C H A P T E R
1
The Pharmacology of Avenanthramides:
Polyphenols
Ilias Marmouzi*, Shahira M. Ezzat†,‡
*Laboratory of Pharmacology and Toxicology, Faculty of Medicine and Pharmacy, Mohammed V University in Rabat,
Rabat, Morocco †Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt ‡Department of
Pharmacognosy, Faculty of Pharmacy, October University for Modern Science and Arts (MSA), Cairo, Egypt
1 INTRODUCTION
According to epidemiological data and experimental
studies, whole grain consumption is associated with a
reduced risk of diseases [1–3]. In fact, the risk for develop-
ing certain diet-related disorders such as type 2 diabetes,
obesity, and cardiovascular disease (CVD) is inversely
correlated with the intake of cereal-based food products
[4]. Among cereals, oats (Avena sativa L.) is a multifunc-
tional crop commonly consumed as whole grains, provid-
ing essential nutrients [5]. The consumption of oat-based
products can lower serum cholesterol levels, reduce
glucose uptake, and decrease plasma insulin response
[3,5]. A. sativa grain contains numerous bioactive phyto-
chemicals [2,6], and exclusively the phenolic amides,
called avenanthramides (AVAs) [7]. AVAs are low mo-
lecular weight cinnamoylanthranilate alkaloid polyphe-
nols composed of an hydroxycinnamic acid derivative
linked to an anthranilic acid derivative through a pseudo
peptide bond [8]. The major AVAs are: N-(40-hydroxy-30-
methoxycinnamoyl)-5-hydroxyanthranilic acid (2f ), N-(40-
hydroxycinnamoyl)-5-hydroxyanthranilic acid (2p), and
N-(30,40-dihydroxycinnamoyl)-5-hydroxyanthranilic acid
(2c) [9]. The concentrations of these three AVAs are highly
dependent on the genotypes and growing environment
[10]. These compounds are antipathogens (phytoalexins),
which are produced by the plant in response to the expo-
sure to pathogens such as fungi [11]. In recent years, there
has been considerable interest in the benefit of phenolic
amides from plant-based foods to human health [12]. Gen-
erally, bioactive phenolic compounds in cereal grains are
mainly located in the bran fraction and covalently bound
to indigestible polysaccharides [13]. However, there are
currently no indications that the AVAs are present as bound
Polyphenols: Prevention and Treatment of Human Disease
https://doi.org/10.1016/B978-0-12-813008-7.00001-1
compounds [14]. AVAs exist in various chemical forms that
determine their gut absorption. Understanding the struc-
tural factors that influence absorption and metabolism is
essential to determine the compounds that are better
absorbed and that lead to the formation of known active
metabolites [15]. However, the data available on phenolics
bioavailability are still limited; Scalbert and Williamson
[15] proposed the possible pathway that allows the predic-
tion of phenolics uptake from the diet [16]. Bioactive com-
pounds are extra nutritional constituents with health
benefits and typically occur in small quantities in foods.
Accordingly, many epidemiologic studies have shown
the protective effects of plant-based diets on CVD [17].
The polyphenolic compounds AVAs are of high interest
regarding their potent antiinflammatory and antioxidant
activities and cardioprotective potential. A more compre-
hensive scheme of AVAs action and biotransformation/
bioavailability can help in using them in therapy and phar-
maceutical formulations. Also, understanding the biosyn-
thetic pathways and the main elicitors will improve
AVAs process and production yield. This chapter aim to
summarize the pharmacological potential and mechanisms
of AVAs, their structure activity/bioavailability relation-
ship, and suggested metabolic pathways.
2 OCCURRENCE AND BIOSYNTHESIS
AVAs are phytoalexins that are generally induced in
oats in response to pathogens [18]. AVA biosynthesis
results from the acylation of anthranilic acid and deriva-
tives by the CoA thioester of p-coumaric, ferulic, or caffeic
acid, catalyzed by hydroxycinnamoyl CoA. AVA were
assigned alphabetic letters, after their first isolation from
3 © 2018 Elsevier Inc. All rights reserved.
4 1. THE PHARMACOLOGY OF AVENANTHRAMIDES: POLYPHENOLS

TABLE 1.1 Collins and Dimberg Nomenclatures for Avenanthramides

Dimberg’s modified Dimberg’s original Collins n R1 R2 R3 R4 R5

1c F 1 H H OH OH H
1f E 1 H H OCH3 OH H
1p D 1 H H H OH H

1s 1 H H OCH3 OH OCH3
2c Bc C 1 H OH OH OH H
2f Bf B 1 H OH OCH3 OH H
2p Bp A 1 H OH H OH H
2s 1 H OH OCH3 OH OCH3

oat groats and hulls [19,20]; later Dimberg developed a
more systematic nomenclature (Table 1.1), assigning the
anthranilate derivatives a number and the accompanying
cinnamate derivatives the following letters: c for caffeic
acid, f for ferulic acid, and p for p-coumaric acid [21].
More than 40 different AVAs have been detected in oats,
although the predominant metabolites are 2c, 2f, and 2p
(Fig. 1.1). AVAs are mainly concentrated in oat grains
with 2–300 mg/kg, with high concentrations in the bran.
However, AVA occurs also in the hulls and leaves with a
concentration range from 5 to 120 mg/kg, highly depen-
dent on cultivars and elicitors [6]. The function of AVAs
in the grains has not been identified, but as the total con-
tent increases during germination [22,23], they might
function as stress protective agents against, for example,
oxidation, or against various pests or pathogens. In the
leaves the AVA levels increase after attacks by incompat-
ible races of the crown rust fungus Puccinia coronata or
treatment with relevant elicitors [24]. Many factors affect
AVA content, including cultivar, year, location, culti-
vation conditions, and the interactions between these fac-
tors [6]. For instance, AVA content is negatively affected
by high nitrogen fertilization but does not differ between
conventional or organic cropping systems [25]. The effect

FIG. 1.1 Structures of major avenanthramides.

