International Journal of Food Sciences and Nutrition

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Metabolites of the ellagitannin, geraniin inhibit

human ACE; in vitro and in silico evidence

Deming Looi , Boon H. Goh , Shafi U. Khan , Nafees Ahemad & Uma D.
Palanisamy

To cite this article: Deming Looi , Boon H. Goh , Shafi U. Khan , Nafees Ahemad &
Uma D. Palanisamy (2020): Metabolites of the ellagitannin, geraniin inhibit human ACE;
in vitro and in�silico evidence, International Journal of Food Sciences and Nutrition, DOI:
10.1080/09637486.2020.1830263

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Published online: 09 Oct 2020.

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INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION
https://doi.org/10.1080/09637486.2020.1830263

BRIEF REPORT

Metabolites of the ellagitannin, geraniin inhibit human ACE; in vitro and in

silico evidence
Deming Looia, Boon H. Gohb, Shafi U. Khanc, Nafees Ahemadc and Uma D. Palanisamya
a
Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Selangor, Malaysia; bSchool of Science, Monash
University Malaysia, Selangor, Malaysia; cSchool of Pharmacy, Monash University Malaysia, Selangor, Malaysia

ABSTRACT ARTICLE HISTORY

Hypertension is defined as the persistence of elevated blood pressure in the circulation system. Received 12 May 2020
The renin-angiotensin-aldosterone system is a major modulator of blood pressure. Among the Revised 16 September 2020
risk factors of cardiovascular disease, hypertension is the most preventable and treatable, with Accepted 21 September 2020
drugs such as ACE inhibitors. Many ACE inhibitors are known to have undesirable side effects
KEYWORDS
and hence, natural alternatives are being sought. Dietary polyphenols, particularly ellagitannins, ACE; ellagitannin geraniin;
are derived from plant products and are known to exhibit a variety of bioactivities. Geraniin, an hypertension; ellagic
ellagitannin has been shown to have antihypertensive activity in animal experiments. It is specu- acid; urolithin
lated that the metabolites of geraniin are responsible for its ACE inhibitory activity. We have per-
formed in vitro ACE inhibition and in silico studies with geraniin and its metabolites (ellagic acid,
urolithins). Our studies confirm that ellagic acid exhibited similar inhibitory potential to ACE as
the positive control captopril.

Introduction remodelling of blood vessels structure. Besides, abnor-

mally high Ang II level may also lead to overactiva-
Hypertension (HTN), also known as high blood pres-
tion of the sympathetic nervous system, further
sure, is a medical condition in which blood pressure
(BP) in the arteries is consistently elevated. exaggerating high blood pressure. Therefore, an effect-
Hypertension is the primary risk factor for cardiovas- ive agent that targets ACE not only benefits normo-
cular disease and chronic renal disease. To date, there tensive BP control but may also ameliorate various
are approximately 874 million adults who suffer from cardiovascular risks. Although several ACE inhibitors
stage-1 hypertension (>140 mmHg) (Oparil et al. have been routinely used in clinical hypertension
2018). BP is regulated by a few systems present in the treatment, some of these agents may come with
body, including the autonomic nervous system, vascu- undesirable side effects. Natural compounds in food
lar tone regulation in blood vessels, and renin-angio- as well as nutraceutical supplements can mimic drugs
tensin-aldosterone system (RAAS). Among these and function in a similar manner to these antihyper-
mechanisms, RAAS plays a pivotal role in controlling tensives. However, they may be less potent and take
the arterial pressure and extracellular fluid homeosta- longer to work but when used together in combin-
sis. Mechanistically, it elevates BP by mediating the ation with other nutrients its effect can be magnified
conversion of Angiotensin I (Ang I) to Ang II, a (Alexander 2014)
potent vasoconstrictor hormone, through angiotensin- Dietary polyphenols, compounds found abundantly
converting enzyme (ACE) (te Riet et al. 2015; Oparil in plants, have become an emerging field of interest
et al. 2018). However, patients with hypertension may in nutritional health in recent decades (Manach et al.
be associated with overactivation of RAAS in their 2004). Polyphenol-containing products are being
bodies. In addition to uncontrolled high blood pres- regarded as functional foods, having potentially posi-
sure, several studies have also indicated that dysregu- tive effects on health. They are classified based on
lated RAAS may contribute to several cardiovascular their chemical structure, ranging from simple mole-
perturbations, such as endothelial dysfunction, vascu- cules, such as phenolic acids, to highly polymerised
lar oxidative stress and inflammation, and abnormal compounds, such as tannins. Ellagitannins (ET),

