PHYTOTHERAPY RESEARCH

Phytother. Res. (2017)

Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.5819

REVIEW
C-Glycosyl Flavonoids from Beta vulgaris Cicla
and Betalains from Beta vulgaris rubra:
Antioxidant, Anticancer and Antiinflammatory
Activities—A Review

Paolino Ninfali,* Elena Antonini, Alessandra Frati and Emanuele-Salvatore Scarpa

Department of Biomolecular Sciences, University of Urbino ‘Carlo Bo’, via Saffi, 261029, Urbino, PU, Italy

The green beet (Beta vulgaris var. cicla L.) and red beetroot (B. vulgaris var. rubra L.) contain phytochemicals
that have beneficial effects on human health. Specifically, the green beet contains apigenin, vitexin, vitexin-2-O-
xyloside and vitexin-2-O-rhamnoside, while the red beetroot is a source of betaxanthins and betacyanins. These
phytochemicals show considerable antioxidant activity, as well as antiinflammatory and antiproliferative
activities. Vitexin-2-O-xyloside, in combination with betaxanthins and betacyanins, exerts antiproliferative
activity in breast, liver, colon and bladder cancer cell lines, through the induction of both intrinsic and extrinsic
apoptotic pathways. A significant body of evidence also points to the role of these phytochemicals in the
downregulation of the pro-survival genes, baculoviral inhibitor of apoptosis repeat-containing 5 and catenin
beta-1, as well as the genes controlling angiogenesis, hypoxia inducible factor 1A and vascular endothelial growth
factor A. The multi-target action of these phytochemicals enhances their anticancer activity. Vitexin-2-O-
xyloside, betaxanthins and betacyanins can be used in combination with conventional anticancer drugs to reduce
their toxicity and overcome the multidrug resistance of cancer cells. In this review, we describe the molecular
mechanisms that enable these dietary phytochemicals to block the proliferation of tumor cells and inhibit their
pro-survival pathways. Copyright © 2017 John Wiley & Sons, Ltd.
Keywords: anticancer; antiinflammatory; antioxidant; betalains; C-glycosyl flavonoids; vitexin-2-O-xyloside.

Abbreviation list: AUC, area under the curve; Bax, Bcl-2-like protein 4; Bcl-2, B-cell leukemia/lymphoma 2; BC, betacyanins; BIRC5,
baculoviral inhibitor of apoptosis repeat-containing 5; BVc, Beta vulgaris var. cicla L.; BVr, Beta vulgaris var. rubra L.; BX,
betaxanthins; CD31, cluster of differentiation 31; COX-2, cyclooxygenase-2; CTNNB1, catenin beta-1; DCFH-DA, 20 , 70 -dichloro
fluorescein diacetate; HIF1A, hypoxia inducible factor 1A; HPLC, high pressure liquid chromatography; IL, interleukin; iNOS,
inducible nitric oxide synthase; Keap1, Kelch-like ECH-associated protein 1; LOX, lypoxygenase; MDR, multi drug resistance; MS,
mass spectrometry; NF-kB, nuclear factor kappa B; Nrf2, nuclear factor (erythroid-derived 2)-like 2; ORAC, oxygen radical
absorbance capacity; PARP1, poly ADP ribosyl polymerase 1; PGE2, prostaglandin E2; ROS, radical oxygen species; TNF-α, tumor
necrosis factor-α; VEGFA, vascular endothelial growth factor A; VOR, vitexin-2-O-rhamnoside; XVX, vitexin-2-O-xyloside

