nutrients

Article
Cyperus esculentus L. and Tetracarpidium conophorum Müll.
Arg. Supplemented Diet Improved Testosterone Levels,
Modulated Ectonucleotidases and Adenosine Deaminase
Activities in Platelets from L-NAME-Stressed Rats
Ayodeji Augustine Olabiyi 1,2,3 , Vera Maria Morsch 3 , Ganiyu Oboh 2 and Maria Rosa Chitolina Schetinger 3, *

1 Functional Foods and Nutraceuticals Unit, Department of Medical Biochemistry, Afe Babalola University,
P.M.B. 5454, Ado Ekiti 360001, Nigeria; ayoaustin@mail.ufsm.br
2 Functional Foods and Nutraceuticals Unit, Department of Biochemistry, Federal University of Technology,
P.M.B. 704, Akure 340001, Nigeria; goboh2001@yahoo.com
3 Center of Natural and Exacts Sciences, Department of Biochemistry and Molecular Biology,
Federal University of Santa Maria, Santa Maria 97105-900, RS, Brazil; veramorsch@gmail.com
* Correspondence: mariachitolina@gmail.com

Abstract: In hypertensive individuals, platelet morphology and function have been discovered
to be altered, and this has been linked to the development of vascular disease, including erectile



dysfunction (ED). The impact of nutritional supplementation with Cyperus esculentus (tiger nut, TN)


and Tetracarpidium conophorum (walnut, WN) on androgen levels, ectonucleotidases, and adenosine
Citation: Olabiyi, A.A.; Morsch, deaminase (ADA) activities in platelets from L-NAME (Nω-nitro-L-arginine methyl ester hydrochlo-
V.M.; Oboh, G.; Schetinger, M.R.C. ride) challenged rats were investigated. We hypothesized that these nuts may show a protective effect
Cyperus esculentus L. and on platelets aggregation and possibly enhance the sex hormones, thereby reverting vasoconstriction.
Tetracarpidium conophorum Müll. Arg.
Wistar rats (male; 250–300 g; n = 10) were grouped into seven groups as follows: basal diet control
Supplemented Diet Improved
group (I); basal diet/L-NAME/Viagra (5 mg/kg/day) as positive control group (II); ED-induced
Testosterone Levels, Modulated
group (basal diet/L-NAME) (III); diet supplemented processed TN (20%)/L-NAME (IV); diet sup-
Ectonucleotidases and Adenosine
plemented raw TN (20%)/L-NAME (V); diet supplemented processed WN (20%)/L-NAME (VI); and
Deaminase Activities in Platelets
from L-NAME-Stressed Rats.
diet supplemented raw WN (20%)/L-NAME (VII). The rats were given their regular diet for 2 weeks
Nutrients 2021, 13, 3529. https:// prior to actually receiving L-NAME (40 mg/kg/day) for ten days to induce hypertension. Platelet
doi.org/10.3390/nu13103529 androgen levels, ectonucleotidases, and ADA were all measured. L-NAME considerably lowers
testosterone levels (54.5 ± 2.2; p < 0.05). Supplementing the TN and WN diets revealed improved
Academic Editor: Giorgio Ivan Russo testosterone levels as compared to the control (306.7 ± 5.7), but luteinizing hormone levels remained
unchanged. Compared to control groups, the L-NAME-treated group showed a rise in ATP (127.5%)
Received: 5 August 2021 hydrolysis and ADA (116.7%) activity, and also a decrease in ADP (76%) and AMP (45%) hydrolysis.
Accepted: 24 September 2021 Both TN and WN supplemented diets resulted in substantial (p < 0.05) reversal effects. Enhanced
Published: 8 October 2021
testosterone levels and modulation of the purinergic system in platelets by TN and WN could be one
of the mechanisms by which they aid in vasoconstriction control.
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
Keywords: TN; WN; L-NAME; erectile dysfunction; platelets; androgen; purinergic
published maps and institutional affil-
iations.

