Flavonoids from Fruit and Vegetables

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Flavonoids from Fruit and Vegetables: A Focus on Cardiovascular Risk Factors

J. Y. Toh, Verena M. H. Tan, Paul C. Y. Lim, S. T. Lim & Mary F. F. Chong

Current Atherosclerosis Reports ISSN 1523-3804 Volume 15 Number 12 Curr Atheroscler Rep (2013) 15:1-7 DOI 10.1007/s11883-013-0368-y

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Author's personal copy Curr Atheroscler Rep (2013) 15:368 DOI 10.1007/s11883-013-0368-y

NUTRITION (BV HOWARD, SECTION EDITOR)

Flavonoids from Fruit and Vegetables: A Focus on Cardiovascular Risk Factors J. Y. Toh & Verena M. H. Tan & Paul C. Y. Lim & S. T. Lim & Mary F. F. Chong

# Springer Science+Business Media New York 2013

Abstract Epidemiological studies suggest that high intakes of dietary flavonoids are associated with decreased cardiovascular disease mortality and risk factors. Less is known about the cardioprotective effects of flavonoids from fruit and vegetables. This review summarizes data from studies which examine the effects of commonly consumed fruit and vegetables on cardiovascular disease risk biomarkers in healthy volunteers or at-risk individuals. Although flavonoids from apples, berries, and onions appear to impact positively on blood pressure, vascular function, and serum lipid levels, further research is required to find out the optimal quantity and food matrix for conferring substantial clinical benefit. The benefits from citrus flavonoids are still inconclusive. Further robust, longer-term dietary intervention studies, with the inclusion of placebo or control arms, are required to improve the credibility of the findings and confirm current observations. An improved understanding of the impact of flavonoids from fruit and vegetables can help one make discerning food choices for optimal cardiovascular health.

Keywords Fruit and vegetables . Flavonoids . Cardiovascular risk . Vascular function . Blood lipids . Blood pressure This article is part of the Topical Collection on Nutrition J. Y. Toh : M. F. F. Chong Singapore Institute for Clinical Sciences, Brenner Centre for Molecular Medicine, 30 Medical Drive, Singapore 117609, Singapore V. M. H. Tan : M. F. F. Chong (*) Clinical Nutrition Research Centre, Singapore Institute for Clinical Sciences, 14 Medical Drive, #07-02, Singapore 117599, Singapore e-mail: [email protected] P. C. Y. Lim : S. T. Lim Department of Cardiology, National Heart Centre Singapore, Mistri Wing, 17 Third Hospital Avenue, Singapore 168752, Singapore

Introduction Flavonoids are nonnutrient phytochemicals that occur naturally in plant-based foods. They are characterized chemically by two benzene rings linked by a linear carbon chain, and come under the broader classification of polyphenols. To date, over 5,000 flavonoid compounds have been identified, and they can be categorized into six classes— flavan-3-ols, flavanones, flavones, isoflavones, flavonols, and anthocyanins [1••]. In the last two decades, epidemiological evidence has consistently demonstrated an inverse relationship between dietary flavonoid intake and cardiovascular disease (CVD) mortality and its risk factors. The Zutphen Elderly Study was one of the first studies that demonstrated increased flavonoid intakes are associated with decreased incidence of CVD mortality [2], whereas proanthocyanidin consumption was shown to be inversely associated with coronary artery disease mortality in postmenopausal women in the Iowa Women’s Health Study [3]. In the Kupio Ischaemic Heart Disease Risk Factor Study, intake of flavan-3-ols was associated with reduction of carotid atherosclerosis in middle-aged men [4]. More recently, data collected from three cohort studies, namely, the Nurses’ Health Study I, the Nurses’ Health Study II, and the Health Professionals Follow-Up Study, further demonstrated that high intakes of anthocyanins could lead to an 8 % reduction in the risk of hypertension [5]. These epidemiological data are further supported by a recent meta-analysis on 184 intervention trials, which revealed that the combined effects of the six flavonoid subgroups significantly improved both acute and chronic flow-mediated dilation (FMD) responses and lowered blood pressure [6]. Dietary flavonoids act via different mechanisms of action to reduce cardiovascular risk. They reduce oxidative stress by upregulating antioxidant enzymes to prevent cholesterol ester accumulation within macrophages [7]. They also exert