2013). As they grow, BVc leaves accumulate minerals,

INTRODUCTION
Red beet (Beta vulgaris var. rubra L., BVr) and green
beet (B. vulgaris var. cicla L., BVc) belong to the same
plant family (Amaranthaceae–Chenopodiaceae) and
are common foods in western countries. Beta vulgaris
var. rubra L. is valued for its root, while BVc is grown
for its leaves. Both plants provide a low caloric intake
and are important sources of several health protective
nutrients, thus complementing staple foods in a
balanced diet (Ninfali et al., 2014).
A large number of selected cultivars can be grown
under conventional, organic or integrated agricultural
conditions, and plants can be uniformly harvested at a
precise stage of growth, when their desired nutrients
reach their maximum values (Ninfali and Angelino,
* Correspondence to: Paolino Ninfali, Department of Biomolecular
Sciences University of Urbino ‘Carlo Bo’ via Saffi, 2, 61029 Urbino (PU),
Italy.
E-mail: paolino.ninfali@uniurb.it
Copyright © 2017 John Wiley & Sons, Ltd.
particularly Fe, K, Ca, Mg, P and several hydro-soluble
vitamins such as C, B3, B5, B9, as well as liposoluble
vitamins such as A, E and carotenoids (Daiss et al.,
2008). Beta vulgaris var. cicla L. is also rich in fiber and
saponins, which allow the clearance of fats and
cholesterol and are able to solve constipation problems.
The content of folates is also considerable
(https://ndb.nal.usda.gov/ndb/foods/show/2917?
manu=&fgcd=&ds=). Moreover, the seeds are rich in
essential fatty acids, with linoleic acid constituting
44% of the total fatty acids (Ninfali and Angelino,
2013).
Green beet extract has been used for the regulation of
the hematic concentration of glucose, counteracting the
physiological damage of diabetes, reducing lipid
peroxidation and enhancing glutathione levels (Li
et al., 2009; Ozsoy-Sacan et al., 2004; Sener et al., 2002).
The health protective phytochemicals found in BVc
have been identified as a class of C-glycosyl flavones,
derived from apigenin, namely vitexin (apigenin 8-C-
glycoside), isovitexin (apigenin-6-C-glycoside), vitexin-
Received 16 February 2017
Revised 28 March 2017
Accepted 29 March 2017
 P. NINFALI ET AL.
2-O-xyloside and vitexin-2-O-rhamnoside. Vitexin and
isovitexin are commercially produced through synthesis,
and interest in their biological activities has grown in the
past decade with investigations providing insights into
their pharmacological properties, as noted by He et al.
(2016). Vitexin-2-O-xyloside and vitexin-2-O-
rhamnoside have been isolated from natural sources
and are less studied.
The most important bioactive phytochemicals in BVr
are betalains, pigments derived from betalamic acid and
grouped into yellow betaxanthins (BX) and red
betacyanins (BC). Betalains hold great interest because
red dye E162, made from beetroot extract, is one of the
few red dyes admitted by the European Food Safety
Authority in food manufacturing (EU, 2015) with
documented biological activity (Esatbeyoglu et al.,
2015).
Only BCs are commercially produced, but several
protocols for the isolation of BX and BC from natural
sources have been published (Rackauskiene et al.,
2015). Beetroot (BVr) is rich in starch, fiber, minerals
and vitamins such as vitamin B6 and folates. Its fiber
content is considerable, and it has been shown to protect
the body from hypercholesterolemia (Bobek et al.,
2000). Moreover, the presence of minerals in BVr, such
as Mg, P and Fe (https://ndb.nal.usda.gov/ndb/foods/
show/2863?manu=&fgcd=&ds=), is useful for bone
health (Pietrzkowski et al., 2010) and counteracts
anemia (Priya et al., 2013).
This review is an update of our previous report on the
biological activities of BVr and BVc phytochemicals
(Ninfali and Angelino, 2013). In this work, we focus on
the anticancer activities and, in particular, on the
molecular basis of the pro-apoptotic effects of BVc
and BVr phytonutrients, which are enhanced when
BVc and BVr phytochemicals are combined. Already
published data have been augmented with new
unpublished results provided by our more recent
investigations.
Chemistry and sources of the beet phytochemicals
Vitexin, vitexin-2-O-rhamnoside and vitexin-2-O-
xyloside are C-glycosyl flavonoids, earlier identified in
hawthorn leaves (Ma et al., 2010) and BVc leaves
(Gennari et al., 2011; Gil et al., 1999; Ma et al., 2010).
The C-glycosyl flavonoids differ from O-glycosyl
flavonoids because their glycosyl moiety is attached,
via anomeric carbon, directly to the flavonoid backbone,
usually at the C-6 or C-8 position of the A ring. These
compounds have higher stability against hydrolysis
compared with O-glycosyl flavonoids (Rauter et al.,
2007), as their C–C linkage is acid resistant and difficult
to cleave (Xiao et al., 2014). The 2-O-glycosyde bond,
by contrast, can be hydrolyzed chemically or
enzymatically, resulting in the aglycone compound
(Xiao et al., 2014).
Vitexin synthesis in plants starts from apigenin
and UDP-glucose or ADP-glucose, in the presence of
C-glycosyl transferase, as described in the cotiledones of
Fagopyrum esculentum M. (Kerscher and Franz, 1986).
The chemical synthesis of vitexin has been performed
successfully in the laboratory using the commercially
available 2,4,6-trihydroxyacetophenone by forming an
O-glycoside intermediate, which was converted via Fries
Copyright © 2017 John Wiley & Sons, Ltd.
rearrangement into a C-glycoside derivative; this
compound, through rearrangement and cyclization,
gave rise to a vitexin derivative, which was completely
de-protected to yield vitexin (Mahling et al., 1995).
The complexity of this pathway, which remains limited
to vitexin, highlights the challenges associated with the
synthesis of vitexin-2-O-rhamnoside and vitexin-2-O-
xyloside, which at present are only purified from
natural sources. In our laboratory, we purify vitexin
and vitexin-2-O-rhamnoside from hawthorn leaf extract
and vitexin-2-O-xyloside from BVc seeds, with a
reproducible method based on liquid–liquid extraction
and two middle pressure chromatographic steps
(Gennari et al., 2011).
Fig. 1 shows the chemical formulae and the
representative high pressure liquid chromatography
(HPLC) analysis of vitexin, vitexin-2-O-rhamnoside
and vitexin-2-O-xyloside, obtained in one of our many
purifications, at 95% purity level.
In addition to BVc and hawthorn leaves, other plant
sources of vitexin and its derivatives are: mung bean
(Cao et al., 2011), pigeon pea (Fu et al., 2008), millet
(Gaitan et al., 1989), mosses (Basile et al., 2003),
passiflora (Zucolotto et al., 2012), bamboo (Wang et al.,
2010), mimosa (Zhang et al., 2011), chastetree (Hajdu
et al., 2007) and Santalum album L. (Yan et al., 2013).
It should be noted that the names vitexin or vitexin 6
are also used for compounds that are chemically
different from the flavonoid C-glycoside, and identified
as lignans (Zhou et al., 2013; Zhou et al., 2009). We must
therefore be careful not to confuse the molecules.
The betalains BX and BC are present in many plants
and 55 well-characterized molecules have been
described to date (Stintzing and Carle, 2004).
Betalains are synthesized in the cytoplasm of beetroot
cells through an enzymatic pathway and then stored in
vacuoles. The starting point of the synthetic pathway is
the amino acid tyrosine, from which the L-3,4-
dihydroxyphenylalanine (L-DOPA) is formed and
converted into a dopaquinone, which spontaneously
produces betalamic acid, the fundamental intermediate
in the synthesis of all betalains (Gandia-Herrero et al.,
2005). Moreover, another synthetic pathway, which
includes tyramine as the starting compound, has been
shown to occur in BVr roots (Kobayashi et al., 2001;
Strack et al., 2003).
The most common red betalain is betanidin,
which forms betanin when glycosylated on the side
of the cyclo-3,4-dihydroxyphenylalanine (cyclo-
DOPA) (Gandia-Herrero et al., 2005).
Betaxanthins are formed in the beetroot by the
spontaneous condensation of betalamic acid with an
amino acid or an ammine, resulting in the formation of
an aldimminic bond, thus generating BX. When
betalamic acid is condensed with proline or glutamine,
indicaxanthin or vulgaxanthin is generated, respectively
(Gandia-Herrero et al., 2005). In BVr, the main BC and
BX that we found were betanin, isobetanin and
vulgaxanthin I (Farabegoli et al., 2017). Fig. 2 shows
the chemical structures of these molecules together with
phenylalanine-betaxanthin and gomphrenin.
Other known sources of betalains include the
following: the fruit of the cactus pear, Opuntia ficus
indica (Stintzing et al., 2005), the dragon fruit from
Hylocerus cacti (Wybraniec et al., 2001), some hybrids
of Swiss chard with red–yellow stems (Kugler et al.,
Phytother. Res. (2017)
 PHARMACOLOGICAL ACTIVITIES OF GREEN AND RED BEETS

Figure 1. HPLC analysis (330 nm) of vitexin-2-O-xyloside (XVX, a), vitexin-2-O-rhamnoside (VOR, b) and vitexin (c), purified from natural
sources. The purity level was about 95%.