CONTACT Uma D. Palanisamy [email protected] School of Medicine and Health Sciences, Monash University Malaysia, Jalan
Lagoon Selatan, Bandar Sunway, Selangor Darul Ehsan, 47500, Malaysia
ß 2020 Taylor & Francis Group, LLC
2 D. LOOI ET AL.

comprises a glucose moiety and a complex derivative circulation (Elendran et al. 2019). We believe that the
of ellagic acid (EA). ET’s form the largest group of metabolites of geraniin play a central role in inhib-
the tannin family with over 500 different characterised ition of ACE. Several studies have also reported the
natural products (Landete 2011). antihypertensive mechanisms of EA evidenced from
ET’s and EA are found in fruits, nuts, and seeds. in vitro and in vivo experimental models; namely dir-
The Rosaceae family of berries (cloudberry, raspberry, ect vasodilation, amelioration of endothelial dysfunc-
rose hip, and strawberry) are known to have a high tion, and suppression of NLRP3 inflammasome
content of EA equivalents (Ascacio-Vald
es et al. activation (Yılmaz and Usta 2013; Berkban et al. 2015;
2011). Another rich source of ET’s is the fruit pom- Tang et al. 2015). However, the effect of EA as well as
egranate (Punica granatum), which has abundant ET, other geraniin-derived metabolites like urolithins, on
punicalagin (Razani et al. 2017). Strawberry and rasp- components of RAAS still remains inconclusive.
berry extracts contain the ET, sanguiin H-6 among Although the BP-lowering activity of geraniin has
other EA derivatives. Walnut extracts have been been previously demonstrated in animal models with
reported to contain pedunculagin, valoneic acid dilac- hypertension, the underlying antihypertensive mech-
tone, and casuarictin. The rambutan (Nephelium lap- anism is largely unknown. Therefore, the objective of
paceum) rind, a common tropical fruit, has been this study is to investigate the ellagitannin geraniin
shown to contain large amounts of the ET geraniin and its metabolites (EA, Urolithin A, & Urolithin B)
(Palanisamy et al. 2008). Several traditional Chinese ability to inhibit ACE activity in both in vitro and in
and Indian herbs (Phyllanthus sp) have also been silico studies.
reported to have varying amounts of geraniin (Perera
et al. 2015). Methodology
Bioavailability of ET’s and EA’s rely on the human
gastrointestinal tract. Initial metabolism of ET’s begins In vitro ACE inhibition studies
in the stomach, where ET is hydrolysed to free EAs ACE activity was carried out using a fluorometric kit
and a small percentage is then absorbed. Further (ACE1 Activity Assay Kit) purchased from Sigma-