I. POLYPHENOLS IN THE PREVENTION AND TREATMENT OF VASCULAR AND CARDIAC DISEASE AND CANCER
 3 IN VITRO PHARMACOLOGICAL ACTIVITIES
of processing on AVA concentrations showed an increase
in AVA 2c, 2p and 2f, possibly through de novo synthesis,
release of insoluble bound forms, increased extractability,
or all factors. The hydrothermal processes, for example,
steaming, autoclaving, and drum drying, also affected
the concentrations of AVA and phenolics in oats [26,27].
3 IN VITRO PHARMACOLOGICAL
ACTIVITIES
3.1 Antiatherosclerosis
Studies have clearly shown that the consumption of
oats lowers total plasma as well as LDL cholesterol,
and it reduces the risk of coronary heart disease (CHD)
[7]. Nie et al. [28] reported that 2c significantly inhibited
the proliferation of serum-induced smooth muscle cells
(SMC). It was noticed that 2c inhibited >50% of SMC pro-
liferation at a concentration of 120 μM. This was mea-
sured by [3H] thymidine incorporation, and increased
the doubling time of rat SMC line from 28 to 48 h. Treating
human SMC with 40, 80, and 120 μM of 2c inhibited cell
number increase by 41%, 62%, and 73% respectively. Fur-
thermore, nitric oxide (NO) production was significantly
increased by treatment of SMC and HAEC with 2c in a
dose-dependent manner. AVA 2c at a concentration of
120 μM increased NO production in SMC and human aor-
tic endothelial cells (HAEC) by three- and ninefold,
respectively. These increases were accompanied by the
up-regulation of mRNA expression for endothelial NO
synthase (eNOS) in both vascular SMC and HAEC. The
obtained results suggested that the AVAs might help in
the prevention of atherosclerosis through inhibition of
SMC proliferation and increasing NO production.
Further work of Nie et al. [29] investigated the cell
cycle inhibitory mechanism of AVA 2c. Rat embryonic
aortic SMC was used, and flow cytometry analysis
revealed that treatment of SMC with 80 μM 2c arrested
the cell cycle in the G1 phase as indicated. The results
demonstrate that AVA 2c arrests SMC proliferation at
G1 phase by up-regulating the p53-p21cip1 pathway
and inhibiting pRB phosphorylation. This inhibitory
effect is an additional indication for the potential
health benefit of oat consumption in the prevention of
CHD beyond its known effect through lowering blood
cholesterol.
The Ldlr
/
mouse is one of the most commonly used
atherosclerosis mouse models with similar cholesterol dis-
tributions to humans. Ldlr
/
mice were fed a low fat,
high fat, high fat containing regular oat brans with low
levels of AVAs (HFLA) or high levels of AVAs (HFHA)
diet [30]. The inclusion of oat bran with high levels of
AVAs in the diet significantly suppresses high fat diet-
induced atherosclerosis in this mouse model. These results
I. POLYPHENOLS IN THE PREVENTION AND TREATMENT OF VASCULAR AND CARDIAC DISEASE AND CANCER
5
provide histological evidence that an oat-based diet is
capable of suppressing aortic fatty lesions induced by con-
suming a high amount of saturated fat in Ldlr
/
mice.
3.2 Antiinflammatory
Yang et al. [21] reported the effectiveness of the major
AVAs 2c, 2f and 2p in inhibiting TNF-α-induced NF-κB
activation in C2C12 cells. The EC50 values were 64.30,
29.30, and 9.10 μM for the three compounds, respectively.
AVA-enriched extract of oats was reported to signifi-
cantly suppress interleukin (IL)-1β-stimulated secretion
of proinflammatory cytokines, such as IL-6, IL-8, and
MCP-1, by HAEC. The inhibitory effect of AVAs on
the expression of proinflammatory cytokines was
studied by Guo et al. [31]. Treatment of HAEC mono-
layers with AVA-enriched extract for 24 h suppressed
IL-β-stimulated activation of NF-κB in a concentration-
dependent manner. CH
3 2c, a synthetically prepared
methyl ester derivative of 2c with a high biological
potency, significantly and dose dependently decreased
mRNA expression and secretion of cytokines IL-6, che-
mokines IL-8, and monocyt chemotactic protein MCP-1
by HAEC as determined by real-time RT-PCR and ELISA,
and it inhibited IL-1β- and TNFα-stimulated NF-κB acti-
vation as determined by a NF-κB DNA binding assay
and a NF-κB luciferase reporter assay. AVA-enriched
extract and 2c as well as CH
3 2c also inhibited the
NF-κB-dependent reporter gene expression activated by
TNFR-associated factor 2 and 6 (TRAF2, TRAF6) and
NFκB-inducing kinase (NIK). CH
3 2c decreased in a
dose-dependent manner the phosphorylation level of
IκB kinase (IKK) and IκB, and prevented IκB degradation
as measured by western blotting. In addition, CH
3 2c
markedly increased the overall levels of high mass
ubiquitin-conjugated protein levels while it mildly inhib-
ited proteasome activity.
Liu et al. [32] also provided evidence for the potential
antiinflammatory activities of AVAs. The authors identi-
fied AVAs 2p, 2f, and 2c as the major constituents of
the total soluble antioxidant phenolic compounds in
oats and assessed the antiatherogenic effect of partially
purified AVAs fraction isolated from oats by testing its
effect on adhesion of monocytes to HAEC monolayers,
expression of adhesion molecules, and production of
proinflammatory cytokines and chemokines by HAEC.
The AVA-enriched mixture (AEM) showed no toxicity
to HAEC in concentrations up to 40 ng/mL. The preincu-
bation of HAEC with 4, 20, and 40 ng/mL AEM for
24 h showed a concentration-dependent reduction of
U937 monocytic cells adhesion to interleukin (IL)-1β-
stimulated HAEC. Twenty-four hours incubation of
HAEC with 20 and 40 μg/mL AEM had a significant sup-
pressing effect on vascular cell adhesion molecule-1
(VCAM-1) IL-1β-stimulated expressions of intracellular
6 1. THE PHARMACOLOGY OF AVENANTHRAMIDES: POLYPHENOLS
adhesion molecule-1 (ICAM-1), and E-selectin and the
secretion of proinflammatory cytokines IL-6, chemokines
IL-8, and monocyte chemoattractant protein (MCP)1.
