nutrients

Review
Phytochemicals as Therapeutic Interventions in Peripheral
Artery Disease
Ahmed Ismaeel 1 , K. Leigh Greathouse 1,2 , Nathan Newton 3 , Dimitrios Miserlis 4 , Evlampia Papoutsi 1 ,
Robert S. Smith 5 , Jack L. Eidson 5 , David L. Dawson 5 , Craig W. Milner 5 , Robert J. Widmer 6 ,
William T. Bohannon 5 and Panagiotis Koutakis 1, *

1 Department of Biology, Baylor University, Waco, TX 76798, USA; ahmed_ismaeel@baylor.edu (A.I.);

Leigh_Greathouse@baylor.edu (K.L.G.); evlampia_papoutsi@baylor.edu (E.P.)
2 Department of Human Sciences and Design, Baylor University, Waco, TX 76798, USA
3 Department of Chemistry and Biochemistry, Baylor University, Waco, TX 76798, USA;
Nathan_Newton1@baylor.edu
4 Department of Surgery, University of Texas Health Science Center San Antonio, San Antonio, TX 78229, USA;
miserlisd@uthscsa.edu
5 Department of Surgery, Baylor Scott & White Medical Center, Temple, TX 76508, USA;
Robert.Smith@BSWHealth.org (R.S.S.); Jack.Eidson@BSWHealth.org (J.L.E.);
David.dawson@bswhealth.org (D.L.D.); craig.milner@bswHealth.org (C.W.M.);
William.Bohannon@BSWHealth.org (W.T.B.)
6 Heart & Vascular Department, Baylor Scott & White Medical Center, Temple, TX 76508, USA;
Robert.Widmer@BSWHealth.org



* Correspondence: Panagiotis_koutakis@baylor.edu



Citation: Ismaeel, A.; Greathouse, Abstract: Peripheral artery disease (PAD) affects over 200 million people worldwide, resulting in
K.L.; Newton, N.; Miserlis, D.; significant morbidity and mortality, yet treatment options remain limited. Among the manifestations
Papoutsi, E.; Smith, R.S.; Eidson, J.L.;
of PAD is a severe functional disability and decline, which is thought to be the result of different
Dawson, D.L.; Milner, C.W.; Widmer,
pathophysiological mechanisms including oxidative stress, skeletal muscle pathology, and reduced
R.J.; et al. Phytochemicals as
nitric oxide bioavailability. Thus, compounds that target these mechanisms may have a therapeutic
Therapeutic Interventions in
effect on walking performance in PAD patients. Phytochemicals produced by plants have been
Peripheral Artery Disease. Nutrients
2021, 13, 2143. https://doi.org/
widely studied for their potential health effects and role in various diseases including cardiovascular
10.3390/nu13072143 disease and cancer. In this review, we focus on PAD and discuss the evidence related to the clinical
utility of different phytochemicals. We discuss phytochemical research in preclinical models of PAD,
Academic Editors: Cristina Nocella and we highlight the results of the available clinical trials that have assessed the effects of these
and Winston Craig compounds on PAD patient functional outcomes.

Received: 27 May 2021 Keywords: polyphenols; beetroot; cocoa; flavonols; claudication; hindlimb ischemia
Accepted: 19 June 2021
Published: 22 June 2021

Publisher’s Note: MDPI stays neutral 1. Introduction

with regard to jurisdictional claims in
Although modern nutritional science is thought to date back to only the 20th century
published maps and institutional affil-
(with the discovery of vitamins), phytochemicals, or plant-derived chemicals, have been
iations.
used traditionally in medicine for thousands of years across the world [1]. In fact, the
Greek physician Dioscorides wrote a pharmacopoeia of medicinal plants, De materia medica,
as early as the year 50 AD [2]. Following the era of nutrient discovery with a focus on
deficiency diseases, the latter half of the 20th century saw nutritional research more focused
Copyright: © 2021 by the authors.
on identifying the relationship between food, nutrition, and chronic diseases [1,3]. Large
Licensee MDPI, Basel, Switzerland.
epidemiologic studies have consistently demonstrated inverse relationships between fruit
This article is an open access article
and vegetable consumption and the risk of a wide range of chronic diseases as well as
distributed under the terms and
all-cause mortality [4–6]. This relationship has been largely studied in the context of
conditions of the Creative Commons
Attribution (CC BY) license (https://
coronary heart disease and stroke, and higher fruit and vegetable intake are consistently
creativecommons.org/licenses/by/
associated with reduced risk of these cardiovascular/cerebrovascular diseases [7]. The most
4.0/).
common underlying cause of cardiovascular and cerebrovascular disease is atherosclerosis,

Nutrients 2021, 13, 2143. https://doi.org/10.3390/nu13072143 https://www.mdpi.com/journal/nutrients

Nutrients 2021, 13, 2143 2 of 23

a disease involving multiple factors including inflammation, oxidative stress, endothelial

dysfunction, and dyslipidemia [8]. Interestingly, subclinical measures of atherosclerosis
are also inversely associated with fruit and vegetable intake [9], and in animal models,
fruit and vegetable supplementation can reduce subclinical markers of atherosclerosis [10].
Another atherosclerotic disease, peripheral artery disease (PAD), affects arteries other
than those supplying the brain or heart, most commonly affecting the femoropopliteal
or infrapopliteal arteries of the legs [11,12]. Although there has been less research on
dietary composition and PAD, the available data suggest a similar association of reduced
PAD risk with higher fruit and vegetable intake. One cross-sectional study including
over 3.6 million participants showed that consumption of 3 or more servings of fruits
and vegetables per day was associated with 18% lower odds of PAD than consuming
≥3 servings of fruits and vegetables less than once a month [13]. Likewise, in a case-cohort
study of 944 patients with type 2 diabetes, a high-score Mediterranean dietary pattern (diet
rich in plant-based foods and polyphenol-rich olive oil) was independently associated with
a 56% reduced risk of PAD [14]. These data suggest that fruits and vegetable consumption
may be a lifestyle and dietary risk factor for PAD.
The mechanisms behind the health benefits associated with fruits and vegetables
are thought to be largely attributable to phytochemical components of plants [15]. There
have been thousands of identified phytochemicals which are divided into classes based on
their chemical structures that largely dictate their function [16]. These categories broadly
include flavonoids, stilbenes, organosulfur compounds, nitrogen-containing compounds,
carotenoids, and diarylheptanoids [16]. The main mechanism by which phytochemicals
are thought to exert their health-promoting effects is via antioxidant effects. Although
some phytochemicals possess free radical scavenging ability in vitro [17], direct reaction
with reactive oxygen species (ROS) in vivo is less likely due to the lower dose of the phy-
tochemicals in vivo. For example, phytochemical concentrations in plasma are around
~1 µM, and the levels in tissue are not believed to exceed the nanomolar range, while
antioxidant enzymes such as superoxide dismutase can be present at concentrations as
high as ~20 µM [18]. Instead, phytochemicals can act to induce de novo expression of
endogenous antioxidant defense genes, such as glutathione peroxidase, superoxide dismu-
tase, NAD(P)H quinone oxidoreductase, and heme oxygenase-1 [16]. These genes contain
a common antioxidant-responsive element in their 50 -flanking regions, which can be bound
and regulated by transcription factors such as nuclear factor erythroid 2-related factor
(Nrf2) or the aryl hydrocarbon receptor [19,20]. Various phytochemicals have been shown
to enhance the expression and activation of these transcription factors, thus inducing the
activation of target antioxidant enzymes [16]. In addition to these mechanisms, organosul-
fur, and nitrogen-containing phytochemicals are thought to exert unique protective effects
by increasing nitric oxide and hydrogen sulfide bioavailability [21]. Both nitric oxide and
hydrogen sulfide are important gaseous transmitters that work together to induce smooth
muscle relaxation, vasodilation, increased blood flow, and angiogenesis [22], and hydrogen
sulfide is also a known activator of Nrf2 [23]. Notably, PAD patients have been shown to
have reduced levels of hydrogen sulfide, reduced Nrf2 activation, and reduced NO bioavail-
ability compared to non-PAD controls [24,25]. Modulation of these pathophysiological
processes by phytochemicals may be viable therapeutic options for PAD patients.
Phytochemicals have been studied in a wide range of conditions ranging from can-
cer [26] to musculoskeletal disorders [27]. Likewise, previous reviews have focused on the
potential cardiovascular and cerebrovascular beneficial effects of phytochemicals [28,29].
However, no comprehensive review article has reported on the preclinical and clinical
vascular beneficial effects of these compounds in the context of limb ischemia, a major com-
plication of PAD. Thus, the purpose of this narrative review is to summarize the research
assessing the effectiveness of different phytochemicals as therapeutic interventions in the
context of PAD. We include studies on phytochemicals that have been studied in preclinical
models of the disease as well as in clinical trials including PAD patients.
Nutrients 2021, 13, 2143 3 of 23

2. Peripheral Artery Disease

PAD is an atherosclerotic condition of the extremities, mostly affecting arteries of
the legs [12]. According to estimates, over 200 million people globally are affected with
PAD [30]. PAD is especially common in older individuals, affecting up to 27% of 45- to
74-year-olds and 1 in 3 individuals in the 91–100-year-old age group [31,32]. Based on the
Fontaine classification system, asymptomatic PAD patients are classified in Stage I, and
patients presenting with the most common symptom of PAD, intermittent claudication,
(walking-induced leg pain) are classified in Stage II. In the later stages of PAD, patients with
foot pain at rest are classified in Stage III, and Stage IV patients with ulcers and gangrene
are considered to have critical limb ischemia [33].
PAD is associated with an elevated risk of all-cause mortality, cardiovascular disease
mortality, myocardial infarction, and stroke [34–36]. In addition, PAD patients experience
exercise limitations and dramatic reductions in walking performance of up to 50% of
non-PAD controls [37–39]. The pathophysiology of the functional impairment found
in PAD is believed to encompass different mechanisms, including oxidative stress [40],
skeletal muscle myopathy [41], and endothelial dysfunction [42]. Increased oxidative stress
markers have been documented in PAD, and oxidative stress markers are correlated with
reduced walking distances of patients [43,44]. Likewise, nitric oxide levels are reduced
in PAD [24], and flow-mediated dilation (FMD), a measure of endothelial function, is
associated with patients’ walking ability [45]. Notably, inflammation is also thought to
play a role in the pathophysiology of PAD [46]. Several inflammatory markers have been
shown to have a strong predictive value for the development and presence of PAD [46].
Large cohort studies have shown that markers of inflammation such as C-reactive protein
(CRP) and cellular adhesion molecules are independently associated with PAD risk [47,48].
In addition to their role as prognostic indicators; however, inflammatory molecules are
also implicated in disease mechanisms. In fact, atherosclerosis has been described as an
“inflammatory disease,” with evidence suggesting that components of inflammation in
the arteries can lead to progressive arterial damage [49]. Inflammatory cytokines such as
tumor necrosis factor alpha (TNF-α) and interleukins (ILs) can lead to arterial stiffening
via increased leukocyte infiltration, enhanced elastin degradation, and increased vascular
smooth muscle cell de-differentiation and collagen synthesis [50]. Immune cells recruited
by inflammatory cytokines can also alter oxidative balance, as these cells are known to
be major sources of ROS [51]. Furthermore, inflammatory molecules such as CRP can
directly inhibit endothelial NO synthase activity [52]. CRP can also promote production of
superoxide, which can result in NO synthase uncoupling by oxidation of the critical NOS
co-factor, tetrahydrobiopterin [53]. Therefore, oxidative stress, inflammation, endothelial
dysfunction, and the interactions between these processes are important considerations in
PAD pathogenesis.
Without therapeutic interventions, PAD also leads to significantly accelerated func-
tional decline over time [54]. Thus, although the primary treatment goals for patients is to
improve cardiovascular risk and limb salvage, improving exercise performance, daily func-
tional and physical activity, and quality of life are also important treatment considerations.
Recent research on PAD interventions have focused on targeting oxidative stress, skele-
tal muscle pathology, and nitric oxide bioavailability to improve functional outcomes in
patients. For practical significance, the minimal clinically important differences in walking
performance following an intervention for PAD patients have been defined. For the 6-min
walk test, a small meaningful change is considered an improvement in walking distance
of 8–20 m, and a large meaningful change has been defined as a change of 50 m [55,56].
During a treadmill test, an increase in peak walking time of at least 38 s and an increase
in claudication onset time of at least 35 s have been suggested as small minimal clinically
important differences [57]. Currently, treatment options for PAD management are limited
with only two modestly effective medications approved for claudication [58,59]. On the
other hand, supervised exercise training has been shown to lead to greater improvements
in walking performance in PAD [60]. Despite the success of individual interventions, the
Nutrients 2021, 13, 2143 4 of 23

long-term efficacy of all treatment options for PAD remains questionable. For example, an
interesting recent network meta-analysis of 46 randomized controlled trials compared the
short-, moderate-, and long-term outcomes of a variety of treatments for PAD including
supervised exercise therapy, endovascular revascularizations, and cilostazol [61]. It was
found that although some treatments effectively improved walking distance at short- or
moderate-term follow-up, none of the treatments improved walking performance of PAD
patients in the long-term (≥2 years) [61]. This highlights the dire need for more durable
PAD treatments.
In addition, exercise bouts have been shown to increase ROS biomarkers in PAD pa-
tients, and some studies have shown that exercise can lead to pathologic changes in skeletal
muscle and skeletal muscle injury [62–64]. This observation has led some researchers to
suggest the addition of antioxidant supplementation along with exercise interventions to
alleviate exercise-induced oxidative stress [65,66]. Although some smaller studies have
suggested that nonspecific antioxidants such as vitamin C and vitamin E may improve
exercise tolerance in PAD patients [65,67,68], large-scale trials such as the “POPADAD”
trial and the “HOPE” trial found no evidence that these antioxidants can improve out-
comes such as revascularizations, amputations, and cardiovascular events [67,68]. Some
researchers argue that this may be due to the fact that the concentration of vitamins C
and E that would be necessary to induce antioxidant effects in the vascular wall may be
unachievable, neither vitamin reacts appreciably with hydrogen peroxide, and the vitamins
themselves may also be oxidized and inactivated by free radicals [69,70]. Instead, a better
strategy may be therapies with antioxidant concepts such as phytochemicals that act to
increase antioxidant capacity by activation of endogenous antioxidant defense systems.
Thus, in recent years, a number of preclinical studies and a few clinical trials have sought
to test the efficacy of individual plant components for the mitigation of the oxidative stress
and oxidative-stress-induced damage and pathology associated with limb ischemia. These
studies are discussed in the next sections.

