Introduction
Several diseases have been linked to the development of ED, such as atherosclerosis,
hypercholesterolaemia, coronary disease, erectile dysfunction, asthma, renal failure,
rheumatoid arthritis, periodontitis, psychiatric disorders, and cancer, as well as
diseases with a high prevalence, such as diabetes (types 1 and 2) and SAH [1], [2], [3], [4], [5], [6], [7], [8], [9].
The preservation of the endothelium is fundamental in maintaining the physiology of
the vascular system (in the regulation of its tonus, the development of immune, structural,
and proliferative functions, and interaction with other cellular types) and also in
the prevention of the development/aggravation of diseases [10], [11], [12].
For example, in SAH, vascular oxidative stress can precede the onset of elevated blood
pressure, which, associated with conditions of hyperlipidaemia, can lead to the rapid
proliferation of endothelial cells. However, the cellular division capacity is limited,
which is caused by a cycle arrest of the endothelial cells. As a consequence, these
senescent cells undergo morphological changes that are responsible for the increased
production of reactive species, which leads to a decrease in the production of NO
and increased sensitivity to apoptotic stimulus. Such events lead to progressive impairment
in vascular responses, with an intensification of ED [10], [13], [14].
Thus, mechanisms such as oxidative stress, eNOS uncoupling, induction of endothelium-dependent
contractile responses, and reduced endothelium-dependent hyperpolarisation can be
related to a decrease in vascular response [15], [16]. However, it is worth noting that although there are several factors involved in
ED, it is strongly marked by the low bioavailability of NO and, therefore, damage
in the NO/cGMP pathway configures one of the most important causes of vascular impairment
[6], [10], [11].
In addition, NO is responsible for vascular smooth muscle relaxation, the inhibition
of adhesion and aggregation of neutrophils and platelets, participation in neurotransmission
and memory processes, the immune system and gene regulation as well as cell cycle
regulation and apoptosis. Due to these important effects, NO deficiency receives much
attention and ED has already been mentioned in more than 20 000 scientific studies,
since both represent risk factors, especially in relation to cardiovascular diseases
([Fig. 1]) [6], [17], [18].
Fig. 1 Number of scientific publications regarding ED, including its causes and consequences,
as well as recent interest in the application of plant-derived substances in the prevention
of its complications. The Pubmed database (December 2016) was used to obtain the data
and “endothelial dysfunction” was applied as the search term. EDRF: endothelium-derived
relaxing factor, ED: endothelial dysfunction, ROS: reactive oxygen species.
The best-characterised endothelium-derived relaxing factor (NO) is synthesised by
NOS from the amino acid L-arginine, while another enzyme, L-arginine-urea hydrolase
arginase, or simply ARG, is responsible for regulating the production of this biological
mediator through substrate competition [6], [18].
A decrease in the formation of NO is a key point in the development of ED because
there is competition for the common substrate, which raises interest in the modulating
role of ARG in decreasing NO levels.
Such a modulating function may culminate in a number of vascular changes, which are
characterised by impairment of vasodilatory response, increased inflammation, vascular
remodelling (collagen deposition and smooth muscle tissue growth), altered platelet
aggregation, and cellular apoptosis ([Fig. 2]) [6], [25], [26].
Fig. 2 Mechanisms underlying ED, highlighting the substrate competition between nitric oxide
synthase and arginase. ARG – arginase, eNOS – endothelial nitric oxide synthase, BH4 – tetrahydrobiopterin, NOHA – Nω-hydroxy-L-arginine, NO – nitric oxide, O2
•- – superoxide anion, ONOO− – peroxynitrite, sGC Fe+2 (red) – reduced soluble guanylate cyclase, sGC Fe+3 (oxide) – oxidised soluble guanylate cyclase, GTP – guanosine triphosphate, cGMP
– cyclic guanosine monophosphate.
It has been suggested that the inhibition of ARG activity may result in increased
NOS substrate availability and, consequently, NO production. This hypothesis has been
confirmed by in vitro and in vivo studies [17], [18], [27], [28], [29], [30].
In this context, research regarding the pharmacological inhibitors of ARG as options
for the development of new molecules to treat metabolic, respiratory, infectious,
and cardiovascular disorders is promising. However, few substances are available for
this purpose, and problems related to the pharmacokinetic and toxicological factors
of these substances have not yet been resolved [25], [26], [27], [31].
Recently, plant extracts and active plant metabolites have emerged as potential alternatives
for therapeutic application in several diseases that affect humans. For example, the
polyphenolic extract of Camellia sinensis (L.) Kuntze (Theaceae) was approved by the U. S. Food and Drug Administration in
2007 for the treatment of genital warts, and in 2012, ingenol mebutate, which is a
tigliane diterpenoid, started to be used in the treatment of actinic keratosis [32], [33]. Ethnopharmacological studies are currently being conducted in order to identify
ARG inhibitory substances for future clinical use in relation to ED, specifically
those related to cardiovascular alterations.
With regard to the latter point, special attention has been paid to substances belonging
to the class of polyphenols [26], [29], [34], [35], [36], [37], [38]. Therefore, this article presents recent research regarding the search for new ARG
inhibitors derived from medicinal plants with a potential therapeutic application
in the fight against diseases related to the development of ED, as well as seeking
to increase interest in the development of promising drugs in this field.
Methods
This systematic review of the literature was based on scientific material that has
already been published in the English language, which was collected from the Pubmed
(US National Library of Medicine – National Institutes of Health) database without
restriction regarding the year of publication. The search terms that were used included
“endothelial dysfunction” and “arginase” or “arginase inhibition”, “nitric oxide”
and “arginase” or “endothelial dysfunction”, and “arginase inhibition” and “plant
derived” or “natural compound” or “natural product” or “polyphenol”.
The research publications that were included provided in vitro or in vivo results (human or rat/mouse) as well as revisions related to the proposed theme.
The following were excluded: in vivo research with species of animals other than those mentioned above (it is important
to note that studies using natural compounds such as inhibitors of Leishmania sp. ARG were not considered), unpublished studies, studies with incomplete information
regarding references, and studies that were not in the format of a scientific article.
The chemical names of the molecules presented in the course of this review are in
agreement with those presented in the original references that were cited, and the
scientific names of the plant species that are mentioned are in accordance with those
mentioned in The Plant List (www.theplantlist.org).
For the in silico analysis, the oral bioavailability and distribution volume data were collected from
ACD/I-Lab (https://ilab.acdlabs.com/iLab2/index.php). The ADME investigation, drug-likeness, and toxicity prediction were obtained through
the PreADMET web programme (https://preadmet.bmdrc.kr/). The MDL molfiles of substances were loaded in these databases for calculations.
Arginase: an overview
ARG (L-arginine-urea hydrolase, or amidinohydrolase – EC 3.5.3.1) is a metalloenzyme
that was first described in 1904 by Kossel and Dakin in mammalian liver samples [25]. Each active unit of the trimer is essentially two Mn+2 ions [6], [39]. The structure and stability of these ions are required for the full catalytic action
of the enzyme [40].
During its catalytic cycle, the guanidine grouping of L-arginine undergoes a nucleophilic
attack from a complex formed by Mn+2 and hydroxide ions from water molecules, forming a neutral, intermediate tetrahedral,
and releasing L-ornithine and urea [6], [27], [40].
Since 1965, different ARG isoforms have been reported in human tissues [41], [42], [43]. In mammals, two of these isoforms are most prominent and, therefore, they are reported
more frequently in the scientific literature, namely, ARG 1 and ARG 2 ([Table 1]) [11], [41].