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anti-inflammatory actions by inhibiting platelet adhesion, secretion, and aggregation [8]. In addition, they have the ability to prevent thrombus formation, improve endothelial function, modify lipid levels, and regulate glucose metabolism [9]. In terms of the dietary sources of flavonoids, a plethora of intervention studies have examined the moderate consumption of tea, red wine, and cocoa and their associated cardiovascular health benefits. The beneficial effects shown with respect to lipid, haemostatic, inflammatory, and vascularrelated biomarkers have been attributed to their high flavonoid contents, mostly from the flavanol subclasses [10–12]. However, less is known about the cardioprotective effects of flavonoids from fruit and vegetables (mostly from the other flavonoid subclasses—see Table 1), even though the benefits of increased consumption of fruits and vegetables on CVDs are well established [13]. Although fruit and vegetables contain a wide range of potentially cardioprotective components such as fiber, folate, nitrate, and vitamins, there is emerging and accumulating evidence to suggest that dietary flavonoids may explain, in large part, the cardiovascular benefits of increased fruit and vegetable intake. Flavonoids are ubiquitously found in a variety of fruit and vegetables. Table 1 shows the various classes of flavonoids and their main food sources. In this review, we have chosen to examine studies which report on the efficacy of commonly consumed and readily available fruit and vegetables or their extracts in reducing the levels of biomarkers of cardiovascular risk, in acute and shortterm interventions with healthy volunteers and at-risk population groups. The primary risk markers selected for this review were blood pressure, vascular function, and blood lipids, this selection being based on their link with the mechanistic actions of flavonoids and their strong, consistent epidemiological and causal relations with CVD [14–17]. The fruits and vegetables examined here are apples, citrus fruits, berries, and onions. The consumption of total fruit and vegetables in the diet was also examined.

Table 1 Subclasses of flavonoids and their food sources [1••, 54, 55]

Apple Flavonoids Apples are one of the most commonly consumed fruits in the human diet, contributing a large percentage of phenolic consumption in the USA and are the third highest contributor of dietary flavonoids in the Netherlands [18, 19]. Most apple flavonoids, namely, quercetin and epicatechin, are found in the skin of apples [20]. The effects of apple flesh compared with apple skin on blood pressure and vascular function were examined in an intervention trial, where 120 g of apple flesh with 80 g of apple skin [Cripps Pink; containing 184 mg of quercetin and 180 mg of (−)-epicatechin] was provided to the intervention group, whereas only apple flesh [containing less than 5 mg of quercetin and (−)-epicatechin] was given to the control group [20]. The amount of apple flesh provided corresponded to approximately one decored apple. In 4 weeks, systolic blood pressure and pulse pressure were significantly lowered to a greater extent in healthy subjects who consumed the apple flesh and skin than in those who consumed only apple flesh (−3.3 mmHg, 95 % confidence interval −4.9 to −1.8, and −1.9 mmHg, 95 % confidence interval −3.2 to −0.3, respectively). In addition, there were significant increases in nitrite (+34 nmol/L) and RXNO (S-nitrosothiols; +18 nmol/L)] production as well as enhanced endothelial function (measured by FMD responses) in the intervention group as compared with the control group [20]. Auclair et al. [21] showed that consumption of either 40 g of high-polyphenol lyophilized apples (Marie Menard; 1,430 mg polyphenol) or 40 g of lowpolyphenol lyophilized apples (Golden Delicious; 214 mg polyphenol) over two periods of 4 weeks did not significantly improve FMD responses in hypercholesterolemic patients. The lyophilized apples were freeze-dried apples ground into powder form, and 40 g of powder corresponded to approximately two fresh apples of 135 g each. Auclair et al. proposed that the lack of effect was the result of catechins in the lyophilized state being less well absorbed than when they are ingested in the aqueous state, in part due to interactions

Flavonoid subclass

Prominent food flavonoids

Food sources

Flavan-3-ols

Black and green teas, red wine, cocoa products

Flavanones

(+)-Catechin, (+)-gallocatechin, (−)-epicatechin, (−)-epigallocatechin, (−)-epicatechin-3-gallate, (−)-epigallocatechin-3-gallate Eriodictyol, hesperetin, naringenin

Flavones

Apigenin, luteolin

Isoflavones

Daidzein, genistein, glycitein, biochanin A, formononentin Isorhamnetin, kaempferol, myricetin, quercetin Cyanidin, delphinidin, malvidin, pelargonidin, petunidin, peonidin