3. Preclinical Research
3.1. Hindlimb Ischemia
Preclinical murine models of hindlimb ischemia (HLI) have long been used to simulate
PAD pathophysiology [71]. The most common method for induction of HLI is by ligation
or excision of the femoral artery [72]. However, this approach is considered an acute limb
ischemia model, and blood flow recovers rapidly due to the quick formation of collateral
circulation [73]. Thus, other approaches have been developed, such as gradual femoral
artery occlusion with ameroid constrictors (subacute) [74] and a two-stage limb ischemia
model (chronic), which are considered more relevant to the human disease [75,76]. HLI
models typically result in reduced limb perfusion, functional deficits, reduced walking
performance, systemic inflammation, and skeletal muscle damage [73]. However, it is
important to note that other manifestations that play an important role in PAD pathogenesis
may not be well-modeled in animal HLI studies. For example, PAD can be accompanied
by ulcers; yet, an animal model of non-healing ulcers in limb ischemia has not yet been
established [77]. In fact, very few studies have demonstrated wound generation in the
clinically relevant peripheral site of the limb or in older animals, and other issues related
to study design and bias reduction also exist [77]. It is also important to note that the
HLI induction technique as well as other factors, including species (rat vs. mouse), strain,
age, and the presence of other comorbid conditions, can affect the resulting ischemic
damage [74]. Regardless of the procedure, HLI models are considered useful for testing
new therapies for PAD treatment [73], and have thus been used to study a wide range of
potentially beneficial phytochemicals.

3.2. Flavonoids
Flavonoids are a category of polyphenolic metabolites found in plants, characterized
by a general structure of a 15-carbon skeleton, consisting of 2 phenyl rings and a hetrocyclic,
Nutrients 2021, 13, 2143 5 of 23

oxygen-containing ring [78]. Flavonoids are further categorized into six subgroups, the
anthocyanidins, anthoxanthins, flavanones, flavanonols, flavans, and isoflavonoids [79].
The results of a meta-analysis of flavonoid effects on aortic atherosclerosis in mice suggest
that flavonoids can significantly reduce aortic atherosclerosis [80]. Interestingly, atheroscle-
rosis area-reducing effects were isolated to flavonols, but not flavan-3-ols, suggesting that
flavonols may hold the most potential therapeutic value in atherosclerosis [80].
Quercetin (Figure 1A), an anthoxanthin, is a flavonoid found in many fruits and veg-
etables, with the most appreciable amounts found in red onions and kale [81]. Quercetin
has been well-studied in basic research due to its antioxidant potential [82]. In rats un-
dergoing acute HLI, 200 mg/kg/day of quercetin, administered for one week prior to
ischemia induction, significantly increased antioxidant enzyme activity and reduced lipid
peroxidation markers [83]. However, in contrast to this study, a more recent study assessed
the effect of quercetin (~185 mg/kg/day for 30 days following HLI) in the sustained, two-
stage model of HLI [84]. These mice also had dyslipidemia (apolipoprotein E-deficient),
which further simulates human PAD [76]. Utilizing this model, quercetin had no effect on
limb perfusion. Despite this, plasma nitrate and nitrite concentrations were significantly
increased in mice that consumed quercetin at the end of the study, although oxidative stress
markers were not measured [84]. Furthermore, this study also measured the clinically
relevant variables of exercise performance and physical activity on a treadmill as well as an
open field test, and quercetin was shown to have no effect on distance travelled or velocity
during either test [84]. Therefore, although it may have some effect on reducing oxidative
stress markers, due to the lack of improvement in functional performance during sustained
HLI, quercetin is unlikely to be effective for PAD treatment. Notably, this ineffectiveness
may be due to its low bioavailability. In another study, quercetin glucosides (enzymatically
trans-glycosylated isoquercitin), which have a higher solubility in water and better bioavail-
ablity compared with quercetin, were administered to mice (100 mg/kg/day) for 2 weeks
prior to acute HLI induction and 2 weeks following surgery [85]. Blood flow recovery
and capillary density were augmented in ischemic limbs of mice that received quercetin
glucosides, along with plasma glutathione levels [85]. Thus, conversion of quercetin into
glucosides may be a promising strategy to enhance its bioavailability and effectiveness in
future interventions.
The flavan subgroup of flavonoids include catechin, epicatechin, and epigallocatechin
3-gallate (EGCG) [79]. EGCG (Figure 1B) is highly abundant in green tea and is believed to
be responsible for much of the effects mediated by tea [86]. Although EGCG administration
(50 mg/kg, administered 30 min prior to reperfusion) was shown to reduce oxidative status
and increase total antioxidant status following acute HLI-reperfusion in rats (2 h), EGCG
had no protective effect on oxidative or antioxidant status following long-term reperfusion
(24 h), which better resembles true ischemia-reperfusion conditions [87]. Furthermore,
serum inflammatory cytokine levels (IL-1, IL-6, IL-8, and TNF-α) were unaffected by EGCG
administration in either short- or long-term reperfusion [87]. EGCG, due to its pyrocatechol
structure, has been shown to coordinate with metal ions, and these metal-EGCG networks
have also been applied to the study of different conditions [88,89]. In mouse HLI models
established by femoral artery ligation and excision, copper-EGCG capsules or zinc-EGCG
capsules injected into ischemic muscles reduced ischemic damage and improved blood
flow recovery, vessel volume, and angiogenic markers [90,91]. However, there were no
significant differences with respect to the levels of the inflammatory cytokines TNF-α
and IL-6. Therefore, although these data suggest that metal-EGCG coordinated capsules
may hold promise as potential medical therapies, their safety and efficacy must first be
established in humans. Additionally, it is unclear how much of the benefit was due to the
metal compared to EGCG. Thus, there is not enough evidence to suggest that EGCG alone
may be an effective treatment for PAD.
Nutrients 2021, 13, 2143 6 of 23

Figure 1. Polyphenol chemical structures. (A) is the chemical structure of quercetin, (B) is the chemical structure of
epigallocatechin gallate (EGCG), (C) is the chemical structure of catechin, (D) is the chemical structure of epicatechin, (E) is
the chemical structure of betanin, (F) is the chemical structure of resveratrol, (G) is the chemical structure of allicin, (H) is
the chemical structure of diallyl disulfide (DADS), (I) is the chemical structure of diallyl trisulfide (DATS), (J) is the chemical
structure of sulforaphane, (K) is the chemical structure of lycopene, and (L) is the chemical structure of curcumin.

Catechin (Figure 1C) and epicatechin (Figure 1D) are found in many different foods
including prune juice, peaches, tea, and açaí. Notably, cocoa is believed to have the highest
content of catechins among all foods (>100 mg/100 g) [78]. Catechin and epicatechin have
been heavily studied in different preclinical models. In one in vitro study, supernatant of
activated platelets from PAD patients were used to increase soluble cell adhesion molecules
(markers of endothelial activation and inflammation) and reduce NO bioavailability in
human umbilical vein endothelial cells [92]. Pretreatment with 0.1–10 µM of catechin and
epicatechin was shown to reduce the increase in adhesion molecules induced by PAD
platelets and increase NO levels [92]. Furthermore, in a mouse HLI model induced by two
ligations proximal and distal to the femoral artery, epicatechin feeding, starting at 5 days
prior to ischemia induction, improved perfusion recovery and capillary density in a dose-
dependent manner, with maximal effects seen at 2 mg/kg per day [93]. These promising
preclinical results have led to a number of clinical trials in PAD patients using catechin-
and epicatechin-rich dark chocolate/cocoa, which will be discussed in later sections.

3.3. Nitrogen-Containing Compounds

Certain phytochemicals that contain nitrogen atoms provide a dietary source of inor-
ganic nirate, which been suggested to have potential beneficial effects related to modulation
of NO levels [94]. Nitrate-containing vegetables, such as spinach, arugula, celery, and
beetroot, have been thought to be key mutual components of diets associated with low in-
cidences of cardiovascular disease (i.e., Mediterranean and Japanese traditional diets [94]).
Notably, nitrate therapy has been showed to induce pro-angiogenic effects in several mod-
els of HLI, including murine [95,96] and swine [97,98]. Of all the studied dietary nitrate
donors, beetroot (Beta vulgaris) is one of the most heavily researched [94]. Aside from the
nitrate-mediated effects of beetroot, associated health benefits may also be a consequence
of the properties of the betanin pigment found in beetroot (Figure 1E). Betanin is one of
the betalain pigments, which are a group of red/yellow pigments found in plants, chemi-
cally characterized as tyrosine-derived aromatic indoles [99]. Compounds with an indole
moiety are known to possess antioxidant activity and likely activate the aryl hydrocarbon
Nutrients 2021, 13, 2143 7 of 23

receptor [100]. A number of studies have also suggested that betalains may possess strong
antioxidant and anti-inflammatory properties [101–103]. During the last 10 years, beetroot
has been extensively studied in preclinical and clinical research for its potential cardiovas-
cular benefits [104]. However, beetroot has yet to be studied in any animal models of limb
ischemia. In a cardiovascular model, mice were exposed to coronary artery occlusion for
30 min followed by 24 h of reperfusion as a model of cardiac ischemia-reperfusion [105].
Interestingly, mice given beetroot juice powder at a dosage containing 0.7 mM nitrate
showed reduced infarct size and improved ventricular function [105]. This suggests that
beetroot may play a protective role in the context of ischemia-reperfusion injury, including
limb ischemia, but studies in a HLI model are required before drawing definite conclusions.

3.4. Stilbenes
Stilbenoids are hydroxylated derivatives of 1,2-diphenylethene (stilbene), charac-
terized by a C6-C2-C6 structure [106]. One of the most widely studied stilbenoids is
3,5,40 -trihydroxy-trans-stilbene, or resveratrol (Figure 1F), which is known for its content
in red wine as well as red or purple grapes. Early in vitro data and in vivo work sug-
gested that resveratrol may hold therapeutic potential for lifespan extension as well as the
prevention or slowed progression of different diseases, including cancer, cardiovascular
disease, and diabetes [107–109]. While the exact mechanisms are unclear, resveratrol has
been shown to exert potent antioxidant effects by modulating antioxidant enzymes and
intrinsic antioxidant capacity [110]. Furthermore, resveratrol is a known activator of sirtuin
1, a histone deacetylase enzyme that regulates expression of genes involved in metabolic
regulation, survival, autophagy, and stress response [110].
In acute rat models of HLI, administration of 1–20 mg/kg/day resveratrol was shown
to reduce oxidative stress markers, improve hindlimb skeletal muscle histology (reduced
myofiber atrophy, segmental necrosis, and edema), and protect femoral artery tissue from
vascular wall thickening, apoptosis, inflammatory cell infiltration, and edema [111–113].
In rabbits with HLI induced by femoral artery excision, resveratrol (2.5 mg/day) has also
been demonstrated to have pro-angiogenic effects by improving endothelial NO synthase
expression, limb blood flow, and capillary density [114]. Similar effects were shown after
injection of bone marrow derived nuclear cells incubated with 100 µM resveratrol into
hindlimb muscles of mice with HLI induced by femoral artery excision [115]. Thus, in
preclinical models, resveratrol has shown efficacy in improving ischemic pathology.

3.5. Organosulfur Compounds

Organosulfides refer to sulfur-containing organic compounds, which are classified
according to the sulfur-containing functional group. One of these compounds is allicin
(Figure 1G), the active flavor compound in chopped or crushed garlic [116]. After formation,
allicin is quickly converted into compounds including diallyl disulfide (DADS) (Figure 1H)
and diallyl trisulfide (DATS) (Figure 1I) [117]. Importantly, both DADS and DATS have
been shown to play a role in cellular detoxification and protection against oxidative stress,
and DATS is further known to be a source of hydrogen sulfide [118]. Thus, DATS has been
widely studied in the context of ischemia-reperfusion injury [119]. In a mouse model of
HLI induced by femoral artery ligation, injection of 500 µg/kg/day i.p. DATS for 10 days
improved blood flow recovery, capillary density, and NO bioavailability [120]. These
results were also duplicated in mice with concomitant HLI and streptozotocin-induced
diabetes [121].
Another promising organosulfur compound is sulforaphane (Figure 1J), a compound
found in cruciferous vegetables such as broccoli that is known to be a potent Nrf2 activa-
tor [122]. Importantly, sulforaphane has been shown to protect against ischemia-reperfusion
injury in preclinical models of cerebral [123], retinal [124], intestinal [125], myocardial [126],
and renal [127] ischemia. Sulforaphane has also recently been studied for its potential
impact on cardiac and skeletal muscle dysfunction associated with aging [128]. In older
mice (21–22 months old), feeding 442.5 mg/kg/day of sulforaphane for 12 weeks restored
Nutrients 2021, 13, 2143 8 of 23

the age-associated loss in mitochondrial function, cardiac function, skeletal muscle satellite
cell activation and differentiation, and skeletal myofiber cross-sectional area [128]. These
changes were also accompanied by improved exercise performance, assessed as improved
running time and work until exhaustion during a treadmill test and improved isometric
grip strength [128]. Sulforaphane may also play a role in the attenuation of atherosclerosis.
In a streptozotocin-induced diabetic mouse model, feeding 0.5 mg/kg/day of sulforaphane
for 12 weeks prevented the diabetes-associated aortic fibrosis [129]. Likewise, in rabbits fed
a high cholesterol diet, supplementation of the diet with 0.25 mg/kg/day of sulforaphane
reduced fibrosis and edema in aortic tissue, normalized NO levels, and improved en-
dothelial function [129]. Although sulforaphane has not been specifically studied in the
context of HLI, studies from other in vivo models of similar pathophysiology suggest that
sulforaphane may be a promising therapeutic in the context of limb ischemia.