2004), the tubers of the Ullucus tuberosus (Svenson
et al., 2008), the Eulichina cacti fruit (Masson et al.,
2011) and the berries of Rivina humilis (Khan et al.,
2011).
Bioavailability
The bioavailability of vitexin, vitexin-2-O-xyloside and
vitexin-2-O-rhamnoside has been investigated in vivo
by several authors using different administration
Copyright © 2017 John Wiley & Sons, Ltd.
approaches: (i) oral gavage (Liang et al., 2007; Ma
et al., 2010; Wang et al., 2012; Yan et al., 2013); (ii)
intravenous administration (Ma et al., 2007; Tong and
Wu, 2012; Xiao et al., 2014; Xue et al., 2014; Zhang
et al., 2010); and (iii) infusion in the target tissue
(Angelino et al., 2013; Xue et al., 2014). In vitro models
for evaluating the transformation of C-glycosyl
flavonoids due to the microbiota have also been
developed (Braune and Blaut, 2012; Kim et al., 2015).
Oral administration of vitexin in rats or mice shows a
distribution of the molecule in various tissues, with the
Phytother. Res. (2017)
 P. NINFALI ET AL.
Figure 2. Chemical structures of the betalains: (a) vulgaxanthin I;
(b) phenylalanine-betaxanthin; (c) betanin and its derivatives,
isobetanin and gomphrenin.
highest concentrations in intestine and liver and very
low levels in brain and adipose tissue (Wang et al., 2012).
Yan et al. (2013) evaluated the plasmatic
concentrations of vitexin and isovitexin in rats after a
single oral administration of a S. album leaf extract
(116 mg/kg), containing 50 and 29.3 mg/kg of vitexin
and isovitexin, respectively. After the administration,
vitexin and isovitexin appeared to be absorbed rapidly,
with the maximal concentration of 19.5 ± 8.85 and
76.0 ± 25.1 ng/mL, respectively, at about 20 min.
However, the compounds were then slowly eliminated
with a t1/2 of 6.37 h for vitexin and 14.6 h for isovitexin.
Also, in this case, the authors did not detect any
metabolic or degradation product.
Oral administration of pure vitexin-2-O-rhamnoside
at 50 mg/kg concentrations was carried out in rats
(Liang et al., 2007). The absolute oral bioavailability of
vitexin-2-O-rhamnoside was 3.57%, showing that the
molecule is hardly absorbed in the gastrointestinal tract
or it undergoes extensive first pass transformation into
metabolic products (Liang et al., 2007). No evaluation
of metabolic phase II products such as glucuronated or
hydroxylated forms was performed by these authors.
Ma et al. (2010) orally administered a single dose of
2 g/kg of hawthorn leaf flavonoids, containing
83 mg/kg vitexin-4-O-glucoside and 342 mg/kg vitexin-
2-O-rhamnoside; the levels of vitexin-4-O-glucoside
and vitexin-2-O-rhamnoside were evaluated in plasma
and tissues, bile, urine and feces. The maximum of
absorption was found at 0.75 h for vitexin-4-O-glucoside
and vitexin-2-O-rhamnoside, and the t1/2 was 2.53 and
2.52 h for vitexin-4-O-glucoside and vitexin-2-O-
rhamnoside, respectively. High levels of tissue
concentrations were observed in liver and kidney. The
total recovery, in 24 h, was 64.9% with 0.6% in urine,
64.3% in feces for vitexin-4-O-glucoside and 89.01%
with 0.72% in urine and 88.29% in feces for vitexin-2-
O-rhamnoside. Hence, it was shown that vitexin-4-O-
glucoside and vitexin-2-O-rhamnoside were not
efficiently absorbed in the rodent gastrointestinal tract.
Intravenous administration of 20 mg/kg of hawthorn
leaf flavonoids, containing the three compounds at the
following concentrations: 1.73 mg/kg vitexin-4-O-
glucoside, 2.63 mg/kg vitexin-2-O-rhamnoside and
0.062 mg/kg vitexin was carried out in rats (Zhang et al.,
2010). The authors showed that vitexin was quickly
eliminated within 4 h and both vitexin-4-O-glucoside
and vitexin-2-O-rhamnoside were eliminated after
12 h. The pharmacokinetic parameters of vitexin-2-O-
rhamnoside and vitexin-4-O-glucoside were found to
Copyright © 2017 John Wiley & Sons, Ltd.
be a little different from those reported in other
investigations, but the authors attributed the differences
to the dosage of the compounds and the route of
administration (Zhang et al., 2010).
Intravenous administration of hawthorn leaf
flavonoids (10–40 mg/kg) was used to investigate the
pharmacokinetics of vitexin-4-O-glucoside and vitexin-
2-O-rhamnoside in rats (Ma et al., 2007). The plasma
concentrations and area under the curve (AUC) of
vitexin-4-O-glucoside and vitexin-2-O-rhamnoside in
rat plasma were proportional to the administrated doses
and dose-linear pharmacokinetics of vitexin-4-O-
glucoside and vitexin-2-O-rhamnoside were shown
(Ma et al., 2007).
Tong and Wu (2012) showed that, when present in
blood, vitexin can bind the human serum albumin, with
a high binding percentage. The glycosylation of
flavonoids can reduce the binding constants for human
serum albumin, according to the conjugation site and
the class of sugar (Xiao et al., 2014).
Infusion of vitexin through intra-portal, intra-
duodenal, intra-gastric and intravenous administration
at 30 mg/kg was performed by Xue et al. (2014). The
authors showed that after intraduodenal administration,
the vitexin concentration peaked at 20 min and
decreased until 180 min. Moreover, it was shown that
vitexin is quickly removed from the blood, its absolute
oral bioavailability is low and the inhibitors of P-
glycoprotein did not increase its absorption in the
intestine. The hepatic, gastric and intestinal first-pass
effects of vitexin in rats were 5.2, 31.3 and 94.1%,
respectively. The authors concluded that the intestinal
first-pass effect of vitexin was considerable and the
gastric and hepatic first-pass effect also contributed to
the low absolute oral bioavailability of vitexin.
After a single dose intra-caecal administration,