Table 1 Some human characteristics of ARG 1 and 2.
|
ARG 1
|
ARG 2
|
|
The presented data does not consider other animal species. LPS: lipopolysaccharide,
TNFα: tumor necrosis factor-alpha, AII: angiotensin II, IL: interleukin, oxLDL: oxidised
low-density lipoprotein
|
|
Amino acids
|
322
|
354
|
|
Weight
|
105 kDa
|
129 kDa
|
|
K
m
|
0.08 at pH 8.5
|
4.8 at pH 7.4
|
|
Tissue distribution
|
Endothelial cells, nephritic glomeruli, macrophages, liver, erythrocyte, coronary
arteries, corpora cavernosal, brain, retinal glia, polymorphonuclear neutrophils,
and saliva.
|
Smooth muscle cells, endothelial cells, normal glomeruli, macrophages, kidney, gastric
cancer tissue, corpora cavernosal, brain, retina, and horizontal cells at heart, placenta,
lung, skeletal muscle, pancreas, and prostate.
|
|
Inducers
|
LPS, TNFα, hyperglycaemia, nitric oxide, AII, IL-1, and glucocorticoids.
|
IL-1, IL-4, IL-13, hypoxia, LPS, TNFα, thrombin, oxLDL and haemodynamic forces.
|
|
Comments
|
Is highly expressed in the cytosol of hepatocytes – catabolic function to convert
L-arginine in ureia (ureia cycle).
|
Is located within the mitochondrial matrix. Has widespread tissue localisation and
a relatively low specific activity (in general, anabolic functions).
|
|
References
|
[26], [27], [39], [41], [42], [44], [45], [46], [47], [48], [49], [50], [51], [52]
|
ARG isoforms are encoded by homologous genes that are mapped in distinct chromosomes
(ARG 1 in chromosome 6q23 and ARG 2 in 14q24) [27], [36], [53], [54], [55], [56]. A genetic sequencing study that was performed with human kidney tissue detected
that the ARG 2 sequence was 58% homologous to that of ARG 1 [49], whereas human and mouse ARG 1 have 87% of the sequence in common [27]. This information is important because it points towards the identification of isoforms
in human samples and makes it possible to investigate enzymatic induction under normal
or pathological conditions.
In eukaryotic organisms, when they are active, both ARG isoforms take the homotrimeric
form (105 kDa – ARG 1 and 129 kDa – ARG 2) [6], [40], [42]. At this point, the maximum activity of ARG is about 1000 times greater than that
of NOS, however, its affinity for L-arginine (K
m
1 – 5 mM) is lower when compared to the same enzyme (K
m
2 – 20 µM) [2], [57].
ARG 1 is the largest fraction of the total ARG expressed in the organism [26]. It is present in the cytosol of liver cells, where it is an integrated part of
the urea cycle (conversion of the L-arginine substrate to L-ornithine and urea) as
well as other enzymes [N-acetylglutamate synthase (NAGS), carbamoylphosphate synthetase
(CPS1), mitochondrial ornithine transporter (OTC), ornithine transcarbamylase (ASL)
and argininosuccinatesynthetase-1 (ASS1)] [2], [47], [53]. ARG 2 is mitochondrial and can be found in several tissues, mainly in the kidney.
This isoform has several roles that have not yet been fully defined, including participation
in the synthesis of polyamines as well as the formation of proline, creatine, glutamate,
agmatine, and γ-amino-butyric acid (GABA) [27], [41], [47], [57].
Both ARG 1 and ARG 2 can be expressed in the vascular endothelium [31]. Despite some controversy about the expression of isoforms in the adjacent smooth
muscle cell layer [2], [58], it has been shown that aortic smooth muscle tissues in rats express ARG 1 [59]. On the other hand, smooth muscle cells of human lung tissue express both isoforms
[26], [55], [59]. In general, ARG expression can be modulated in different sites, depending on the
stimulus that is applied [7], [41], [55], [60].
Furthermore, it has been demonstrated that iNOS-derived NO can nitrosate the sulphur
of the cysteine residue 303 of ARG, activating the enzyme [61]. However, reduction in the levels of L-arginine caused by ARG activity may cause
decreased iNOS activity [62]. These data suggest a bidirectional relationship between ARG 1 and iNOS that could
play an important role in vascular diseases [2].
In vitro and in vivo studies have demonstrated that LPSs increase the mRNA of ARG 1/ARG 2 and iNOS in
different tissues, such as the lung, heart, liver, and endothelial cells of rats [41]. In parallel, other substances, such as THF-α, high glucose concentrations, oxidised low-density lipoprotein, hydrogen peroxide
or peroxynitrite, and thrombin may induce increased ARG expression ([Fig. 3]) [63]. Thus, inflammatory mediators modulate the expression of iNOS and ARG, depending
on the cellular system that is involved [41], [64], [65].
Fig. 3 Pathways involved in arginase expression. Several humoral and haemodynamic factors,
including intracellular pathogens and ROS, are part of the mechanism of ARG activation
[26], [41], [46]. Intracellular pathogens (e.g., Mycobacterium tuberculosis) induced ARG expression through the toll-like receptor pathway [66]. The formation of pores in the endothelium and hyperpermeability in the lungs (as
occurs in severe pneumonia) can increase intracellular calcium concentration, activating
protein kinase C (PKCα), which activates RhoA/ROCK to elevate ARG expression [67]. Similarly, the atherogenic stimulus oxLDL acts via the RhoA effectors ROCK and
mDia1 to activate L-arginine catabolism by augmenting ARG levels [50]. Microgravity conditions activate the p38 MAPK (mitogen-activated protein kinase)-C/EBPβ pathway [68]. Furthermore, the induction of tyrosine phosphorylation of proteins, like the Janus
kinase family (JAK1, JAK2) and tyrosine kinases (TyK-2), leads to adenylyl cyclase
activity through a cAMP (cyclic adenosine monophosphate)/PKA or Epac pathway [45], [69]. The ability of p38 MAPK to phosphorylate the activation transcription factor (pATF)
suggests that p38 MAPK may modulate the expression of cAMP – responsive-elements (CRE).
Furthermore, CRE-binding protein (CREB) can be activated by PKA and bind to pATF as
a heterodimer to facilitate ARG transcription via CRE [70]. LPS – lipopolysaccharide, GLI – glucose, TNFα – tumor necrosis factor alpha, TGF-β – transforming growth factor beta, PGE2 – prostaglandin E2, IL – interleukin, H2O2 – hydrogen peroxide, ONOO•- – peroxynitrite, NO – nitric oxide, NADPH – dihydronicotinamide-adenine dinucleotide
phosphate, AII – angiotensin II.
Interestingly, Nelin et al. [71] showed that an increase in ARG expression, whilst not affecting NOS levels, can
result from the activation of the EGFR (expressed in endothelial cells). Likewise,
it has been demonstrated that AII led to an increase in ARG expression and activity
in the mouse aorta [51]. Furthermore, the increased expression and stimulation of AII receptors is associated
with alterations in the activity of ARG [72].
Other conditions, such as hypertension, ischemia-reperfusion, intima layer hyperplasia,
and aging, can elevate ARG levels, which is expressed in vivo in endothelial tissue [63]. Thus, in addition to its interaction with iNOS, ARG is also closely related to
the maintenance of the functions of eNOS, which is an important enzyme isoform for
the preservation of vascular homeostasis because the eNOS-derived NO acts to inhibit
the vascular tonus, platelet aggregation, and inflammation [1], [2]. Consequently, any alteration of the system orchestrated by NO may cause what is
known as ED, and although the main effect of this disorder is damage to vasodilation
mechanisms, it has also been reported that local inflammation, lipoperoxidation, SMC
proliferation, deposition of extracellular matrix, and platelet and thrombotic activation
can occur ([Fig. 2]) [10].