1. Introduction
Platelets are one of the most essential biological cell fragments in maintaining vascu-
Copyright: © 2021 by the authors.
lar integrity and facilitating main and secondary haemostasis following vessel injury [1].
Licensee MDPI, Basel, Switzerland.
This article is an open access article
Platelet formation and function have been found to be altered in hypertensive people,
distributed under the terms and
which may be linked to increased vascular disease [2,3]. In hypertension, enhanced platelet
conditions of the Creative Commons activation and aggregation has also been linked to cardiovascular changes [4]. Other
Attribution (CC BY) license (https:// studies [5,6], as well as studies by our group [7–9], have employed the nitric oxide syn-
creativecommons.org/licenses/by/ thase (NOS) inhibitor, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) as an
4.0/). experimental hypertension model. Due to the emergence and success of sildenafil citrate

Nutrients 2021, 13, 3529. https://doi.org/10.3390/nu13103529 https://www.mdpi.com/journal/nutrients

Nutrients 2021, 13, 3529 2 of 12

(Viagra) in 1998, the overall general information available regarding erectile dysfunction
(ED) has increased [10–12] over the past decades. Viagra (VG) operates by increasing nitric
oxide (NO) actions on vascular or corpus cavernosum, thereby causing smooth muscle
cell relaxation [10,11,13]. As a result of the therapeutic use of VG, the key role of NO
in mediating normal erection and in the aetiology of erectile dysfunction (ED) has been
highlighted. However, many side effects have been reported on the use of VG, therefore
consumers seeking complementary medicines and/or alternatives has provoked a great
interest in research into medicinal plant/food products which are cheaper and safer. ED
can have psychological, neurological, hormonal, vascular, cavernosal, or a mixed patho-
physiological basis [14]. Furthermore, platelets from hypertensive individuals have been
shown to have a higher proclivity for spontaneous aggregation and are extremely hypersen-
sitive to agonists like adenosine diphosphate (ADP) [15,16]. The hydrolysis of extracellular
nucleotides is carried out by a multi-enzymatic component on the surface of platelets. This
enzyme complex includes the enzymes ectonucleoside triphosphate phosphohydrolase
(E-NTPDase), ecto-50 -nucleotidase, and ecto-adenosine deaminase (ADA) [17]. ATP and
ADP are converted to AMP [18] by E-NTPDase, while AMP is converted to adenosine by
ecto-50 -nucleotidase [19]. The irreversible deamination of adenosine to inosine is catalysed
by adenosine deaminase (ADA) [20]. These ecto-enzymes constitute a well-organized
enzymatic series that controls the extracellular levels of adenine nucleotides and nucleo-
sides, which are critical in maintaining good haemostasis and thrombogenesis, principally
through controlling platelet aggregation [21]. In a number of studies [22,23], androgen
levels and substitution have been associated with sexual function, most notably erection
quality, desire, and ejaculatory function. Hypogonadism, however, has been linked to ED.
According to anatomic studies, androgen receptors are abundantly produced in the male
genital tract, the spinal nucleus, the medial preoptic nucleus of the hypothalamus, and the
bulbocavernosus muscle [24,25].
The tiger nut (TN) tuber is often referred to as Zulu nut, yellow nut grass, ground
almond, edible rush, and rush nut, and it belongs to the Cyperaceae grass family. The
amino acid arginine [26], which is a contributing factor to nitric oxide bioavailability, has
been found to be one of the components of tiger nut tuber. In addition, the nut has been
reported to be a natural therapy for treating inflammatory disorders like atherosclero-
sis [27], preventing heart attacks, and improving blood circulation [28]. Walnut (WN) is a
tropical tree in the Euphorbiaceae family [29]. It is widely distributed in Nigeria’s south-
ern region [29]. Walnut nuts have been used locally for decades, and they have several
nutritional and therapeutic benefits. Due to the polyphenolics that make up the active
ingredients in these nuts, TN and WN supplementation diet has been detected to improve
sexual behaviour [30], modulate biochemical indices related to erectile function [31,32], and
affect extracellular metabolism of ATP and adenosine via the NOS/cGMP/PKG signalling
pathway in the kidney [32]. Our hypothesis in this study is that these nuts will show a
protective effect on platelets aggregation and possibly enhance the sex hormones, thereby
reverting the effect of hypertension. Therefore, considering ethno-medicinal qualities of TN
and WN in the vascular system, as well as the importance of enzymes that degrade adenine
nucleotides in the mechanism of thromboregulation, the current study aims to look at the
activity of E-NTPDase, ecto-50 -nucleotidase, and ADA in platelets, as well as sex hormone
levels from L-NAME (Nω-nitro-L-arginine methyl ester hydrochloride)-stressed rats.