Moreover, the results from a Sur et al. [33] study
demonstrated that AVAs could be considered potent
antiinflammatory agents that appear to mediate the anti-
irritant effects of oats. It was reported that AVAs at 1 part
per billion inhibited the degradation of inhibitor of
nuclear IκB-α in keratinocytes; this effect could be attrib-
uted to the reduction of p65 subunit phosphorylation of
NF-κB. Moreover, the AVAs-treated cells showed a sig-
nificant reduction of the IL-8 as a result of the inhibition
of TNF-α induced NF-κB luciferase activity. Furthermore,
AVA topical application (1–3 ppm) eliminated the inflam-
mation produced in murine models as a result of contact
hypersensitivity and neurogenic inflammation, and
also reduced pruritogen-induced scratching in a murine
itch model.
3.3 Antioxidant
In a study done by Peterson et al. [34] 2p, 2f, and 2c
were synthesized, purified, and tested for antioxidant
effect using two in vitro assays: inhibition of β-carotene
bleaching and scavenging of the free radical 2,2-
diphenyl-1-picrylhydrazyl (DPPH). All the tested AVAs
possessed antioxidant activity in both systems, with 2c
showing the strongest activity compared to 2p and 2f in
both DPPH and β-carotene bleaching with EC50 (0.074
and 0.0029 μmoles, respectively). 2c was almost as active
as the standard synthetic antioxidant, butylated hydroxyto-
luene (BHT), in the β-carotene bleaching system (EC50
0.0012 μmoles). In the DPPH system, 2c and 2f were more
active than the Trolox (EC50 0.074, 0.105, and 0.160 μmol,
respectively).
In another study by Cai et al., [35], oats were extracted
with 80% aqueous ethanol and successively fractionated
by liquid-liquid partition to yield n-hexane, ethyl
acetate, n-butanol, and water layers. The ethyl acetate
fraction exhibited the highest total phenolic content
(3764.00  29.90 mg gallic acid equivalents/100 g dry
weight fraction). This fraction had the strongest DPPH
radical scavenging activity with EC50 (2.50  0.40 mg/
mL) and also showed the highest inhibitory activity on
the oleic acid-induced fatty liver model in vitro. The
SF3 subfraction showed the highest activity in DPPH
scavenging and oleic acid-induced fatty liver assays.
HPLC analysis of SF3 revealed that its major components
are AVAs 2c, 2p, and 2f, which constituted 5.20%, 9.19%,
and 8.06% of SF3 dry weight, respectively. Also, those
compounds had significant inhibitory effects on oleic
acid-induced fatty liver.
Bratt et al. [22] synthesized eight AVAs from anthranilic
acid derived 2-methylbenzoxazin-4-ones and 5-
hydroxyanthranilic acid, for identification in oat extracts
I. POLYPHENOLS IN THE PREVENTION AND TREATMENT OF VASCULAR AND CARDIAC DISEASE AND CANCER
and evaluation of their antioxidant activity against DPPH
and linoleic acid peroxidation; 2f and 2c possessed high
free radical scavenging activity toward DPPH, while 2p
had no activity. Initially, the antioxidant activity of the
AVAs decreased in a similar order as for the correspond-
ing cinnamic acids, that is: sinapic > caffeic > ferulic > p-
coumaric acid. It was noted also that the AVAs derived
from 5-hydroxyanthranilic acid were somewhat more
active than those derived from anthranilic acid derived
2-methylbenzoxazin-4-ones. All AVAs inhibited azo-
initiated peroxidation of linoleic acid.
Fagerlund et al. [36] investigated the effect of the
structure-activity relationship on the antioxidant potenti-
ality of 15 AVAs with various substitution patterns in their
two aromatic rings. The compounds have their A ring
derived from anthranilic [1], 5-hydroxy anthranilic [2] or
5-hydroxy-4-methoxyanthranilic [3] acids, and their
B ring derived from cinnamic (a), p-coumaric (p), ferulic
(f ), sinapic (s), or caffeic (c) acids. Radical-scavenging
activity was tested against DPPH. No activity was exerted
by the AVAs containing no hydroxy group in either of the
aromatic rings. The presence of a single p-hydroxy group
in either of the rings had a minor influence on the activity.
Addition of one methoxy group ortho to the hydroxy
group in either of the rings resulted in increasing activity,
such as the 5-hydroxy-4-methoxyanthranilic derivatives
and ferulic acid derivatives. Addition of a second methoxy
group also ortho to the hydroxy group, as in sinapic acid,
enhanced the activity. Thus, among the noncatecholic
compounds, the AVA with three methoxy groups (with
ring A derived from 4-methoxyanthranilic acid and ring
B from sinapic acid) had the highest activity. Comparison
of the catecholic (caffeic acid) with the ferulic acid series
showed that a hydroxy group adjacent to the para
hydroxyl in the B ring increased the activity more than
when a methoxy group was present in the same position.
No information is yet available on whether the same is
valid for the A ring. To sum it up, the activity increased
with increasing the groups of radical-stabilizing ortho to
the phenolic hydroxy group. Both aromatic rings were
independently important for activity, while conjugation
across the amide bond was of minor importance. In con-
trast to the radical-scavenging activity, the inhibition of
linoleic acid oxidation was observed for most AVAs,
and also for compounds with only one hydroxy group
in either of the aromatic rings. Compared with α-tocoph-
erol, the AVAs protected linoleic acid from oxidation to
a smaller extent initially, but the effect lasted for a
longer time.