hydrolysis of ET’s occurs in the small intestine and Aldrich and the protocol described by the manufac-
duodenum and the presence of the gut microbiome turer was followed. Enzyme was provided with the
aids in the further breakdown of ET’s into EA’s (Ito assay kit and was derived from human recombinant
2011). ET and EA’s are further metabolised by gut ACE 1 (non-tagged). Activity was measured at kex
bacteria into smaller metabolites such as urolithins A, 320 nm/kem 405 nm. One unit of ACE (mU) is defined
B and C, which are then absorbed into the circulation. as the amount of enzyme that releases 1 nmol of fluor-
The metabolites of ETs have been shown to exhibit escent product from the substrate, in 1 minute, under
beneficial bioactivities in animal models as well as in assay conditions at 37  C. Captopril (CAS 62571-86-2)
human trials (Istas et al. 2018). purchased from Santa Cruz Biotechnology was used
The ET of interest in this study is geraniin, a com- as the positive control. Geraniin was prepared as pre-
pound that has been shown to possess innumerable viously described (Perera et al 2012). Ellagic acid (EA)
biological activities (Perera et al. 2015; Cheng et al. was obtained from Sigma-Aldrich (CAS Number 476-
2017). We recently reported geraniin’s ability to lower 66-4) whereas urolithins A (CAS Number:1143-70-0)
systolic blood pressure in a high-fat diet-induced and B (CAS Number:1139-83-9) were purchased from
obese Sprague-Dawley (SD) rats (Phang et al. 2019) Toronto Research Chemicals. The inhibition potential
while others have reported BP reduction in spontan- of captopril, geraniin, EA and urolithins at various
eous hypertensive rats (SHR) after both oral consump- concentrations (0.01-0.0001 mM) were compared
tion and intravenous (i.v) administration of geraniin against the negative control (assay without the pres-
(Cheng et al. 1994). Furthermore, consumption of ence of inhibitor). Due to the commercial unavailabil-
fruits known to contain ETs or EAs in randomised ity of urolithin glucuronides, we were unable to
controlled trials have been associated with reduction investigate its in-vitro ACE inhibition.
in blood pressure in pre-hypertensive patients (Jeong
et al. 2016) and metabolic syndrome related hyperten-
Molecular docking studies
sive participants (Moazzen and Alizadeh 2017). In
vitro studies have indicated geraniin’s ability to inhibit Preparation of protein structure
ACE (Lin et al. 2008) but it is understood that gera- The three-Dimensional structure of Human ACE was
niin does not get absorbed into the systemic retrieved from the RCSB database (PDB ID: 1O86).
 INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION 3