3.6. Carotenoids
Carotenoids are yellow, orange, or red pigments produced mainly by plants and algae.
Their general structure is characterized by a polyene hydrocarbon chain, consisting of
4 terpene units each containing 10 carbon atoms [130]. Carotenoids are further divided
into oxygenated (xanthophylls) and oxygen-free subclasses (carotenes). Carotenes include
α-carotene, β-carotene, and lycopene. Both α- and β-carotene are considered precursors
to Vitamin A (provitamin A) [130]. Interestingly, despite the antioxidant properties of
carotenes, two large cross-sectional studies (The Rotterdam study, N = 4367 and The
Edinburgh Artery Study, N = 1592) both showed no association between dietary intake of
β-carotene and risk of PAD or lower extremity blood flow [131,132]. Although no studies
have assessed the effectiveness of α- or β-carotene supplementation in preclinical or clinical
limb ischemia, lycopene (Figure 1K) has been studied in this context. In rats with HLI,
induced by clamping of the abdominal aortae, feeding 10 mg/kg/day for 15 days prior to
ischemic induction protected hindlimb skeletal muscles from atrophy and necrosis [133].
Lycopene has also been shown to protect against injury due to other forms of ischemia,
including myocardial [134] and cerebral [135] ischemia. However, it is important to note
that issues related to lycopene tissue bioavailability may limit its clinical utility [136,137].
The low bioactivity may explain the lack of effectiveness of lycopene consumption on
cardiovascular markers in humans [138]. Future studies should take into account factors
that may affect the absorption, bioavailability, and bioconversion of specific carotenoids.

3.7. Diarylheptanoids
Diarylheptanoids are a group of secondary metabolites of plants, consisting of two
aromatic rings connected by a seven carbon chain [139]. Curcuminoids are linear diarylhep-
tanoids, and the most widely studied is curcumin (Figure 1L), isolated from the turmeric
flowering plant of the ginger family. Curcumin has been studied in numerous laboratory
and clinical studies of several different diseases, including HLI models. In mice with
HLI induced by a double ligation of the femoral, great saphenous, and iliac circumflex
arteries with a suture, injection of 100 mg/kg i.p. of curcumin 1 h prior to ligation reduced
skeletal muscle fibrosis and enhanced muscle fiber density [140]. These effects also trans-
lated to enhanced running capacity during a treadmill test in curcumin-treated mice [140].
Curcumin has also been shown to elicit pro-angiogenic effects in HLI. In mice with HLI
induced by femoral artery ligation and excision, feeding 1000 mg/kg/day of curcumin for
2 weeks improved perfusion recovery and increased capillary density [141]. This was also
demonstrated in mice exposed to the same HLI conditions and concomitant streptozotocin-
induced diabetes [142]. However, despite these promising preclinical findings, curcumin’s
low bioavailability and limited tissue distribution limit its bioactivity and clinical effec-
tiveness [143]. This has led researchers to call curcumin “deceptive” and “a cautionary
tale” [144]. In fact, despite >120 clinical trials of curcumin in several different disease
conditions, no double-blinded, placebo controlled clinical trial has been successful [143].
Nutrients 2021, 13, 2143 9 of 23

Thus, curcumin is likely not a viable approach for PAD treatment. Table 1 summarizes the
results of the preclinical studies discussed.

Table 1. Preclinical studies of phytochemicals in HLI models.

Dosage and Treatment

Compound Species HLI Induction Results Reference
Administration Schedule
Reduced lipid peroxidation
200 mg/kg/day 1 week prior to
Rat Tourniquet and increased antioxidant [83]
(orally) HLI
enzyme activity
Glucosides: 2 weeks prior to
Resection of femoral Enhanced blood flow recovery,
100 mg/kg/day HLI and 2 weeks Mouse [85]
Quercetin and saphenous artery increased capillary density
(gavage) following
2-stages: 1st stage:
No effect on exercise
ameroid constrictors
30 days, performance or limb
185 mg/kg/day around femoral
beginning 5 days Mouse perfusion, but increased [84]
(orally) artery; 2nd stage:
after HLI plasma nitric oxide
resection of femoral
metabolites
artery
50 mg/kg reduced total
oxidative status and increased
total antioxidant status of
skeletal muscle during acute
25 or 50 mg/kg 30 min prior to Tourniquet (ischemia-
Rat reperfusion (2 h); neither dose [87]
(i.p. injection) reperfusion reperfusion)
affected oxidant/antioxidant
status long-term (24 h); no
effect on inflammatory
cytokines
EGCG
Copper-EGCG Increased angiogenic markers,
(2 µg) 1, 3, and 5 days Ligation and excision enhanced blood flow recovery,
Mouse [91]
(intramuscular after HLI of femoral artery reduced ischemic damage,
injection) improved vessel volume
Zinc-EGCG Increased angiogenic markers,
(0.682 µg) 1, 3, and 5 days Ligation and excision enhanced blood flow recovery,
Mouse [90]
(intra-muscular after HLI of femoral artery reduced ischemic damage,
injection) improved vessel volume
Improved perfusion recovery
1–10 mg/kg/day 5 days prior to Ligation of femoral
Catechin Mouse and increased capillary [93]
(orally) HLI artery
density
Alleviated femoral artery
1 mg/kg/day 8 weeks following tissue damage, reduced lipid
Rat Tourniquet [111]
(orally) HLI peroxidation, and increased
antioxidant expression
Immediately Improved skeletal muscle
10 mg/kg (i.p. Tourniquet (ischemia-
prior to Rat histopathology, reduced lipid [112]
injection) reperfusion)
reperfusion peroxidation
Reduced markers of muscle
20 mg/kg/day 2 weeks prior to Tourniquet (ischemia-
Rat damage and oxidative stress [113]
(orally) HLI reperfusion)
markers
2.5 mg/kg
Resveratrol (loading Resection of femoral,
subcutaneous popliteal, and Increased limb blood pressure
4 weeks following
dose), followed Rabbit saphenous arteries, ratio, angiographic score, and [114]
HLI
by and ligation of angiogenic markers
2.5 mg/kg/day external iliac artery
(orally)
Intramuscular
injection of bone
Enhanced blood flow recovery,
marrow
Immediately Ligation and excision increased capillary density
mononuclear cells Mouse [115]
following HLI of femoral artery and angiogenic markers,
incubated with
reduced ROS production
100 µM
resveratrol
Nutrients 2021, 13, 2143 10 of 23

Table 1. Cont.

Dosage and Treatment

Compound Species HLI Induction Results Reference
Administration Schedule
Enhanced blood flow recovery
500 µg/kg/day 10 days following Ligation of femoral
Mouse and capillary density, reduced [120]
(i.p. injection) HLI artery
oxidative stress
Enhanced blood flow recovery
DATS and capillary density,
increased angiogenic markers,
500 µg/kg/day 2 weeks following Ligation of femoral
Mouse increased NO reduced [121]
(i.p. injection) HLI artery
oxidative stress markers,
increased tissue nitric oxide
metabolites
Reduced lipid peroxidation,
15 days prior to Tourniquet (ischemia- increased antioxidant levels,
Lycopene 10 mg/kg/day Rat [133]
HLI reperfusion) improved skeletal muscle
histopathology

3.8. Phytochemical Bioavailability

One of the issues related to the clinical utility of phytochemicals is that of bioavail-
ability. While certain compounds can demonstrate promising results in vitro or in animal
models, these effects do not always translate to human studies [145]. Cell culture studies
only assess biological effects, without taking into account the important factors of digestion,
absorption, metabolism, and tissue distribution [146]. Moreover, animal studies often use
supra-physiological dosages of phytochemicals that are beyond what can be achieved
with normal human dietary intakes [147]. In addition, differences in animal and human
metabolism may also lead to differential results. For example, there are known variations
in the metabolites that result from the consumption of phenols between rodents and hu-
mans [148]. Importantly, many factors can influence bioavailability in vivo. One of the
most important determinants for a compound’s bioavailability is related to its chemical
structure. For example, according to Lipinski et al.’s ‘rule of 50 , compounds with more than
five hydrogen-bond donors, 10 hydrogen-bond acceptors, a molecular weight over 500 dal-
tons, and a common logarithm of the partition coefficient (logP, a measure of lipophilicity)
greater than 5, can be considered to have poor absorption [149]. Certain phytochemicals,
such as curcumin, fall into this category. Still, other compounds that would appear to
have better bioavailability can demonstrate high excretion rates, low stability in the gastric
environment, increased oxidation, rapid first-pass metabolism or high hepatic uptake,
extensive metabolism by the gut microbiome, or poor absorption across the intestinal
wall [150,151]. Finally, in addition to these factors, the amount of the compound that
accumulates at the target site (for example, in the arterial wall) is also another important
factor for consideration [152]. Taken together, these issues urge caution when interpreting
preclinical studies, and highlight the need for well-controlled human intervention trials to
corroborate findings from in vitro and animal models.

4. Clinical Research
4.1. Flavonoids
Flavonoid intake has been inversely associated with coronary heart disease in several
studies [153,154]. Total flavonoid intake is also associated with a lower risk of PAD hos-
pitalizations, atherosclerosis, and aneurysm, and a lower incidence of revascularizations,
endovascular surgery, and amputations [155]. Due to the high flavonoid content of cocoa
and dark chocolate, the effects of dietary consumption of cocoa and cocoa products on
human health has been widely researched. Three previous studies have tested cocoa in the
setting of PAD. In one of these studies, the primary outcome measure assessed was flow-
mediated dilation (FMD) [156]. In a randomized, controlled, cross-over design, 21 Stage II
PAD patients were randomized to either 50 g dark chocolate (70% cocoa content, 13.5 mg
catechin, and 45 mg epicatechin) or cocoa-free chocolate (control). FMD was measured
Nutrients 2021, 13, 2143 11 of 23

at baseline and 2 h after ingestion. There were no significant changes in FMD in either
group after chocolate ingestion, and there was no difference in the change in FMD between
study arms [156]. The other two studies assessed walking performance as the primary
outcome measures following cocoa consumption. Specifically, the first study sought to
assess the acute effect of cocoa on walking ability [157]. Twenty Stage II PAD patients
received either 40 g dark chocolate (>85% cocoa) or 40 g milk chocolate (control, <35%
cocoa) in a randomized, controlled, cross-over design. The maximum walking distance
and maximum walking time until maximum claudication pain were assessed during a
treadmill test at baseline and 2 h after chocolate ingestion. Maximum walking distance
improved significantly (p < 0.001) by 11.5 m (+11%) and maximum walking time increased
(p < 0.001) by 20 s (+15%) following dark chocolate ingestion, but not after milk chocolate
ingestion [157]. Additionally, in contrast to the previous study demonstrating no acute
change in FMD, in this study FMD was shown to increase significantly after dark chocolate
compared to control.
Recently, the results of the COCOA-PAD phase 2 randomized clinical trial were also
published [158]. In this study, 44 PAD patients were randomized to either a cocoa beverage
containing 15 g of cocoa (75 mg epicatechin) or a placebo beverage daily for 6 months. The
primary outcome measures assessed were the change in the maximum walking distance
during a 6-min walk test, measured 2.5 h and 24 h after beverage consumption at the
6-month follow-up. At the end of the study, compared with placebo, the 6-min walk
distance significantly improved in the cocoa-treated group by 42.6 m at 2.5 h. The 6-min
walk distance also increased by 18 m at 24 h [158]. However, there was no significant
effect of cocoa on FMD [158]. Notably, increases of 18 m and 42.6 m are both considered
meaningful changes in walking performance. However, the data regarding the effect
of cocoa flavonoids on FMD are less consistent. For example, although Loffredo et al.
demonstrated an increase in FMD following acute dark chocolate consumption [157],
Hammer et al. showed no change [156]. One possible explanation may be the higher
cocoa content in Loffredo et al.’s study (>85%) compared to Hammer et al.’s study (70%).
However, another potential explanation is the lower baseline FMD in Loffredo et al.’s study
(2.3%) compared to Hammer et al.’s study (5.1%). Notably, in McDermott et al.’s study,
6 months of cocoa also did not affect FMD, and the mean baseline FMD was 6.47% [158].
Therefore, dark chocolate/cocoa may improve PAD patients’ FMD only when baseline
FMD is low (<3%). Overall, the results of these studies suggest a possible therapeutic effect
of cocoa flavonoids on walking performance in PAD patients. Future studies in larger
samples should be conducted to definitively determine the effect of cocoa flavonoids on
endothelial function and walking performance in PAD patients.

4.2. Nitrogen-Containing Compounds

In recent years, there has been a specific interest in studying the potential health effects
of beetroot. In fact, beetroot has been recommended as an adjunct treatment that may
improve clinical outcomes in cardiovascular disease management [159]. Two previous
studies have investigated the use of beetroot in the setting of PAD. The first study was
a randomized, cross-over study in which 8 Stage II PAD patients consumed 500 mL of
beetroot juice or placebo [160]. The primary outcome measures assessed were claudication
onset time and peak walking time during a Gardner treadmill test performed 3 h after
beverage consumption. Participants walked for 32 s (18%) longer before the onset of
claudication following beetroot juice consumption, which was a significant difference
compared to placebo. Peak walking time also increased by 65 s (17%) after beetroot
juice compared to placebo. [160]. The NO-PAD trial was an extension of this previous
preliminary study [161]. In a randomized, per-protocol study design, 24 Stage II PAD
patients were assigned to a 36-session (12 week) exercise rehabilitation program along with
an oral inorganic nitrate beverage (4.2 mmol beetroot juice), or the exercise program and a
placebo. The exercise program consisted of supervised treadmill training for 30 min three
times per week. The beetroot juice or placebo were consumed 3 h before training [161].
Nutrients 2021, 13, 2143 12 of 23

The primary outcome measures were claudication onset time during a maximal treadmill
test and 6-min walk distance. Although the claudication onset time improved for both
groups after 12 weeks, the increase was statistically significant in only the exercise +
beetroot juice group, resulting in a medium-large effect size of 0.62 [162]. Similarly, the
6-min walk distance improved in both groups, but the increase was only statistically
significant in the exercise + beetroot juice group, resulting in an effect size of 0.43 [162].
Notably, a combination of beetroot and exercise improved claudication onset time by
121.1 s (more than one full stage of the graded treadmill test), which was an increase of
200% compared to exercise alone [162]. The increase of over 2 min can be considered
a moderate minimal clinically important difference. Thus, based on these pilot studies,
beetroot may be a viable treatment option for improving functional performance in PAD
patients, especially in combination with exercise. Future study should be conducted in
a larger sample to confirm whether beetroot therapy may provide clinically meaningful
changes in functional performance.