Therefore, ARG is a regulator of the bioavailability of NO by competing with eNOS
for the L-arginine substrate, and an increase in ARG activity and a consequent decrease
in NO bioavailability are linked to the development of ED and its complications in
the various diseases in which it is present. This emphasises why ARG has become the
subject of studies regarding the development of inhibitors as new pharmaceutical tools
[17], [18], [61].
As previously mentioned, changes in NO bioavailability constitute the key event in
the development of ED. Many mechanisms are involved in the decompensation of the NO
supply, especially its inactivation due to oxidative stress (mitochondrial respiration,
arachidonic acid cascade, cytochrome p450 complex, xanthine oxidase, NADH/NADPH oxidase,
iNOS, peroxidases, and haemoproteins), which is associated with eNOS uncoupling and
a decrease in the expression of this same enzyme, with or without a shortage of enzymatic
or substrate cofactors (L-arginine) [13], [73].
Several studies have shown that blocking the advancement of ED is a powerful tool
in reducing cardiovascular risks and, thus, many strategies have been investigated
in order to prevent the development of ED or complications associated with it [16].
Compounds of natural origin, especially polyphenols with antioxidant activity, have
been successfully tested in relation to ED [12], [74], [75], [76], [77]. Dal-Ros et al. [35] showed that the consumption of polyphenols in red wine protected against aging-related
ED by normalising the oxidative stress that was induced in the animal model that was
tested. Similarly, natural products have a recognised stabilising or stimulating effect
on eNOS, which promotes an increase in NO levels, which are lower in ED [12], [30], [65], [78], [79].
Another strategy that has been evaluated in studies regarding the treatment of diseases
associated with ED is an attempt to provide physiological supplementation with L-arginine
substrate, although this has produced controversial results that are related to limiting
factors such as the consumption of this amino acid via alternative metabolic pathways,
rapid metabolisation after oral administration, the need to screen patients who would
clinically require L-arginine replacement, and the difficulties in determining individual
levels of active ARG [4], [6], [55]. A controlled study of oral L-arginine supplementation conducted with patients with
a history of myocardial infarction had to be discontinued because of the excessive
mortality rate of the participants [6], [25]. It has also been observed that the exposure of cell cultures to arginine may even
precipitate endothelial senescence [15].
Furthermore, it should be taken into account that the intracellular level of arginine
is higher (more than 800 µM) than the extracellular level (50 – 200 µM). Given that
the affinity of eNOS purified by this substrate is in the micromolar range of K
m
= 2.9 µM, it is suggested that eNOS operates below its saturation concentration,
and therefore would not respond to changes in the concentration of circulating L-arginine,
which would theoretically refute the alternative of supplementation with the semi-essential
amino acid against ED [6], [12], [80].
In fact, the chronic intake of L-arginine offers minimal therapeutic outcomes in vascular
disease, showing that this substance is probably not a limiting factor regarding NO
production. The exception may be when ARG is more active, reinforcing the competition
with eNOS for the common substrate [15].
Thus, because a decrease in the bioavailability of NO has a central role in the mechanism
of ED, and due to the fact that competition between eNOS and ARG for L-arginine can
intensify this process, scientific efforts were concentrated in order to better investigate
the role of ARG in this mechanism.
Scientific evidence began to emerge in the 2000s that ARG activity limited NO production
by NOS, and that this was closely related to the depletion of endothelium-dependent
vasodilation [81]. These results revealed the importance of ARG as a regulator of the process of the
development of ED and transformed it into a new issue of interest for the scientific
community regarding the search for new ways to block the degradation caused by ED
in the various diseases in which it occurs ([Fig. 4]) [82], [83].
Fig. 4 Development of research regarding the following: the involvement of arginase (ARG)
in endothelial dysfunction (ED) (purple line), the perception of the existence of
competition between nitric oxide synthase (NOS) and ARG (black line), and the consequent
reduction of the bioavailability of NO (orange line). This resulted in increased interest
in the research and development of ARG inhibitors with therapeutic appeal in relation
to ED. The Pubmed database (December 2016) was used to obtain the data and “nitric
oxide and arginase”, “nitric oxide synthase and arginase”, and “endothelial dysfunction
and arginase” were applied as search terms.
In the period 1990 – 2011, more than 500 patents were registered in the field of new
synthetic ARG inhibitors (425 were registered in the USA), most of which were boronic
derivatives. Nevertheless, this constitutes a vast field of research, since many of
the patented products still present problems related to pharmacodynamic and kinetic
action (factors such as the lack of selectivity in relation to ARG 1 and ARG 2 isoforms,
short half-life, loss of potency in physiological pH, and intrinsic toxicity) [6], [25], [27], [63], [84].
In 2003, the U. S. Food and Drug Administration gave approval for a representative
of boronic acid derivatives (bortezomib) to be used to treat multiple myeloma and
mantle cell lymphoma [85], [86]. However, toxicological tests on rats and monkeys have indicated haematological,
lymphoid, cardiac, renal, gastrointestinal, and neurological problems linked to its
use, and data on its genotoxicity have not yet been published [6], [25].
Thus, plants are a resource that is still little explored, but which have great potential.
Research into new agents of natural origin has been gaining prominence as a source
of interesting substances that can be used to develop new therapeutic options with
low NO bioavailability [5], [11].
Plants as a new source of arginase-inhibiting molecules: in vitro and in vivo evidence
Different methods have been developed to study the inhibition of natural products
in relation to ARG activity. In vitro techniques include a micro-immobilised enzyme reactor (IMER), which uses ARG that
is covalently bound to an ethylenediamine monolithic convective interaction media
disk submitted to an HPLC system. Using this procedure, a procyanidin-enriched extract
of the stem bark from Ficus glomerata Roxob was assessed by simultaneous injection with an enzyme substrate (nitro guanidine
benzene). As a result, the enzyme K
m
values did not change, but the Vmax decreased due to a high quantity of polymers that affected the enzyme proximity and
orientation. This demonstrated, for the first time, the direct action of plant-derived
compounds on ARG activity and the modifications induced on it [58].
Interestingly, the hypothesis of a molecular interaction effect between isolated substances,
or plant-derived extracts and ARG, has been little explored in the literature. From
this point of view, polyphenolics have an important role to play due to their ability
to alter the active conformation of enzymes by destabilising the bonds between hydrogen
bonds and water molecules [27].
Akanni et al. [87] tested the effects of the methanolic extracts of the African species Artocarpus altilis (Parkinson ex F. A.Zorn) Fosberg (stem bark), Ficus exasperata Vahl (leaves), Kigelia africana (Lam.) Benth. (fruits), and catechin in relation to samples of cardiac ARG. The in vitro results indicated that F. exasperata and K. africana were not effective, whereas A. altilis and catechin (both tested at 500 and 700 µg/mL) inhibited enzymatic activity in 63,
67, 42, and 52% of cases, respectively, when compared to the control.
The rhizomes of ginger [Zingiber officinale Roscoe (Zingiberaceae)] and saffron, which is better known as red ginger [Curcuma longa L. (Zingiberaceae)] (2 and 4%) were included in a diet that was rich in cholesterol
(2%) that was given to rats for 14 days. At the end of the trial period, it was shown
that there was a significant reduction in the ARG activity measured in the plasma
and liver of the treated animals when compared to the control. In addition, the presence
of gallic acid, catechin, caffeic acid, epicatechin, rutin, quercetin, quercetrin,
campherol, luteolin, and curcumin in samples of the rhizomes was noted, and the results
that were obtained were attributed to these substances because an inverse correlation
was observed between the consumption of phenolics (flavonoids) and the total concentration
of plasma cholesterol [17], [88].