vitexin-2-O-xyloside was shown to be quickly absorbed
in the first minutes of administration, without any
changes caused by enterocytes and gut microbiota
(Angelino et al., 2013). However, once it reaches the
liver through the portal vein, the compound seems to
undergo enterohepatic recirculation several times until
it is totally metabolized (Angelino et al., 2013). In fact,
HPLC-MS analyses showed that vitexin-2-O-xyloside
undergoes hydrolysis to monoglucoside, as well as
reduction and conjugation, leading to the formation of
a bioavailable glucuronide (Angelino et al., 2013). These
results were confirmed by analyzing bile and urine,
which showed the presence of the vitexin-2-O-xyloside
metabolites (Angelino et al., 2013).
Vitexin and orientin have been treated with intestinal
bacteria in order to verify their transformation in the
colon (Braune and Blaut, 2012).
Kim et al. (2015) isolated different strains of intestinal
bacteria from the feces of 43 individuals and incubated
various flavonoid-C-glycosides, including vitexin, to
verify if the bacteria were able to cleave the bond
between sugar and apigenin. The authors showed that
puerarin, daidzein, genistein and apigetrin were
converted into an aglyconic form, but vitexin, rutin,
hesperidin and naringenin were not. Two out of three
bacterial strains were able to deglicosilate the C-
glycosidic bonds, but the study was not conclusive, as it
was limited to only three bacterial strains.
In conclusion, vitexin is absorbed well by the
intestine, very likely by passive diffusion; the sugar
Phytother. Res. (2017)
 PHARMACOLOGICAL ACTIVITIES OF GREEN AND RED BEETS
moiety influences the absorption, distribution and
metabolism of the C-glycosides due to steric hindrance
and polarity in biological fluids. Once it enters the
enterocytes, vitexin generates an intense entero-hepatic
recirculation that leads to its partial secretion in the bile
in a modified, but still active form. Another aliquot is
degraded by the phase I and II metabolic reactions
and finally eliminated in the urine and feces.
The entero-hepatic recirculation and metabolism do
not allow the maintenance of a advantageous AUC
following oral administration, but this may be an
advantage if these molecules are being used for a
therapeutic function in the entero-hepatic compartment.
With respect to betalains, there is an extensive
amount of literature on their in vitro bioavailability
and fewer reports in vivo (Clifford et al., 2015; Clifford
et al., 2017).
An in vitro model of the human intestinal epithelium
was used to mimic the gut functional barrier and to
demonstrate that betanin and indicaxanthin are
absorbed well through a CaCo-2 cell monolayer, mostly
in their unmetabolized forms via paracellular transport
(Tesoriere et al., 2013).
Regarding the in vivo studies, Kanner et al. (2001)
investigated the presence of betanin and betanidin in
human urine after 2–4 h from the consumption of
300 mL of red beet juice, containing 120 mg of betalains.
The authors found 0.5–0.9% of the ingested BC in the
urine (Kanner et al., 2001). Similarly, Frank et al.
(2005) showed that in healthy humans, consuming red
beet juice (500 mL containing 362.7 mg of betalains),
the recovered amount of betalains in the urine was
0.28 ± 0.08% of the administered dose, assuming that
either the bioavailability of the betalains is low or that
renal clearance is a minor route of systemic elimination
for these compounds.
Recently, Clifford et al. (2017) confirmed the poor
bioavailability of BC, as these molecules could not be
detected in the human plasma within 8 h post-ingestion
of 250 mL of beetroot juice or 300 g of whole beetroot,
containing 194 and 66 mg of betanin, respectively.
In conclusion, considering the low bioavailability of
vitexin and betalains, it is important to detect their
metabolites, in order to establish if these molecules
could exploit significant biological activities. Moreover,
extensive investigations of vitexin bioavailability in
humans must be performed before any conclusion on
its biological effects can be drawn.
Pharmacological activities
Recent studies, reviewed by Xiao et al. (2014), deal with
the in vivo benefits of flavonoid-glycosides, especially
the C-glycosides. Vitexin, vitexin-2-O-rhamnoside and
vitexin-2-O-xyloside are flavonoid-C-glycosides
characterized by antioxidant, antiinflammatory and
anticancer properties (Ninfali et al., 2007; Ninfali and
Angelino, 2013). This is also true of the betalains, which
possess notable antioxidant (Kugler et al., 2007),
antiinflammatory (Vidal et al., 2014) and anticancer
activities (Nowacki et al., 2015). It is therefore important
to make a detailed analysis of the three main
pharmacological activities of the BVc flavonoids and
BVr betalains (Fig. 3).
Copyright © 2017 John Wiley & Sons, Ltd.
Antioxidant activity
Fig. 4 shows that per gram, apigenin has the highest
oxygen radical absorbance capacity (ORAC) value,
followed by vitexin, vitexin-2-O-xyloside and vitexin-2-
O-rhamnoside. On mmol basis, apigenin remains the
most efficient antioxidant, followed by vitexin-2-O-
xyloside, vitexin and vitexin-2-O-rhamnoside (Fig. 4).
The ORAC of phytochemical nutrients is generally
considered a useful protective tool against radical
oxygen species (ROS) dependent damage (Ninfali and
Bacchiocca, 2003). Once ingested and absorbed,
antioxidants protect cells against lipid peroxidation of
cytoplasm and mitochondrial membranes, as well as
from oxidative damage to proteins and nucleic acids
(Lobo et al., 2010).
The vitexin, vitexin-2-O-xyloside and vitexin-2-O-
rhamnoside antioxidant activities measured inside