Another study performed by Ishihara et al. [26]
showed that N-feruloyl-4,5-dihydroxyanthranilic acid,
N-p-coumaroyl-4,5-dihydroxyanthranilic acid, and
N-caffeoyl-4,5-dihydroxyanthranilic acid showed stron-
ger DPPH radical-scavenging activity than the corre-
sponding AVAs with 5-hydroxyanthranilic acid. One
 3 IN VITRO PHARMACOLOGICAL ACTIVITIES
mole of N-p-coumaroyl, N-feruloyl, and N-caffeoyl-4,5-
dihydroxyanthranilic had the same activity as
0.76  0.03, 0.84  0.04, 2.08  0.22 mol of Trolox, respec-
tively, while 1 mol of each of their corresponding
5-hydroxyanthranilic acid derivatives were equivalent
in activity to 0.38  0.03, 0.66  0.05, and 1.06  0.09 mol
of Trolox, respectively. These results indicate the partici-
pation of 4,5-dihydroxyanthranilic acid moiety in
the scavenging of DPPH radicals. Eight AVAs (1c, 1f,
1p, 1s, 2c, 2f, 2p, and 2s) were synthesized and tested
for their antioxidant activity using DPPH and FRAP
(ferric reducing antioxidant potential), and also tested
for antigenotoxicity using the Comet assay with stressed
human adenocarcinoma colon cells. Among the
tested compounds, N-(30 ,40 -dihydroxy-(E)-cinnamoyl)-5-
hydroxyanthranilic acid (2c), the abundant oat AVA, gen-
erally had the highest activity in all three assays [37].
Moglia et al. [38] used mouse embryonic fibroblasts
cells characterized by high steady-state levels of intracel-
lular reactive oxygen species (ROS) as a cellular model
to test the antioxidant activity of AVAs. When AVAs
were applied to mouse fibroblasts, the ROS were
monitored and quantitatively measured. The N-(E)-p-
coumaroyl-3-hydroxyanthranilic acid induced a reduc-
tion of intracellular ROS, and revealed its strong antiox-
idant activity and indicated a potential therapeutic value.
Moreover, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid
and its caffeoyl variant showed radical scavenging activ-
ity against ABTS.
Fu et al. [39] studied the effect of AVAs 2c, 2f, and 2p
on HK-2 human renal proximal tubule cells with aim of
identifying their mechanism of antioxidant action.
HK-2 cells are immortalized cells from normal adult
human kidney injury via nuclear factor-E2-related factor
2 (Nrf2) pathway. AVAs could significantly up-regulate
heme oxygenase-1(HO-1) expression both in a dose-
and time-dependent manner. Moreover, the AVA-
induced HO-1 expression was mediated through translo-
cation of Nrf2. HK-2 cells were pretreated with pharma-
cological inhibitors of different protein kinases such as
PI3K (LY294002), MEK1 (PD098059), or p38 (SB202190)
for 30 min, and then exposed to 2c, 2f, or 2p (100 μM).
After the cells were lysed and analyzed by Western
blotting, it was found that HO-1 expression was not
blocked by any of these inhibitors. These indicated that
2c, 2f, or 2p induced HO-1 expression was not associated
with protein kinases. In contrast, with the addition of
N-acetylcysteine, which suppressed ROS mediation in
the cells, AVA-induced HO-1 expression was highly
attenuated; this indicated the important role for ROS, in
activating the HO-1 pathway. Moreover, hydrogenation
of the double bond of the functional, β-unsaturated car-
bonyl group of AVAs eliminated their effects on HO-1
expression, suggesting that this group is crucial for the
antioxidant activity of AVAs.
I. POLYPHENOLS IN THE PREVENTION AND TREATMENT OF VASCULAR AND CARDIAC DISEASE AND CANCER
7
Yang et al. [21] performed a study to investigate the
free radical scavenging activities of three AVAs: 2c, 2f,
and 2p. The total antioxidant capacity of 2c was approx-
imately 1.5-fold those of 2f and 2p. Total antioxidant
capacity was primarily attributable to superoxide anion
scavenging assay and ORAC for 2c, and to ORAC and
Singlet oxygen scavenging assay for 2f. ORAC accounted
for approximately 32% of total antioxidant capacity in 2p.
Variation of the antioxidant capacities among the AVAs
could be attributable to structural differences between the
three compounds.
AVAs might enhance the endogenous antioxidant
cellular response by activation Nrf2. In this respect Pelle-
grini et al. [40] investigated the ability of the synthetic
AVAs 2c, 2f, and 2p to regulate gene expression in bone
cells; to affect the viability of osteoblasts, osteocytes
and osteoclasts and to generate osteoclasts from their
precursors; and finally to examine the role of Nrf2 tran-
scription in these actions. Collagen 1A expression was
up-regulated by 2c in all concentrations and also by 2p
at 1 and 5 μM. OPG (osteoprotegerin) in OB-6 osteoblastic
cells was upregulated by lower doses of AVAs, whereas
100 μM of 2f and all concentrations of 2c down-regulated
RANKL gene expression in MLO-Y4 osteocytic cells.
AVAs did not affect apoptosis of OB-6 osteoblastic cells
or MLO-Y4 osteocytic cells; however, they prevented
apoptosis induced by the DNA topoisomerase inhibitor
etoposide, the glucocorticoid dexamethasone, and hydro-
gen peroxide. Apoptosis of both wild type (WT) and Nrf2
Knockout (KO) osteoblasts was prevented by AVAs; this
demonstrated that AVAs-induced survival is not Nrf2-
dependent. Furthermore, KO osteoclast precursors pro-
duced more mature osteoclasts than WT, and KO cultures
exhibited less apoptotic osteoclasts than WT cultures.
Although AVAs did not affect WT osteoclasts, AVA 2p
reversed the low apoptosis of KO osteoclasts. These
in-vitro results demonstrate that AVAs regulate, in part,
the function of osteoblasts and osteocytes and prevent
osteoblast/osteocyte apoptosis and increase osteoclast
apoptosis; further, these regulatory actions are indepen-
dent of Nrf2.
Liu et al. [41] studied the effects of genotype (G) and
growing environment (E) on AVAs and antioxidant
activity of oats through the investigation of 39 cultivars
of oat collected from four different locations in northwest-
ern China (Inner Mongolia, Qinghai, Shanxi, and Gansu).