2. Materials and Methods

2.1. Chemicals and Reagents
Sigma-Aldrich, Inc. (St Louis, MO, USA) provided L-NAME, adenosine, Coomassie
brilliant blue G, Trizma base, sodium azide HEPES, nucleotides, and Percoll reagent, while
Reagen (Colombo, PR, Brazil) provided bovine serum albumin, K2 HPO4 . All of the other
chemical substances used in this experiment were of the purest quality, and the water was
distilled in glass.
Nutrients 2021, 13, 3529 3 of 12

2.2. Sample Preparation

TN and WN were purchased from the Nigerian city of Akure in May of 2016. Au-
thentication was conducted at the Department of Biology at the Federal University of
Technology in Akure, Nigeria. In order to remove stones and other dirt, the nuts were
carefully washed under running water. The edible component of the walnut was separated
from the inedible section, and the whole tiger nut was used. Following that, a fraction of
the tiger nut was roasted in an electric oven for about thirty minutes at 120 ◦ C (categorized
as tiger nut processed), which is the manner in which it is commonly consumed. The raw
tiger nut component was dried in an oven to a uniform weight at 45 ◦ C (categorized as raw
tiger nut), and the two samples were pulverized, defatted in cold n-hexane, and stored in
an airtight container until use. As is conventional, a portion of the walnut was also roasted
over an electric gas stove for 45 min at 100 ◦ C (shell removed after cooking). Both the raw
(uncooked) and cooked (shell removed) walnut fractions were dried in a vacuum oven to
a uniform weight at 45 ◦ C. Additionally, proximate analyses were performed on the nut
samples (unpublished data) to establish the nutritional composition of the nuts for feed
formulation relative to the amount of protein (Table 1).

Table 1. Feed formulation chat for basal, tiger nut (TN) and walnut (WN) dietary supplementation
for both control and test groups.

Groups
Materials I II III IV V VI VII
Skimmed milk 37.5 37.5 37.5 33.1 33.1 21.3 26.3
Oil 10.0 10.0 10.0 10.0 10.0 10.0 10.0
Vitamin mix. 4.0 4.0 4.0 4.0 4.0 4.0 4.0
Corn Starch 48.5 48.5 48.5 32.9 32.9 44.7 39.7
Tig 1 - - - 20.0 - - -
Tig 2 - - - - 20.0 - -
Wal 1 - - - - - 20.0 -
Wal 2 - - - - - - 20.0
VIAGRA - + - - - - -
L-NAME - + + + + + +
Total (g) 100 100 100 100 100 100 100
Wal 1: processed walnut; Wal 2: raw walnut, Tig 1: processed tiger nut; Tig 2: raw tiger nut. Skimmed milk
contains 32% protein. 1 g vitamin mixture Vitamin A: 3200 IU, 600 IU Vitamin D3, 2.8 mg vitamin E, 0.6 mg vitamin
K3, 0.8 mg vitamin B1, 1 mg vitamin B2, 6 mg niacin, 2.2 mg pantothenic acid, 0.8 mg vitamin B6, 0.004 mg vitamin
B12, 0.2 mg folic acid, 0.1 mg biotin H2, 70 mg choline chloride, 0.08 mg cobalt, 1.2 mg copper, 0.4 mg iodine,
8.4 mg iron, 16 mg manganese, 0.08 mg selenium, 12.4 mg zinc, and 0.5 mg antioxidant. Group I: Normal control
placed on a basal diet; Group II: Positive control placed on a basal diet/L-NAME/Viagra (5 mg/kg/day); Group
III: ED-induced group placed on a basal diet/L-NAME (40 mg/kg/day); Group IV: Processed TN supplemented
diet (20%)/L-NAME; Group V: Raw TN supplemented diet (20%)/L-NAME (20%); Group VI: Processed WN
supplemented diet (20%)/L-NAME; Group VII: Raw WN supplemented diet (20%)/L-NAME.

2.3. Animals
Wistar rats (male; 80–100 days; 250–300 g) were used in this experiment. The Central
Animal House provided all of the animals (Federal University of Santa Maria, Brazil). The
rats were kept at a constant temperature of 23 ± 1 ◦ C with a 12 h dark/light cycle and
were given unlimited food and drink. The rats were used in accordance with the rules
of the National Council for the Control of Animal Experiments (CONCEA) as well as
international standards. All experimental protocols were authorized by the Animal Ethics
Committee of the Federal University of Santa Maria, Brazil (Project Ethical Approved
number: CEUA 7019030816).

