International Food Research Journal 24(2): 471-482 (April 2017)

Journal homepage: http://www.ifrj.upm.edu.my

Mini Review
Plant-derived foods containing polyphenols with endothelial
protective effects

Mokhtar, S. S. and *Rasool, A. H.

Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, Health

Campus, 16150, Kota Bharu, Malaysia

Article history Abstract

Received: 20 November 2015 Cardiovascular disease (CVD) is the leading cause of death and disability in the world. The
Received in revised form: primary cause of CVD is development of atherosclerosis resulting from chronic inflammation
4 April 2016 and endothelial dysfunction. Indeed, endothelial dysfunction is considered to be the earliest
Accepted: 15 April 2016
stage in the process of atherosclerosis development. There is great interest in discovering
strategies to inhibit endothelial dysfunction and atherosclerosis progression. The role of plant
constituents routinely consumed have attracted much attention as preventive health approaches
Keywords due to their availability and perceived safety. Accumulating studies suggest that constituents
present in tea, grape, cocoa, soy and pomegranate are associated with reduced risks of CVD.
Tea In this review, we discuss the potential of the above mentioned dietary ingredients to improve
Cocoa endothelial function in vivo and in vitro.
Polyphenols
Endothelial function © All Rights Reserved
Cardiovascular diseases

Introduction beverages. Polyphenols is the subject of increasing

scientific interest because of their possible beneficial
Endothelium, the inner monolayer of the blood effects on human health (Pandey and Rizvi, 2009).
vessel, regulates vascular tone and permeability, Polyphenol molecules and components typically
the balance between coagulation and fibrinolysis, carry several hydroxyl groups; and more than 4000 to
inflammatory activity as well as cell proliferation. 7000 varieties are present in plants. Polyphenols can
Alterations to these functions lead to endothelial be categorized into flavonoids and non-flavonoids.
dysfunction (Vanhoutte et al., 2009). Endothelial The flavonoid group has a phenyl chroman frame
dysfunction has been considered to be an early event (C6-C3-C6) and based on differences in side-chain
of pathophysiologic importance in the atherosclerotic structures can be classified into 6 subclasses: flavones,
process. Endothelial dysfunction is associated with isoflavones, flavanones, flavonols, anthocyanidins and
most forms of cardiovascular diseases (CVD) such flavanols. Typical non-flavonoids include phenolic
as hypertension, coronary artery disease (CAD), acids, tannins, curcumins and resveratrol (Table 1)
chronic heart failure and peripheral artery disease. (Habauzit and Morand, 2012; Del Rio et al., 2013;
The hallmark of endothelial dysfunction is reduced Yamagata et al., 2015).
endothelial nitric oxide synthase (eNOS) expression This paper aimed to review available evidence
and/or impaired nitric oxide (NO) availability on use of polyphenol-containing foods (tea, grapes,
(Felaco et al., 2001; Mokhtar et al., 2013). In blood cocoa, soy and pomegranate) on endothelial function
vessels, NO is synthesized by the eNOS enzyme in humans. Possible mechanistic principles involving
in endothelial cells and diffuses into vascular the effects of these dietary ingredients on endothelial
smooth muscle cells, leading to vasodilatation. NO function are also discussed using experimental and in
is a major anti-atherogenic factor due to a number vitro studies. A table summarising the effects of these
of vasoprotective effects; thus decreased NO natural products on endothelial function are given as
availability in the vasculature is likely to promote Table 2.
the progression of vascular diseases. Thus improved
NO bioavailability would be a promising step in the Black and green tea
therapy and prevention of cardiovascular disorders.
Polyphenols are naturally occurring compounds Intervention study
found largely in fruits, vegetables, cereals and Tea, a product made up from the leaf and bud of
the plant Camellia sinensis, is the second most widely

*Corresponding author.
Email: aida.rasool@yahoo.com
472 Mokhtar, S. S. and Rasool, A. H./IFRJ 24(2): 471-482