The results demonstrated that E, G, and the interaction of
both factors significantly affected the total phenolic con-
tent, the concentrations of different AVAs. The results
suggest that oats containing more AVAs that exhibited
high levels of antioxidant activity could be obtained by
selecting an appropriate genotype and growth location.
In order to determine the effect of two defatting
methods on antioxidant activities and polyphenol con-
tents of oat milling fractions, whole flour (WF), medium
8 1. THE PHARMACOLOGY OF AVENANTHRAMIDES: POLYPHENOLS
oat bran (MB), fine bran (FB), and low bran (LB) were
defatted with hexane or supercritical carbon dioxide
(SC-CO2) fluid and then extracted with aqueous metha-
nol. AVA contents of three of the defatted flour/bran
samples (WF, LB, FB) were 1.7- to 2.4-fold higher when
SC-CO2 was used instead of hexane. However, for the
MB sample, using SC-CO2 resulted in lower concentra-
tion of AVAs compared to hexane, implying that its
larger particle size was a limiting factor for AVAs only
during the SC-CO2 step [42].
3.4 Anticancer
A high dietary intake of whole grains in food helps in
reducing the risk of colon cancer; however, the mecha-
nism behind this effect hasn’t yet been elucidated.
Chronic inflammation is always associated with
increased expression of cyclooxygenase-2 (COX-2) in
the epithelium of the colon and this condition may induce
epithelial carcinogenesis, proliferation, and tumor
growth [43]. AVAs are a relatively unstudied family of
phytochemicals that could be novel chemotherapeutics.
These compounds, found in oats, are nontoxic to healthy
cells and have been shown to reduce viability of human
colon and liver cancers in vitro. However, these studies
do not elucidate a molecular mechanism for individual
AVA. Guo et al. [43] examined the effect of AVAs with
antiinflammatory properties on COX-2 expression in
macrophages, colon cancer cell lines, and on proliferation
of human colon cancer cell lines. AVAs-enriched
extract of oats (AvExO) showed no activity on the expres-
sion of COX-2, but it has no effect on the activity of COX
enzyme and the production of prostaglandin E2 (PGE2)
in lipopolysaccharide stimulated mouse peritoneal mac-
rophages. AVAs (AVAs-ExO, 2c, and the methylated
form of 2c (CH
3 2c)) significantly inhibited cell prolifera-
tion of both COX-2-positive human colon adenocarci-
noma cancer cells: HT29, Caco-2, and LS174T, and
COX-2-negative HCT116 human colon cancer cell lines.
The CH
3 2c showed the highest inhibitory activity. How-
ever, AVAs showed no activity on expression of COX-2
and production of PGE2 in Caco-2 and HT29. These
results indicated that the inhibitory activity of AVAs on
the proliferation of colon cancer cell may not be related
to inhibition of COX-2 expression and PGE2 production.
Thus, AVAs might reduce colon cancer risk through inhi-
bition of macrophage PGE2 production and non-COX-
related antiproliferative effects in colon cancer cells. It
is worthy also to notice that AVAs had no effect on cell
viability of confluence-induced differentiated normal
colonic epithelial Caco-2 cells. Oats and oat bran con-
sumption can reduce the risk of colon cancer because of
its high fiber and AVAs content, as the latter has a nega-
tive effect on colon cancer proliferation.
I. POLYPHENOLS IN THE PREVENTION AND TREATMENT OF VASCULAR AND CARDIAC DISEASE AND CANCER
Moglia et al. [44] used two plant genes 4cl-2 from
tobacco and hct from globe artichoke in the engineering
of Saccharomyces cerevisiae for producing two novel pheno-
lic compounds, N-(E)-p-coumaroyl-3-hydroxyanthranilic
(Yav I) acid and N-(E)-caffeoyl-3-hydroxyanthranilic acid
(Yav II). These two compounds have a structural similarity
with AVAs. The potential antioxidant and antiprolifera-
tive activities of the two compounds were evaluated on
two models: immortalized mouse embryonic fibroblast
cell lines and HeLa cancer cells. The results showed that
both Yav I and Yav II entered the cell and caused the
down-regulation of Cyclin D1. Intriguingly, these effects
were also demonstrated in cellular models of the human
genetic disease cerebral cavernous malformation, suggest-
ing that the novel phenolic compounds Yav I and Yav II
are endowed with bioactive properties relevant to biomed-
ical applications.
Scarpa et al. [45] evaluated the antiproliferative effects
exerted by Vitexin-2-O-xyloside (XVX) and AVAs,
individually and in combination, in CaCo-2 and HepG2
cancer cells using sulforhodamine B method. The pro-
apoptotic activities of the two compounds were also
assessed using caspase activity assays. XVX and AVAs,
both individually and in combination, inhibited the
proliferation of CaCo-2 and HepG2 cancer cells, through
activation of caspases 9, 8, and 3.
The effects of AVAs on breast cancer cells was studied
using an MTT assay to determine cell viability [46]. Stain-
ing and analysis with a flow cytometer was used to iden-
tify cell cycle progression and apoptosis. FloJo software
was used to analyze the cytometric data. The study dem-
onstrates that AVA 2p, 2f, and 2c individually reduce via-
bility in the MDA-MB-231 breast cancer cell line. 2c has
the most potent decrease in tumor cell viability, decreas-
ing viable cells to below 25% at 400 μM when compared to
control after 96 h. We demonstrate that treatment with 2c
causes DNA fragmentation and accumulation of over
90% of cells into a sub G1 cell cycle population. Further,
we conclude that 2c-treated cells activate apoptosis
because 97% of treated cells stain positive for annexin
V while 91% have caspase-3/7 activity, a late marker of
apoptosis. Breast cancer cells treated with 2c have a
decrease in cell viability, an increase in the sub G1 popu-
lation, and stain positive for both annexin V and caspase
activity, indicating that AVN-C induces apoptosis in
breast cancer cells.