These results have contributed to the study of the application of new ARG inhibitors
in cardiovascular alterations, since the ED involved in these situations would be
impeded by the inhibition of the enzyme, resulting in a greater blood supply (NO-mediated
vasodilatation) to the tissues. Spontaneously hypertensive rats showed low pressure
rates and improved endothelial function when submitted to ARG inhibition [58].
Other studies have also tested dietary supplementation with plant extracts to inhibit
in vivo ARG activity. Wistar rats (male, adults) were sprayed with 400 µL (200 mg/Kg) of
the aqueous extract of Yucca schidigera Roezl ex Ortgies (Asparagaceae) (Mohave yucca) and the fractions were obtained by
the partition of the extract with n-butanol. At the end of the 76 days of the experiment, a significant decrease in hepatic
ARG activity was observed in the animals treated with the total aqueous extract of
Y. schidigera and with its n-butanolic fraction (p = 0.03) [89].
Similarly, Schnorr et al. [29] performed a study regarding the action of a cocoa drink that was either poor (< 90 mg)
or rich (985 mg) in flavanols. This mixture provided (−)-epicatechin (0.1 µM) and
catechin (0.4 µM) as well as the metabolites epicatechin-7-β-glucuronide (0.25 µM), 4′-O-methyl-epicatechin (0.2 µM), and 4′-O-methyl-epicatechin-7-β-glucuronide (1.7 µM) (values of plasma concentration measured after 2 h of consumption
of 200 mL of cocoa beverage that provided 2.6 µM of flavonoids) in healthy humans
(2 days). A protein diet containing 0 or 4% cocoa powder was provided to male rats
(28 days). As a result, in the samples of erythrocytes taken 24 h after the end of
the experiment, those that belonged to the flavonoid-rich cocoa beverage group showed
a decrease in the active ARG portion. A reduction in the enzymatic activity of the
renal ARG in the rats was also observed.
Corroborating this, in vitro testing of ARG inhibition in HUVEC cells shows that both (−)-epicatechin and its
mixture of flavanol metabolites exhibited effects, suggesting that after metabolisation,
polyphenols can retain anti-ARG activity (at least under controlled conditions) [29].
Taken together, these results show that the in vivo inhibition of both isoforms of the enzyme is possible, which is represented by the
previously cited results regarding ARG 1 and ARG 2 obtained in different tissues,
where each of them are mostly expressed and active. Furthermore, this demonstrates
that at this level it is important to understand the biological effects of low levels
of enzymatic activity and its correlation with the responses that are obtained. On
the other hand, research regarding ARG activity using in vitro techniques is still valuable because it makes it possible to predict behaviours and
mechanisms for the models on which therapeutic applications are based.
The ethyl acetate extract of the lignum of Caesalpinia sappan L. (Leguminosae), which is used in Asian culture to promote improved circulation
and also to prevent blood stasis, was evaluated in relation to ARG 2 of the kidney
lysate of C57BL/6 mice as well as in HUVEC cells. As a result, residual activity in
ARG 2 was observed (31%) at the highest concentration of the extract used (50 µg/mL),
and the calculated IC50 was 36.82 µg/mL. In the other experiment that was conducted, after 18 h of incubation
with 20 µg/mL of the extract, a significant decrease in enzymatic activity was observed
when compared to the untreated control [90].
The aforementioned study also demonstrated that with the inhibition of the ARG in
the HUVEC cells there was a dose-dependent increase in NO production, with a maximum
level of 130% at 50 µg/mL. This data highlights the relationship between decreased
levels of active ARG and increases in NO, which serves as a basis for ethnopharmacological
applications of C. sappan, given the antithrombotic and provascular properties of NO [90].
A further two published studies that evaluated the use of the aqueous extract of Korean
red ginseng [Panax ginseng C. A.Mey (Araliaceae)] to improve endothelial function impairment associated with
age (in atherosclerosis models) reached similar conclusions; the extract (10 – 20 mg/mouse/day
during 4 – 6 weeks) inhibited ARG activity in a nonselective manner, causing an increase
in eNOS dimerisation and a consequent increase in NO levels, which strengthened the
vasodilatation dependent on this mediator. Moreover, active components of Korean red
ginseng (ginsenoside Rb1 and Rg3) have been linked to increased NO production in endothelial
cells by the activation of the phosphoinositide 3-kinase (PI3K)/PKB intracellular
pathway (also known as Akt, which is a serine/threonine-specific protein kinase) [91], [92].
Concrete evidence supports the involvement of ARG 1 and ARG 2 in the pathophysiology
of erectile dysfunction. Because NO serves to relax the smooth muscles of the corpus
cavernosum, inhibition of ARG, at this time, is useful for increasing the supply of
the substrate to the action of eNOS [93].
Oboh et al. [38] found that extracts of the leaves of Moringa oleifera Lam. inhibited ARG from rat penis homogenates in a dose-dependent manner (IC50 of 159.59 µg/mL). In the aforementioned study, the authors identified the polyphenol
composition of the extract (gallic acid, catechin, chlorogenic acid, ellagic acid,
epicatechin, rutin, quercitrin, isoquercitrin, quercetin, kaempferol), which, in their
opinion, contributed greatly to the mechanism of action against erectile dysfunction.
Of the secondary metabolites that have been isolated from plants, polyphenols have
been extensively tested against ARG as a tool to control diseases attributed with
the advancement of ED [11], [25], [27], [29], [36], [38].
Using an indirect technique (the quantification of urea produced), Reis et al. [94] found that at a concentration of 1 mM, the polyphenols (−)-epigallocatechin-3-gallate,
(+)-catechin, (−)-epicatechin, and gallic acid were able to inhibit the activity of
ARG isolated from rat liver by 29, 26, 22, and 20%, in that order.
Nelin et al. [71] used immunoblotting and Real-Time PCR methods in relation to ARG 1 (bovine pulmonary
arterial endothelial cells) and ARG 2 respectively, to demonstrate that the induction
of the expression of these enzymes by a mixture of LPS/TNF-α partially depended on the activity of the EGFR, and that the flavonoid genistein
acted indirectly on the expression of ARG as an EGFR inhibitor.
Using a low-cost in vitro colorimetric technique with commercially available b-ARG 1, Bordage et al. [34] determined the ARG inhibitory potential of a range of polyphenols. Other studies,
which used some changes in this technique, also evaluated the anti-ARG action of several
phenolics in vitro ([Table 2]).