Flavonols Anthocyanins

Oranges, grapefruits, citrus fruits Green leafy vegetables, spices and herbs Soybeans, tofu, soy products, miso, legumes Apples, onions, kale, leeks, broccoli Strawberries, blueberries, cranberries, blackcurrants

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with the apple matrix. In addition, FMD measurements were taken 10 h after the consumption of the last apple, and the lack of an effect could stem from the rapid elimination of cathechin monomers from the body system [21]. For the effects on serum lipid concentrations, outcomes have been mixed. Consumption of 300 g of Golden Delicious apples (corresponding to two apples and containing a total of 145.5 mg of polyphenols) daily for 8 weeks in one study did not significantly reduce serum levels of total cholesterol (TC), LDL cholesterol, HDL cholesterol, lipoprotein a, and apolipoprotein B or the LDL/HDL ratio in hyperlipidemic and overweight men [22]. However, in another study, daily intake of 600 mg of apple polyphenol extract (consisting mainly of procyanidins) led to significant decreases in serum TC and LDL cholesterol levels. In subjects who consumed apple polyphenol extracts, the TC level dropped significantly by about 15 mg/dL at the end of the 12-week intervention period and decreased by an additional 5 mg/dL at the end of a further 4-week follow-up. LDL cholesterol levels were reduced by 13.11 mg/dL at the end of the 12-week intervention and further decreased by 3 mg/dL after the 4-week follow-up. [23]. Conversely, the placebo-controlled group had only slight decreases in TC and LDL cholesterol levels, which were not significantly different from baseline levels. In a study comparing intake of whole apples and apple juice, intake of whole apples resulted in a better lipid profile than did consumption of apple juice. As the total polyphenol content for both treatments was fairly similar (145 mg/day vs 108 mg/day), it was concluded that the lack of fiber (pectin) in the apple juice was responsible for the cholesterol-lowering effects instead of the effects of polyphenols [24•]. In general, apple flavonoids may have beneficial effects on blood pressure, vascular function, and blood lipid levels, but the quantity administered is the key to determining the level of the effects. This quantity is, in turn, highly dependent on the variety of apple, the part of the apple (skin or flesh), and the food matrix, which is how the apple is consumed (aqueous or lyophilized or juice or extract). It appears that apple polyphenol extracts give the best effects, followed by consumption of whole apple with skins. A minimum period of 4 weeks of consumption appears to be necessary for there to be observable effects.

Citrus Flavonoids Citrus fruits such as oranges and grapefruits provide an abundant source of flavonoids, of which naringin and hesperidin are the main components under the class of flavanones. Naringin is mainly present in grapefruits and sour oranges, and hesperidin is present in sweet oranges, mandarins, and lemons [25]. Hydrolysis by gut microflora to their bioactive compounds, naringenin and hesperetin, is required for absorption by the body.

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To examine the effects of hesperidin in orange juice on vascular function, moderately overweight subjects were provided with 500 mL (292 mg of hesperidin and 47. 5 mg of nairutin) of orange juice daily for 4 weeks. With use of Doppler flowmetry, significant improvement in postprandial microvascular endothelial reactivity (+105.3±25.48 %) and a significant decrease in diastolic blood pressure (4.5 ± 2.0 mmHg) were observed in subjects who consumed orange juice as compared with those who consumed the control drink [26]. Similarly, results from FMD reflected significant improvements in endothelial function [27] when 500 mg of hesperidin (in capsule form) was provided daily for 3 weeks to subjects with metabolic syndrome. To our knowledge, there have not been any human studies examining grapefruit or naringin and their effects on vascular function. At least two studies have demonstrated that consumption of one to two grapefruits (red grapefruit, blond grapefruit, and pomelo) a day for 30 days can improve the lipid profile of hypercholesterolemic patients diagnosed with coronary artery disease [28, 29], although this was not attributed to any particular compound. To further investigate the effects of hesperidin and naringin on blood lipids, glucosyl hesperidin, a soluble derivative of hesperidin, was administered to hypertriglyceridemic patients in the form of two 250-mg tablets per day for 24 weeks. This resulted in significant reduction of plasma triglyceride and apolipoprotein B levels [30]. Similarly, 400 mg of naringin, in capsule form, was given every day for 8 weeks to hypercholesterolemic subjects. At the end of the trial, a reduction in LDL cholesterol levels by 17 % and triglyceride levels by 14 % was noted [31]. The aforementioned trials were, however, not placebo-controlled. In contrast, in a placebo-controlled, randomized trial conducted by Demonty et al. [32], the effect of pure hesperitin (800 mg/day) or naringin (500 mg/day) capsules provided for 4 weeks did not have a cholesterol-lowering effect on hypercholesterolemic patients, despite the high dose used and even after adjustment for baseline LDL levels. Grapefruit juice has been shown experimentally to decrease the presystemic metabolism of simvastatin and atorvastatin, thus increasing drug bioavailability. Although there have been concerns of drug–grapefruit juice interactions in some patients, most patients receiving these drugs can consume grapefruit juice without adverse effects. There is presently no contraindication for grapefruit juice consumption in those taking pravastatin, fluvastatin, or rosuvastatin. Although simvastatin is stated by the British National Formulary to have a potentially serious interaction with grapefruit juice, this is the case only when the juice is consumed in high concentrations [33]. There are currently limited human studies investigating the effects of consumption of citrus fruits containing hesperidin and nariginin. Rather, hesperidin and nariginin are more commonly administered in the form of extracts provided as tablets