4 IN VIVO STUDIES ON
AVENANTHRAMIDES
A few studies have explored the in vivo activity of oat
phenolics [1] using an oat bran phenol-rich powder. An
in vivo study used the hamster model groups [1], divided
according to their body weight to six time point groups 0,
 4 IN VIVO STUDIES ON AVENANTHRAMIDES
20, 40, 60, 80, and 120 min. The hamsters were anesthe-
tized with Aerrane and received oat bran phenol-rich
powder (which contains 40 μmol phenolics, 6.8 mg) using
stomach gavage. The chosen dose 45 mg/kg body weight
was equal to 5 times the required daily dose for a 70-kg
body human, which is 14 mg/kg; this is due to the fact
that rodents consume five–six times more food-based
energy than humans [47]. Absorbed oat phenolics were
tested ex vivo to evaluate their antioxidant capacity on
the resistance of hamster LDL to Cu-induced oxidation.
The phenolics showed no change in the resistance of ham-
ster LDL to Cu-induced oxidation at 40 and 60 min. How-
ever, after 5 μmol/L ascorbic acid was added to the assay
mixture, LDL collected at 60 min had a 58% longer lag
time than that collected at baseline (216 and 137 min,
respectively; P  .05). The oat phenolics had no change
on both the ORACtotal and ORACpca in the plasma sam-
ples collected. Because the amount of LDL available from
hamsters is limited, human LDL was used to confirm the
observed synergistic relation between oat phenolics and
vitamin C. The antioxidant activity of oat phenolics
in vitro was apparent through a dose-dependent increase
in the resistance of human LDL against Cu-induced oxi-
dation. Meanwhile, the addition of ascorbic acid in a con-
centration of 5 μmol/L caused an observed value twice
the expected value from additive calculation.
Hassanein et al. [48] evaluated the protective effect
of oats AVA enriched extract on toxicity and oxidative
stress caused by titanium dioxide nanoparticles (TiO2
NPs) in Sprague-Dawley rats. TiO2 NPs toxicity induced
oxidative stress and different inflammatory responses,
such as the elevation in TNF-α, in addition to increase
in the liver enzyme markers and DNA damage.
The study showed that administration of AVA (20 mg/
kg b.w.) TiO2 NPs (in a dose of 150 mg/kg b.w) together
by gastric tube for 6 weeks improved the values of serum
AST, ALT, serum lipid peroxidation, glutathione, total
antioxidants, and TNF-α, when compared to the group
treated with TiO2 NPs. AVA also restored the level of tes-
tosterone, which was decreased due to TiO2 NPs treat-
ment. Furthermore, AVA significantly reversed the
DNA damage compared to TiO2 NPs treated group.
The histopathological study has shown that AVA
reduced congestion, inhibited the necrosis and mononu-
clear infiltration caused in the liver by TiO2 NPs, and pre-
vented the lesion formation in the brain, testes, and lungs.
An interesting study was performed on AVA by Koe-
nig et al. [49] with the following objectives: first, to assess
the concentration of various fractions of AVA and the
time intervals of their detection following their oral
administration in rats; second, to evaluate the content
of various AVA fractions in the liver, heart, and skeletal
muscle and their time courses; finally, to detect the extent
and time course of the conjugation of AVA in the post-
prandial period. In this study three synthetic AVAs 2p,
I. POLYPHENOLS IN THE PREVENTION AND TREATMENT OF VASCULAR AND CARDIAC DISEASE AND CANCER
9
2f, and 2c were tested. First, the three derivatives were
administered by oral gavage in a dose of 20 mg/kg/body
weight. The rats were sacrificed at 1, 2, 4, or 12 h postin-
gestion. 2p was detected in plasma in a percent of 91%,
95%, 91%, and 94% of the total administered 2p, at 1, 2,
4, and 12 h, respectively. 2f was detected in 72%, 83%,
50%, and 77% of total 2f at 1, 2, 4, and 12 h, respectively,
while 2c was detected in plasma at a concentration of
94%, 95%, 100%, and 100% of total 2c at 1, 2, 4, and
12 h postgavage, respectively. Concerning the liver, the
three fractions of AVA were detected in the liver of
AVA-fed rats. 2p was detected at a percentage of 23%,
41%, 52%, and 64% of total 2p at 1, 2, 4, and 12 h, respec-
tively. Conjugated 2f concentrations were 92%, 87%, 85%,
and 90% of total 2f at 1, 2, 4, and 12 h, respectively. Total
2c was detected at a percentage of 47%, 55%, 47%, and
49% of total 2c at 1, 2, 4, and 12 h postgavage, respec-
tively. The 2p was not detected at statistically significant
levels at any time point in the heart tissue, 2f was not
detected at 2 h postgavage, but 79%, 100%, and 99% of
total 2f were detected at 1, 4, and 12 h. On the other hand,
66%, 86%, 67%, and 73% of total 2c were detected in the
heart tissues at 1, 2, 4, and 12 h, respectively. No signifi-
cant changes in liver glutathione concentration, GSSG
concentration, or GSH:GSSG ratio were detected.
ROS can be generated in the body through different
mechanisms, one of them being infection with aerobic
organisms [50]. When the produced cytotoxic agents
exceed the capability of natural antioxidant defence
mechanisms, serious damage due to oxidative stress
may occur as a reflection of the oxidative modification
of macromolecules like lipids, proteins and DNA [51].