2.4. Design of Experiments

The rats were randomly separated into seven groups of ten animals after two weeks of
acclimatization (n = 10) as follows: basal diet control group (I); basal diet/L-NAME/Viagra
(5 mg/kg/day) as positive control group (II); ED-induced group (basal diet/L-NAME) (III);
diet supplemented processed TN (20%)/L-NAME (IV); diet supplemented raw TN (20%)/L-
Nutrients 2021, 13, 3529 4 of 12

NAME (V); diet supplemented processed WN (20%)/L-NAME (VI); and diet supplemented
raw WN (20%)/L-NAME (VII). The rats were fed their normal food for two weeks before
being given L-NAME to induce vasoconstriction for ten days. Throughout the experiment,
daily feed intake was monitored. Gavage route of administration was used to produce
ED with L-NAME (40 mg/kg/day) [8,17]. In order to expose the animals in the normal
control group (I) to the same stress as the others, they were given water via gavage during
the experiment. These rats were euthanized 24 h after the last treatment session, and sex
hormone levels (testosterone and luteinizing) were measured using commercially available
kits (Randox Laboratories Ltd.—Crumlin, Dublin, Northern Ireland, UK) in accordance
with the manufacturer’s instructions. Additionally, relevant biochemical parameters were
evaluated and analysed.

2.5. Hormonal Assays

Serum testosterone and luteinizing (LH) hormone levels were measured using an
enzyme-linked immunosorbent assay (ELISA) kit. The testosterone equine testosterone
antigen (ELA) was based on the principle of competitive binding between testosterone
in the test specimen and testosterone-horseradish peroxidase (HRP) conjugate for a con-
stant amount of anti-testosterone. For the incubation, anti-testosterone-coated wells were
incubated with 25 µL of testosterone standards, control, samples, and 100 µL testosterone-
HRP conjugate reagent at room temperature for 60 min. During the incubation, a fixed
number of wells were HRP-labelled. Testosterone hydrogen-filled molecular graph (HFG)
competes with the endogenous testosterone in the standard, sample, or quality control
serum for a fixed number of binding sites of the specific testosterone antibody. Thus, the
amount of testosterone peroxidase conjugate immunologically bound to the well progres-
sively decreases as the concentration of testosterone in the specimen increases. Unbound
testosterone peroxidase conjugate was then removed, and the wells were washed. Next, a
solution of 3,30 ,5,50 -tetramethylbenzidine (TMB) reagent was then added and incubated at
room temperature for 15 min, resulting in the development of a blue colour. The colour
development was stopped with the addition of a stop solution, and the absorbance was
measured using a spectrophotometer at 450 nm. Additionally, for the LH hormonal assay,
sixteen 12 × 75 mm disposable plastic test tubes were labelled for the standards. Two tubes
were used for each sample. Blank reagent (300 µL), standards (200 µL), controls (200 µL),
and clinical samples (200 µL) were added to the sample bottles. One hundred microlitres
of LH antiserum were added to the tubes and vortexed. The tubes were incubated for
30 min at room temperature. One millilitre of precipitating reagent was added to all sample
tubes, and the sample tubes were then vortexed and centrifuged at 1500× g for 15 min.
The supernatant was carefully decanted from all tubes, except tubes 1 and 2, immediately
after centrifugation, inverting the tubes gently to avoid disturbing the precipitate. Then,
the supernatant was discarded properly. The radioactivity in the pellets and tubes 1 and 2
was measured in a gamma counter for 1 min to obtain at least 40,000 counts for (57Co) and
75,000 counts for (1251) in the total count tubes. The total counts in tubes 1 and 2 depended
upon the efficiency of the scintillation counter in use and the age of the tracer.

2.6. Platelet Preparation

The following minor modifications were made to the method used by Lunkes et al. [33]
for preparing platelet-rich plasma (PRP). Whole blood was collected using a cardiac punc-
ture and 0.120 M sodium citrate was used as an anticoagulant. The blood–citrate system
was centrifuged at 1600× g for 15 min. PRP was centrifuged at 14,000× g for 30 min before
being cleaned twice in 3.5 mM HEPES buffer, pH 7.0, comprising 142 mM NaCl, 2.5 mM
KCl, and 5.5 mM glucose. The enzymatic activities of platelet pellets were measured after
they were re-suspended in HEPES buffer.
Nutrients 2021, 13, 3529 5 of 12