Table 2 ARG inhibition of important polyphenols from a medicinal chemistry point of view.
|
Substance
|
Structure
|
IC50 (µM)
|
|
a
[34], Mammal bovine liver arginase (b-ARG 1);
b
[36] and
c
[95], arginase 2 from the kidney of C57BL/6 mice
|
|
Chlorogenic acid
a
|
 
|
10.6
|
|
Piceatannol
a
|
|
12.1
|
|
Resveratrol
a
|
|
18.2
|
|
(−)-Epicatechin
a
|
|
19.9
|
|
Taxifolin
a
|
|
23.2
|
|
Quercetin
a
|
|
31.2
|
|
Fisetin
a
|
|
82.9
|
|
Kaempferol
a
|
|
179.1
|
|
Caffeic acid
a
|
|
86.7
|
|
(2R,4S)-4,5,6,7,8,4′-Hexamethoxylflavan
b
|
|
> 200
|
|
Wogonin
b
|
|
> 200
|
|
(2S)-5,7-Dihydroxy-8,2′-dimethoxyflavanone
b
|
|
25.1
|
|
Apigenin
b
|
|
> 200
|
|
(2S)-5,2′,5′-Trihydroxy-7,8-dimethoxyflavanone
b
|
|
11.6
|
|
Naringenin
b
|
|
> 200
|
|
Naringenin-5-O-β-d-glucopyranoside
b
|
|
> 200
|
|
(2S)-5,5′-Dihydroxy-7,8-dimethoxyflavanone-2′-O-β-d-glucopyranoside
b
|
|
> 200
|
|
7-Hydroxysauchinone
c
|
|
89.6
|
|
Sauchinone
c
|
|
61.4
|
|
meso-Dihydroguaiaretic acid
c
|
|
> 200
|
|
Guaiacin
c
|
|
> 200
|
|
(7S,8R)-4-Hydroxy-3,7-dimethoxy-1′,2′,3′,4′,5′,6′,7′-heptanorlign-8′-one
c
|
|
> 200
|
|
(E)-7-(4-Hydroxy-3-methoxyphenyl)-7-methylbut-8-en-9-one
c
|
|
> 200
|
|
Licarin A
c
|
|
> 200
|
As can be seen in [Table 2], the most active phenolics were chlorogenic acid and piceatannol, and the efficacy
was similar to the positive control that was used, with Emax values of 81 and 98% for the phenolics respectively, and an Emax of 97% for the BEC. It was also observed that there was competitive inhibition behaviour
between these phenolics and b-ARG 1.
In relation to the study of the activity structure relationship, according to the
IC50 data obtained in two recent studies, the caffeoyl (3,4-dihydroxycinnamoyl) group
appears to be essential, since both chlorogenic acid and piceatannol have this substituent
in their structure. This is reinforced by the fact that in isolation, caffeic and
quinic acids did not present satisfactory enzymatic inhibition when compared to the
whole molecule. In relation to the derivatives of the flavonoid class, whose prototype
is quercetin, pertinent observations include the importance of hydroxyl in C5 for
the maintenance of activity, while the presence of the carbonyl group in C4 and the
unsaturation at the C2 – C3 bond exerted less significant influence, as well as the
fact that the substitutions of hydroxyl, glucose, or acetate at the C3, C7, C8, and
C2′ positions appear to have had no positive influence on the inhibition of arginase.
Furthermore, the hydroxyl in C5′ (catechol group) is essential to the inhibitory activity
as well as the α bond between C2-C1′, which increases the activity ([Fig. 5]) [34], [36].
Fig. 5 Structure-activity relationships of flavonoid-type polyphenols as arginase inhibitors.
Highlights in red lines indicate important parts of the molecule in relation to anti-arginase
action; the hydroxyl group (–OH) at C5′ and C5 and the α bond between C2-C1′ are essential for the activity.
Based on the in vitro results obtained by Kim et al. [36], who tested eight flavonoid-type substances isolated from a methanolic extract of
Scutellavia indica L. in relation to ARG 2 from mouse kidney homogenate, another group of researchers
sought to perform more in-depth in vivo research regarding the anti-ARG properties of the substance TDF, which had been previously
isolated. In that study, the authors used a hyperlipidemia model to demonstrate that
TDF inhibited both ARG 1 (IC50 of 12.18 µM) and ARG 2 (IC50 of 11.86 µM) in a noncompetitive manner, simultaneously increasing NO levels by the
phosphorylation and dimerisation of eNOS, as well as indicating an improvement in
vascular function in normal mice that received a standard diet, and also ApoE–/– mice fed on a high cholesterol diet [96].
In the study by Kim et al. [36], referred to above, PG was used as a positive control (IC50 of 1.0 µM).
Piceatannol (3,3′,4′,5-transtetrahydroxystilbene) is naturally found in rhubarb rhizomes
[Rheum undulatum L. (Polygonaceae)] and can be metabolised from resveratrol through hydroxylation
by the action of cytochrome P4501B1 [97]. The stilbene derivative PG was first evaluated by Woo et al. [65] and it showed antioxidant capacity and important inhibitory in vitro action in relation to ARG 1 and ARG 2, which was associated with the dose-dependent
increase in NO levels. In the experiments, PG behaved as a nonselective ARG inhibitor
in C57BL/6 mice (IC50 of 11.22 µM for liver lysate and IC50 of 11.06 µM for kidney lysate) and was able to increase NO production and decrease
ROS in isolated aortic fragments.
Inspired by these results regarding the potential of PG, Frombaum et al. [98] compared the behaviour of resveratrol and piceatannol in relation to BAEC. The effects
were measured in BAEC that was stimulated by high concentrations of glucose (25 mM)
for 24 h in order to mimic the hyperglycaemic conditions observed in the diabetes
state. As a result, both resveratrol (10 µM) and PG aglycone (1 µM) were shown to
produce enzymatic inhibition in the experiments; the efficacy of the latter was considered
to be greater, sustaining its therapeutic potential for application in relation to
ED.
The research group led by Woo et al. [99] subsequently proved that the administration of PG (~ 500 µg/mouse/day for 6 weeks)
was able to improve ED in an animal model of hyperlipidaemia via ARG inhibition and,
reciprocally, eNOS activation through enhanced stability of the eNOS dimer.
Based on these results, a review was published regarding the effects of piceatannol
on the diversity of cardiovascular impairment, including the prevention of hypercholesterolaemia,
cardiac arrhythmia, monocyte adhesion to the endothelium, proliferation and migration
of SMCs, ED, and angiogenesis, as well as its anti-inflammatory, vasorelaxant, and
antioxidant effects [97].
However, the application of piceatannol, or its derivative glucopyranoside, as a pharmaceutical
product to reduce cardiovascular risks is limited due to its low oral bioavailability
and a lack of studies regarding its pharmacokinetic profile [84], [97].
In an attempt to contribute to resolving these problems, Nguyen and Ryoo [100] proposed a study regarding the intravenous administration of piceatannol in mice
with endothelial function compromised by old age. The animals (C57BL/6, male, 65 weeks)
received injections of piceatannol (30 mg/Kg body weight/day) over 4 consecutive days,
after which time the tissues of interest were properly treated for subsequent analysis.
In conclusion, the in vivo potential for ARG inhibition of piceatannol as well as its ability to improve the
vascular function of senescent mice was reinforced by the increase in NO production
by the phosphorylation of eNOS Ser1177 and the stabilisation of its dimer, strengthening
the results obtained by Woo et al. [99] with the glucuronidated form of the stilbene derivative.
Thus, according to the promising results that have been obtained with piceatannol
and PG, and in view of its structural similarity to resveratrol, Yi et al. [30] identified a new substance, THSG, ([Fig. 6]) from the Polygonum multiflorum Thunb (Polygonaceae) rhizome and tested it as an ARG inhibitor and eNOS activator.
According to the authors, the mechanisms by which THSG acts are similar to those found
for TDF, i.e., the restoration of vasculature function by the inhibition of ARG 1
and ARG 2 (25 and 38%, respectively, at 50 µM), the increase of NO, and the decrease
in ROS formation by the phenomenon of uncoupled eNOS. In addition, it was identified
that THSG presented noncompetitive inhibition in relation to ARG 2 [96].
Fig. 6 Molecular structure of 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside isolated from the rhizome of P. multiflorum Thunb. (Polygonaceae).
According to a survey, in vitro and in vivo research carried out in recent years supports the fact that numerous polyphenols
that are derived from the most diverse plants are active in improving endothelial
function by increasing NO bioavailability. In accordance with epidemiological investigations,
basic and clinical research studies suggest that polyphenols demonstrate beneficial
effects for the maintenance of vascular homeostasis in animal models as well as in
humans [24].