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or capsules. Whereas orange juice consumption (approximately 500 mL/day) or hesperidin taken in the form of 500-mg tablets appears to exhibit positive effects on blood pressure and endothelial function, the lipid-lowering effects of hesperidin and naringin are still inconclusive, in part owing to the limitations of the study design. Further randomized, placebo-controlled studies examining hesperidin and naringin in different food matrices may help confirm the above-mentiuoned findings and improve our understanding of the effects of flavonoids from citrus fruits on cardiovascular risk.

Berry Flavonoids Berry fruits such as strawberries, blueberries, and cranberries contain abundant levels of anthocyanins and ellagitannins in their skin and flesh, and these flavonoids also gives the berries their brightly colored exterior [34]. It is estimated that 100 g of strawberries contains 33.63 mg of anthocyanidin, whereas 100 g of blueberries contains 163.52 mg of anthocyanidin [34]. Besides providing good source of flavonoids, berries are also rich in micronutrients such as vitamins C and E, folate, potassium, a-carotene, and b-carotene, some of which exhibit high antioxidant activity [34, 35]. Several studies have consistently reported a reduction in blood pressure after the consumption of berries [35–37]. Healthy subjects consuming mixed berries (bilberries 50 g/ day, lingonberries 25 g/day, blackcurrant and strawberry purée 50 g/day, chokeberry and raspberry juice 35 g/day) equivalent to 515 mg of anthocyanins daily for 8 weeks showed a decrease in systolic blood pressure of up to 1.5 mmHg [35]. Obese subjects with metabolic syndrome experienced a 7.8 % (± SD 2.5) decrease in systolic blood pressure and a 2.5 % (± SD 1.10) decrease in diastolic blood pressure after consuming 50 g of freeze-dried blueberries (742 mg anthocyanins) reconstituted in 480 mL of water daily for 8 weeks [36]. Berries have also been demonstrated to be effective in reducing the levels of proinflammatory markers and adhesion molecules, which are surrogate markers of endothelial function. Compared with control subjects given placebo capsules, a significant decrease in the levels of proinflammatory markers such as IL-4, IL-13, IL-8, and interferon-α (60 %, 38 %, 45 %, and 40 %, respectively) was observed in healthy subjects given capsules of anthocyanin extract (300 mg/day) obtained from billberries and black currant, over a period of 3 weeks. [38]. In a placebo-controlled intervention study, intake of 500 mL of cranberry juice per day, equivalent to 20.8 mg of anthocyanins, over a period of 4 weeks significantly decreased the levels of oxidized LDL (−20.7±21.1 %) and cell adhesion molecules such as soluble vascular cell adhesion molecule 1 (−6.4±11.5 %) and soluble intercellular adhesion molecule 1 (−7.5± 15.4 %) [39]. In a separate placebo-controlled study, intake of 50 g of freeze-dried