2.7. Determination of E-NTPDase and Ecto-50 -Nucleotidase Activities

The platelets were enzymatically tested for E-NTPDase (EC 3.6.1.5) in a reaction
medium comprising 5 mM KCl, 1.5 mM CaCl2, 0.1 mM EDTA, 10 mM glucose, 225 mM
sucrose, and 45 mM Tris–HCl buffer, pH8.0, as described in a previous study [34]. The
reaction mixture was added to 20 microliters of enzyme preparation (8–12 g protein) and
incubated for 10 min at 37 ◦ C. In either case, the reaction was started by adding 1.0 mM
ATP or ADP to a final concentration of 1.0 mM and incubating for 20 min. In a reaction
medium containing 10 mM MgSO4 and 100 mM Tris–HCl buffer, pH7.5, in a final volume
of 200 µL, the activity of 5’-nucleotidase (EC 3.1.3.5) was evaluated following the method
of Heymann et al. [35]. The reaction mixture was pre-incubated at 37 ◦ C for 10 min with
20 microliters of enzyme preparation (8–12 g of protein). The reaction was started by
adding AMP to a final concentration of 2.0 mM, and it took 20 min to complete. In both
cases, the reaction was stopped by adding 200 µL of 10% trichloroacetic acid to achieve a
final concentration of 5%. The tubes were then refrigerated for 10 min on ice. This method
was followed by Chan et al. [36], who used malachite green as a colorimetric reagent and
KH2 PO4 as a standard to evaluate the amount of liberated inorganic phosphate (Pi).
The synaptosomal fraction was added after the trichloroacetic acid addition to adjust for
non-enzymatic nucleotide hydrolysis. Enzyme activities were measured in nanomoles of
Pi released per minute per milligram of protein for each sample examined in triplicate.

2.8. Adenosine Deaminase Activity Determination

Guisti and Galanti [37] established a method for determining ADA activity that
is based on the reliable detection of ammonia production when ADA acts in excess of
adenosine. In brief, 50 µL of enzyme preparation was incubated with 21 mmol/L of
adenosine at pH 6.5 for 60 min at 37 ◦ C. In the experiment, the protein concentration was
set to be between 0.7 and 0.9 mg/mL. Units per litre (U/L) were used to calculate the
results. One unit (1 U) of ADA is defined as the quantity of enzyme necessary to release 1
mmol of ammonia per minute from adenosine under normal test conditions.

2.9. Protein Determination

Bradford’s Coomassie blue technique [38] and serum albumin as a standard were
used to determine protein levels.

2.10. Statistical Evaluations

One-way ANOVA was performed first, followed by Tukey’s multiple range tests; a
significant difference was defined as p < 0.05 in both analyses. All data were provided as a
mean ± standard error of mean (SEM). The statistical studies were performed using the
GraphPad Prism 5 software tool.

3. Results
3.1. Feed Intake Measurement and the Impact of TN and WN Supplementation in
L-NAME-Stressed Rats
After gavage administration of L-NAME (40 mg/kg/day), no significant (p > 0.05)
changes in body weight or feed intake were found among the test groups (data not published).

3.2. Impact of TN and WN Supplementation on the Activity of EC 3.6.1.5, EC 3.1.3.5, and ADA
in the Platelets of L-NAME-Stressed Rats
In the L-NAME-treated group, the results for E-NTPDase (EC 3.6.1.5; ATP and ADP, as
substrates) and ecto-50 -nucleotidase (EC 3.1.3.5) activities in platelets revealed an increase
in ATP (127.5%; p < 0.01) hydrolysis with a reduction in ADP (72%; p < 0.05) and AMP (68%;
p < 0.05) hydrolysis when compared to the normal and positive control groups. However,
it is interesting to note that both TN (raw = 78%; processed = 80%) and WN (raw = 79.8%;
processed = 75%) supplemented diets caused a decrease in E-NTPDase using ATP as
substrate (F(4,49) = 2.840; p < 0.01). Moreover, a non-significant increase in the hydrolysis
 In the L-NAME-treated group, the results for E-NTPDase (EC 3.6.1.5; ATP and ADP,
as substrates) and ecto-5’-nucleotidase (EC 3.1.3.5) activities in platelets revealed an in-
crease in ATP (127.5%; p < 0.01) hydrolysis with a reduction in ADP (72%; p < 0.05) and
AMP (68%; p < 0.05) hydrolysis when compared to the normal and positive control groups.
Nutrients 2021, 13, 3529 However, it is interesting to note that both TN (raw = 78%; processed = 80%) and WN6(raw of 12
= 79.8%; processed = 75%) supplemented diets caused a decrease in E-NTPDase using ATP
as substrate (F(4,49) = 2.840; p < 0.01). Moreover, a non-significant increase in the hydrol-
ysis of platelet’s ADP (F(4,49) = 0.8351; p < 0.05) for TN (raw = 68%; processed = 66.4%)
of
andplatelet’s
WN (raw ADP (F(4,49)
= 69.1%; = 0.8351;=p67.3%)
processed < 0.05)and
for TN (raw = 68%;
a significant processed
decrease in AMP= 66.4%) and=
(F(4,49)
WN (raw = 69.1%; processed = 67.3%) and a significant decrease in AMP (F(4,49)
4.759; p < 0.05], for TN (raw = 67%; processed = 65%) and WN (raw = 66%; processed = = 4.759;
p68%)
< 0.05],
werefor TN (raw(Figures
observed. = 67%; 13).
processed = 65%) the
Furthermore, andL-NAME-treated
WN (raw = 66%;group
processed = 68%)
(116.7%) had
were observed. (Figures 1–3). Furthermore, the L-NAME-treated group
higher ADA activity (p < 0.001) than the control (66.7%) group, whereas the TN (raw (116.7%) had=
higher ADA activity
67%; processed (p < 0.001)
= 66.8%) and WN than (raw
the control (66.7%)
= 69.3%; group,=whereas
processed the TN (raw = 67%;
71.2%) supplemented diet
processed = 66.8%) and WN (raw = 69.3%; processed = 71.2%) supplemented diet
caused a significant (p < 0.05) reduction in ADA activity (F(4,49) = 3.835; p < 0.001) com- caused
apared
significant (p < 0.05)
to controls reduction
(Figure 4). in ADA activity (F(4,49) = 3.835; p < 0.001) compared to
controls (Figure 4).