Table 1. Classification of phenolic compounds

consumed drink in the world after water. Tea is a studied in 14 healthy individuals who took either
rich source of polyphenolic compounds, particularly six grams of green tea, 125 milligrams of caffeine
flavonoids. The major flavonoids present in green tea or hot water. This study showed that FMD increased
are catechin (under the subclass of flavanols) such significantly with green tea consumption, but was not
as epicatechin (EC), epicatechin-3-gallate (ECG), affected by caffeine or hot water (Alexopoulos et al.,
epigallocatechin (EGC) and epigallocatechin-3- 2008). In a randomized study of 66 patients with pre-
gallate (EGCG). In black tea, the major flavonoids existing CAD, acute (450 millilitres for two hours)
present are polymerized catechins such as theflavins and chronic consumption (900 ml/day for four weeks)
and thearubigens (McKay and Blumberg, 2002; of black tea resulted in significant improvements in
Cabrera et al., 2006; Chacko et al., 2010; Khan and endothelium-dependent FMD (Duffy et al., 2001).
Mukhtar, 2013; Fuchs et al., 2014). Consumption of green tea (eight g/day) for two
A number of studies have investigated the effects weeks also improved FMD in young healthy smokers
of black and green tea, or tea flavonoids on flow- (Nagaya et al., 2004; Kim et al., 2006). In patients
mediated dilatation (FMD) of the human brachial with chronic kidney disease, consumption of green
arteries. FMD represents endothelium-dependent tea (five g/day) for four weeks improved FMD (Park
relaxation of the brachial artery, mediated via release et al., 2010). In a cross-over study performed in 19
of NO. Improvement in FMD has been observed healthy men, twice daily intake of black tea (0, 100,
in human trials with the consumption of green or 200, 400 and 800 mg/day) for a period of one week
black teas (Duffy et al., 2001; Nagaya et al., 2004; increased FMD and decreased peripheral arterial
Hodgson 2006; Kim et al., 2006; Alexopoulos et al., stiffness in a dose-dependent manner (Grassi et al.,
2008; Jochmann et al., 2008; Grassi et al., 2009). The 2009). Another study in 21 healthy women showed
effect of green tea consumption on FMD has been a significant increase in FMD after two hours of
 Mokhtar, S. S. and Rasool, A. H./IFRJ 24(2): 471-482 473

Table 2. Natural products and its effects on endothelial-dependent vasodilatation

474 Mokhtar, S. S. and Rasool, A. H./IFRJ 24(2): 471-482
 Mokhtar, S. S. and Rasool, A. H./IFRJ 24(2): 471-482 475

CAD, coronary artery disease; DBP, diastolic blood pressure; eNOS, endothelial nitric oxide
synthase; EC, epicatechin; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EGCG,
epigallocatechin-3-gallate;FMD, flow-mediated dilatation; HUVEC, human umbilical vein
endothelial cells; NADPH, nicotinamide adenine dinucleotide phosphate ; NO, nitric oxide;
PPARα, peroxisome proliferator-activated receptor α; RCT, randomized controlled trial; SBP,
systolic blood pressure; SHR, spontaneously hypertensive rat; SOD, superoxide dismutase

green and black tea consumption (Jochmann et al., demonstrated that both black and green teas promoted
2008). Meta-analysis from nine human intervention both endothelial NOS activity and NO bioavailability
studies illustrated that moderate consumption of tea in cultured endothelial cells (Anter et al., 2004; Lorenz
substantially enhanced FMD, in which the overall et al., 2004; Jochmann et al., 2008; Siamwala et al.,
increase in FMD with daily dose of 500 ml of tea (2-3 2013). In addition, incubation of human endothelial
cups) compared to placebo was 2.6% of the arterial cells with four derivatives of tea flavanols; EC, ECG,
diameter (Ras et al., 2011). EGC, and EGCG showed dose-dependent increases
in NO production (Persson et al., 2006). In diabetic
Experimental / In vitro study rats, treatment with green tea improved uncoupling
The underlying molecular mechanisms for tea- of eNOS, thus increased NO bioavailability (Faria et
induced effects on endothelial function may be due al., 2012). Uncoupling of eNOS is characterized by
to its direct effect on the NO system. It has been a reduction in tetrahydrobiopterin (BH4) levels and
476 Mokhtar, S. S. and Rasool, A. H./IFRJ 24(2): 471-482