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strawberries (154 mg of anthocyanins) also lowered vascular cell adhesion molecule 1 levels significantly by 18 % in subjects with metabolic syndrome [40•]. Similarly, serum lipid profiles have been shown to be consistently improved in intervention trials involving the consumption of berries. In a trial conducted by Basu et al. [40•], 50 g of freeze-dried strawberries (equivalent to 154 mg of anthocyanins) was blended in water to make a drink and was provided daily to subjects with metabolic syndrome for 8 weeks. The control group was given water instead of the strawberry beverage. Significant relative reductions in mean serum TC levels (−5 %) and LDL cholesterol levels (−6 %) from the baseline were observed. In another study, daily consumption of 1.5 g of cranberry extract in the form of capsules (amount of flavonoids unspecified) over a period of 12 weeks also significantly lowered the LDL cholesterol level (by 0.4 mmol/L) and the TC to HDL cholesterol ratio (by 0.3 mmol/L) compared with placebo capsules, which did not lead to significant changes in the lipid profile from the baseline [41]. Compared with apple and citrus flavonoids, flavonoids derived from berries have been more extensively investigated. Evidence suggests that berries are effective in reducing the levels of proinflammatory markers and blood pressure and improving the lipid profile, regardless of the form in which they are administered e.g., fresh, dried, juice, or extract. The effective concentration of anthocyanins to confer such benefits appear to range from as low as 20.8 mg (approximately 500 mL of cranberry juice) to as high as 742 mg (approximately 50 g of freeze-dried blueberries). In addition to the flavonoids, the berry sources administered in these studies were significant sources of several micronutrients and/or fiber, which may synergistically contribute to the observed health outcomes. Studies examining the dose–response effect of berries consumed in their various forms and the effects of specific flavonoids will be required to further our understanding in this area. The literature on the effects of berries on endothelial function is sparse and is an area which warrants further investigation.

Onion Flavonoids Research on specific high-flavonoid vegetables in the literature is scarce. The most commonly cited flavonoid-rich vegetable is onion, which is predominantly rich in the flavonoid quercetin. Up to 22 mg of quercetin can be found in about 100 g of onions [42]. To examine the effects on cardiovascular risk factors, most studies administer quercetin in the form of tablets or capsules, instead of providing onions in the form of food. A summary of the key findings is described as follows. In a study on subjects with stage 1 hypertension, intake of 730 mg of quercetin (in the form of tablets) daily for 4 weeks led to significant decreases in systolic (−7±2 mmHg) and

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diastolic (−5±2 mmHg) blood pressure as compared with controls, who showed insignificant slight decreases in blood pressure [43]. Egert et al. [44] also showed that ingestion of 150 mg of quercetin (in the form of capsules) daily for 6 weeks by overweight subjects significantly lowered systolic blood pressure by 2.6 mmHg. Additionally, serum levels of the inflammatory marker TNF-α and oxidized LDL were reduced by 0.25 pg/mL and 170.8 ng/mL, respectively, compared with the baseline values [44]. With regard to the effects on vascular function markers, an acute intervention study showed that consumption of 200 mg of quercetin dissolved in water by healthy male adults resulted in significant increases in the 2-h postprandial plasma levels of S-nitrosothiols (approximately + 16.25 nmol/L) and plasma nitrite (approximately +2.25umol/L) as compared with controls, who experienced no change [45]. The level of plasma endothelin-1 was also significantly lowered, by approximately 0.06 pg/mL [45]. Although 150 mg of quercetin appears to confer some cardiovascular benefits, on the basis of these limited studies, the effects of quercetin on cardiovascular risk factors is still inconclusive, and more studies are required to confirm the above-mentioned findings.

Flavonoid-Rich Fruit and Vegetables Evidence from clinical trials demonstrating the combined effects of the various flavonoids from fruit and vegetables in the diet on cardiovascular risk markers, in particular vascular function, is limited. Dose–response relationships between the quantity of fruit and vegetables consumed and cardioprotective outcomes are also poorly defined. In a randomized controlled study designed to delineate the effects of minerals and fiber from other components of the DASH diet on blood pressure, it was found that a 3-week DASH diet (consisting of 3.3 servings of vegetables and 4.5 servings of fruits) was more effective than potassium, magnesium, and fiber supplements in lowering blood pressure (−6.2±1.4 and −3.7±1.4 mmHg, respectively) and improving small artery elasticity in obese hypertensive subjects. These findings highlight the additive beneficial effects of other nutritional factors found in fruits and vegetables [46]. In a parallel-design 8-week observational study, McCall et al. reported a 6.2 % improvement in forearm blood flow responses to intra-arterial administration of the endotheliumdependent vasodilator acetylcholine for every one-portion increase in reported fruit and vegetable consumption in subjects who consumed one, three, or six mixed fruit and vegetables portions daily. Similarly, studies using mixed concentrated fruit and vegetable supplements, in the form of either capsules or fruit and vegetable liquid concentrates, have demonstrated positive effects on reducing oxidative stress or that they were associated with significant reductions in the oxidation of