human erythrocytes provided the evidence of
intracellular protection against ROS-dependent damage
(Blasa et al., 2011; Ninfali and Angelino, 2013). In fact,
when erythrocytes are challenged by the action of
strong oxidants, such as H2O2, vitexin, as well as
isovitexin, are able to protect cells from oxidative
damage (An et al., 2015; Kim et al., 2005). The
protection was likely due to the maintenance of the
sulfidryl groups of the proteins of the red cell
membranes (Praveena et al., 2013). On the contrary,
when the CaCo-2 colon cancer cells and HepG2 liver
cancer cells were incubated with vitexin-2-O-xyloside,
ROS production was revealed through the 20 ,70 -
dichloro fluorescein diacetate (DCFH-DA) assay
(Fig. 5). As recently demonstrated by us, 30-μM
vitexin-2-O-xyloside is in the range of concentration
able to induce a remarkable increase in the
endogenous ROS levels, in both CaCo-2 and HepG2
cancer cells, leading to the reduction of their
proliferation rate (Scarpa et al., 2017).
These apparently contradictory results in human
erythrocytes and human cancer cells could be explained
by the high proliferation rate of cancer cells, which is
associated with a high aerobic metabolism and high
levels of oxidative stress (Cairns et al., 2011). If a
phytochemical inhibits one enzyme of the mitochondrial
respiratory chain, such as the complexes I, II, III and IV
inside the cancer cells (Kowaltowski et al., 2009; Scatena
et al., 2007), the blockade in the electron transfer chain
increases ROS production. Indeed, vitexin-2-O-xyloside
can increase ROS production in cancer cells by the
inhibition of the enzymes that synthesize reduced
glutathione (GSH), as demonstrated for apigenin, the
vitexin aglycone, which is able to generate intracellular
ROS and inhibit the synthesis of GSH (Kachadourian
and Day, 2006). Therefore, a strong antioxidant
molecule, such as vitexin-2-O-xyloside, becomes a pro-
oxidant when it enters cancer cells in certain
concentrations, and it may kill the cancer cell by
exacerbating oxidative stress and interfering with cell
signaling pathways at different levels (Harris and
Brugge, 2015).
As regards to betalains, BC showed a higher
antioxidant capacity, assessed using the ORAC method,
compared with BX (Farabegoli et al., 2017; Kugler et al.,
2007). Indeed, the uptake of dietary betalains provides
intracellular antioxidant protection in human red blood
cells (Tesoriere et al., 2005).
Phytother. Res. (2017)
 P. NINFALI ET AL.
Figure 3. Summary of pharmacological activities of BVc and BVr phytochemicals considered in this review.
Regarding cancer cells, BX and BC were able to
reduce oxidative damage caused directly by H2O2 in
CaCo-2 colon cancer cells (Farabegoli et al., 2017). It is
highly likely that betalains exert their antioxidant effect
at a genomic level by increasing the expression and
transcriptional activity of the redox-sensitive
transcription factor nuclear factor (erythroid-derived
2)-like 2 (Nrf2) (Na and Surh, 2014), which is normally
bound to the cytosolic protein Kelch-like ECH-
associated protein 1 (Keap1). When betalains react with
the redox-reactive cysteine residues of Keap1, the
connection Nrf2–Keap1 is disrupted and Nrf2
translocates into the nucleus, where it binds to the
antioxidant responsive elements and initiates the gene
transcription of phase-II and antioxidant enzymes
(Esatbeyoglu et al., 2012; Esatbeyoglu et al., 2014;
Krajka-Kuzniak et al., 2013; Saw et al., 2011).
In vivo studies of antioxidant activity of betalains
have been performed in rat (Kujawska et al., 2009) and
mice models (Cho et al., 2017). Kujawska et al. (2009)
concluded that beetroot juice pretreatment (8 mL/kg/
day for 28 days, containing 79.3 mg/100 mL of
betaxanthines and 159.6 mg/100 mL of betacyanins)
can counteract xenobiotic-induced oxidative stress in
Male Wistar rats. Cho et al. (2017) documented the
association between the antioxidant and radioprotective
capacities of beetroot extracts in C57BL/6 mice exposed
to γ-ray irradiation for 10 days. These authors showed
that the administration of beetroot extracts induced
significant decrease in the DNA damage and increase
in proliferation of hematopoietic progenitor cells (Cho
et al., 2017).
Copyright © 2017 John Wiley & Sons, Ltd.
In short, betalains differ from vitexin and its
glucosides, as the former behave as antioxidants in both
human erythrocytes and cancer cells, the latter as
antioxidants in human erythrocytes and as pro-oxidants
in cancer cells. Greater efforts must be done to fill in the
gaps of the absence of studies regarding the in vivo
antioxidant activity of vitexin.
Antiproliferative activity
The antineoplastic activity of vitexin and isovitexin has
been reviewed recently (He et al., 2016). Several papers
demonstrated the pro-apoptotic effect of vitexin in
different cancer cell lines. For instance, Lee et al.
(2012) showed that vitexin is able to decrease the
mitochondrial membrane potential in U937 human
leukemia cells, leading to the activation of the caspases
9/3-mediated apoptosis.
We focused here on the anticancer activity of vitexin-
2-O-xyloside, vitexin-2-O-rhamnoside and betalains, as
well as their combinations.
Our results and those of other authors, regarding
different types of cancer cell lines, are listed in Table 1,
which also refers to the molecular mechanisms under
investigation.
Vitexin-2-O-xyloside and vitexin-2-O-rhamnoside
were able to inhibit DNA synthesis and reduce the
proliferation rate of MCF-7 breast cancer cells (Ninfali
et al., 2007) and RKO cancer cell lines (Gennari et al.,
2011).
Phytother. Res. (2017)
 PHARMACOLOGICAL ACTIVITIES OF GREEN AND RED BEETS