a decrease in the eNOS dimer-to-monomer ratio. and protein expression and stimulated the synthesis of
BH4 is a critical cofactor for the production of NO. NO in human endothelial cells (Leikert et al., 2002;
When BH4 is limited, eNOS becomes uncoupled, Wallerath et al., 2003; Rathel et al., 2007; Simoncini
and superoxide ion is produced instead of NO. Thus et al., 2011). Simoncini et al. (2011) reported that the
BH4 availability is essential for normal endothelial compounds derived from wine enhanced expression
function. Hence, Faria et al. (2012) demonstrated of eNOS in human endothelial cells, indicating that the
that green tea reversed diabetes-induced reduction of wine extract acts at the transcriptional level. Isometric
BH4 levels, decreased eNOS uncoupling, leading to study using rat femoral artery reported that the red
increased NO production. wine polyphenol, Provinol elicited endothelium-
dependent relaxation by stimulating NOS activity
Grape and/ or wine concomitantly with scavenging free radicals, which
led to the enhancement of NO bioavailability
Intervention study (Zenebe et al., 2003). In spontaneously hypertensive
Recently, it has been suggested that grape rats (SHR), treatment with Alibernet red wine extract
polyphenols including epicatechin, catechin, (24.2 mg/kg/day) for three weeks contributed to an
quercetin, gallic acid and resveratrol have increase in NOS and superoxide dismutase (SOD)
vasculoprotective effects and can improve endothelial activities in left ventricle, aorta and kidney tissues
function. In CAD patients, consumption for 14 days (Kondrashov et al., 2012).
of four ml/kg grape juice improved FMD (Stein et al., It has been established that the compounds
1999). Similarly, Agewall et al. (2000) demonstrated contained in wine behave like estrogens. Wine
improvement of FMD one hour after consumption extract induces NO synthesis in vascular endothelial
of de-alcoholized red wine (250 ml) among healthy cells through the activation of estrogen receptors
volunteers (Agewall et al., 2000). Karatzi et al. expressed in vascular cells. These estrogen receptors
(2004) assessed the acute effects of 250 ml of either play important vascular regulatory actions. Indeed,
red wine or de-alcoholized red wine consumption on similar to estradiol, exposure of endothelial cells to
FMD in 15 males with CAD. FMD was shown to be wine polyphenols activates estrogen receptor alpha.
higher following the consumption of de-alcoholized The activation of estrogen receptors leads to the
red wine compared to regular red wine, suggesting recruitment of the mitogen-activated protein kinase
that the beneficial effects may be attributed to ERK 1/2 and phosphatidylinositol-3-OH kinase/Akt
the presence of polyphenols in wine without the pathways at the cell membrane or within the cell’s
presence of alcohol (Karatzi et al., 2004). Meta- cytosoplasm resulting in the eNOS activation that
analysis of nine studies revealed that intake of grape eventually increases NO production (Chalopin et
polyphenols increased FMD levels in both healthy al., 2010). Indeed, similar observations have been
and subjects with high cardiovascular risk (smoker demonstrated in a study using resveratrol treatment,
and CAD); the increase in FMD appeared to be much a polyphenol present in wine. Repeated treatment
more obvious in the latter subject groups. The effect with resveratrol for five days increased eNOS mRNA
of grape polyphenols on FMD in healthy subjects and protein expression in cultured human endothelial
was observed at 30 minutes after ingestion; the effect cells, possibly by activating the estrogen receptors
was delayed in subjects with high cardiovascular and PPARα in endothelial cells (Takahashi and
(CVS) risk, which was at 60 minutes after ingestion. Nakashima, 2012). Another mechanism by which
The difference in timing of the acute effects of grape grape polyphenols can exerts it cardioprotective
polyphenols between the two groups may be due benefit is through it antioxidant effects. In a study
to impaired endothelial function in the high CVS using aorta from female SHR, treatment with red wine
risk group. This is supported by the observation polyphenols (40 mg/kg for five weeks) improved
that baseline FMD in the group with high CVS risk endothelial function which was associated with
ranged from 2.6% to 5.65%, which were lower than reduced vascular oxidative stress (Lopez-Sepulveda
in the healthy group (5.4% to 7.4%) (Li et al., 2013). et al., 2011).

Experimental / In vitro study Cocoa

In vitro and animal studies have indicated that
grape polyphenols enhance eNOS activity and Intervention study
increase NO production in endothelial cells. The Cocoa is derived from the seeds of the fruit from
compounds obtained by purification of the non- Theobroma cacao tree. Human trials suggested that
alcoholic fraction of wine enhanced eNOS mRNA cocoa or chocolate consumption were correlated
 Mokhtar, S. S. and Rasool, A. H./IFRJ 24(2): 471-482 477