Production of ROS is responsible for the incidence of
many diseases and also induction of aging [52]. More-
over, excessive physical exercises have been shown to
raise oxidative stress in skeletal muscle and myocardium
as a result of increased generation of ROS [53]. In this
respect, AVA proven to possess in vitro antioxidant activ-
ity [22] was tested in vivo to evaluate its antioxidant
potentiality. The study, made by Ji et al., [54] was
designed to evaluate the ability of AVA supplementation
to attenuate the exercise-induced ROS generation and
oxidative tissue damage, and to increase the endogenous
antioxidant enzyme activities in the various tissues of
rats. First, 2c was synthesized from caffeic acid and
5-hydroxyanthranilic acid and supplemented in the diet
(0.1 g/kg) to female Sprague-Dawley rats. Each group
of rats was fed the respective diet for 50 days, before they
were randomly divided into exercised (E) and rested
(R) treatment groups. AVA supplementation did not
affect ROS concentration in the liver. E rats had signifi-
cantly higher ROS levels in the liver than R rats. Among
the exercised groups of rats, those whose diet was supple-
mented with AVA had a greater ROS concentration in the
heart tissues than the control rats. ROS in the kidney was
10 1. THE PHARMACOLOGY OF AVENANTHRAMIDES: POLYPHENOLS
not affected by either AVA or E. In soleus muscle, E rats
had significantly higher ROS levels than R rats. AVA
decreased ROS generation and attenuated exercise
induced ROS. AVA supplementation increased SOD
activity in the liver, kidney, the deep portion of vastus
lateralis DVL and soleus muscle. In the heart; however,
AVA-supplemented exercised rats showed significantly
lower SOD activity than any other group. Glutathione
peroxidase (GPX) activity in the liver was increased in
E versus R rats, but unaffected by AVA treatment. GPX
activity tended to be higher in the heart of AVA-treated
rats. In the kidney, DVL, and soleus, GPX activity was
not altered with either AVEN or exercise. Tissue lipid per-
oxidation using MDA content as a marker was elevated
in the liver, heart, and DVL of E vs. R rats. AVA supple-
mentation did not influence MDA levels in the liver or
DVL, but decreased exercise-induced lipid peroxidation
in the heart.
Another study was made by Ren et al. [55] in which the
authors used the avenanthramide-rich extract (ARE) from
oat bran and evaluated its effect on the antioxidant
enzymes in D-galactose-induced oxidative stresses in mice.
High-performance liquid chromatography (HPLC) analy-
sis of ARE showed that its major components are 2c
(6.07%), 2f (5.36%) and 2p (4.37%), in addition to the fol-
lowing phenolic acids: vanillic acid (0.60%), caffeic acid
(0.50%), syringic acid (0.54%), p-coumaric acid (0.16%),
ferulic acid (0.08%), and sinapic acid (0.03%). The admin-
istration of D-galactose to mice could significantly reduce
the activity of SOD and GPx; moreover it could also lower
the gene expression of Mn-SOD, copper-zinc SOD,
GPx, and lipoprotein lipase (LPL) mRNA. Oral admin-
istration of ARE in different doses of 250, 500, and
1000mg/kg body weight/day by intragastric gavage for
2 weeks after injection of D-galactose significantly reversed
the D-galactose-induced oxidative stress by restoring the
activity of the hepatic antioxidant enzymes, where ARE
caused up-regulation of SOD and GPx activities, especially
at a dose of 1000mg/kg of body weight. Furthermore,
ARE at the two doses 500 and 1000 mg/kg also increased
the expression of Mn SOD, Cu-Zn SOD, GPx, and LPL
mRNA expression. Administration of ARE at the doses
of 500 or 1000 mg/kg in mice could significantly reverse
the rise in hepatic MDA level caused by D-galactose com-
pared to the control. The obtained results confirmed that
ARE exerted antioxidant activity which was apparent
through reversing D-galactose-induced oxidative stress.
5 PHARMACOKINETICS,
BIOTRANSFORMATION, AND
BIOAVAILIBILITY
Oat AVAs are of great benefit to human health
(Fig. 1.2). A diet supplementation with oats-derived
I. POLYPHENOLS IN THE PREVENTION AND TREATMENT OF VASCULAR AND CARDIAC DISEASE AND CANCER
AVA capsules or placebo in healthy randomized people
increased the levels of SOD and GSH by 8.4% and 17.9%,
respectively, while MDA level significantly decreased, by
28.1%. The total cholesterol, triglyceride, and low density
lipoprotein cholesterol levels were lowered, and the high
density blood lipoprotein cholesterol levels in the same
treatment were increased. It appears that oats extract con-
taining AVAs possessed a high antioxidative activity on
humans, suggesting their indication to prevent hyperli-
pemic and angiocardiopathy [56]. Additionally, the
AVAs increase antioxidant protection and reduce inflam-
mation after a bout of downhill walking in postmeno-
pausal women. Thus, dietary supplementation of AVA
appeared to be useful in reducing inflammation after
demanding physical exercise [57]. Moreover, long-term
AVA supplementation can attenuate blood inflammation
markers, decrease ROS generation and NFκB activation,
and increase antioxidant capacity during an eccentric
exercise bout [58]. Phenolic compounds are inversely cor-
related to the risk of heart disease; however, limited data
are available on those compounds’ bioavailability. AVA
bioavailability is directly linked to structural differences.
The 2p is less hydrophilic and less readily eliminated in
the urine, correlating with its increased plasma concen-
tration. In fact, 2p is unsubstituted at the carbon-3 posi-
tion of the cinnamic acid, while -B and -C contains a
methoxy and hydroxy group, respectively, at this posi-
tion. Regardless of the high concentrations of AVA deriv-
atives in oat bran phenol-rich powder, those compounds
were bioavailable with low concentrations in hamster’s
plasma. In the hamster, the pharmacokinetic profile of
2p and 2f was marked with a maximum plasma concen-
tration (Cmax) of 0.04 and 0.03 μmol/L, respectively; the
Cmax was reached at 40 min (Tmax) and essentially elimi-
nated by 120 min. On the other hand, their apparent rel-
ative bioavailability was only 5% of the least bioavailable
phenolic acid (vanillic acid) [1]. Data from another study
suggested that the elimination kinetics of plasma AVAs
appeared to follow first-order kinetics in healthy older
adults. The study found that these compounds are bio-
available and increase some biomarkers of antioxidant
capacity without apparent adverse side effects. In
contrast to hamsters, the Tmax in humans was 1.5–2.3 h,
indicating that absorption and metabolism of AVAs are
species dependent. These findings are possibly a result
of species’ innate differences in detoxification pathways.