Preparation of protein was carried out by using

Discovery Studio Client 16.1.0. In the protein prepar-
ation step, all water molecules were removed, and
hydrogen atoms were added to protein structure for
correct ionisation and tautomeric states. The active
site was selected around the co-crystal ligand using
the Make Receptor 3.3.0 tool in OpenEye
Scientific Software.
Figure 1. Inhibition pattern of captopril, geraniin, EA, urolithin
Preparation of ligand structures A, and urolithin B against negative control. Concentrations pre-
Structures of all eight compounds were generated sented in descending order from 0.01 to 0.0001 mM.
using the ChemDraw professional v15 and then 3D Significantly different from positive control (p < 0.05), #
optimisation and minimisation of the compounds Significantly different from captopril (p < 0.05)
were carried out using ligand preparation utilities of control (Figure 1). The concentration of geraniin was
Discovery Studio Client 16.1.0 and maximum con- initially chosen based on a previous study by Lin et al.
formers were generated using OMEGA 2.5.1 tools of who reported an IC50 of 13.22 uM using a 20 mU
OpenEye Suit. ACE from rabbit lungs. The current study uses an
ACE concentration of 2mU. Further, preliminary
Molecular docking protocol inhibition studies of the tested compounds were also
After the preparation of protein and compound struc- carried out before finally deciding on doses mentioned
tures, docking calculations were performed using in this study. The results indicate that there is a sig-
HYBRID utility within the OEDocking tool of OpenEye nificant difference (p < 0.05) in enzyme activity
Suit. In the first step, the docking protocol was validated between compounds and ACE negative control.
by redocking the crystal ligand lisinopril present within A significant inhibition of ACE activity was
the target protein and was found to have RMSD lower observed in all compounds, similar to that of the posi-
than 2 Å. Furthermore, all six compounds were docked tive control, captopril. Captopril and EA exhibited a
into the active site of the target protein and a minimum similar dose response inhibitory pattern, with a 75%
of 10 poses were generated. Generally, it is accepted that inhibition at 0.0001 mM. However, geraniin and the
the top 10 most favourable poses are taken into consid- urolithins A and B, although displaying inhibitory
eration for analysis of interaction. The best poses of activity of about 55%, 75%, and 75% respectively
each compound were selected based on the lowest (0.0001 mM), did not exhibit a typical dose dependent
Chemguass4 score (binding affinity). Visualisation of response. We performed a one-way ANOVA statistical
binding interaction of all compounds was carried out analysis to compare the inhibition pattern between the
using Discovery Studio visualise. metabolites against captopril. The analysis showed
that a significant difference in inhibition pattern was
Statistical analysis observed between geraniin, urolithin A, and urolithin
All values were expressed as mean ± standard devi- B against captopril (# p < 0.05). No significant differ-
ation (SD) and all experiments were carried out in ence was observed between captopril and ellagic acid.
duplicates. Data were analysed with one-way ANOVA, It should be noted that the positive control capto-
followed by Tukey’s range test to determine the sig- pril did not display a distinct dose-response curve, as
nificant difference between the means using GraphPad reported by others. Several reasons are suggested for
Prism 8.1.1. p-values of <0.05 was considered statis- the results we observed; apart from experimental
tically significant.
error, the source of ACE in our study was a human
recombinant enzyme as opposed to the rabbit lung
Results ACE used in the other studies (Ueno et al. 1988; Lin
et al. 2008).
Inhibition of human recombinant ACE activity by
geraniin and its metabolites
In silico molecular docking of geraniin and its
The ACE inhibitory potential of the positive control
metabolites against human ACE
captopril, geraniin, EA, urolithin A, and urolithin B at
various concentrations (0.01, 0.005, 0.001, 0.0005, In silico prediction of ACE inhibition was carried out
0.0001 mM) were compared against the negative after the in vitro study with human recombinant
4 D. LOOI ET AL.