with improvement in NO-mediated FMD and on eNOS expression and activity, cocoa flavanols
increased plasma or urine NO-derived species also exert antioxidant effects in vitro. Treatment
(S-nitrosothiols). Faridi et al. (2008) showed that of cultured human endothelial cells with cocoa-
acute ingestion of solid dark chocolate (22 g cocoa derived epicatechin increased NO concentration
powder) and liquid cocoa improved FMD in 45 via inhibition of nicotinamide adenine dinucleotide
overweight adults (Faridi et al., 2008). Recently, West phosphate (NADPH) oxidase, which is an enzyme
et al. demonstrated enhanced vasodilatation in both that catalyzes the production of superoxide anion
conduit and resistance arteries of overweight women (Steffen et al., 2007).
after consumption of high-flavanol cocoa drink (814
mg/day) and dark chocolate (37 g/day) (West et al., Soy
2014). Ingestion of high-flavanol cocoa drink (917
mg of flavonols) increased NO metabolites in plasma Intervention studies
and urine of healthy subjects, and this was associated There is a growing interest on the effects of soy
with improvement of FMD (Schroeter et al., 2006). It isoflavones on endothelial dysfunction in animals
has been demonstrated that administration of a high and humans. Human trial in healthy, postmenopausal
flavonoid-cocoa drink transiently improved NO- women with hypercholesterolemia showed that
dependent FMD in the presence of pre-existing CVS four weeks treatment with soy protein (40 g soy
risk, including hypertension and diabetes (Heiss et al., protein powder; 80 mg isoflavones) increased FMD
2003; Grassi et al., 2005; Balzer et al., 2008). In pre- indicating the improvement in endothelial function
hypertensive and hypertensive patients, consumption (Cuevas et al., 2003). Hall et al. (2008) demonstrated
of 6.3 g of chocolate (30 mg of polyphenols) daily for that ingestion of isoflavone-enriched low-fat meal
18 weeks showed increased plasma NO metabolites increased endothelium-dependent vasodilatation
(Taubert et al., 2007). In healthy individuals, two in postmenopausal women (Hall et al., 2008). In
weeks ingestion of flavanoid-rich dark chocolate bar another study on healthy postmenopausal women,
(213 mg procyanidins, 46 mg epicatechin) improved treatment with soy isoflavone tablets (30 mg
FMD compared to ingestion of low-flavanoid dark genistin and 30 mg daidzein) for 6 months showed
chocolate bars (46 g, 1.6 o.z) (Engler et al., 2004). significant improvement in endothelium-dependent
Similarly, consumption of cocoa-derived beverage vasodilatation among these women (Colacurci et
over five days in 27 healthy volunteers improved al., 2005). Recently, a 12 months human trial was
NO-dependent vasodilatation (Fisher et al., 2006). conducted among 182 Indonesian postmenopausal
A meta-analysis of 11 chronic and 11 acute clinical women to determine the effect of 100 mg/day soy
studies suggested improvement in FMD after acute isoflavone tablets on vascular endothelial function.
and chronic ingestion of chocolate (Hooper et al., This study showed a reduction in oxidative stress
2012). through lowering of malondialdehyde (MDA)
concentration, which is a marker of lipid oxidation.
Experimental / In vitro study However there was no improvement in vascular
Animal study showed that treatment of rabbit endothelial function observed (Pusparini et al., 2013).
aorta with the cocoa extract, procyanidin caused The different results obtained between Pusparini et
endothelium-dependent vasodilatation, which was al. (2013) and other clinical studies may be due to
likely dependent on the activation of eNOS and few factors such as including duration and dosage of
increased NO production (Karim et al., 2000). soy isoflavones, as well as subjects’ characteristics.
Increased NO bioavailability is dependent on the The subjects in the study by Pusparini et al. (2013)
availability of the substrate for eNOS, L–arginine. consumed relatively high daily soy isoflavones (100
In mammals, arginase catalyzes the conversion mg/day) for a year, whereas other human trials that
of L-arginine to urea. Vascular arginase competes showed significant results only consumed low soy
with eNOS for their common substrate L-arginine, isoflavone for shorter durations. In addition, the study
and this may impair NO production. Schnorr et al. by Pusparini et al. (2013) was performed in an Asian
(2008) demonstrated that treatment with cocoa- country, whereas the other studies were conducted in
flavanols decreased arginase activity in humans Western countries, suggesting that ethnic influences
and reduced arginase mRNA expression in cultured may affect the different outcomes. Yildirir et al.
human endothelial cells. This finding suggested that (2001) also reported improvement in endothelial
cocoa-flavanols may contribute to the regulation function in 20 males with hypercholesterolemia
of L-arginine concentration and NOS substrate after soy ingestion (Yildirir et al., 2001). In patients
supply (Schnorr et al., 2008). Besides their effects with renal transplant, soy protein diet consumption
478 Mokhtar, S. S. and Rasool, A. H./IFRJ 24(2): 471-482