The elimination of AVAs from plasma followed first-
order kinetics, further suggesting the dependence of Tmax
and T1/2 on the specific AVA structure. Although plasma
AVAs were higher after the 1.0 g than the 0.5 g dose,
adjusting for dose with the use of the AUC to oral dose
ratio revealed that the larger dose had a disproportion-
ately greater bioavailability. This might be due to an
insufficient capacity of the phase II metabolism toward
AVA; for example, less 2p and 2f may be conjugated
 5 PHARMACOKINETICS, BIOTRANSFORMATION, AND BIOAVAILIBILITY
FIG. 1.2 Main effects of avenanthramides on organs
and cells.
through glucuronidation and sulfation for urinary and/
or biliary excretion. However, it is not clear why 2c did
not show a similar differentiation between doses [59].
Koenig and collaborators [49] elaborated a protocol to
study the distribution of AVA at the organ and tissue
level and the extent of conjugation following ingestion
of a synthetic AVA in rats and plasma. The concentra-
tions of this AVA were measured in liver, heart, and gas-
trocnemius obtained over time points, and the results
demonstrated that the AVA was bioavailable in the blood
circulation following oral ingestion in the rat and reach
peripheral tissues, remaining in the organs for up to
12 h. The group suggested a possible increase in the level
of AVA in the rat via repeated feedings. Notably, in
humans, 2f was the slowest fraction to be eliminated,
while it was eliminated most quickly in rats. These dis-
crepancies may be the result of differences in phase
I and II metabolism between species. The study also sug-
gested that the differences in absorption via the gut and/
or differences in the method of delivery may play a role,
as glycosylated AVA may need to be cleaved to the agly-
cone form before it can be absorbed from the gut. Further-
more, the presence of the gallbladder in humans may
affect the biliary excretion compared to rats. At the
end, a plausible high concentration of AVA reached
before the first measurement at 1 h may explain the rela-
tively low concentrations measured. In contrast to
plasma, liver showed increased concentrations of 2f, with
a large percentage of conjugated 2f. On the other hand,
the conjugated 2p and 2c peaks demonstrated low
contents in plasma. In summary, the data suggest a
disconnect between plasma and liver AVA concentration,
a phenomenon previously described in flavanone
I. POLYPHENOLS IN THE PREVENTION AND TREATMENT OF VASCULAR AND CARDIAC DISEASE AND CANCER
11
aglycones and glucuronides [49]. Although the bioavail-
ability of AVAs has been investigated, little is known
about their metabolism. Recently, the metabolism of
AVA 2c was investigated in mice and the human micro-
biota. This study demonstrated the biotransformation
of AVA 2c to eight major metabolites in mice, and that
the composition of gut microbiota may influence its
metabolism and bioavailability. The identified meta-
bolites detected from the 2c-treated mouse urine samples
were: 5-hydroxyanthranilic acid (M1), dihydrocaffeic
acid (M2), caffeic acid (M3), dihydroferulic acid (M4),
ferulic acid (M5), dihydroavenanthramide-C (M6),
dihydroavenanthramide-B (M7), and avenanthramide-B
(M8). The major metabolic routes found were the reduc-
tion of 2c_s C7#-C8# double bond and the cleavage of its
amide bond. In the human microbiota study, 2c was con-
verted into M1–M3 and M6. Moreover, interindividual
differences in 2c metabolism were observed among the
human subjects. To link the gut microbial diversity to
nutritional phenotypes and bioactivity of the 2c and its
analogs, the study proposed to analyze the microbial
composition of 2c-active vs. 2c-inactive cultures from dif-
ferent human subjects. Furthermore, DH-2c showed
higher bioactivities than that of 2c in the inhibition of
growth of human cancer cells and triggering of apoptosis
in human colon cancer cells. These findings indicate that
this biotransformation of 2c retains its pharmacologic
activities.
The absorption of AVAs in humans after oral consump-
tion of natural oat flour is unknown. The apparent AVA
concentration in plasma after oral ingestion of oat cookies
was calculated and key pharmacokinetic parameters were
estimated in male and female nonobese participants
12 1. THE PHARMACOLOGY OF AVENANTHRAMIDES: POLYPHENOLS
consuming cookies made with oat flour containing high
(229.6 mg/kg, H-AVA) or low (32.7 mg/kg, L-AVA)
amounts of AVAs, including 2p, 2f, and 2c. AVAs found
naturally in oats were absorbed in the plasma after oral
administration in humans; 2f has the slowest elimination
rate and the longest half-life compared to 2p and 2c, while
2c demonstrated the lowest plasma concentrations [60].
6 CONCLUSION
Oats are a healthful cereal grain not only because of
their β-glucan ability to reduce blood cholesterol but also
because of the strong antioxidant activity of their unique
polyphenols. The polyphenolic AVAs are exclusively
present in oats and present an interesting profile as poten-
tial biomarkers for the intake of A. sativa. However, AVAs
are poorly absorbed and extensively metabolized in vivo.
Therefore, further pharmacokinetic study of these metab-
olites is needed to explore their bioavailability/biotrans-
formation in vivo. Among AVAs, the interindividual
variation in the metabolism of AVA 2c may be important
in future studies of the use of oats in targeted nutritional
therapies. Current research has demonstrated that AVAs
exert antioxidant, antiinflammatory, cardioprotective,
and anticancer actions in vitro and in vivo, which attest
to the health benefits of oats intake. These biological
activities may explain the well-known dermatoprotective
activities of oats. Thus the biological functions of AVAs
warrant further exploration. Compared to drugs, dietary
phenolics are usually consumed in amounts lower than
hundreds of milligrams in a diluted dose. Therefore,
drugs can readily saturate the metabolic pathways,
whereas AVAs cannot. Research directed to AVAs encap-
sulation, metabolism, delivery, pharmacokinetics, and
toxicokinetics will allow the integration of AVAs-based
products for nutritional therapies.
Acknowledgments
The author Ilias Marmouzi is thankful to Dr. Houda Serrar for Fig. 1.1
and to M. Mehdi Allaoui for Fig. 1.2.
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