Figure 4. Antioxidant capacity, measured with the ORAC method of apigenin and its derivatives glycosides: vitexin, vitexin-2-O-xyloside
(XVX) and vitexin-2-O-rhamnoside (VOR). ORAC values, expressed on gram basis, i.e. μmol TE/g of pure phytochemical (a). ORAC values,
expressed on mmol basis, i.e. μmol TE/mmol of pure phytochemical (b). Different letters indicate statistically significant differences,
evaluated by one-way ANOVA (p < 0.05). The dark column highlights how the ORAC of XVX changes when this value is calculated on gram
or mmol basis.

In Hep3B liver cancer cells, vitexin-2-O-xyloside had
a greater cytotoxic effect than vitexin-2-O-rhamnoside
(p < 0.05), as shown by the IC50 values of
64.9 ± 3.2 μM and 72.5 ± 3.6 μM, respectively
(unpublished results).
In CaCo-2 cancer cells, characterized by mutated
TP53 and overexpression of multidrug resistance
(MDR) proteins, vitexin-2-O-xyloside was shown to
activate the mitochondrial intrinsic apoptotic pathway
by increasing the levels of cleaved caspase 3 and poly
ADP ribosyl polymerase 1 (PARP1) (Farabegoli et al.,
2017; Papi et al., 2013).
In T24 bladder cancer cells, which are resistant to
several anticancer drugs (Massari et al., 2015), due to
Copyright © 2017 John Wiley & Sons, Ltd.
over expression of the MDR (Shimada et al., 2011) and
mutated TP53 gene (Savio et al., 2014), vitexin-2-O-
xyloside showed a remarkable dose–response
anticancer effect, with an IC50 of 8.8 ± 0.8 μM. This
effect was exerted through an activation of the intrinsic
pathway of apoptosis and downregulation of the mRNA
level of baculoviral inhibitor of apoptosis repeat-
containing 5 (BIRC5) (Scarpa et al., 2016), the gene that
codifies for survivin, a member of the inhibitor of
apoptosis proteins, which allow the survival of the
cancer cells (Samali and Jager, 2014).
In CaCo-2 colon cancer cells and HepG2 liver cancer
cells, vitexin-2-O-xyloside is able to induce
downregulation in the expression levels of both hypoxia
Phytother. Res. (2017)
 P. NINFALI ET AL.
Figure 5. Representative images of DCFH-DA assay in CaCo-2 (a) and HepG2 (b) cancer cells treated with vitexin-2-O-xyloside (XVX).
(a, b) CTRL, untreated cells; XVX (30 μM), cells treated with 30 μM XVX for 24 h. Quantitative analysis (%) of DCF fluorescent positive
**
CaCo-2 (c) and HepG2 (d) cells. p < 0.01. [Colour figure can be viewed at wileyonlinelibrary.com]
inducible factor (HIF) 1A and vascular endothelial
growth factor (VEGF) A genes, leading to a reduction
of their pro-survival skills and proliferation rates
(Scarpa et al., 2017).
In rat neuroblastoma PC12 cells, vitexin was found to
downregulate the protein levels of the transcription
factor HIF1α as well as of VEGFA, with a reduction in
migration and invasiveness rates (Baba et al., 2010; Choi
et al., 2006).
The link between HIFs and the cell proliferation rate
can be ascribed to the fact that HIFs are heterodimers
having a constitutively expressed HIF1β subunit and
an oxygen responsive HIF1α subunit (Brahimi-Horn
et al., 2007). In hypoxic conditions, HIF1α escapes
proteasomal degradation, migrates to the nucleus, binds
HIF1β and stimulates the target gene expression to
increase the proliferation rate of cancer cells (Klimova
and Chandel, 2008).
Vascular endothelial growth factor A is one of the key
genes controlled by HIF1α, as it encodes for VEGF, the
principal regulator of angiogenesis during tumor growth
and development (Ellis et al., 2000; Ferrara, 2005). The
downregulation of VEGFA inhibits the angiogenesis
process, thus blocking the oxygen supply to the cancer
cells. Vascular endothelial growth factor is also able to
inhibit apoptosis through upregulation of anti-apoptotic
proteins (Doll et al., 2007).
In brief, vitexin and vitexin-2-O-xyloside behave as
multitarget anticancer agents, which activate the
intrinsic pathway of apoptosis, as well as downregulate
survival, hypoxia and angiogenesis factors in the tumor
cells.
Table 1 also lists the individual antiproliferative
activity of BX and BC on cancer cells.
In a recent investigation, Clement et al. (2016)
assessed that beetroot, juiced or blended, was the
second most popular functional food used among a
Copyright © 2017 John Wiley & Sons, Ltd.
group of 150 prostate, breast and colorectal cancer
patients in Trinidad, in combination with conventional
anticancer drugs.
In CaCo-2 colorectal cancer cells, cytotoxicity of BX
and BC showed a hormetic pattern, with the highest
cytotoxicity exerted through the activation of apoptosis,
in the range 0.25–0.35 μg/mL (Farabegoli et al., 2017).
At higher concentrations, the cytotoxic effect
disappears, possibly due to their antioxidant activity
(Farabegoli et al., 2017), which helps the cells to
counteract the oxidative stress.
Recently, Nowacki et al. (2015) investigated the
antiproliferative effect of BC in MCF-7 breast cancer
cells, showing that a mixture of betanin/isobetanin is
able to induce apoptosis in these cancer cells, through
the activation of p53, the increase of the protein levels
of pro-apoptotic factors Bcl-2-associated death
promoter (Bad), TNF-related apoptosis-inducing ligand
receptor 4 (TRAILR4), Fas and the induction of
autophagic cell death.
In T24 bladder cancer cells, BC displayed a dose–
response antiproliferative effect, with an IC50 of
99.8 ± 19.9 μg/mL, while the BX did not show any effect
(Scarpa et al., 2016). Most of the studies in literature
show that BC have a higher antitumor effect than BX
(Clifford et al., 2015). In animal models, the antitumor
effect of BC has been observed in lung, skin and liver
cancers (Lechner et al., 2010), and recently, it has been
investigated in human prostate, skin, breast and
pancreatic tumor cells, both in the presence and in the
absence of doxorubicin (Kapadia et al., 2011; Kapadia
et al., 2013).
Moreover, Das et al. (2016) showed that BC from beet
root juice can increase the doxorubicin-mediated
apoptosis of MDA-MB-231 breast cancer cells and are
also able to decrease the doxorubicin toxicity in
cardiomyocytes.
Phytother. Res. (2017)
 Table 1. Molecular targets and mechanisms of anticancer and antiinflammatory effects exerted by vitexin-2-O-xyloside (XVX), vitexin-2-O-rhamnoside (VOR), betaxanthins (BX), betacyanins (BC),
XVX + BC, XVX + BX and XVX + BC + BX on different cancer cell lines