(25 g/day) for five weeks improved FMD and the 2013; Asgary et al., 2014). Furthermore, two studies
effects disappeared after soy withdrawal, suggesting demonstrated that consumption of pomegranate
that the improvement was dependent on soy intake juice (240 ml/day) for 30 days improved FMD in 30
(Cupisti et al., 2007). A meta-analysis of nine human adolescents with metabolic syndrome (Hashemi et
trials found that soy isoflavones increased FMD al., 2010; Kelishadi et al., 2011).
in postmenopausal women with low baseline, but
not in high baseline FMD levels (Li et al., 2010). Experimental / In vitro study
Recently, a Bayesian meta-analysis by Beavers et The principal mechanisms of action of
al. (2012) accumulated evidence from 17 human pomegranate juice on vasodilatation may be due
trials and revealed that exposure to soy isoflavones to enhanced expression of eNOS protein and NO
improved endothelial function as measured by FMD production. Treatment with pomegranate juice
(Beavers et al., 2012). The difference between these increased eNOS protein expression in cultured
two meta-analysis were that the first analysis omitted human endothelial cells and carotid arteries of
studies which included men and used soy protein, hypercholesterolemic mice (De Nigris et al., 2007).
while the latter expands the study inclusion criteria Besides, pomegranate juice also possesses potent
to accommodate a larger sample size without regard antioxidant activity. Pomegranate juice has been
to gender. shown to protect NO against oxidative damage, thus
resulting in increased bioavailability of NO in bovine
Experimental / In vitro study pulmonary artery (Ignarro et al., 2006).
The vascular effects of soy isoflavones were often
studied in animal models which have reduced levels Conclusion
of circulating estrogens and eNOS activity, such as
ovariectomized animals. It was demonstrated that diet Endothelial dysfunction is the precursor and
enriched in soy isoflavones improved acetylcholine- early marker in the development and progression of
induced endothelium-dependent vasodilatation in atherosclerosis. There is increasing evidence showing
aorta from ovariectomized rats (Catania et al., 2002) that several plant-derived foods taken in the diet
and coronary arteries from atherosclerotic female influences endothelial NO production and improves
monkeys (Honore et al., 1997). Besides, a diet rich in endothelial function. Thus, consumption of these
soy isoflavones also induces increase in antioxidant natural products as dietary supplements may serve as
and eNOS expression in animal aortas, leading to a preventive measure to prevent vasculopathy. They
improved endothelium-dependent vasodilatation and may also serve as an adjunct to routine conventional
reduced blood pressure (BP) (Mahn et al., 2005). treatments in patients with CVD.
Despite progress seen in the field of natural
Pomegranate products and their cardioprotective effects, further
research is still needed, especially in terms of
Intervention study phytochemical analyses of these active extracts and
Pomegranate (Punica granatum L. Punicaceae) is their pharmacological activities. In addition, more
a seeded or granular apple, a delicious fruit consumed detailed work needs to be performed on the synergistic
worldwide. Pomegranate contains substantial amounts effects of plant constituents, to understand whether it
of phenolic compounds, including flavonoids and can exert maximum therapeutic efficacy individually
hydrolysable tannins especially punicalagin. Most or in combination with other plant constituents. As
studies have demonstrated antioxidant, anticancer and most natural products have not been scientifically
anti-inflammatory properties of pomegranate (Faria evaluated, the information of their efficacy, safety
and Calhau, 2011; Ismail et al., 2012). Although and potential interaction with conventionally used
several studies have demonstrated cardioprotective drugs are limited. Thus, research aimed to fill the
role of pomegranate extracts such as attenuation of current gaps in knowledge about potential harm and
atherosclerosis development and reduction of in BP effectiveness of the plant-derived foods is needed to
(Aviram and Dornfeld, 2001; Aviram et al., 2004), translate their applications into the clinical setting.
there seemed to be very scarce literature that reports
the effect of pomegranate on endothelium-mediated Acknowledgements
vasodilatation.
In hypertensive patients, ingestion of pomegranate We thank Malaysian Ministry of Higher Education
juice (150 ml/day) for two weeks reduced BP and for providing a scholarship to SSM. We also thank
showed increasing trends in FMD (Asgary et al., USM Postgraduate Research Grant Scheme (1001/
 Mokhtar, S. S. and Rasool, A. H./IFRJ 24(2): 471-482 479

PPSP/8145001). Sturiale, A., Squadrito, F., Caputi, A. P. and Calapai,

G. 2002. Oral administration of a soy extract improves
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