ATP
60
*
ηmol Pi/ mg protein/ min

#
40

20

0
I II III IV V VI VII
Groups
Figure 1.
Figure 1. E-NTPDase
E-NTPDase (ATP(ATP as
as substrate)
substrate) activity
activity in
in platelets
platelets of
of L-NAME-stressed
L-NAME-stressed rats
rats treated
treated with
with
tiger nut and walnut supplemented diets. Data are presented as mean ± SEM (n
tiger nut and walnut supplemented diets. Data are presented as mean ± SEM (n = 10). The = 10). The bars with
various symbols are statistically different (p < 0.05). * represents a significant difference from the
bars with various symbols are statistically different (p < 0.05). * represents a significant difference
control (I) group. # represents a significant difference from the ED (III) group (one-way ANOVA
from the control (I) group. # represents a significant difference from the ED (III) group (one-way
followed by post hoc Tukey, *, # denote p < 0.05, Key: Group I: Normal control placed on a basal
ANOVA followed
diet; Group by post
II: Positive hoc Tukey,
control placed *,on# adenote p < 0.05, Key: Group (5
basal diet/L-NAME/Viagra I: Normal control
mg/kg/day); placed
Group III:
on a basal diet; Group II: Positive control placed on a basal diet/L-NAME/Viagra (5 mg/kg/day);
Group III: ED-induced group placed on a basal diet/L-NAME (40 mg/kg/day); Group IV: Processed
TN supplemented diet (20%)/L-NAME; Group V: Raw TN supplemented diet (20%)/L-NAME (20%);
Group VI: Processed WN supplemented diet (20%)/L-NAME; Group VII: Raw WN supplemented
diet (20%)/L-NAME.

3.3. Impact of TN and WN Supplementation on Testosterone and Luteinizing Hormone (LH)

Levels in L-NAME-Stressed Rats
Result from this study showed that L-NAME reduces testosterone levels (54.5 ± 2.2)
significantly (p < 0.05). However, supplementation with TN (raw = 118.2 ± 9.3;
processed = 88.7 ± 0.7) and WN (raw = 123.8 ± 5.7; processed = 145 ± 6.0) was shown to
increase testosterone levels in the serum as compared to the control (306.7 ± 5.7), while LH
levels were not altered across the groups, as shown in Table 2.
 Nutrients
Nutrients 2021,
2021, 13,
13, xx FOR
FOR PEER
PEER REVIEW
REVIEW 77 of
of 12
12

Nutrients 2021, 13, 3529 ED-induced

ED-induced group
group placed
placed on
on aa basal
basal diet/L-NAME
diet/L-NAME (40 (40 mg/kg/day);
mg/kg/day); Group
Group IV:
IV: Processed
Processed TN
TN sup-
sup- 7 of 12
plemented
plemented dietdiet (20%)/L-NAME;
(20%)/L-NAME; Group
Group V: V: Raw
Raw TNTN supplemented
supplemented diet
diet (20%)/L-NAME
(20%)/L-NAME (20%);
(20%);
Group
Group VI:VI: Processed
Processed WNWN supplemented
supplemented dietdiet (20%)/L-NAME;
(20%)/L-NAME; Group
Group VII:
VII: Raw
Raw WNWN supplemented
supplemented
diet
diet (20%)/L-NAME.
(20%)/L-NAME.