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protein, lipids, and DNA. Findings reflecting endothelial function [47], inflammatory biomarkers, and immune function [48–51] have, however, been inconsistent, and larger randomized controlled trials are required to ascertain their effects. It is recognized that by the nature of the study designs described above, it would not be possible to single out a specific nutrient or phytochemical compound responsible for the aforementioned health effects. Instead, nutrient biomarkers, selected on the basis of their antioxidant properties, were commonly used in these studies to assess the compliance of the study participants to fruit and vegetable intake. A few examples include ascorbic acid and a range of carotenoids such as α-carotene, β-carotene, β-cryptoxanthin, and lutein. It is, however, increasingly acknowledged that these biomarkers measured may themselves have physiological effects, and their increased levels could be mediators of the other beneficial components of fruit and vegetables [52]. Flavonoids were more recently examined as possible biomarkers of fruit and vegetable intake. An early study examining a low-vegetable versus a high-vegetable diet for two 5-week periods found that although hesperetin, naringenin, and quercetin were bioavailable, plasma concentrations of hesperetin and naringenin were poor biomarkers of intake [53]. Recent studies have demonstrated that urinary flavonoids are comparatively better biomarkers of flavonoid intakes [11, 36, 54]. With the aim of delineating the effects of flavonoids from those of the other nutrient and phytochemicals in fruit and vegetables on cardiovascular risk factors, a uniquely designed, parallel controlled dose–response dietary intervention study was recently conducted. Participants at risk of CVD were assigned to three diets: control diet, high-flavonoid fruit and vegetable diet, or low-flavonoid fruit and vegetable diet. Whereas the groups consuming the high-flavonoid fruit and vegetable diet and the low-flavonoid fruit and vegetable diet sequentially increased their assigned daily fruit and vegetable intake by an additional two, four, and six portions for 6-week periods during the 18-week study, the control group was asked to consume their habitual diet throughout the study. Participants were also asked to limit their intake of other rich sources of flavonoids in the diet. Good compliance in the study was reflected by the dose-dependent increase in dietary and urinary flavonoids in the group consuming the high-flavonoid fruit and vegetable diet, with no changes seen in other groups. The pending results from this study should help shed light on the effects of flavonoid-rich fruit and vegetables and CVD risk [17].

Conclusion To our knowledge, no randomized interventional trials has yet been conducted to establish the effects of flavonoids on cardiovascular events or mortality [55]. However, a number of

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trials involving flavonoid-rich fruit and vegetables have provided evidence demonstrating beneficial effects toward blood pressure control, FMD response indicative of endothelial function, and serum lipid levels. These are important cardiovascular risk factors, which have been shown to predict cardiovascular morbidity and mortality. Although the effects of flavonoids from apples, berries, and onions on the various biomarkers are generally positive, further research is still needed on the optimal doses in various food matrices in order to confer substantial clinical benefit. Although some studies suggest citrus flavonoids improve vascular function, the lipid-lowering benefit of citrus flavonoids is still uncertain. More robust, well-designed studies with the inclusion of placebo or control arms are required to improve the credibility of the findings and to confirm current observations. Current studies examine flavonoids in flavonoid-rich food or as isolated flavonoid compounds, but it remains unclear whether the beneficial effects on cardiovascular biomarkers could be attributed to a specific flavonoid compound or a particular food source. It is important to consider that fruit and vegetables also contain other nutrients and/or phytochemicals that could confer cardiovascular risk reduction benefits. Improved understanding of how flavonoids from different fruits and vegetables consumed in the diet can affect cardiovascular risk is imperative as it can help the general population make discerning food choices for optimal cardiovascular health. Studies using a longer-term dietary intervention design to examine the effects of individual flavonoid compounds and flavonoid-rich food sources on CVD end points are required to establish a causal relationship between intake of flavonoids and CVD risk.

Conflict of Interest J.Y. Toh, Verena M.H. Tan, Paul C.Y. Lim, S.T. Lim, and Mary F.F. Chong declare that they have no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

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