ACE. The active binding site of ACE was used in the Urolithin A-glucuronide was observed to form
docking studies where lisinopril (in the x-crystal struc- three hydrogen bonds with amino acid residues His
ture) binds. ACE has one domain responsible for con- 383, Lys 511, and Tyr 520 followed by one hydropho-
version of angiotensin I to angiotensin II. The same bic interaction with His 513. Urolithin B-glucuronide
domain was used to design new ACE inhibitors. This formed two hydrogen bonds with His 383 and Tyr
domain is responsible for conversion of normal sub- 520 followed by a hydrophobic bond at amino acid
strate (angiotensin I) to angiotensin II. A detailed 3D residue His 513.
binding model of interactions with geraniin, EA, uro- The binding affinity (ChemGuass4 score) of the
lithin A, B and urolithin A and B glucuronides, and compounds and positive control is shown in Table 1.
positive controls with human ACE (Figure 2) was car- It was observed that all compounds had bound to the
ried out. The glucuronides of Urolithin A and B were active site of ACE with comparable binding affinity.
also modelled against ACE as these compounds have Captopril, a drug used to treat hypertension, has been
been shown to be present in circulation after the con- shown to bind specifically to the active site of ACE
sumption of ET rich food (Esp
ın et al. 2013). (Natesh et al. 2004).
In the x-ray crystal structure (PDB ID: 1O86),
Lisinopril is bound to active site. We initially docked Discussion
Lisinopril to validate the docking protocol, and RMSD
Food rich in ET and EA have been reported to have
was found to be less than 2 Å (Figure 2). After which
HTN lowering ability (Lin et al. 2008; Istas et al.
captopril and the metabolites were docked into the
2018; Phang et al. 2019). Of late there has been grow-
active binding site. Captopril is anchored onto the
ing evidence that alludes to the fact that ETs are not
binding site of ACE by two moieties, a carbonyl group
orally bioavailable and that they are hydrolysed to EA
and a proline carboxylate group. The carbonyl group
in the duodenum and finally metabolised by gut
forms two strong hydrogen bonds with His 513 and
microbes to various urolithins. Urolithin conjugates
His 353 at a distance of 2.69 Å and 2.54 Å, respect-
have been predominantly found in the plasma, tissue
ively. The oxygen molecules of the proline carboxylate
and urine, indicating that the metabolites undergo
group 46 are positioned and held by interactions with
Phase II metabolism and may even exert various
Tyr 520 (2.66 Å), Gln 281 (3.1 Å) and Lys 511
health benefits (Nu~ nez-S
anchez et al. 2014; Piwowaski
(2.73 Å). These electrostatic interactions prevent the et al. 2017; Garcia-Villalba et al. 2019). This is the first
binding of Angiotensin I to the C-domain site of study that describes the in-silico interaction of gera-
ACE. The C-domain of ACE is primarily involved in niin and its metabolites (EA, urolithin A, B and its
blood pressure regulation and it is a target of interest glucuronides) with the human ACE enzyme, as well
to inhibit the activity of ACE. Similarly, lisinopril is as the in vitro ACE inhibition by the metabolites (EA,
also bound to the binding site of ACE by these Urolithin A and B). Due to the high cost of the uroli-
two moieties. thin glucuronides, we were unable to carry out its
Geraniin, on the other hand, was observed to form in vitro inhibition of ACE, while only its in silico
multiple hydrogen and hydrophobic interactions with interaction with ACE is reported here. When compar-
the active site of the enzyme. Interactions of hydrogen ing our in vitro studies, it is evident that among the
bonds were observed in amino acid residues Tyr523, compounds tested, EA had the most efficacious
Tyr520, Glu162, and Thr160. A hydrophobic bond inhibitory property towards ACE. Moreover, it also
was observed with amino acid residue Val379. bound well to the active site in in-silico study. It dis-
EA, urolithins A, B, and its glucuronides had simi- played similar binding interaction to key amino acids
lar docking positions. EA formed two hydrogen bonds as captopril and in terms of binding affinity had the
with Ala 354 amino acid residue and Phe 512 fol- lowest ChemGuass4 score.
lowed by three hydrophobic interactions with amino Earlier in vitro studies have reported geraniin’s
acid residues His 513, His 383, and Val 518. Urolithin inhibitory activity against ACE (Lin et al. 2008; Ueno
A formed two hydrogen bonds with amino acid resi- et al. 1988); however, both our in vitro and in silico
dues Val 518 and Glu 162. Urolithin B was observed studies suggest a non-specific pattern of inhibition.
to form multiple hydrophobic interactions with amino The non-dose-dependent inhibition of geraniin
acid residue His 383 and Val 518 followed by two observed (Figure 1) is evidence of its non-specific
hydrogen bonds with amino acid residues Ala 354 binding. This is further substantiated by the fact that
and His 513. geraniin is not orally bioavailable (Ito 2011; Elendran
 INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION 5