4.3. Stilbenes
Despite many promising preclinical studies, there is little evidence of health benefits
of resveratrol in humans due to the poor bioavailability of the compound [163]. Still, some
clinical studies have shown some cardioprotective effects of resveratrol, such as improved
endothelial function, ventricular and diastolic function, and improved blood lipids [164].
Resveratrol was also studied for its potential impact on walking performance in PAD
patients during the RESTORE randomized clinical trial [165]. In this study, 66 patients
were randomized to receive 125 mg/day resveratrol, 500 mg/day resveratrol, or placebo
for 6 months. The maximum walking time during a 6-min walking test increased only
in the group receiving 125 mg/day by 4.6 ± 8.1 m, which was a statistically significant
difference based on the defined level of statistical significance (although not considered to
be a clinically meaningful improvement). Further, there was no change in the maximum
walking time during a treadmill test in either group [165]. Thus, there seems to be no
consistent evidence that resveratrol improves walking performance in PAD patients to a
clinically relevant extent.

4.4. Organosulfur Compounds

Organosulfur compounds, including garlic-derived allicin/DATS, as well as isoth-
iocyanates such as sulforaphane, have been studied in a variety of conditions ranging
from autism to metabolic disease and diabetes. However, these compounds have not been
well studied in PAD patients. Only one early randomized clinical trial in 80 Stage II PAD
patients investigated the effect of garlic powder (800 mg diallyl dose) in combination with
a physical therapy training program for 12 weeks on walking performance, compared with
the training program and a placebo [166]. Although pain-free walking distance during a
treadmill test increased in both groups (physical therapy + garlic: 46 m [28.5%] increase,
physical therapy + placebo: 31 m [18.1%] increase), the improvement was significantly
greater in the group receiving the garlic powder along with the physical therapy train-
ing [166]. The increase of walking distance of 46 m following physical therapy + garlic
is considered a clinically meaningful change. Additionally, even the difference in the im-
provement in walking performance between the patients randomized to physical therapy
+ garlic compared to physical therapy + placebo of 15 m is a meaningful difference [166].
Table 2 summarizes the results of the discussed clinical trials.
Nutrients 2021, 13, 2143 13 of 23

Table 2. Clinical studies of phytochemicals in PAD patients.

Sample Intervention/ Outcome Difference or Change in Outcome

Study (Ref.) Compound Dosage
Size Study Type Intervention Length Measure(s) Measure(s) p-Value

treadmill claudication onset time:

treatment: treadmill: placebo: 183 ± 84 s; beetroot: treadmill claudication onset time:
Kenjale et al., beetroot juice randomized claudication onset 215 ± 99 s p < 0.01 *
Nitrate content: N=8 time, peak walking
18,181 µmol/L control 3 h (acute) treadmill peak walking time: treadmill peak walking time:
2011 (160) placebo: orange cross-over trial
juice time; flow-mediated
dilation placebo: 467 ± 223 s; beetroot: p < 0.05 *
533 ± 233 s
treadmill claudication onset time:
change in exercise + placebo group:
Treatment: treadmill: 59.2 ± 57.3 s; change in exercise +
Woessner beetroot juice exercise (3×/wk) + claudication onset beetroot group: 180.3 ± 46.6 s treadmill claudication onset time:
et al., 2018 Placebo: Nitrate content: N = 24 randomized daily beverage, time; 6-min walking
4.2 mmol controlled trial 6-min walking distance: p < 0.05 *
(162) nitrate-depleted 12 weeks test: 6-min walking 6-min walking distance: p < 0.05 *
identical drink distance change in exercise + placebo group:
24.6 ± 12.1 m; change in exercise +
beetroot group: 53.4 ± 19.6 m
treadmill maximum walking distance:
placebo: 109.1 ± 65.1 m; Cocoa:
treadmill: maximum 122.2 ± 61.5 treadmill maximum walking distance:
Treatment: 40 g dark walking distance,
Loffredo Cocoa randomized treadmill maximum walking time: p = 0.01 *
et al., 2014 Placebo: Milk chocolate N = 20 control 2 h (acute) maximum walking placebo: 125.4 ± 64.1 s; Cocoa: treadmill maximum walking time:
(157) chocolate (>85% cocoa) cross-over trial time; flow-mediated 142.2 ± 62.0 s p = 0.006 *
dilation flow-mediated dilation: flow-mediated dilation: p = 0.003 *
placebo: 3.6 ± 3.2%; Cocoa:
6.3 ± 2.7%

Hammer Treatment: 50 g dark flow-mediated dilation:

Cocoa randomized flow-mediated
et al., 2015 chocolate (70% N = 21 control 2 h (acute) change in placebo group: −2.0%; flow-mediated dilation: p = 0.18
(156) Placebo: Milk dilation
chocolate cocoa content) cross-over trial change in Cocoa group: 0.4%

6-min walking test:

6-min walking 6-min walking distance 2.5 h: 6-min walking distance 2.5 h:
distance 2.5 h, 6-min change in placebo group: from p = 0.005 *
Treatment: walking distance 24 337.3 ± 85.2 m to 322.0 ± 96.4 m; 6-min walking distance 24 h: p = 0.12
McDermott Cocoa 15 g cocoa h, change in Cocoa group: from
Placebo: randomized Daily beverage, treadmill: pain-free 348.6 ± 74.2 m to 356.6 ± 64.0 m flow-mediated dilation: 2.5 h:
et al., 2020 Identical, (75 mg N = 44 p = 0.84; 24 h: p = 0.30
(158) control trial 6 months walking time, 6-min walking distance 24 h:
cocoa-free epicatechin) treadmill pain-free walking time:
maximum walking change in placebo group: from
beverage p = 0.68
time; flow-mediated 329.1 ± 90.4 m to 335.4 ± 92.5 m; treadmill maximum walking time:
dilation 2.5 h; change in Cocoa group: from
flow-mediated 347.7 ± 74.3 m to 353.0 ± 76.9 m p = 0.62
dilation 24 h
Nutrients 2021, 13, 2143 14 of 23

Table 2. Cont.

Sample Intervention/ Outcome Difference or Change in Outcome

Study (Ref.) Compound Dosage
Size Study Type Intervention Length Measure(s) Measure(s) p-Value

physical therapy treadmill pain-free walking distance:

Kieswetter Treatment:
Garlic Garlic coated randomized (2×/wk) + daily treadmill: pain-free change in placebo group: treadmill pain-free walking distance:
et al., 1993 Placebo: coated N = 80 172 ± 60.9 m to 203.1 ± 72.8 m
(166) tablets (800 mg) control trial supplement, walking distance p < 0.038 *
cellulose tablets change in Garlic group: from
12 weeks 161 ± 65.1 m to 207.1 ± 85 m
6-min walking distance:
change in placebo group:
−12.3 ± 7.9 m; change in 125 mg 6-min walking distance: 125 mg vs.
6-min walking test: placebo: p = 0.07 *; 500 mg vs.
Treatment: group: 4.6 ± 8.1 m; change in 500 mg
McDermott Resveratrol 125 mg/day or Daily supplement, 6-min walking group: −12.8 ± 7.5 m placebo: p = 0.96
et al., 2017 N = 66 randomized
Placebo: Placebo 500 mg/day control trial 6 months distance; treadmill: treadmill maximum walking time: treadmill maximum walking time:
(165) pill maximum walking
time change in placebo group: 125 mg vs. placebo: p = 0.18; 500 mg
0.4 ± 2.1 min; change in 125 mg vs. placebo: p = 0.12
group: 0.5 ± 2.3 min; change in
500 mg group: −0.6 ± 2.1 min
Note: * indicates significant difference, as defined by study.
Nutrients 2021, 13, 2143 15 of 23

Although sulforaphane has not been specifically tested in PAD patients, clinical trials
from other conditions have shown improvements in atherosclerosis-related biomarkers
following supplementation. Specifically, two independent randomized controlled trials
(N = 130) reported that consumption of high sulforaphane-content broccoli (400 g broccoli,
containing 24.83 µmol/g glucoraphanin, the glucosinolate of sulforaphane) for 12 weeks
reduced plasma LDL-C [167]. Likewise, in 40 overweight participants, consumption of
30 g/day of sulforaphane-containing broccoli sprouts (51.08 mg glucoraphanin) reduced
the inflammatory markers interleukin-6 and C-reactive protein [168]. Additionally, one
of the most promising applications of sulforaphane to date has been in the treatment of
type 2 diabetes. An earlier 4-week randomized controlled trial (N = 63) administering
112.5 or 225 µmol/d sulforaphane to patients with diabetes showed significant reductions
in oxidative stress markers and oxidized LDL [169]. This was followed by other random-
ized controlled trials (N = 72) that demonstrated that the same protocol of sulforaphane
administration reduced serum triglycerides and the atherogenic index of plasma [170],
and decreased serum insulin concentration and improved insulin resistance [171]. Most
recently, a 12-week study administering 150 µmol sulforaphane/day in 103 obese patients
with type 2 diabetes showed that sulforaphane improved fasting glucose and glycated
hemoglobin [172]. One of the aspects related to potential organosulfur compound benefits
may be the comparatively high bioavailability. For example, in contrast to a number of
molecules with low bioavailability (i.e., quercetin, ECGC, lycopene, and curcumin), sul-
foraphane exhibits a high bioavailability (80% compared to 4% for quercetin and 1% for
curcumin) [173,174]. Thus, based on these promising findings, future studies should assess
the clinical relevance of these organosulfur compounds in the treatment of PAD.

4.5. Dietary Interventions

In addition to testing individual phytochemicals, the Prevención con Dieta Mediterránea
(PREDIMED) randomized trial assessed the effect of three different dietary interventions:
a Mediterranean diet supplemented with olive oil, a Mediterranean diet supplemented
with nuts, or a control low-fat diet [175,176]. At baseline, participants (N = 7447) aged
55 to 80 years had no clinical PAD or cardiovascular disease. In an exploratory analysis,
both Mediterranean diet interventions were associated with a reduced PAD risk compared
to the control diet at 5-year follow-up [177]. Furthermore, the reduction in PAD risk
was greater in the group randomized to the Mediterranean diet supplemented with olive
oil (hazard ratio of 0.34) compared to the Mediterranean diet supplemented with nuts
(hazard ratio of 0.50), although there was no statistically significant difference between
the two groups [177]. These results provide evidence for a relationship between diet and
incident PAD. Consumption of a wide range of phytochemicals and antioxidants from
fruits, vegetables, nuts, and olive oil may be beneficial for the prevention of PAD.

5. Conclusions
Phytochemicals have long been studied for their antioxidant activities, both as direct
scavengers of ROS as well as by inhibition of pro-oxidant enzymes and upregulation of
antioxidant enzymes [178]. More recently, phytochemicals have also been studied for the
potential to counter endothelial dysfunction by increasing NO bioavailability [178]. While
a large body of research has developed surrounding the cardioprotective effects of different
phytochemicals, fewer studies are available in the context of PAD. The functional impair-
ment characteristic of PAD is associated with increased oxidative stress and endothelial
dysfunction [42]; thus, treatments targeting these mechanisms may be helpful in improving
patients’ walking performance. Despite the promising findings of a number of preclinical
studies using several phytochemical compounds, clinical trials in patients have been less
successful. The poor bioavailability exhibited by several of the molecules discussed in the
preclinical section may be the reason for significant laboratory potential but limited clinical
usefulness. However, from the studies included in this review, it appears that beetroot and
cocoa may be promising phytochemicals for improving functional status in PAD patients,
Nutrients 2021, 13, 2143 16 of 23

with two studies for each compound demonstrating some degree of clinically significant
improvements in walking performance. Since the compounds likely act via both shared
and distinct mechanisms, it would be interesting to assess the utility of a combined beetroot
+ cocoa intervention. Furthermore, based on the findings of a number of preclinical studies
as well as clinical trials in other diseases, organosulfur compounds may be promising
therapeutic approaches for PAD treatment as well. Future studies should test sulforaphane
as a potentially clinically relevant nutraceutical in the treatment of PAD. Additionally, none
of the human studies assessed the effects on inflammatory molecules; since phytochemicals
may have anti-inflammatory effects, future studies should also include inflammation mark-
ers as outcome measures. Another important point to consider is the potential long-term
beneficial effects of these compounds, as the longest trial included in this review was only
6 months. Since data suggest a lack of durable PAD treatments [61], future studies should
consider whether beneficial effects of phytochemicals can be sustained in the long-term.
Finally, aside from the specific actions of certain phytochemicals, due to the broad health
benefits of fruits and vegetables, consumption of a wide variety of fruits and vegetables
(i.e., Mediterranean diet) should be recommended and encouraged for PAD patients.

