanthocyanidins, isoflavones.


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