Other phenolic substances have also been tested as inhibitors of ARG activity or expression
with a view of developing new pharmaceutical products to be used regarding ED-related
problems.
Quercetin is widely known for its multifaceted biological action and has shown promising
anti-ARG results, although only in a limited fashion thus far (only one scientific
publication was located). Nikolić et al. [88] induced a model of acute renal failure in adult male rats by the intramuscular injection
of 50% glycerol (8 mL/Kg) with pretreatment (2 h) of subcutaneous quercetin (20 mg/Kg).
As a result, the flavonoid was able to decrease levels of plasma urea and creatinine,
as well as decreasing hepatic ARG activity when compared to the control group (glycerol
only). According to the researchers, the established antioxidant action of quercetin,
combined with the inhibition of L-arginine consumption (anti-ARG effect), may have
contributed to the provision of a substrate for the synthesis of NO, whose vasorelaxant
power contributed to decreasing vascular resistance and restoring renal function.
Other substances with important action against ARG include the polyphenolics salvianolic
acid B [isolated from Salvia miltiorrhiza Bunge (Lamiaceae)] [101] and sauchinone [isolated lignan from Saururus chinensis (Lour.) Baill. (Saururaceae)] [95] ([Fig. 7]).
Fig. 7 Molecules of salvianolic acid B (A), and sauchinone (B).
Both of these substances are active in inhibiting ARG, particularly salvianolic acid
B, which also decreased the expression of iNOS in RAW 264.7 macrophages that were
induced by LPS [84], [101].
Such decreased levels of iNOS provide protection from the toxic effects of high NO
concentrations derived from this high throughput isoform and, together with reduced
ARG activity, this enhances the potential of salvianolic acid B against cardiovascular
diseases associated with ED.
It is also worth mentioning that ellagic acid has received special attention from
researchers because of its pluripotent biological activity and the multiple molecular
targets that it acts upon [102]. Based on this, an animal model of hepatocellular carcinoma demonstrated that the
oral administration of ellagic acid (50 mg/Kg/day), 7 days before and 14 days after
tumour induction (N-nitrosodiethylamine and CCl4), provided 23.6% of inhibition of ARG activity when compared to the negative control
group (healthy rats). In that particular study, the elevation of ARG levels after
the injection of the tumour agent was considered a marker of disease progression,
and other studies have also attributed a biomonitoring function to this enzyme in
the most varied clinical conditions, such as the oxidative stress observed in pregnant,
overweight women and their neonates [103], [104].
It is interesting to note that ARG activity is related to tumour progression, since
the formation of polyamines and proline that are the result of enzyme action can contribute
to cell proliferation and tumour growth, as shown by studies that have found a relationship
of risk between the increased expression of ARG 2 and the appearance of disease [93], [105]. Thus, ARG inhibition has the potential to curb this process, which might work in
favour of the action of other anticancer substances.
Stolarczyk et al. [105] studied the aqueous and ethyl acetate extracts (aerial parts) of three species of
Epilobium sp. [Epilobium angustifolium L., Epilobium parviflorum Schreb, and Epilobium hirsutum L. (Onagraceae)], as well as polyphenols isolated from these species, in relation
to the ARG of prostate cancer (LNcaP) cells and demonstrated that almost all the extracts
(50 and 70 µg/mL) and phenolics that were tested, which included quercetin-3-O-glucuronide and oenothein B (20 and 40 µM), were able to significantly inhibit enzymatic
action.
Furthermore, the same authors provided valuable data regarding anti-ARG research.
They made an incubation of E. hirsutum herb extract, which contains high concentrations of oenothein B (dimeric macrocyclic
ellagitannin), with human gut flora (final concentration 1.6 mg/mL) for 48 h. After
this time, the metabolites urolithins A, B, and C, which can be detected in plasma
(0.5 – 18.6 µM), were produced and then tested for anti-ARG potential in LNcaP cells.
The results showed that both urolithin A (ARG activity of 39.8 ± 2.5 mUnits of urea/mg
protein) and C (ARG activity of 27.9 ± 3.3 mUnits of urea/mg protein) were active
as enzyme inhibitors compared with the control cells (65.2 ± 1.1 mUnits of urea/mg
protein), whereas urolithin B was inactive. Thus, these data suggest that anti-ARG
activity remains in metabolites as well as in its precursor compound, at least under
in vitro conditions.
Indeed, the amount of ellagitannins in systemic circulation and tissues is virtually
undetectable, whereas urolithins and their conjugates can be found in higher levels
(µM). It has been reported that ellagitannin metabolites can be detected in the liver
and kidneys [106], urolithins are enhanced in the prostate, intestinal tissue, and colon in mice,
and urolithin A-glucuronide is the main metabolite found in the human prostate (> 2 ng/g
tissue) as well as traces of urolithin B-glucuronide and ellagic acid-dimethyl ether
[102], [107].
Regarding the plasma concentration, the level of polyphenols and their metabolites
found in vivo needs to be biologically applicable and should also be taken into account. Engler
et al. [108] found that the consumption of chocolate containing high levels of flavonoids improved
endothelial function and increased the plasma concentrations of epicathecin (already
reported as an ARG inhibitor) in healthy adults, with a marked increase after 2 weeks
(204.4 ± 18.5 nmol/L).
Other studies have been performed to better characterise the absorption and metabolism
of polyphenols, which would help to shed light on the pivotal relationship between
the bioavailable amount and the biological effect. In an ex vivo experiment to measure NO-dependent vasodilation, Schroeter et al. [109] performed an incubation of preconstricted rabbit aortic rings with a mixture of
flavanols and their metabolites (catechin, epicatechin, 4′-methyl-epicatechin, epicatechin-O-β-D-glucuronide, and 4′-O-methyl-epicatechin-O-β-D-glucuronide) in the same higher plasma concentration achieved after 2 hours of
administration, resulting in relaxation (74.2 ± 14.5%).
However, there is still a lack of data about the pharmacokinetics of plant-derived
compounds. Characterisation of factors such as absorption, distribution, metabolism,
excretion (ADME), and toxicological parameters may help to improve the evaluation
of the drug-likeness features of plant-derived substances. For this purpose, methods
of drug-likeness prediction have been developed (drug database screens, knowledge-based
methods, and functional group filters) and they serve as valuable tools, especially
in the pharmaceutical field [110] ([Tables 3] and [4]).
Table 3 Pharmacokinetic properties of revised polyphenol compounds with anti-ARG potential.
|
Substance
|
MF
|
MW
|
OB
|
HIA (%)
|
Caco2 (nm/s)
|
MDCK (nm/s)
|
PPB (%)
|
BBB (%)
|
Pgp inhibition
|
V
d
(L/Kg)
|
Inhibitor (CYP)
|
Substrate (CYP)
|
|
2C19
|
2C9
|
2D6
|
3A4
|
2D6
|
3A4
|
|
MF: Molecular formula, WM: molecular weight. OB: Oral bioavailability (p: poor, less
than 30%; m: moderate, between 30 – 70%; g: good, more than 70%). HIA: Human intestinal
absorption (less than 20%: weakly absorbed; between 30 – 70%: moderately absorbed;
more than 70%: well absorbed). Caco2: In vitro Caco2 cell permeability (human colorectal carcinoma) (less than 4: weakly permeable;
between 4 – 70: moderately permeable; more than 70: highly permeable). MDCK: In vitro MDCK cell permeability (mandin darby canine kidney) (less than 25: weakly permeable;
between 25 – 500: moderately permeable; more than 500: highly permeable). PPB: In vitro plasma protein binding (less than 90%: weakly bound; more than 90%: strongly bound).