Author Contributions: A.I.: Conceptualization, Methodology, Investigation, Writing—Original

Draft, Writing—Review and Editing; K.L.G.: Conceptualization, Writing—Review and Editing;
N.N.: Visualization, Investigation, Writing—Original Draft; D.M.: Investigation, Writing—Original
Draft; E.P.: Investigation, Writing—Original Draft; R.S.S.: Methodology, Investigation, Writing—
Original Draft; J.L.E.: Methodology, Investigation, Writing—Original Draft; D.L.D.: Methodology,
Investigation, Writing—Original Draft; C.W.M.: Methodology, Investigation, Writing—Original Draft;
R.J.W.: Methodology, Investigation, Writing—Original Draft; W.T.B.: Conceptualization, Writing—
Review and Editing; P.K.: Conceptualization, Resources, Funding acquisition, Writing—Review and
Editing. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the National Institute on Aging at the National Institutes of
Health under (grant number R01AG064420) to (P.K.). The content is solely the responsibility of the
authors and does not necessarily represent the official views of the National Institutes of Health.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments: Graphical Abstract created with BioRender.com (accessed on 27 February 2021).
Figure 1 created with ChemDraw (accessed on 8 April 2021).
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Mozaffarian, D.; Rosenberg, I.; Uauy, R. History of modern nutrition science-implications for current research, dietary guidelines,
and food policy. BMJ 2018, 361, k2392. [CrossRef]
2. Staub, P.O.; Casu, L.; Leonti, M. Back to the roots: A quantitative survey of herbal drugs in Dioscorides’ De Materia Medica (ex
Matthioli, 1568). Phytomedicine 2016, 23, 1043–1052. [CrossRef]
3. Carpenter, K.J. A short history of nutritional science: Part 4 (1945–1985). J. Nutr. 2003, 133, 3331–3342. [CrossRef] [PubMed]
4. Wang, X.; Ouyang, Y.Y.; Liu, J.; Zhu, M.M.; Zhao, G.; Bao, W.; Hu, F.B. Fruit and vegetable consumption and mortality from all
causes, cardiovascular disease, and cancer: Systematic review and dose-response meta-analysis of prospective cohort studies.
BMJ 2014, 349. [CrossRef] [PubMed]
5. Bellavia, A.; Larsson, S.C.; Bottai, M.; Wolk, A.; Orsini, N. Fruit and vegetable consumption and all-cause mortality: A dose-
response analysis. Am. J. Clin. Nutr. 2013, 98, 454–459. [CrossRef] [PubMed]
6. Hung, H.C.; Joshipura, K.J.; Jiang, R.; Hu, F.B.; Hunter, D.; Smith-Warner, S.A.; Colditz, G.A.; Rosner, B.; Spiegelman, D.; Willett,
W.C. Fruit and vegetable intake and risk of major chronic disease. JNCI 2004, 96, 1577–1584. [CrossRef] [PubMed]
7. Aune, D.; Giovannucci, E.; Boffetta, P.; Fadnes, L.T.; Keum, N.; Norat, T.; Greenwood, D.C.; Riboli, E.; Vatten, L.J.; Tonstad, S.
Fruit and vegetable intake and the risk of cardiovascular disease, total cancer and all-cause mortality-a systematic review and
dose-response meta-analysis of prospective studies. Int. J. Epidemiol. 2017, 46, 1029–1056. [CrossRef]
8. Momiyama, Y.; Adachi, H.; Fairweather, D.; Ishizaka, N.; Saita, E. Inflammation, Atherosclerosis and Coronary Artery Disease.
Clin. Med. Insights Cardiol. 2014, 8, 67–70. [CrossRef]
Nutrients 2021, 13, 2143 17 of 23

9. Blekkenhorst, L.C.; Bondonno, C.P.; Lewis, J.R.; Woodman, R.J.; Devine, A.; Bondonno, N.P.; Lim, W.H.; Zhu, K.; Beilin, L.J.;
Thompson, P.L.; et al. Cruciferous and Total Vegetable Intakes Are Inversely Associated with Subclinical Atherosclerosis in Older
Adult Women. J. Am. Heart Assoc. 2018, 7. [CrossRef]
10. Guo, W.; Kim, S.H.; Wu, D.; Li, L.; Ortega, E.F.; Thomas, M.; Meydani, S.N.; Meydani, M. Dietary Fruit and Vegetable
Supplementation Suppresses Diet-Induced Atherosclerosis in LDL Receptor Knockout Mice. J. Nutr. 2021. [CrossRef]
11. Kullo, I.J.; Rooke, T.W. CLINICAL PRACTICE. Peripheral Artery Disease. N. Engl. J. Med. 2016, 374, 861–871. [CrossRef]
[PubMed]
12. Chen, Q.; Smith, C.Y.; Bailey, K.R.; Wennberg, P.W.; Kullo, I.J. Disease Location Is Associated with Survival in Patients with
Peripheral Arterial Disease. J. Am. Heart Assoc. 2013, 2, e000304. [CrossRef] [PubMed]
13. Heffron, S.P.; Rockman, C.B.; Adelman, M.A.; Gianos, E.; Guo, Y.; Xu, J.F.; Berger, J.S. Greater Frequency of Fruit and Vegetable
Consumption Is Associated with Lower Prevalence of Peripheral Artery Disease. Arterioscl. Throm. Vas. 2017, 37, 1234–1240.
[CrossRef] [PubMed]
14. Ciccarone, E.; Di Castelnuovo, A.; Salcuni, M.; Siani, A.; Giacco, A.; Donati, M.B.; De Gaetano, G.; Capani, F.; Iacoviello, L.; Inv, G.
A high-score Mediterranean dietary pattern is associated with a reduced risk of peripheral arterial disease in Italian patients with
Type 2 diabetes. J. Thromb. Haemost. 2003, 1, 1744–1752. [CrossRef]
15. Howard, B.V.; Kritchevsky, D. Phytochemicals and cardiovascular disease. A statement for healthcare professionals from the
American Heart Association. Circulation 1997, 95, 2591–2593. [CrossRef] [PubMed]
16. Chen, H.; Liu, R.H. Potential Mechanisms of Action of Dietary Phytochemicals for Cancer Prevention by Targeting Cellular
Signaling Transduction Pathways. J. Agric. Food Chem. 2018, 66, 3260–3276. [CrossRef]
17. Roleira, F.M.; Tavares-da-Silva, E.J.; Varela, C.L.; Costa, S.C.; Silva, T.; Garrido, J.; Borges, F. Plant derived and dietary phenolic
antioxidants: Anticancer properties. Food Chem. 2015, 183, 235–258. [CrossRef] [PubMed]
18. Owens, D.J.; Twist, C.; Cobley, J.N.; Howatson, G.; Close, G.L. Exercise-induced muscle damage: What is it, what causes it and
what are the nutritional solutions? Eur. J. Sport Sci. 2019, 19, 71–85. [CrossRef] [PubMed]
19. Itoh, K.; Chiba, T.; Takahashi, S.; Ishii, T.; Igarashi, K.; Katoh, Y.; Oyake, T.; Hayashi, N.; Satoh, K.; Hatayama, I.; et al. An
Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements.
Biochem. Biophys. Res. Commun. 1997, 236, 313–322. [CrossRef] [PubMed]
20. Fukunaga, B.N.; Probst, M.R.; Reisz-Porszasz, S.; Hankinson, O. Identification of functional domains of the aryl hydrocarbon
receptor. J. Biol. Chem. 1995, 270, 29270–29278. [CrossRef]
21. Gu, X.; Zhu, Y.Z. Therapeutic applications of organosulfur compounds as novel hydrogen sulfide donors and/or mediators.
Expert Rev. Clin. Pharmacol. 2011, 4, 123–133. [CrossRef]
22. Altaany, Z.; Yang, G.; Wang, R. Crosstalk between hydrogen sulfide and nitric oxide in endothelial cells. J. Cell Mol. Med. 2013, 17,
879–888. [CrossRef]
23. Corsello, T.; Komaravelli, N.; Casola, A. Role of Hydrogen Sulfide in NRF2- and Sirtuin-Dependent Maintenance of Cellular
Redox Balance. Antioxidants 2018, 7, 129. [CrossRef]
24. Ismaeel, A.; Papoutsi, E.; Miserlis, D.; Lavado, R.; Haynatzki, G.; Casale, G.P.; Bohannon, W.T.; Smith, R.S.; Eidson, J.L.; Brumberg,
R.; et al. The Nitric Oxide System in Peripheral Artery Disease: Connection with Oxidative Stress and Biopterins. Antioxidants
2020, 9, 590. [CrossRef]
25. Islam, K.N.; Polhemus, D.J.; Donnarumma, E.; Brewster, L.P.; Lefer, D.J. Hydrogen Sulfide Levels and Nuclear Factor-Erythroid
2-Related Factor 2 (NRF2) Activity Are Attenuated in the Setting of Critical Limb Ischemia (CLI). J. Am. Heart Assoc. 2015, 4.
[CrossRef]
26. Ranjan, A.; Ramachandran, S.; Gupta, N.; Kaushik, I.; Wright, S.; Srivastava, S.; Das, H.; Srivastava, S.; Prasad, S.; Srivastava, S.K.
Role of Phytochemicals in Cancer Prevention. Int. J. Mol. Sci. 2019, 20, 4981. [CrossRef] [PubMed]
27. Di Meglio, F.; Sacco, A.; Belviso, I.; Romano, V.; Sirico, F.; Loiacono, C.; Palermi, S.; Pempinello, C.; Montagnani, S.; Nurzynska, D.
Influence of Supplements and Drugs used for the Treatment of Musculoskeletal Disorders on Adult Human Tendon-Derived
Stem Cells. Muscles Ligaments Tendons J. (MLTJ) 2020, 10, 376–384. [CrossRef]
28. Pagliaro, B.; Santolamazza, C.; Simonelli, F.; Rubattu, S. Phytochemical Compounds and Protection from Cardiovascular Diseases:
A State of the Art. Biomed. Res. Int. 2015, 2015, 918069. [CrossRef] [PubMed]
29. Kumar, G.P.; Khanum, F. Neuroprotective potential of phytochemicals. Pharmacogn. Rev. 2012, 6, 81–90. [CrossRef]
30. Fowkes, F.G.; Rudan, D.; Rudan, I.; Aboyans, V.; Denenberg, J.O.; McDermott, M.M.; Norman, P.E.; Sampson, U.K.; Williams, L.J.;
Mensah, G.A.; et al. Comparison of global estimates of prevalence and risk factors for peripheral artery disease in 2000 and 2010:
A systematic review and analysis. Lancet 2013, 382, 1329–1340. [CrossRef]
31. Mortality, G.B.D.; Causes of Death, C. Global, regional, and national life expectancy, all-cause mortality, and cause-specific
mortality for 249 causes of death, 1980-2015: A systematic analysis for the Global Burden of Disease Study 2015. Lancet 2016, 388,
1459–1544. [CrossRef]
32. Savji, N.; Rockman, C.B.; Skolnick, A.H.; Guo, Y.; Adelman, M.A.; Riles, T.; Berger, J.S. Association Between Advanced Age and
Vascular Disease in Different Arterial Territories A Population Database of Over 3.6 Million Subjects. J. Am. Coll. Cardiol. 2013, 61,
1736–1743. [CrossRef]
Nutrients 2021, 13, 2143 18 of 23