ADP
25
25

ηmol Pi/ mg protein/ min

20
20

15
15

10
10

5
5

0
0
II II
II III
III IV
IV V
V VI
VI VII
VII
Groups
Groups
Figure
Figure 2.2. NTPDase (ADP
(ADP as as substrate)
substrate) activity
activity in in platelets
platelets of
of L-NAME-stressed
L-NAME-stressed rats rats treated
treated with
Figure 2.NTPDase
NTPDase (ADP as substrate) activity in platelets of L-NAME-stressed with
rats treated with
tiger
tiger nut
nut and
and walnut
walnut supplemented
supplemented diets. diets. Data
Data are
are presented
presented as as mean
mean ±± SEM
SEM (n (n == 10).
10). The
The bars
bars with
with
tiger
various nut and
symbols walnut
are supplemented
statistically different (pdiets.
< Data
0.05). * are presented
represents a as
significant mean
difference±
various symbols are statistically different (p < 0.05). * represents a significant difference from the= 10). The barsSEM
from (n
the
control
with (I)
(I) group.
controlvariousgroup. ## represents
symbols aa significant
significant difference
are statistically
represents differentfrom
difference (p <the
from ED
0.05).
the ED (III)
(III) group
Key: Group
group (one-way ANOVA
I: Normal
(one-way ANOVA control placed on
followed
followed by
by post
post hoc
hoc Tukey,
Tukey, *,*, ## denote
denote pp << 0.05;
0.05; Key:
Key: Group
Group I:I: Normal
Normal control
control placed
placed onon aa basal
basal
a basal diet; Group II: Positive control placed on a basal diet/L-NAME/Viagra (5 mg/kg/day);
diet;
diet; Group
Group II: II: Positive
Positive control
control placed
placed on
on aa basal
basal diet/L-NAME/Viagra
diet/L-NAME/Viagra (5 (5 mg/kg/day);
mg/kg/day); Group Group III:III:
Group
ED-induced
ED-induced III: group
ED-induced
group placed
placed ongroup
on aa basalplaced
basal on a basal
diet/L-NAME
diet/L-NAME (40diet/L-NAME
(40 mg/kg/day);
mg/kg/day); Group (40 IV:
Group mg/kg/day);
IV: Processed
Processed TN TN Group
sup- IV: Processed
sup-
TN supplemented
plemented
plemented diet diet (20%)/L-NAME;
diet (20%)/L-NAME;
(20%)/L-NAME; Group
Group V: V: RawGroup
Raw TN V:
TN Raw TN supplemented
supplemented
supplemented diet
diet (20%)/L-NAME
(20%)/L-NAME diet (20%)/L-NAME
(20%);
(20%); (20%);
Group
Group VI:
Group VI:
VI:Processed
Processed
Processed WN
WNWN supplemented
supplemented
supplemented diet
diet (20%)/L-NAME;
(20%)/L-NAME;
diet (20%)/L-NAME;Group
Group VII: Raw
Raw WN
VII:Group WN supplemented
supplemented
VII: Raw WN supplemented
diet
diet (20%)/L-NAME.
(20%)/L-NAME.
diet (20%)/L-NAME.

Figure 3. 50 -nucleotidase (AMP as substrate) activity in platelets of L-NAME-stressed rats treated

with tiger nut and walnut supplemented diets. Data are presented as mean ± SEM (n = 10). The
bars with various symbols are statistically different (p < 0.05). * represents a significant difference
from the control (I) group. # represents a significant difference from the ED (III) group (one-way
ANOVA followed by post hoc Tukey, *, # denote p < 0.05; Key: Group I: Normal control placed on a
basal diet; Group II: Positive control placed on a basal diet/L-NAME/Viagra (5 mg/kg/day); Group
III: ED-induced group placed on a basal diet/L-NAME (40 mg/kg/day); Group IV: Processed TN
supplemented diet (20%)/L-NAME; Group V: Raw TN supplemented diet (20%)/L-NAME (20%);
Group VI: Processed WN supplemented diet (20%)/L-NAME; Group VII: Raw WN supplemented
diet (20%)/L-NAME.
 with various symbols are statistically different (p < 0.05). * represents a significant difference from
the control (I) group. # represents a significant difference from the ED (III) group (one-way ANOVA
followed by post hoc Tukey, *, # denote p < 0.05; Key: Group I: Normal control placed on a basal
diet; Group II: Positive control placed on a basal diet/L-NAME/Viagra (5 mg/kg/day); Group III:
ED-induced group placed on a basal diet/L-NAME (40 mg/kg/day); Group IV: Processed TN sup-
Nutrients 2021, 13, 3529 plemented diet (20%)/L-NAME; Group V: Raw TN supplemented diet (20%)/L-NAME (20%); 8 of 12
Group VI: Processed WN supplemented diet (20%)/L-NAME; Group VII: Raw WN supplemented
diet (20%)/L-NAME.