BBB: In vivo blood-brain barrier penetration (C. brain/C. blood) (less than 0.1: weak penetration;
between 0.1 – 2.0: moderate penetration; more than 2.0: high penetration). Pgp: In vitro P-glycoprotein inhibition. V
d
: Distribution volume (less than 1: small V
d
value; between 1 – 10: moderate V
d
value). CYP: Cytochrome P-450 enzymes (~: weakly)
|
|
Chlorogenic acid
|
C17H20O8
|
352.3359
|
p
|
29.77
|
17.43
|
1.98
|
47.03
|
0.035
|
no
|
0.25
|
yes
|
yes
|
no
|
yes
|
no
|
~
|
|
Piceatannol
|
C14H12O4
|
244.2426
|
m
|
81.95
|
2.37
|
258.17
|
100
|
1.013
|
no
|
1.58
|
yes
|
yes
|
no
|
yes
|
no
|
~
|
|
Resveratrol
|
C14H12O3
|
228.2432
|
g
|
88.47
|
5.19
|
76.74
|
100
|
1.738
|
no
|
1.84
|
yes
|
yes
|
no
|
yes
|
no
|
no
|
|
(−)-Epicatechin
|
C15H14O6
|
290.2680
|
p
|
66.70
|
0.65
|
44.38
|
100
|
0.394
|
no
|
1.36
|
yes
|
yes
|
no
|
yes
|
no
|
~
|
|
Taxifolin
|
C15H12O7
|
304.2515
|
p
|
60.16
|
3.42
|
9.56
|
95.16
|
0.166
|
no
|
0.64
|
yes
|
yes
|
no
|
yes
|
no
|
~
|
|
Quercetin
|
C15H10O7
|
302.2357
|
p
|
63.48
|
3.41
|
13.35
|
93.23
|
0.172
|
no
|
0.6
|
yes
|
yes
|
no
|
yes
|
no
|
~
|
|
Fisetin
|
C15H10O6
|
286.2363
|
p
|
79.43
|
9.57
|
68.19
|
88.72
|
0.316
|
no
|
0.6
|
yes
|
yes
|
no
|
yes
|
no
|
no
|
|
Kaempferol
|
C15H10O6
|
286.2363
|
p
|
79.43
|
9.57
|
29.61
|
89.60
|
0.286
|
no
|
0.61
|
yes
|
yes
|
no
|
yes
|
no
|
no
|
|
Caffeic acid
|
C9H8O4
|
180.1574
|
m
|
82.30
|
21.10
|
109.43
|
40.29
|
0.497
|
no
|
0.31
|
no
|
yes
|
no
|
yes
|
no
|
no
|
|
(2R,4S)-4,5,6,7,8,4′-Hexamethoxylflavan
|
C21H26O7
|
390.4269
|
g
|
98.48
|
55.24
|
1.25
|
85.65
|
0.069
|
yes
|
1.62
|
yes
|
yes
|
no
|
yes
|
no
|
yes
|
|
Wogonin
|
C16H12O5
|
284.2634
|
g
|
93.03
|
4.28
|
152.11
|
90.44
|
0.724
|
no
|
0.64
|
yes
|
yes
|
no
|
yes
|
no
|
no
|
|
(2S)-5,7-Dihydroxy-8,2′-dimethoxyflavanone
|
C17H16O6
|
316.3053
|
p
|
92.97
|
16.82
|
75.20
|
89.55
|
0.681
|
no
|
0.62
|
yes
|
yes
|
no
|
yes
|
no
|
~
|
|
Apigenin
|
C15H10O5
|
270.2369
|
g
|
88.12
|
10.54
|
44.30
|
97.25
|
0.565
|
no
|
0.91
|
yes
|
yes
|
no
|
yes
|
no
|
no
|
|
(2S)-5,2′,5′-Trihydroxy-7,8-dimethoxyflavanone
|
C17H16O7
|
332.3047
|
p
|
86.48
|
16.18
|
35.62
|
91.45
|
0.061
|
no
|
1.46
|
yes
|
yes
|
no
|
yes
|
no
|
~
|
|
Naringenin
|
C15H12O5
|
272.2527
|
p
|
87.31
|
10.52
|
44.63
|
100
|
0.596
|
no
|
0.65
|
yes
|
yes
|
no
|
yes
|
no
|
no
|
|
Naringenin-5-O-β-d-glucopyranoside
|
C21H22O10
|
434.3933
|
p
|
42.26
|
4.93
|
0.91
|
66.78
|
0.037
|
no
|
0.67
|
yes
|
yes
|
no
|
yes
|
no
|
~
|
|
(2S)-5,5′-Dihydroxy-7,8-dimethoxyflavanone-2′-O-β-d-glucopyranoside
|
C23H26O12
|
494.4453
|
p
|
32.36
|
6.53
|
0.14
|
55.26
|
0.034
|
no
|
0.9
|
yes
|
yes
|
no
|
yes
|
no
|
~
|
|
7-Hydroxysauchinone
|
C18H16O7
|
344.3254
|
m
|
95.35
|
21.78
|
2.54
|
75.16
|
0.475
|
no
|
1.1
|
no
|
yes
|
no
|
yes
|
no
|
~
|
|
Sauchinone
|
C18H16O6
|
328.3160
|
m
|
98.41
|
38.62
|
27.86
|
87.26
|
1.401
|
no
|
1.28
|
yes
|
yes
|
no
|
yes
|
no
|
~
|
|
meso-Dihydroguaiaretic acid
|
C20H26O4
|
330.4180
|
p
|
93.35
|
35.17
|
57.64
|
100
|
5.286
|
yes
|
2.49
|
yes
|
yes
|
no
|
yes
|
no
|
~
|
|
Guaiacin
|
C20H24O4
|
328.4021
|
p
|
93.35
|
27.16
|
105.31
|
100
|
2.609
|
yes
|
2.67
|
yes
|
yes
|
no
|
yes
|
no
|
~
|
|
(7S,8R)-4-Hydroxy-3,7-dimethoxy-1′,2′,3′,4′,5′,6′,7′-heptanorlign-8′-one
|
C13H18O4
|
238.2796
|
g
|
94.55
|
37.07
|
51.68
|
74.29
|
0.641
|
no
|
1.27
|
yes
|
yes
|
no
|
yes
|
no
|
yes
|
|
(E)-7-(4-Hydroxy-3-methoxyphenyl)-7-methylbut-8-en-9-one
|
C12H14O3
|
206.2377
|
g
|
94.73
|
29.92
|
352.94
|
77.91
|
0.661
|
no
|
1.33
|
yes
|
yes
|
no
|
no
|
~
|
~
|
|
Licarin A
|
C19H20O4
|
312.6597
|
p
|
95.65
|
55.84
|
123.41
|
98.77
|
1.206
|
yes
|
2.58
|
yes
|
yes
|
no
|
yes
|
no
|
yes
|
Table 4 Toxicity features and drug-likeness properties of revised polyphenol compounds with
anti-ARG potential.