33. Norgren, L.; Hiatt, W.R.; Dormandy, J.A.; Nehler, M.R.; Harris, K.A.; Fowkes, F.G.; Group, T.I.W.; Bell, K.; Caporusso, J.; Durand-
Zaleski, I.; et al. Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II). Eur. J. Vasc. Endovasc. Surg.
2007, 33 (Suppl. 1), S1–S75. [CrossRef]
34. Criqui, M.H.; Langer, R.D.; Fronek, A.; Feigelson, H.S.; Klauber, M.R.; McCann, T.J.; Browner, D. Mortality over a period of 10
years in patients with peripheral arterial disease. N. Engl. J. Med. 1992, 326, 381–386. [CrossRef] [PubMed]
35. Caro, J.; Migliaccio-Walle, K.; Ishak, K.J.; Proskorovsky, I. The morbidity and mortality following a diagnosis of peripheral arterial
disease: Long-term follow-up of a large database. BMC Cardiovasc. Disord 2005, 5, 14. [CrossRef] [PubMed]
36. Steg, P.G.; Bhatt, D.L.; Wilson, P.W.; D’Agostino, R., Sr.; Ohman, E.M.; Rother, J.; Liau, C.S.; Hirsch, A.T.; Mas, J.L.; Ikeda, Y.; et al.
One-year cardiovascular event rates in outpatients with atherothrombosis. JAMA 2007, 297, 1197–1206. [CrossRef]
37. Gardner, A.W. Claudication pain and hemodynamic responses to exercise in younger and older peripheral arterial disease
patients. J. Gerontol. 1993, 48, M231–M236. [CrossRef]
38. Gardner, A.W.; Killewich, L.A.; Katzel, L.I.; Womack, C.J.; Montgomery, P.S.; Otis, R.B.; Fonong, T. Relationship between
free-living daily physical activity and peripheral circulation in patients with intermittent claudication. Angiology 1999, 50, 289–297.
[CrossRef]
39. Hiatt, W.R.; Armstrong, E.J.; Larson, C.J.; Brass, E.P. Pathogenesis of the limb manifestations and exercise limitations in peripheral
artery disease. Circ. Res. 2015, 116, 1527–1539. [CrossRef] [PubMed]
40. Koutakis, P.; Ismaeel, A.; Farmer, P.; Purcell, S.; Smith, R.S.; Eidson, J.L.; Bohannon, W.T. Oxidative stress and antioxidant
treatment in patients with peripheral artery disease. Physiol. Rep. 2018, 6, e13650. [CrossRef] [PubMed]
41. McDermott, M.M.; Ferrucci, L.; Gonzalez-Freire, M.; Kosmac, K.; Leeuwenburgh, C.; Peterson, C.A.; Saini, S.; Sufit, R. Skeletal
Muscle Pathology in Peripheral Artery Disease: A Brief Review. Arterioscler. Thromb. Vasc. Biol. 2020, 40, 2577–2585. [CrossRef]
42. Ismaeel, A.; Brumberg, R.S.; Kirk, J.S.; Papoutsi, E.; Farmer, P.J.; Bohannon, W.T.; Smith, R.S.; Eidson, J.L.; Sawicki, I.; Koutakis, P.
Oxidative Stress and Arterial Dysfunction in Peripheral Artery Disease. Antioxidants 2018, 7, 145. [CrossRef] [PubMed]
43. Dopheide, J.F.; Doppler, C.; Scheer, M.; Obst, V.; Radmacher, M.C.; Radsak, M.P.; Gori, T.; Warnholtz, A.; Fottner, C.; Munzel,
T.; et al. Critical limb ischaemia is characterised by an increased production of whole blood reactive oxygen species and expression
of TREM-1 on neutrophils. Atherosclerosis 2013, 229, 396–403. [CrossRef] [PubMed]
44. Weiss, D.J.; Casale, G.P.; Koutakis, P.; Nella, A.A.; Swanson, S.A.; Zhu, Z.; Miserlis, D.; Johanning, J.M.; Pipinos, I.I. Oxidative
damage and myofiber degeneration in the gastrocnemius of patients with peripheral arterial disease. J. Transl. Med. 2013, 11, 230.
[CrossRef] [PubMed]
45. Kraiss, L.; Chong, K. Walking disability in patients with peripheral artery disease is associated with arterial endothelial function
DISCUSSION. J. Vasc Surg 2014, 59, 1033–1034.
46. Brevetti, G.; Giugliano, G.; Brevetti, L.; Hiatt, W.R. Inflammation in peripheral artery disease. Circulation 2010, 122, 1862–1875.
[CrossRef]
47. Ridker, P.M.; Cushman, M.; Stampfer, M.J.; Tracy, R.P.; Hennekens, C.H. Plasma concentration of C-reactive protein and risk of
developing peripheral vascular disease. Circulation 1998, 97, 425–428. [CrossRef]
48. Tzoulaki, I.; Murray, G.D.; Lee, A.J.; Rumley, A.; Lowe, G.D.; Fowkes, F.G. Inflammatory, haemostatic, and rheological markers
for incident peripheral arterial disease: Edinburgh Artery Study. Eur. Heart J. 2007, 28, 354–362. [CrossRef] [PubMed]
49. Ross, R. Atherosclerosis–an inflammatory disease. N. Engl. J. Med. 1999, 340, 115–126. [CrossRef]
50. Zanoli, L.; Rastelli, S.; Inserra, G.; Castellino, P. Arterial structure and function in inflammatory bowel disease. World J.
Gastroenterol. 2015, 21, 11304–11311. [CrossRef]
51. Weber, C.; Zernecke, A.; Libby, P. The multifaceted contributions of leukocyte subsets to atherosclerosis: Lessons from mouse
models. Nat. Rev. Immunol. 2008, 8, 802–815. [CrossRef]
52. Jialal, I.; Verma, S.; Devaraj, S. Inhibition of endothelial nitric oxide synthase by C-reactive protein: Clinical relevance. Clin. Chem.
2009, 55, 206–208. [CrossRef] [PubMed]
53. Barbato, J.E.; Tzeng, E. Nitric oxide and arterial disease. J. Vasc. Surg. 2004, 40, 187–193. [CrossRef] [PubMed]
54. McDermott, M.M.; Liu, K.; Greenland, P.; Guralnik, J.M.; Criqui, M.H.; Chan, C.; Pearce, W.H.; Schneider, J.R.; Ferrucci, L.; Celic,
L.; et al. Functional decline in peripheral arterial disease: Associations with the ankle brachial index and leg symptoms. JAMA
2004, 292, 453–461. [CrossRef] [PubMed]
55. McDermott, M.M.; Guralnik, J.M.; Criqui, M.H.; Liu, K.; Kibbe, M.R.; Ferrucci, L. Six-Minute Walk Is a Better Outcome Measure
Than Treadmill Walking Tests in Therapeutic Trials of Patients With Peripheral Artery Disease. Circulation 2014, 130, 61–68.
[CrossRef]
56. McDermott, M.M.; Tian, L.; Criqui, M.H.; Ferrucci, L.; Conte, M.S.; Zhao, L.; Li, L.; Sufit, R.; Polonsky, T.S.; Kibbe, M.R.; et al.
Meaningful change in 6-min walk in people with peripheral artery disease. J. Vasc. Surg. 2021, 73, 267–276.e1. [CrossRef]
57. Gardner, A.W.; Montgomery, P.S.; Wang, M. Minimal clinically important differences in treadmill, 6-min walk, and patient-based
outcomes following supervised and home-based exercise in peripheral artery disease. Vasc. Med. 2018, 23, 349–357. [CrossRef]
58. Salhiyyah, K.; Forster, R.; Senanayake, E.; Abdel-Hadi, M.; Booth, A.; Michaels, J.A. Pentoxifylline for intermittent claudication.
Cochrane Database Syst. Rev. 2015, 9, CD005262. [CrossRef]
59. Dawson, D.L.; Cutler, B.S.; Hiatt, W.R.; Hobson, R.W., 2nd; Martin, J.D.; Bortey, E.B.; Forbes, W.P.; Strandness, D.E., Jr. A
comparison of cilostazol and pentoxifylline for treating intermittent claudication. Am. J. Med. 2000, 109, 523–530. [CrossRef]
Nutrients 2021, 13, 2143 19 of 23

60. Treat-Jacobson, D.; McDermott, M.M.; Bronas, U.G.; Campia, U.; Collins, T.C.; Criqui, M.H.; Gardner, A.W.; Hiatt, W.R.;
Regensteiner, J.G.; Rich, K.; et al. Optimal Exercise Programs for Patients with Peripheral Artery Disease: A Scientific Statement
From the American Heart Association. Circulation 2019, 139, E10–E33. [CrossRef]
61. Thanigaimani, S.; Phie, J.; Sharma, C.; Wong, S.; Ibrahim, M.; Huynh, P.; Moxon, J.; Jones, R.; Golledge, J. Network Meta-Analysis
Comparing the Outcomes of Treatments for Intermittent Claudication Tested in Randomized Controlled Trials. J. Am. Heart Assoc.
2021, 10, e019672. [CrossRef] [PubMed]
62. Li, S.; Myers, S.A.; Thompson, J.; Kim, J.; Koutakis, P.; Williams, M.; Zhu, Z.; Schieber, M.; Lackner, T.; Willcockson, G. Different
Outcomes after Revascularization or Standard Supervised Exercise Treadmill Training of Claudicating Patients with Peripheral
Artery Disease. JVS-Vasc. Sci. 2020, 1, 255. [CrossRef]
63. Hiatt, W.R.; Regensteiner, J.G.; Wolfel, E.E.; Carry, M.R.; Brass, E.P. Effect of exercise training on skeletal muscle histology and
metabolism in peripheral arterial disease. J. Appl. Physiol. 1996, 81, 780–788. [CrossRef]
64. Hickman, P.; Harrison, D.K.; Hill, A.; McLaren, M.; Tamei, H.; McCollum, P.T.; Belch, J.J. Exercise in patients with intermittent
claudication results in the generation of oxygen derived free radicals and endothelial damage. Adv. Exp. Med. Biol. 1994, 361,
565–570. [CrossRef]
65. Silvestro, A.; Scopacasa, F.; Oliva, G.; de Cristofaro, T.; Iuliano, L.; Brevetti, G. Vitamin C prevents endothelial dysfunction
induced by acute exercise in patients with intermittent claudication. Atherosclerosis 2002, 165, 277–283. [CrossRef]
66. Hamburg, N.M.; Balady, G.J. Exercise rehabilitation in peripheral artery disease: Functional impact and mechanisms of benefits.
Circulation 2011, 123, 87–97. [CrossRef]
67. Belch, J.; MacCuish, A.; Campbell, I.; Cobbe, S.; Taylor, R.; Prescott, R.; Lee, R.; Bancroft, J.; MacEwan, S.; Shepherd, J.; et al. The
prevention of progression of arterial disease and diabetes (POPADAD) trial: Factorial randomised placebo controlled trial of
aspirin and antioxidants in patients with diabetes and asymptomatic peripheral arterial disease. BMJ 2008, 337, a1840. [CrossRef]
[PubMed]
68. Lonn, E.; Yusuf, S.; Hoogwerf, B.; Pogue, J.; Yi, Q.; Zinman, B.; Bosch, J.; Dagenais, G.; Mann, J.F.; Gerstein, H.C.; et al. Effects of
vitamin E on cardiovascular and microvascular outcomes in high-risk patients with diabetes: Results of the HOPE study and
MICRO-HOPE substudy. Diabetes Care 2002, 25, 1919–1927. [CrossRef] [PubMed]
69. Cobley, J.N.; McHardy, H.; Morton, J.P.; Nikolaidis, M.G.; Close, G.L. Influence of vitamin C and vitamin E on redox signaling:
Implications for exercise adaptations. Free Radic. Biol. Med. 2015, 84, 65–76. [CrossRef]
70. Drummond, G.R.; Selemidis, S.; Griendling, K.K.; Sobey, C.G. Combating oxidative stress in vascular disease: NADPH oxidases
as therapeutic targets. Nat. Rev. Drug. Discov. 2011, 10, 453–471. [CrossRef]
71. Couffinhal, T.; Silver, M.; Zheng, L.P.; Kearney, M.; Witzenbichler, B.; Isner, J.M. Mouse model of angiogenesis. Am. J. Pathol. 1998,
152, 1667–1679. [PubMed]
72. Krishna, S.M.; Omer, S.M.; Golledge, J. Evaluation of the clinical relevance and limitations of current pre-clinical models of
peripheral artery disease. Clin. Sci. 2016, 130, 127–150. [CrossRef]
73. Niiyama, H.; Huang, N.F.; Rollins, M.D.; Cooke, J.P. Murine model of hindlimb ischemia. J. Vis. Exp. 2009. [CrossRef] [PubMed]
74. Padgett, M.E.; McCord, T.J.; McClung, J.M.; Kontos, C.D. Methods for Acute and Subacute Murine Hindlimb Ischemia. J. Vis. Exp.
2016. [CrossRef]
75. Pipinos, I.I.; Swanson, S.A.; Zhu, Z.; Nella, A.A.; Weiss, D.J.; Gutti, T.L.; McComb, R.D.; Baxter, B.T.; Lynch, T.G.; Casale, G.P.
Chronically ischemic mouse skeletal muscle exhibits myopathy in association with mitochondrial dysfunction and oxidative
damage. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2008, 295, R290–R296. [CrossRef]
76. Krishna, S.M.; Omer, S.M.; Li, J.; Morton, S.K.; Jose, R.J.; Golledge, J. Development of a two-stage limb ischemia model to better
simulate human peripheral artery disease. Sci. Rep. 2020, 10, 3449. [CrossRef]
77. Thanigaimani, S.; Phie, J.; Golledge, J. Animal models of ischemic limb ulcers: A systematic review and meta-analysis. BMJ Open
Diabetes Res. Care 2020, 8. [CrossRef] [PubMed]
78. Kwik-Uribe, C.; Bektash, R.M. Cocoa flavanols-Measurement, bioavailability and bioactivity. Asia. Pac. J. Clin. Nutr. 2008, 17
(Suppl. 1), 280–283.
79. Panche, A.N.; Diwan, A.D.; Chandra, S.R. Flavonoids: An overview. J. Nutr. Sci. 2016, 5, e47. [CrossRef] [PubMed]
80. Phie, J.; Krishna, S.M.; Moxon, J.V.; Omer, S.M.; Kinobe, R.; Golledge, J. Flavonols reduce aortic atherosclerosis lesion area in
apolipoprotein E deficient mice: A systematic review and meta-analysis. PLoS ONE 2017, 12, e0181832. [CrossRef]
81. Manach, C.; Scalbert, A.; Morand, C.; Remesy, C.; Jimenez, L. Polyphenols: Food sources and bioavailability. Am. J. Clin. Nutr.
2004, 79, 727–747. [CrossRef]
82. Williams, R.J.; Spencer, J.P.; Rice-Evans, C. Flavonoids: Antioxidants or signalling molecules? Free Radic. Biol. Med. 2004, 36,
838–849. [CrossRef] [PubMed]
83. Ekinci Akdemir, F.N.; Gulcin, I.; Karagoz, B.; Soslu, R. Quercetin protects rat skeletal muscle from ischemia reperfusion injury. J.
Enzyme Inhib. Med. Chem. 2016, 31, 162–166. [CrossRef] [PubMed]
84. Sumi, M.; Tateishi, N.; Shibata, H.; Ohki, T.; Sata, M. Quercetin glucosides promote ischemia-induced angiogenesis, but do not
promote tumor growth. Life Sci. 2013, 93, 814–819. [CrossRef] [PubMed]
85. Phie, J.; Krishna, S.M.; Kinobe, R.; Moxon, J.V.; Andrade-Lima, A.; Morton, S.K.; Lazzaroni, S.M.; Huynh, P.; Golledge, J. Effects of
quercetin on exercise performance, physical activity and blood supply in a novel model of sustained hind-limb ischaemia. BJS
Open 2021, 5. [CrossRef]
Nutrients 2021, 13, 2143 20 of 23