Phytochemical Concentration Cancer type Cell line Molecular target/mechanism Effect Reference

XVX 9 μg/mL Breast MCF-7 DNA synthesis inhibition Proliferation inhibition (Ninfali et al., 2007)
90 μg/mL Colon RKO ANNEXIN V, G1/S BLOCK Apoptosis induction (Gennari et al., 2011)

Copyright © 2017 John Wiley & Sons, Ltd.

40 μg/mL LoVo ANNEXIN V, Bax, Bcl-2, CASPASE 3, PARP1 Apoptosis induction (Papi et al., 2013)
25 μg/mL CaCo-2 CASPASE 3, PARP1 Apoptosis induction (Farabegoli et al., 2017)
25 μg/mL CaCo-2 COX-2, IL-8 Inflammation inhibition (Farabegoli et al., 2017)
2.5 μg/mL Bladder T24 ANNEXIN V, CASPASE 3, BAX, BIRC5 Apoptosis induction (Scarpa et al., 2016)
2.5 μg/mL T24 CTNNB1 Pro-survival pathways inhibition (Scarpa et al., 2016)
64.9 μM Liver Hep3B CASPASES 9/3 Proliferation inhibition (Unpublished results)
50 μM HepG2 CASPASES 9/3 Apoptosis induction (Scarpa et al., 2017)
50 μM HepG2 BIRC5, HIF1A, VEGFA Pro-survival pathways inhibition (Scarpa et al., 2017)
VOR 17.5 μg/mL Breast MCF-7 DNA synthesis inhibition Proliferation inhibition (Ninfali et al., 2007)
72.5 μM Liver Hep3B Induction of cell mortality Proliferation inhibition (Unpublished results)
BX 0.35 μg/mL Colon CaCo-2 Bcl-2, CASPASE 3, PARP1 Apoptosis induction (Farabegoli et al., 2017)
0.35 μg/mL CaCo-2 COX-2, IL-8 Inflammation inhibition (Farabegoli et al., 2017)
BC 40 μM Leukemia K562 Bax, Bcl-2, PARP1 Apoptosis induction (Sreekanth et al., 2007)
30 μM Breast MCF-7 ANNEXIN V, Bad, TRAIL, Fas, p53 Apoptosis induction (Nowacki et al., 2015)
30 μM MCF-7 Autophagic vacuoles Autophagy induction (Nowacki et al., 2015)
0.25 μg/mL Colon CaCo-2 Bax, CASPASE 3, PARP1 Apoptosis induction (Farabegoli et al., 2017)
0.25 μg/mL CaCo-2 COX-2, IL-8 Inflammation inhibition (Farabegoli et al., 2017)
50 μg/mL Bladder T24 CASPASES 8/3 Apoptosis induction (Scarpa et al., 2016)
50 μg/mL T24 CTNNB1 Pro-survival pathways inhibition (Scarpa et al., 2016)
200 μg/mL Lung A549 CASPASES 9/3/7, PARP1 Apoptosis induction (Zhang et al., 2013)
100 μg/mL A549 CD31 Angiogenesis inhibition (Zhang et al., 2013)
XVX + BC 2.5 + 50 μg/mL Bladder T24 ANNEXIN V, CASPASES 8/3, BAX, BIRC5 Apoptosis induction (Scarpa et al., 2016)
PHARMACOLOGICAL ACTIVITIES OF GREEN AND RED BEETS

2.5 + 50 μg/mL T24 CTNNB1 Pro-survival pathways inhibition (Scarpa et al., 2016)
25 + 0.25 μg/mL Colon CaCo-2 CASPASE 3 Proliferation inhibition (Farabegoli et al., 2017)
XVX + BX 25 + 0.35 μg/mL Colon CaCo-2 CASPASE 3 Proliferation inhibition (Farabegoli et al., 2017)
XVX + BC + BX 25 + 0.25 + 0.35 μg/mL Colon CaCo-2 Bax, Bcl-2, CASPASE 3, PARP1 Apoptosis induction (Farabegoli et al., 2017)
25 + 0.25 + 0.35 μg/mL CaCo-2 COX-2, IL-8 Inflammation inhibition (Farabegoli et al., 2017)

Phytother. Res. (2017)