|
Substance
|
Mutagenicity
|
Carcinogenity (mouse)
|
Carcinogenity (rat)
|
hERG inhibition (risk)
|
Lipinskiʼs rule
|
|
Mutagenicity: based on the Ames test; Carcinogenity: 2-year bioassay in the mouse/rat;
hERG: in vitro human ether-a-go-go-related gene channel inhibition; Lipinskiʼs rule: hydrogen bond
donors less than 5, hydrogen bond acceptor less than 10, molecular weight less than
500 Da; CLogP less than 5 (MlogP less than 4.15)
|
|
Chlorogenic acid
|
negative
|
positive
|
negative
|
medium
|
suitable
|
|
Piceatannol
|
positive
|
negative
|
negative
|
medium
|
suitable
|
|
Resveratrol
|
positive
|
negative
|
negative
|
medium
|
suitable
|
|
(−)-Epicatechin
|
positive
|
negative
|
negative
|
medium
|
suitable
|
|
Taxifolin
|
positive
|
negative
|
positive
|
medium
|
suitable
|
|
Quercetin
|
positive
|
negative
|
positive
|
medium
|
suitable
|
|
Fisetin
|
positive
|
negative
|
positive
|
medium
|
suitable
|
|
Kaempferol
|
positive
|
negative
|
positive
|
medium
|
suitable
|
|
Caffeic acid
|
positive
|
negative
|
positive
|
medium
|
suitable
|
|
(2R,4S)-4,5,6,7,8,4′-Hexamethoxylflavan
|
positive
|
negative
|
positive
|
low
|
suitable
|
|
Wogonin
|
positive
|
negative
|
positive
|
medium
|
suitable
|
|
(2S)-5,7-Dihydroxy-8,2′-dimethoxyflavanone
|
negative
|
negative
|
positive
|
medium
|
suitable
|
|
Apigenin
|
positive
|
positive
|
positive
|
medium
|
suitable
|
|
(2S)-5,2′,5′-Trihydroxy-7,8-dimethoxyflavanone
|
negative
|
negative
|
positive
|
low
|
suitable
|
|
Naringenin
|
positive
|
negative
|
positive
|
medium
|
suitable
|
|
Naringenin-5-O-β-d-glucopyranoside
|
positive
|
negative
|
negative
|
high
|
suitable
|
|
(2S)-5,5′-Dihydroxy-7,8-dimethoxyflavanone-2′-O-β-d-glucopyranoside
|
negative
|
negative
|
negative
|
–
|
violated
|
|
7-Hydroxysauchinone
|
negative
|
negative
|
negative
|
low
|
suitable
|
|
Sauchinone
|
positive
|
negative
|
positive
|
low
|
suitable
|
|
meso-Dihydroguaiaretic acid
|
negative
|
negative
|
negative
|
medium
|
suitable
|
|
Guaiacin
|
positive
|
negative
|
negative
|
medium
|
suitable
|
|
(7S,8R)-4-Hydroxy-3,7-dimethoxy-1′,2′,3′,4′,5′,6′,7′-heptanorlign-8′-one
|
positive
|
negative
|
positive
|
low
|
suitable
|
|
(E)-7-(4-Hydroxy-3-methoxyphenyl)-7-methylbut-8-en-9-one
|
positive
|
negative
|
positive
|
low
|
suitable
|
|
Licarin A
|
positive
|
negative
|
positive
|
medium
|
suitable
|
The potential therapeutic properties of bioactive substances depend on their bioavailability
after oral administration. Therefore, matrix effects (for example, the vehicle for
solubilisation or composition of the diet), the physical and chemical properties of
the substance (degree of glycosylation/acylation, basic structure [benzene or flavones],
conjugation with other phenolics, molecular size, degree of polymerisation, solubility/partition
coefficient), interindividual variations (gastrointestinal secretions, motility, blood/lymph
flow, etc.), and other interactions (alcohol or the presence of macronutrients like
fat, protein, and carbohydrates) can be important factors to be considered in relation
to the bioavailability of natural substances as well as the dosage used. Furthermore,
gastric pH, enterocyte metabolism, digestive enzyme activity, first pass metabolism,
and mechanisms of resistance (expression of apical multidrug resistance-associated
proteins such as P-glycoprotein 1) should all be considered [111], [112], [113], [114].
Aglycones, simple phenolic acids, and flavonoids can be absorbed in the stomach or
small bowel mucosa. If this does not occur, the phenolic substance will be carried
to the colon, which contains catalytic and hydrolytic potential that is powered by
microorganisms. This colonic microflora transforms polyphenols (glycoside derivates
with a hydrophilic nature and relatively high molecular weight) into more simple substances,
such as phenolic acids (aglycone) [115]. In addition, bile plays a pivotal role in the adsorption of plant-derived polyphenols
from the gastrointestinal tract (enterohepatic cycle) [116].
As shown in [Table 3], all the reviewed polyphenols with anti-ARG potential are moderately or well absorbed
(human intestinal absorbed and Caco2 permeability), but this inversely correlates
with oral bioavailability (a minority have good parameters). It is suggested that
this is due to first-pass metabolism, which extensively alters the quantities of substances
in plasma.
Manach et al. [117] evaluated data from 97 studies about kinetics and the absorption of polyphenols
among adults (the ingestion of a single dose of the substance). They found that gallic
acid was better absorbed than other phenolic substances (the Cmax values reached 4 µmol/L with a 50-mg dose), followed by isoflavones, catechins, flavanones,
quercetin glucosides, proanthocyanidins, galloylated tea catechins, and anthocyanins
[118]. Additionally, the time to Cmax varied from approximately 1.5 h to 5.5 h, taking into account the site of intestinal
absorption [117].
After absorption, molecules are distributed from plasma to other compartments of the
body. In relation to anti-ARG polyphenols, approximately half of them occur in free
state in the circulation (weakly bound to plasma proteins) and they can reach several
parts of the peripheral system to achieve their potential enzyme inhibition (V
d
value). In addition, only two of these anti-ARG polyphenols have the ability to cross
the blood-brain barrier, which could result in biological or toxicological effects.
For most of the polyphenols that are absorbed, the plasma concentration quickly decreases.
The metabolism mainly occurs in the liver (methylation and/or conjugation with glucuronic
acid or sulphate), supported by the metabolism of the kidneys and intestinal mucosa.
Thus, achieving elevated levels of polyphenols in plasma requires repeated ingestion
over time. However, catechins, gallic acid, and flavanones seem to have no chance
to accumulate, even with sequential administrations. On the other hand, quercetin
exhibits a high affinity for plasmatic albumin, which might explain its higher elimination
half-life (24 h) [115], [117].
Taking this into consideration, the excretion of polyphenols occurs mainly in urine
or feces (especially phenols that are resistant to microflora degradation, such as
condensed tannins and those linked to macromolecules) [113], and can be expressed as MDCK cell permeability, which predicts renal excretion
ability. In this context, most of the anti-ARG phenolics reviewed present moderate
permeability capability, suggesting a moderate to high maintenance of these substances
in the organism. Additionally, attention should be paid to those phenolics that are
highly bound to plasma proteins due to the risk of toxicity from long-term use and
accumulated doses [110].
Regarding toxicity ([Table 4]), plant-derived substances have a favourable spectrum in most cases, which is very
important during drug development. Only one of the reviewed polyphenols presents a
high risk of inhibiting hERG (a gene that encodes a potassium ion channel expressed
in the heart and when inhibited can produce a long QT syndrome that results in potentially
fatal arrhythmias) [119], although almost all the phenolics presented positive predictions regarding mutagenic
or carcinogenic (mouse and/or rat) action. These points are relevant since they can
determine the final outcome of new therapeutic approaches.
Concerning the predicted toxicological potential, the dosage must be considered because
some effects only appear at higher doses. For this purpose, daily dietary reference
intakes of polyphenols are required and are highly desirable, although data are currently
insufficiently available to establish how to avoid upper doses with possible toxic
effects [120].
Finally, completing the prediction analysis, the drug-likeness investigation of polyphenols
with potential activity as ARG inhibitors showed that only one substance violated
Lipinkiʼs rule and therefore could not be recommended as an emergent drug in the management
of ED.