86. Lorenz, M.; Urban, J.; Engelhardt, U.; Baumann, G.; Stangl, K.; Stangl, V. Green and black tea are equally potent stimuli of NO
production and vasodilation: New insights into tea ingredients involved. Basic Res. Cardiol. 2009, 104, 100–110. [CrossRef]
87. Ergun, Y.; Kilinc, M.; Aral, M.; Hedef, A.; Kaya, E. Protective Effect of Epigallocatechin Gallate in Ischemia-Reperfusion Injury of
Rat Skeletal Muscle. J. Surg. Res. 2020, 247, 1–7. [CrossRef]
88. Rahim, M.A.; Ejima, H.; Cho, K.L.; Kempe, K.; Mullner, M.; Best, J.P.; Caruso, F. Coordination-Driven Multistep Assembly of
Metal-Polyphenol Films and Capsules. Chem. Mater. 2014, 26, 1645–1653. [CrossRef]
89. Ejima, H.; Richardson, J.J.; Liang, K.; Best, J.P.; van Koeverden, M.P.; Such, G.K.; Cui, J.W.; Caruso, F. One-Step Assembly of
Coordination Complexes for Versatile Film and Particle Engineering. Science 2013, 341, 154–157. [CrossRef]
90. Chen, Z.; Duan, J.; Diao, Y.; Chen, Y.; Liang, X.; Li, H.; Miao, Y.; Gao, Q.; Gui, L.; Wang, X.; et al. ROS-responsive capsules
engineered from EGCG-Zinc networks improve therapeutic angiogenesis in mouse limb ischemia. Bioact. Mater. 2021, 6, 1–11.
[CrossRef]
91. Duan, J.; Chen, Z.; Liang, X.; Chen, Y.; Li, H.; Tian, X.; Zhang, M.; Wang, X.; Sun, H.; Kong, D.; et al. Construction and application
of therapeutic metal-polyphenol capsule for peripheral artery disease. Biomaterials 2020, 255, 120199. [CrossRef]
92. Carnevale, R.; Loffredo, L.; Nocella, C.; Bartimoccia, S.; Bucci, T.; De Falco, E.; Peruzzi, M.; Chimenti, I.; Biondi-Zoccai, G.;
Pignatelli, P.; et al. Epicatechin and catechin modulate endothelial activation induced by platelets of patients with peripheral
artery disease. Oxid. Med. Cell Longev. 2014, 2014, 691015. [CrossRef]
93. Schuler, D. (−)-Epicatechin increases angiogenesis after hindlimb ischemia. Nitric. Oxide Biol. Ch. 2013, 31, S35–S36. [CrossRef]
94. Lidder, S.; Webb, A.J. Vascular effects of dietary nitrate (as found in green leafy vegetables and beetroot) via the nitrate-nitrite-nitric
oxide pathway. Br. J. Clin. Pharmacol. 2013, 75, 677–696. [CrossRef]
95. Hendgen-Cotta, U.B.; Luedike, P.; Totzeck, M.; Kropp, M.; Schicho, A.; Stock, P.; Rammos, C.; Niessen, M.; Heiss, C.; Lundberg,
J.O.; et al. Dietary nitrate supplementation improves revascularization in chronic ischemia. Circulation 2012, 126, 1983–1992.
[CrossRef]
96. Kumar, D.; Branch, B.G.; Pattillo, C.B.; Hood, J.; Thoma, S.; Simpson, S.; Illum, S.; Arora, N.; Chidlow, J.H., Jr.; Langston, W.; et al.
Chronic sodium nitrite therapy augments ischemia-induced angiogenesis and arteriogenesis. Proc. Natl. Acad. Sci. USA 2008, 105,
7540–7545. [CrossRef]
97. Bradley, J.M.; Islam, K.N.; Polhemus, D.J.; Donnarumma, E.; Brewster, L.P.; Tao, Y.X.; Goodchild, T.T.; Lefer, D.J. Sustained release
nitrite therapy results in myocardial protection in a porcine model of metabolic syndrome with peripheral vascular disease. Am.
J. Physiol Heart Circ. Physiol. 2015, 309, H305–H317. [CrossRef]
98. Polhemus, D.J.; Bradley, J.M.; Islam, K.N.; Brewster, L.P.; Calvert, J.W.; Tao, Y.X.; Chang, C.C.; Pipinos, I.I.; Goodchild, T.T.; Lefer,
D.J. Therapeutic potential of sustained-release sodium nitrite for critical limb ischemia in the setting of metabolic syndrome. Am.
J. Physiol. Heart Circ. Physiol. 2015, 309, H82–H92. [CrossRef] [PubMed]
99. Stafford, H.A. Anthocyanins and Betalains-Evolution of the Mutually Exclusive Pathways. Plant. Sci. 1994, 101, 91–98. [CrossRef]
100. Hubbard, T.D.; Murray, I.A.; Perdew, G.H. Indole and Tryptophan Metabolism: Endogenous and Dietary Routes to Ah Receptor
Activation. Drug. Metab. Dispos. 2015, 43, 1522–1535. [CrossRef] [PubMed]
101. Tesoriere, L.; Allegra, M.; Butera, D.; Livrea, M.A. Absorption, excretion, and distribution of dietary antioxidant betalains in
LDLs: Potential health effects of betalains in humans. Am. J. Clin. Nutr. 2004, 80, 941–945. [CrossRef] [PubMed]
102. Vulic, J.J.; Cebovic, T.N.; Canadanovic-Brunet, J.M.; Cetkovic, G.S.; Canadanovic, V.M.; Djilas, S.M.; Saponjac, V.T.T. In vivo and
in vitro antioxidant effects of beetroot pomace extracts. J. Funct. Foods 2014, 6, 168–175. [CrossRef]
103. Vidal, P.J.; Lopez-Nicolas, J.M.; Gandia-Herrero, F.; Garcia-Carmona, F. Inactivation of lipoxygenase and cyclooxygenase by
natural betalains and semi-synthetic analogues. Food Chem. 2014, 154, 246–254. [CrossRef] [PubMed]
104. Traverse, J.H. “Beet It”. Circ. Res. 2018, 123, 635–637. [CrossRef]
105. Salloum, F.N.; Sturz, G.R.; Yin, C.; Rehman, S.; Hoke, N.N.; Kukreja, R.C.; Xi, L. Beetroot juice reduces infarct size and improves
cardiac function following ischemia-reperfusion injury: Possible involvement of endogenous H2S. Exp. Biol. Med. 2015, 240,
669–681. [CrossRef]
106. Akinwumi, B.C.; Bordun, K.M.; Anderson, H.D. Biological Activities of Stilbenoids. Int. J. Mol. Sci. 2018, 19, 792. [CrossRef]
107. Baur, J.A.; Pearson, K.J.; Price, N.L.; Jamieson, H.A.; Lerin, C.; Kalra, A.; Prabhu, V.V.; Allard, J.S.; Lopez-Lluch, G.; Lewis, K.; et al.
Resveratrol improves health and survival of mice on a high-calorie diet. Nature 2006, 444, 337–342. [CrossRef]
108. Howitz, K.T.; Bitterman, K.J.; Cohen, H.Y.; Lamming, D.W.; Lavu, S.; Wood, J.G.; Zipkin, R.E.; Chung, P.; Kisielewski, A.; Zhang,
L.L.; et al. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 2003, 425, 191–196. [CrossRef]
109. Milne, J.C.; Lambert, P.D.; Schenk, S.; Carney, D.P.; Smith, J.J.; Gagne, D.J.; Jin, L.; Boss, O.; Perni, R.B.; Vu, C.B.; et al. Small
molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes. Nature 2007, 450, 712–716. [CrossRef]
110. Baur, J.A.; Sinclair, D.A. Therapeutic potential of resveratrol: The in vivo evidence. Nat. Rev. Drug. Discov. 2006, 5, 493–506.
[CrossRef]
111. Song, X.; Liu, Z.; Zeng, R.; Shao, J.; Zheng, Y.; Ye, W. Resveratrol Alleviates Vascular Endothelial Damage Caused by Lower-
Extremity Ischemia Reperfusion (I/R) through Regulating Keap1/Nrf2 Signaling-Mediated Oxidative Stress. Evid. Based
Complement. Alternat. Med. 2021, 2021, 5556603. [CrossRef]
112. Elmali, N.; Esenkaya, I.; Karadag, N.; Tas, F.; Elmali, N. Effects of resveratrol on skeletal muscle in ischemia-reperfusion injury.
Ulus. Travma Acil Cerrahi Derg. 2007, 13, 274–280.
Nutrients 2021, 13, 2143 21 of 23

113. Ikizler, M.; Ovali, C.; Dernek, S.; Erkasap, N.; Sevin, B.; Kaygisiz, Z.; Kural, T. Protective effects of resveratrol in ischemia-
reperfusion injury of skeletal muscle: A clinically relevant animal model for lower extremity ischemia. Chin. J. Physiol. 2006, 49,
204–209.
114. El Eter, E.A.; Al Zamil, H.; Deif, M. Enhancement of Collaterals by Resveratrol in a Rabbit Model of Hind Limb Ischemia.
Circulation 2013, 128, A18567.
115. Gan, L.; Matsuura, H.; Ichiki, T.; Yin, X.; Miyazaki, R.; Hashimoto, T.; Cui, J.; Takeda, K.; Sunagawa, K. Improvement of
neovascularization capacity of bone marrow mononuclear cells from diabetic mice by ex vivo pretreatment with resveratrol.
Hypertens Res. 2009, 32, 542–547. [CrossRef]
116. El-Awady, M.H.; El-Ghetany, H.H.; Aboelghait, K.M.; Dahaba, A.A. Bioactivities of Allicin and Related Organosulfur Compounds
from Garlic: Overview of the Literature Since 2010. Egypt J. Chem. 2019, 62, 37–45. [CrossRef]
117. Gunther, W.H.H. Garlic and other alliums-The lore and the science. J. Sulfur. Chem. 2013, 34, 208. [CrossRef]
118. Chiang, Y.H.; Jen, L.N.; Su, H.Y.; Lii, C.K.; Sheen, L.Y.; Liu, C.T. Effects of garlic oil and two of its major organosulfur compounds,
diallyl disulfide and diallyl trisulfide, on intestinal damage in rats injected with endotoxin. Toxicol. Appl. Pharmacol. 2006, 213,
46–54. [CrossRef] [PubMed]
119. Predmore, B.L.; Kondo, K.; Bhushan, S.; Zlatopolsky, M.A.; King, A.L.; Aragon, J.P.; Grinsfelder, D.B.; Condit, M.E.; Lefer, D.J. The
polysulfide diallyl trisulfide protects the ischemic myocardium by preservation of endogenous hydrogen sulfide and increasing
nitric oxide bioavailability. Am. J. Physiol. Heart Circ. Physiol. 2012, 302, H2410–H2418. [CrossRef] [PubMed]
120. Hayashida, R.; Kondo, K.; Morita, S.; Unno, K.; Shintani, S.; Shimizu, Y.; Calvert, J.W.; Shibata, R.; Murohara, T. Diallyl Trisulfide
Augments Ischemia-Induced Angiogenesis via an Endothelial Nitric Oxide Synthase-Dependent Mechanism. Circ. J. 2017, 81,
870–878. [CrossRef] [PubMed]
121. Yang, H.B.; Liu, H.M.; Yan, J.C.; Lu, Z.Y. Effect of Diallyl Trisulfide on Ischemic Tissue Injury and Revascularization in a Diabetic
Mouse Model. J. Cardiovasc. Pharmacol. 2018, 71, 367–374. [CrossRef] [PubMed]
122. Ma, Q. Role of nrf2 in oxidative stress and toxicity. Annu. Rev. Pharmacol. Toxicol. 2013, 53, 401–426. [CrossRef]
123. Yu, C.; He, Q.; Zheng, J.; Li, L.Y.; Hou, Y.H.; Song, F.Z. Sulforaphane improves outcomes and slows cerebral ischemic/reperfusion
injury via inhibition of NLRP3 inflammasome activation in rats. Int. Immunopharmacol. 2017, 45, 74–78. [CrossRef] [PubMed]
124. Ambrecht, L.; McDonnell, J.F.; Perlman, J.I.; Bu, P. Protected Retinal Function by Sulforaphane on Retinal Ischemic Injury. Invest.
Ophth. Vis. Sci. 2014, 55, 1891.
125. Chen, Z.Q.; Mohr, A.; Heitplatz, B.; Hansen, U.; Pascher, A.; Brockmann, J.G.; Becker, F. Sulforaphane Elicits Protective Effects in
Intestinal Ischemia Reperfusion Injury. Int. J. Mol. Sci. 2020, 21, 5189. [CrossRef] [PubMed]
126. Silva-Palacios, A.; Ostolga-Chavarria, M.; Sanchez-Garibay, C.; Rojas-Morales, P.; Galvan-Arzate, S.; Buelna-Chontal, M.; Pavon,
N.; Pedraza-Chaverri, J.; Konigsberg, M.; Zazueta, C. Sulforaphane protects from myocardial ischemia-reperfusion damage
through the balanced activation of Nrf2/AhR. Free Radical. Biol. Med. 2019, 143, 331–340. [CrossRef]
127. Shokeir, A.A.; Barakat, N.; Hussein, A.M.; Awadalla, A.; Harraz, A.M.; Khater, S.; Hemmaid, K.; Kamal, A.I. Activation of Nrf2 by
Ischemic Preconditioning and Sulforaphane in Renal Ischemia/Reperfusion Injury: A Comparative Experimental Study. Physiol.
Res. 2015, 64, 313–323. [CrossRef]
128. Bose, C.; Alves, I.; Singh, P.; Palade, P.T.; Carvalho, E.; Borsheim, E.; Jun, S.R.; Cheema, A.; Boerma, M.; Awasthi, S.; et al.
Sulforaphane prevents age-associated cardiac and muscular dysfunction through Nrf2 signaling. Aging Cell 2020, 19, e13261.
[CrossRef]
129. Shehatou, G.S.; Suddek, G.M. Sulforaphane attenuates the development of atherosclerosis and improves endothelial dysfunction
in hypercholesterolemic rabbits. Exp. Biol. Med. 2016, 241, 426–436. [CrossRef]
130. Britton, G. Structure and properties of carotenoids in relation to function. FASEB J. 1995, 9, 1551–1558. [CrossRef]
131. Donnan, P.T.; Thomson, M.; Fowkes, F.G.R.; Prescott, R.J.; Housley, E. Diet as a Risk Factor for Peripheral Arterial-Disease in the
General-Population—The Edinburgh-Artery-Study. Am. J. Clin. Nutr. 1993, 57, 917–921. [CrossRef] [PubMed]
132. Klipstein-Grobusch, K.; den Breeijen, J.H.; Grobbee, D.E.; Boeing, H.; Hofman, A.; Witteman, J.C. Dietary antioxidants and
peripheral arterial disease: The Rotterdam Study. Am. J. Epidemiol. 2001, 154, 145–149. [CrossRef]
133. Kirisci, M.; Guneri, B.; Seyithanoglu, M.; Kazanci, U.; Doganer, A.; Gunes, H. The protective effects of lycopene on is-
chemia/reperfusion injury in rat hind limb muscle model. Ulus. Travma Acil Cerrahi Derg. 2020, 26, 351–360. [CrossRef]
[PubMed]
134. Li, X.; Jia, P.; Huang, Z.; Liu, S.; Miao, J.; Guo, Y.; Wu, N.; Jia, D. Lycopene protects against myocardial ischemia-reperfusion
injury by inhibiting mitochondrial permeability transition pore opening. Drug. Des. Devel. Ther. 2019, 13, 2331–2342. [CrossRef]
135. Hsiao, G.; Fong, T.H.; Tzu, N.H.; Lin, K.H.; Chou, D.S.; Sheu, J.R. A potent antioxidant, lycopene, affords neuroprotection against
microglia activation and focal cerebral ischemia in rats. In Vivo 2004, 18, 351–356. [PubMed]
136. Borel, P.; Desmarchelier, C.; Dumont, U.; Halimi, C.; Lairon, D.; Page, D.; Sebedio, J.L.; Buisson, C.; Buffiere, C.; Remond, D.
Dietary calcium impairs tomato lycopene bioavailability in healthy humans. Br. J. Nutr. 2016, 116, 2091–2096. [CrossRef]
137. Moran, N.E.; Clinton, S.K.; Erdman, J.W., Jr. Differential bioavailability, clearance, and tissue distribution of the acyclic tomato
carotenoids lycopene and phytoene in mongolian gerbils. J. Nutr. 2013, 143, 1920–1926. [CrossRef]
138. Collins, J.K.; Arjmandi, B.H.; Claypool, P.L.; Perkins-Veazie, P.; Baker, R.A.; Clevidence, B.A. Lycopene from two food sources
does not affect antioxidant or cholesterol status of middle-aged adults. Nutr. J. 2004, 3. [CrossRef] [PubMed]
Nutrients 2021, 13, 2143 22 of 23