Figure 2. 3D Interactions of geraniin, its metabolites and positive controls with target human Angiotensin Converting Enzyme.
Hydrophobic interactions are shown as green dashed lines while hydrogen bonds are shown as light pink dashed lines.
6 D. LOOI ET AL.

Table 1. HYBRID ChemGuass4 Score. On the other hand, urolithins A and B, and uroli-
Compound HYBRID ChemGuass4 Score thins A glucuronide were seen to form multiple
EA 7.84 hydrogen bonds with several other non-similar amino
Captopril (positive control) 7.56
Uro B-Glucuronide 6.94 acids. This may be indicative of non-specific binding,
Urolithin A 6.13 however more in-depth in silico studies and in vitro
Geraniin 6.09
Uro A-glucuronide 5.98 experimentation will be required to confirm this.
Urolithin B 5.81 Several studies have documented the promising car-
diovascular benefits of EA and urolithins. EA not only
et al. 2019), and as such its inhibition to ACE in sys-
mediates vasorelaxation via endothelium-dependent
temic circulation is highly unlikely. It can therefore be
mechanisms in rat aortic rings, but also restores endo-
inferred that geraniin’s antihypertensive activity
thelial nitric oxide synthase (eNOS) for nitric oxide
observed in in vivo studies may perhaps be due to the
production while suppressing NADPH oxidase-induced
metabolites of geraniin; EA and/or urolithins (Cheng
reactive oxygen species (ROS) production in rodent
and Hsu 1993; Cheng et al. 2017; Phang et al. 2019).
model with L-NAME-induced hypertension (Yılmaz
In fact, our ACE inhibition studies showed that EA
and Usta 2013). Besides, NLRP3 inflammasome activity
exhibited a similar dose-dependent inhibitory poten-
was also significantly ameliorated in a rodent model
tial as captopril, the positive control. Furthermore,
with pulmonary artery hypertension following EA
fruits rich in EA have also been documented to show
administration (Berkban et al. 2015; Tang et al. 2015).
inhibitory action against ACE such as grapefruit peel
Glucuronides, the conjugates of urolithins following
extract (Ademosun et al. 2015), pomegranate juice
phase II metabolism, may also be beneficial for vascular
(Aviram and Dornfeld 2001). In contrast, Kwon et al.
(2006) had reported that EA (1050 mM) did not show health. Spigoni et al. 2016 showed that a combination
any ACE inhibition via a high-performance liquid of urolithin B glucuronide, urolithins A, and B
chromatography (HPLC) method. It should be noted improves aortic endothelial cell function by increasing
that the concentration range of test compounds used secretion of endothelial nitric oxide in a human aortic
in our assay was 10 2 to 10 4 mM while we also used cell model. Istas et al. (2018) also showed that plasma
a sensitive fluorometric assay. The variation of artifi- urolithins A-3 glucuronide correlated with the
cial substrate specificity, inhibitor concentration and improvements of flow mediated dilation in a rando-
detection methods used could explain the difference mised controlled trial. Studies have also established the
in our results. However, further studies will be association between overactivation of RAAS and endo-
required to establish this. thelial dysfunction (Pacurari et al. 2014). Dysregulated
The active site of ACE is composed of a zinc ion Ang II level contributes to impaired vasorelaxation due
and HEXXH E motif domain where ACE inhibitors to the loss of NO bioavailability and/or NADPH oxi-
bind. The substrate, angiotensin I, selectively binds to dase-mediated ROS production in the vascular system.
the amino acid residues His353, Ala354, Tyr520 and Our current findings on geraniin’s metabolites (ellagic
His513 at this site, and allows the conversion of acid and urolithins)-mediated ACE inhibition, together
angiotensin I to angiotensin II (Natesh et al. 2004; with the observed vascular benefits of ET’s metabolites
Fang et al. 2019). In the presence of captopril, hydro- documented in previous studies, signifies that geraniin-
gen bonds are formed with His383, His513 and derived metabolites may inhibit ACE activity to reduce
Glu384 at the active site hence inhibiting the conver- Ang II production, subsequently ameliorating all the
sion of angiotensin I to angiotensin II. Similarly, EA possible Ang II-induced vascular damage in hyperten-
(Figure 2) was also seen to form hydrogen bonds with sion. However, the effect of urolithins on BP regulation
the same amino acids; His383, His513, and Glu384, is yet to be known. Therefore, further studies are war-
and a hydrophobic bond at Tyr520 but in a different ranted to address the BP-lowering activity of geraniin’s
pose. Urolithin B-glucuronide has the same pose as metabolites, particularly ellagic acid and urolithins, and
EA and similar bonding to the key amino acids but their promising protective effect against Ang II-associ-
displays different bond types; His383 and Glu384 ated cardiovascular complications.
(hydrophobic bonds). By comparing the HYBRID
ChemGuass 4 score (Table 1), EA had the lowest
Conclusion
score followed by captopril and Urolithin B-glucuro-
nide. As such, we argue that the binding of EA is fav- Of late there has been growing evidence that alludes
oured at the ACE active site followed by captopril and to the fact that ETs are not orally bioavailable and
urolithin B-glucuronide. that they are hydrolysed to EA in the duodenum and
 INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION 7