 P. NINFALI ET AL.
Figure 6. Proposed mechanisms by which vitexin and vitexin-2-O-xyloside (XVX) are able to trigger (a) apoptosis and (b) antiinflammatory
effect. The molecular markers modulated by vitexin and XVX are written in full in the abbreviation list.
Effects of betalains combined with vitexin-2-O-xyloside
(XVX) in cancer cells
The pairs XVX + BX, XVX + BC and the triple
combination XVX + BX + BC showed a significantly
greater antiproliferative effect compared with the
individual compounds on several cell culture models.
In CaCo-2 colon cancer cells, in the presence of
XVX + BC + BX, a downregulation of the anti-
apoptotic protein B-cell leukemia/lymphoma 2 (Bcl-2)
was observed, together with an overexpression of the
pro-apoptotic Bcl-2-like protein 4 (Bax) protein
(Table 1). The decrease in Bcl2/Bax ratio has been
proposed as an indicator of the activation of the intrinsic
(mitochondrial) pathway of apoptosis (Khan et al.,
2008). Indeed, the triple combination induced an
increase of the late apoptosis signaling molecules, i.e.
the cleaved Caspase 3 and cleaved PARP1, a nuclear
enzyme involved in DNA repair and stability, which is
cleaved and inactivated by the Caspase 3.
In T24 bladder cancer cells, the combination
XVX + BC induced a significant increase in the number
of apoptotic cells due to the vitexin-2-O-xyloside-
mediated induction of the intrinsic pathway of apoptosis
and downregulation of the anti-apoptotic gene BIRC5
(survivin) (Khan et al., 2007); activation of the extrinsic
apoptotic pathway, through an increase in caspase 8
activity levels due to BC (Scarpa et al., 2016);
downregulation of the expression levels of Catenin
beta-1 (CTNNB1), the gene that encodes for β-catenin
(Table 1).
Regarding the reduction of the β-catenin expression
levels, other authors have shown that the sole reduction
of CTNNB1 mRNA levels caused a considerable
reduction in the proliferation rate of T24 bladder cancer
Copyright © 2017 John Wiley & Sons, Ltd.
cells (Yang et al., 2011). Therefore, the remarkable
reduction in the expression levels of the pro-survival
gene CTNNB1, exerted by XVX + BC, very likely
contributes to the reduction in the proliferation rate in
T24 cancer cells (Scarpa et al., 2016).
Importantly, vitexin-2-O-xyloside and BC, neither
individually nor in combination, were able to exert
cytotoxic effects on normal human skin NCTC 2544
keratinocytes (Scarpa et al., 2016), thus demonstrating
that their cytotoxic and pro-apoptotic effects specifically
target the tumor cells.
Antiinflammatory activity
The antiinflammatory effects of vitexin and isovitexin
have been documented in vitro (Huang et al., 2005; Lin
et al., 2005; Rosa et al., 2016) and in vivo (Borghi et al.,
2013; De Melo et al., 2005; Dong et al., 2011; Dos Reis
et al., 2014; Kang et al., 2015; Rosa et al., 2016) and
recently reviewed (He et al., 2016).
Our in vitro investigations showed that vitexin-2-O-
xyloside, when used individually, inhibits the expression
levels of interleukin (IL)-8 and cyclooxygenase-2
(COX-2) genes in LPS-treated CaCo-2 cells. This effect
is enhanced when vitexin-2-O-xyloside is combined with
BX and BC (Farabegoli et al., 2017).
Regarding the in vivo antiinflammatory activity,
Borghi et al. (2013) showed that vitexin (10 mg/kg,
intra-peritoneally administered) is able to reduce the
levels of the pro-inflammatory cytokines (tumor necrosis
factor (TNF)-α, IL-1β, IL-6, and IL-33) and increase the
antiinflammatory cytokine (IL-10) production, in Swiss
mice. In addition, Dos Reis et al. (2014) showed a
reduction in the nitric oxide and IL-17 production, in
Phytother. Res. (2017)
 PHARMACOLOGICAL ACTIVITIES OF GREEN AND RED BEETS
the same mouse model. When orally administered,
vitexin (5, 15, 30 mg/kg) reduced prostaglandin E2 (PG
E2), TNF-α, IL-1β, nitric oxide release in the peritoneal
cavity of LPS-challenged Swiss–Webster mice (Rosa
et al., 2016).
The antiinflammatory effects of betalains have been
documented in vitro (Reddy et al., 2005; Vidal et al.,
2014) and in vivo (Asgary et al., 2016; El Gamal et al.,
2014; Krajka-Kuzniak et al., 2013; Pietrzkowski et al.,
2010; Tan et al., 2015) and recently reviewed by Clifford
et al. (2015).
Regarding molecular mechanisms, the betalains are
able to reduce the protein levels of the pro-
inflammatory cytokines TNF-α, IL-6, IL-8 and IL-1β,
the reactive oxygen and nitrogen species levels and also
the activity of COX-2 and lypoxygenase (LOX)
enzymes, leading to the reduction of PGE2 and LOX-5
levels. Moreover, it has been shown that BX and BC
can reduce the activity of the transcription factors
activator protein-1 and nuclear factor kappa B (NF-
kB), blocking the positive feedback loop, which can lead
to a further increase in the levels of pro-inflammatory
cytokines (Clifford et al., 2015).
Regarding the in vivo antiinflammatory activity of
betalains, Pietrzkowski et al. (2010) showed that the
therapeutic administration of betalain enriched capsules
can reduce the inflammation state and osteoarthritis
pain in patients, reducing the levels of the pro-
inflammatory markers IL-6 and TNF-α.
El Gamal et al. (2014) demonstrated that rats treated
with gentamicin and fed with a beetroot extract had
significantly lower concentrations of several
inflammatory markers, including IL-6, TNF-α,
myeloperoxidase and NF-kB compared with rats
treated with the nephrotoxic drug gentamicin. Asgary
et al. (2016) showed that in 24 hypertensive subjects
who consumed 250 mL/day of red beet juice or
250 g/day of cooked beet for 2 weeks, the levels of pro-
inflammatory markers, like IL-6 and TNF-α, were
significantly decreased.
In conclusion, the in vivo antiinflammatory effects of
betalains are widely documented, with the effects on
humans clearly illustrated. Some important papers
documenting the in vivo antiinflammatory activities of
vitexin have been published in animals, providing an
important contribution for future studies on humans.
CONCLUSIONS
In this review, we have illustrated the ability of vitexin,
vitexin-2-O-rhamnoside and vitexin-2-O-xyloside,
together with BC and BX to influence the expression
of several genes, in such a way as to contrast cancer cell
proliferation and induce apoptosis (Fig. 6a), as well as to
provide antiinflammatory activity (Fig. 6b).
Vitexin-2-O-xyloside and betalains, when used in
combination, are effective in inhibiting the proliferation
of some cancer cell lines that possess mutated and
inactive p53 and show high resistance against
conventional anticancer drugs. No toxic side effect was
assessed in normal cells. In the tumor cells, vitexin-2-
O-xyloside triggers intrinsic apoptosis, acting as
intracellular pro-oxidant; the betalains and, particularly,
BC trigger the extrinsic apoptotic pathway and act as
Copyright © 2017 John Wiley & Sons, Ltd.
antioxidants. The two compounds behave in opposite
ways towards intracellular ROS but behave in a
complementary fashion in providing pro-apoptotic
signaling, which is corroborated by inhibition of the
pro-survival markers: survivin and β-catenin.
The combined effect of vitexin-2-O-xyloside and
betalains seems particularly effective in colon and
bladder cancer cells and could be useful for
chemoprevention in post-surgical oncological patients.
Despite the remarkable progress that has been made
in our understanding of the molecular mechanisms of
the anticancer effects of these phytochemicals, some
critical aspects require further investigation.
First, the appropriate formulation of the combination
of vitexin-2-O-xyloside with BC and BX able to attain
optimal chemopreventive efficacy must be investigated.
Second, the use of these phytochemicals in
combination with conventional anticancer drugs must
be explored. Preliminary results are promising (Kapadia
et al., 2013), but long-term studies to evaluate their
potential beneficial effects are needed.
Third, a higher number of tumor cell lines should be
investigated to find an appropriate combination of
vitexin-2-O-xyloside and BC or BX phytochemicals able
to obtain the right cytotoxic effect for specific tumor
cells and tissues. Moreover, the ability of the
phytochemicals to block the invasiveness and metastasis
development should be exploited (Wu et al., 2017).
Fourth, the effect of the tumor microenvironment on
the cancer cells should be explored in order to
understand if the phytochemicals could influence the
pro-survival and pro-inflammatory cell signaling
between the tumor cells and the microenvironment (de
Barrios et al., 2017).
Finally, green and red beet phytochemicals are
derived from natural sources, as the chemical synthesis
of vitexin-2-O-xyloside, vitexin-2-O-rhamnoside and
betalains is complex and unresolved at this time.
Therefore, efficient methods of purification must be
developed and upgraded to industrial scale. Starting
from the chemical structure of natural compounds,
future molecules able to further promote apoptosis in
tumor cells must be designed.
In conclusion, further studies are necessary to reveal
the complete antitumoral potential of the
phytochemicals of the genus Beta, which has been part
of the food supply chain and traditional medicine for
millennia among Mediterranean populations.
Acknowledgements
The authors wish to thank Dr. Donato Angelino from the Department
of Food Sciences, University of Parma, Italy, for useful suggestions
during the preparation of the manuscript. The authors also wish to
thank the Suba Seeds Company, Longiano (FC), Italy, for providing
seeds of Beta vulgaris var. cicla L. and Beta vulgaris var. rubra L. and
the Company ABOCA S.p.A. Soc. Agr., San Sepolcro (AR), Italy,
for providing lyophilized extract of hawthorn leaves. The help of Dr.
Timothy Bloom for assistance in the English language is gratefully
acknowledged.
Conflict of Interest
The authors declare no conflict of interest.
Phytother. Res. (2017)
 P. NINFALI ET AL.
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