139. Cikrikci, S.; Mozioglu, E.; Yilmaz, H. Biological Activity of Curcuminoids Isolated from Curcuma longa. Rec. Nat. Prod. 2008, 2,
19–24.
140. Liu, Y.; Chen, L.Y.; Shen, Y.; Tan, T.; Xie, N.Z.; Luo, M.; Li, Z.H.; Xie, X.Y. Curcumin Ameliorates Ischemia-Induced Limb Injury
Through Immunomodulation. Med. Sci. Monitor 2016, 22, 2035–2042. [CrossRef]
141. Zhang, J.; Wang, Q.; Rao, G.; Qiu, J.; He, R. Curcumin improves perfusion recovery in experimental peripheral arterial disease by
upregulating microRNA-93 expression. Exp. Ther. Med. 2019, 17, 798–802. [CrossRef] [PubMed]
142. You, J.; Sun, J.; Ma, T.; Yang, Z.; Wang, X.; Zhang, Z.; Li, J.; Wang, L.; Ii, M.; Yang, J.; et al. Curcumin induces therapeutic
angiogenesis in a diabetic mouse hindlimb ischemia model via modulating the function of endothelial progenitor cells. Stem. Cell
Res. Ther. 2017, 8, 182. [CrossRef]
143. Nelson, K.M.; Dahlin, J.L.; Bisson, J.; Graham, J.; Pauli, G.F.; Walters, M.A. The Essential Medicinal Chemistry of Curcumin. J.
Med. Chem. 2017, 60, 1620–1637. [CrossRef]
144. Baker, M. Deceptive curcumin offers cautionary tale for chemists. Nature 2017, 541, 144–145. [CrossRef] [PubMed]
145. Karas, M.; Jakubczyk, A.; Szymanowska, U.; Zlotek, U.; Zielinska, E. Digestion and bioavailability of bioactive phytochemicals.
Int. J. Food Sci. Tech. 2017, 52, 291–305. [CrossRef]
146. Carbonell-Capella, J.M.; Buniowska, M.; Barba, F.J.; Esteve, M.J.; Frigola, A. Analytical Methods for Determining Bioavailability
and Bioaccessibility of Bioactive Compounds from Fruits and Vegetables: A Review. Compr. Rev. Food Sci. Food Saf. 2014, 13,
155–171. [CrossRef]
147. Shin, S.A.; Joo, B.J.; Lee, J.S.; Ryu, G.; Han, M.; Kim, W.Y.; Park, H.H.; Lee, J.H.; Lee, C.S. Phytochemicals as Anti-Inflammatory
Agents in Animal Models of Prevalent Inflammatory Diseases. Molecules 2020, 25, 5932. [CrossRef]
148. D’Archivio, M.; Filesi, C.; Vari, R.; Scazzocchio, B.; Masella, R. Bioavailability of the polyphenols: Status and controversies. Int. J.
Mol. Sci. 2010, 11, 1321–1342. [CrossRef]
149. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv. Drug. Deliv. Rev. 2001, 46, 3–26. [CrossRef]
150. Yang, C.S.; Sang, S.; Lambert, J.D.; Lee, M.J. Bioavailability issues in studying the health effects of plant polyphenolic compounds.
Mol. Nutr. Food Res. 2008, 52 (Suppl. 1), S139–S151. [CrossRef]
151. Gao, S.; Hu, M. Bioavailability challenges associated with development of anti-cancer phenolics. Mini Rev. Med. Chem. 2010, 10,
550–567. [CrossRef]
152. Aqil, F.; Munagala, R.; Jeyabalan, J.; Vadhanam, M.V. Bioavailability of phytochemicals and its enhancement by drug delivery
systems. Cancer Lett. 2013, 334, 133–141. [CrossRef] [PubMed]
153. Hertog, M.G.; Feskens, E.J.; Hollman, P.C.; Katan, M.B.; Kromhout, D. Dietary antioxidant flavonoids and risk of coronary heart
disease: The Zutphen Elderly Study. Lancet 1993, 342, 1007–1011. [CrossRef]
154. Hertog, M.G.; Kromhout, D.; Aravanis, C.; Blackburn, H.; Buzina, R.; Fidanza, F.; Giampaoli, S.; Jansen, A.; Menotti, A.;
Nedeljkovic, S.; et al. Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study. Arch.
Intern. Med. 1995, 155, 381–386. [CrossRef] [PubMed]
155. Bondonno, N.P.; Murray, K.; Cassidy, A.; Bondonno, C.P.; Lewis, J.R.; Croft, K.D.; Kyro, C.; Gislason, G.; Torp-Pedersen, C.;
Scalbert, A.; et al. Higher habitual flavonoid intakes are associated with a lower risk of peripheral artery disease hospitalizations.
Am. J. Clin. Nutr. 2020. [CrossRef]
156. Hammer, A.; Koppensteiner, R.; Steiner, S.; Niessner, A.; Goliasch, G.; Gschwandtner, M.; Hoke, M. Dark chocolate and vascular
function in patients with peripheral artery disease: A randomized, controlled cross-over trial. Clin. Hemorheol. Microcirc. 2015, 59,
145–153. [CrossRef]
157. Loffredo, L.; Perri, L.; Catasca, E.; Pignatelli, P.; Brancorsini, M.; Nocella, C.; De Falco, E.; Bartimoccia, S.; Frati, G.; Carnevale,
R.; et al. Dark chocolate acutely improves walking autonomy in patients with peripheral artery disease. J. Am. Heart Assoc.
2014, 3. [CrossRef]
158. McDermott, M.M.; Criqui, M.H.; Domanchuk, K.; Ferrucci, L.; Guralnik, J.M.; Kibbe, M.R.; Kosmac, K.; Kramer, C.M.; Leeuwen-
burgh, C.; Li, L.; et al. Cocoa to Improve Walking Performance in Older People with Peripheral Artery Disease: The COCOA-PAD
Pilot Randomized Clinical Trial. Circ. Res. 2020, 126, 589–599. [CrossRef]
159. Clifford, T.; Howatson, G.; West, D.J.; Stevenson, E.J. The potential benefits of red beetroot supplementation in health and disease.
Nutrients 2015, 7, 2801–2822. [CrossRef]
160. Kenjale, A.A.; Ham, K.L.; Stabler, T.; Robbins, J.L.; Johnson, J.L.; Vanbruggen, M.; Privette, G.; Yim, E.; Kraus, W.E.; Allen,
J.D. Dietary nitrate supplementation enhances exercise performance in peripheral arterial disease. J. Appl. Physiol. 2011, 110,
1582–1591. [CrossRef]
161. Woessner, M.N.; VanBruggen, M.D.; Pieper, C.F.; O’Reilly, E.K.; Kraus, W.E.; Allen, J.D. Combined Dietary Nitrate and Exercise
Intervention in Peripheral Artery Disease: Protocol Rationale and Design. JMIR Res. Protoc. 2017, 6, e139. [CrossRef]
162. Woessner, M.; VanBruggen, M.D.; Pieper, C.F.; Sloane, R.; Kraus, W.E.; Gow, A.J.; Allen, J.D. Beet the Best? Circ. Res. 2018, 123,
654–659. [CrossRef]
163. Tome-Carneiro, J.; Gonzalvez, M.; Larrosa, M.; Yanez-Gascon, M.J.; Garcia-Almagro, F.J.; Ruiz-Ros, J.A.; Tomas-Barberan, F.A.;
Garcia-Conesa, M.T.; Espin, J.C. Resveratrol in primary and secondary prevention of cardiovascular disease: A dietary and
clinical perspective. Ann. Ny Acad. Sci. 2013, 1290, 37–51. [CrossRef] [PubMed]
Nutrients 2021, 13, 2143 23 of 23

164. Berman, A.Y.; Motechin, R.A.; Wiesenfeld, M.Y.; Holz, M.K. The therapeutic potential of resveratrol: A review of clinical trials.
NPJ Precis Oncol. 2017, 1. [CrossRef] [PubMed]
165. McDermott, M.M.; Leeuwenburgh, C.; Guralnik, J.M.; Tian, L.; Sufit, R.; Zhao, L.; Criqui, M.H.; Kibbe, M.R.; Stein, J.H.; Lloyd-
Jones, D.; et al. Effect of Resveratrol on Walking Performance in Older People with Peripheral Artery Disease: The RESTORE
Randomized Clinical Trial. JAMA Cardiol. 2017, 2, 902–907. [CrossRef] [PubMed]
166. Kiesewetter, H.; Jung, F.; Jung, E.M.; Blume, J.; Mrowietz, C.; Birk, A.; Koscielny, J.; Wenzel, E. Effects of garlic coated tablets in
peripheral arterial occlusive disease. Clin. Investig. 1993, 71, 383–386. [CrossRef]
167. Armah, C.N.; Derdemezis, C.; Traka, M.H.; Dainty, J.R.; Doleman, J.F.; Saha, S.; Leung, W.; Potter, J.F.; Lovegrove, J.A.; Mithen,
R.F. Diet rich in high glucoraphanin broccoli reduces plasma LDL cholesterol: Evidence from randomised controlled trials. Mol.
Nutr. Food Res. 2015, 59, 918–926. [CrossRef]
168. Lopez-Chillon, M.T.; Carazo-Diaz, C.; Prieto-Merino, D.; Zafrilla, P.; Moreno, D.A.; Villano, D. Effects of long-term consumption
of broccoli sprouts on inflammatory markers in overweight subjects. Clin. Nutr. 2019, 38, 745–752. [CrossRef]
169. Bahadoran, Z.; Mirmiran, P.; Hosseinpanah, F.; Hedayati, M.; Hosseinpour-Niazi, S.; Azizi, F. Broccoli sprouts reduce oxidative
stress in type 2 diabetes: A randomized double-blind clinical trial. Eur. J. Clin. Nutr. 2011, 65, 972–977. [CrossRef]
170. Bahadoran, Z.; Mirmiran, P.; Hosseinpanah, F.; Rajab, A.; Asghari, G.; Azizi, F. Broccoli sprouts powder could improve serum
triglyceride and oxidized LDL/LDL-cholesterol ratio in type 2 diabetic patients: A randomized double-blind placebo-controlled
clinical trial. Diabetes Res. Clin. Pr. 2012, 96, 348–354. [CrossRef]
171. Bahadoran, Z.; Tohidi, M.; Nazeri, P.; Mehran, M.; Azizi, F.; Mirmiran, P. Effect of broccoli sprouts on insulin resistance in type 2
diabetic patients: A randomized double-blind clinical trial. Int. J. Food Sci. Nutr. 2012, 63, 767–771. [CrossRef] [PubMed]
172. Axelsson, A.S.; Tubbs, E.; Mecham, B.; Chacko, S.; Nenonen, H.A.; Tang, Y.; Fahey, J.W.; Derry, J.M.J.; Wollheim, C.B.; Wierup,
N.; et al. Sulforaphane reduces hepatic glucose production and improves glucose control in patients with type 2 diabetes. Sci.
Transl. Med. 2017, 9. [CrossRef] [PubMed]
173. Peluso, I.; Villano Valencia, D.; Chen, C.O.; Palmery, M. Antioxidant, Anti-Inflammatory, and Microbial-Modulating Activities of
Nutraceuticals and Functional Foods 2019. Oxid. Med. Cell Longev. 2020, 2020, 6981542. [CrossRef] [PubMed]
174. Houghton, C.A. Sulforaphane: Its “Coming of Age” as a Clinically Relevant Nutraceutical in the Prevention and Treatment of
Chronic Disease. Oxidative Med. Cell. Longev. 2019, 2019. [CrossRef]
175. Estruch, R.; Ros, E.; Salas-Salvado, J.; Covas, M.I.; Corella, D.; Aros, F.; Gomez-Gracia, E.; Ruiz-Gutierrez, V.; Fiol, M.; Lapetra,
J.; et al. Primary Prevention of Cardiovascular Disease with a Mediterranean Diet Supplemented with Extra-Virgin Olive Oil or
Nuts. N. Engl. J. Med. 2018, 378, e34. [CrossRef]
176. Martinez-Gonzalez, M.A.; Corella, D.; Salas-Salvado, J.; Ros, E.; Covas, M.I.; Fiol, M.; Warnberg, J.; Aros, F.; Ruiz-Gutierrez, V.;
Lamuela-Raventos, R.M.; et al. Cohort profile: Design and methods of the PREDIMED study. Int. J. Epidemiol. 2012, 41, 377–385.
[CrossRef]
177. Ruiz-Canela, M.; Estruch, R.; Corella, D.; Salas-Salvado, J.; Martinez-Gonzalez, M.A. Association of Mediterranean diet with
peripheral artery disease: The PREDIMED randomized trial. JAMA 2014, 311, 415–417. [CrossRef]
178. Tangney, C.C.; Rasmussen, H.E. Polyphenols, inflammation, and cardiovascular disease. Curr. Atheroscler. Rep. 2013, 15, 324.
[CrossRef]