finally metabolised by gut microbes to various uroli- grapefruit peels inhibit HMG-CoA reductase and angio-
thins. Urolithin conjugates are predominantly found tensin-I converting enzyme and show antioxidative prop-
in the plasma, tissue and urine, indicating that the erties in endothelial EA. Hy 926 cells. Food Sci Hum
Wellness. 4(2):80–85.
metabolites undergo phase II metabolism. Several Alexander W. 2014. Hypertension: is it time to replace
studies have suggested that these ET-derived metabo- drugs with nutrition and nutraceuticals? P T. 39(4):
lites are clinically beneficial for human vascular health 291–295.
(Spigoni et al. 2016; Istas et al. 2018; Andreux et al. Andreux PA, Blanco-Bose W, Ryu D, Burdet F, Ibberson
2019). Our current work is the first study that shows M, Aebischer P, Auwerx J, Singh A, Rinsch C. 2019. The
mitophagy activator urolithin A is safe and induces a
the in-silico interaction of geraniin and its metabolites
molecular signature of improved mitochondrial and cel-
with ACE enzyme, as well as the in vitro inhibition of lular health in humans. Nat Metab. 1(6):595–603.
ACE by these metabolites. While our results demon- Ascacio-Vald
es JA, Buenrostro-Figueroa JJ, Aguilera-Carbo
strate the inhibitory nature of EA against ACE, indi- A, Prado-Barrag
an A, Rodr
ıguez-Herrera R, Aguilar CN.
cating its potential anti-hypertensive role, it should be 2011. Ellagitannins: biosynthesis, biodegradation and bio-
noted that this may not truly reflect in vivo situations. logical properties. J. Med. Plants Res. 5(19):4696–4703.
Aviram M, Dornfeld L. 2001. Pomegranate juice consump-
An important fact to consider will be the poor oral tion inhibits serum angiotensin converting enzyme activ-
bioavailability of EA. It has been reported that a small ity and reduces systolic blood pressure. Atherosclerosis.
proportion of EA from EA-containing food occurs in 158(1):195–198.
the stomach while the basic conditions in the small Berkban T, Boonprom P, Bunbupha S, Welbat J,
intestine facilitates its further release and absorption Kukongviriyapan U, Kukongviriyapan V, Pakdeechote P,
Prachaney P. 2015. Ellagic acid prevents L-NAME-
(Kang et al. 2016). Gonz
alez-Sarr
ıas et al. (2015) in a
induced hypertension via restoration of eNOS and
crossover pharmacokinetic study with healthy volun- p47phox expression in rats. Nutrients. 7(7):5265–5280.
teers, indicated that EA bioavailability was limited by Cheng JT, Chang SS, Hsu FL. 1994. Antihypertensive action
the ellagitannin, pH and protein environment. They of geraniin in rats. J Pharm Pharmacol. 46(1):46–49.
mentioned that a higher free EA intake does not Cheng HS, Ton SH, Kadir KA. 2017. Ellagitannin geraniin:
enhance its bioavailability but promotes urolithin pro- a review of the natural sources, biosynthesis, pharmaco-
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the circulatory levels of EA and the urolithin conju- derivatives and their use for treatment of hypertension.
gates will provide valuable input. U.S. Patent 5,266,319.
In addition, the effect of EA and its derived metab- Elendran S, Muniyandy S, Lee WW, Palanisamy UD. 2019.
Permeability of the ellagitannin geraniin and its metabo-
olites on RAAS-mediated signalling messengers and
lites in a human colon adenocarcinoma Caco-2 cell cul-
the possible cardiovascular consequences will be a ture model. Food Funct. 10(2):602–615.
worthy study. Evaluation on protein crystallisation of Esp
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geraniin and/or geraniin-derived metabolites may also F. 2013. Biological significance of urolithins, the gut
add value to further understand how these com- microbial ellagic acid-derived metabolites: the evidence so
pounds interact with ACE. A limitation in this study, far. Evidence-Based Complement Altern Med. 2013:1–15.
Fang L, Geng M, Liu C, Wang J, Min W, Liu J. 2019.
is that the dose response results were not able to be
Structural and molecular basis of angiotensin-converting
presented as a logarithmic scale. enzyme by computational modeling: insights into the
mechanisms of different inhibitors. PLoS One. 14(4):
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Disclosure statement Garcia-Villalba R, Selma MV, Esp
ın JC, Tom
as-Barber
an
No potential conflict of interest was reported by FA. 2019. Identification of novel urolithin metabolites in
the author(s). human feces and urine after the intake of a pomegranate
extract. J Agric Food Chem. 67(40):11099–11107.
Gonz
alez-Sarr
ıas A, Garc
ıa-Villalba R, N
~ez-S
anchez MA,
un

ORCID Tom
e-Carneiro J, Zafrilla P, Mulero J, Tom
as-Barber
an
FA, Esp
ın JC. 2015. Identifying the limits for ellagic acid
Uma D. Palanisamy http://orcid.org/0000-0002-
bioavailability: a crossover pharmacokinetic study in
8615-8241
healthy volunteers after consumption of pomegranate
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