40
*

(Units/mg protein)
30 # #

ADA activity
20

10

0
I II III IV V VI VII
Groups
Figure 4. Adenosine deaminase (ADA) activity in platelets of L-NAME-stressed rats treated with
Figure
tiger nut 4.
andAdenosine deaminasediets.
walnut supplemented (ADA)Dataactivity in platelets
are presented as meanof±L-NAME-stressed rats
SEM (n = 10). The bars treated with
with
tiger nut
various and walnut
symbols supplemented
are statistically differentdiets. Data* represents
(p < 0.05). are presented as mean
a significant ± SEM from
difference (n = 10).
the The bars
control (I) group.
with various # represents
symbols a significantdifferent
are statistically difference(pfrom the ED
< 0.05). (III) groupa(one-way
* represents ANOVA
significant difference from
followed
the controlby post hoc Tukey,
(I) group. *, # denote
# represents p < 0.05; Key:
a significant Group I:from
difference Normalthe control
ED (III)placed
groupon(one-way
a basal ANOVA
diet; Group II: Positive control placed on a basal diet/L-NAME/Viagra (5 mg/kg/day); Group III:
followed by post hoc Tukey, *, # denote p < 0.05; Key: Group I: Normal control placed on a basal
ED-induced group placed on a basal diet/L-NAME (40 mg/kg/day); Group IV: Processed TN sup-
diet; Group
plemented dietII:(20%)/L-NAME;
Positive control placed
Group V: on
Raw a basal diet/L-NAME/Viagra
TN supplemented (5 mg/kg/day);
diet (20%)/L-NAME (20%); Group
III: ED-induced
Group VI: Processed group
WN placed on a basal
supplemented diet/L-NAME Group
diet (20%)/L-NAME; (40 mg/kg/day);
VII: Raw WNGroup IV: Processed TN
supplemented
diet (20%)/L-NAME.
supplemented diet (20%)/L-NAME; Group V: Raw TN supplemented diet (20%)/L-NAME (20%);
Group VI: Processed WN supplemented diet (20%)/L-NAME; Group VII: Raw WN supplemented
3.3. Impact of TN and WN Supplementation on Testosterone and Luteinizing Hormone (LH)
diet (20%)/L-NAME.
Levels in L-NAME-Stressed Rats
Result
Table from this study
2. Testosterone showed that
and luteinizing L-NAME
hormone reduces
levels fromtestosterone levels (54.5
L-NAME-stressed ± 2.2) with TN
rats treated
significantly (p < 0.05). However, supplementation with TN (raw = 118.2 ± 9.3; processed
and WN supplemented diets.
= 88.7 ± 0.7) and WN (raw = 123.8 ± 5.7; processed = 145 ± 6.0) was shown to increase
testosterone levels in the serum as compared
Testosterone to the control (306.7 ± 5.7),
Hormone while LHHormone
Luteinizing levels
were notGroups
altered across the groups, as shown in Table 2.
(ng/dL) (mIU/mL)
Table 2. Testosterone
I and luteinizing hormone 5.7 a from L-NAME-stressed rats treated
306.7 ±levels 0.1 a TN
28.5 ± with
and WN supplemented
II diets. 102.4 ± 7.4 d 27.8 ± 0.9 a
III 54.5 ± 2.2 f Hormone
Testosterone Luteinizing27.1 ± 0.2
Hormone
Groups
IV 88.7 ± 0.7 e 27.5 ± 0.6 a
(ng/dL) (mIU/mL)
V 118.2 ± 9.3 c a 29.2 ± 0.7 a
I 306.7 ± 5.7 28.5 ± 0.1 a
VI 145.5 ± 6.0 b 29.8 ± 0.4 a
VII 123.8 ± 5.7 c 29.5 ± 0.3 a
The results are presented (mean ± SD) in triplicates. Different letters indicate a significant difference taken into
account by Tukey post hoc test at p < 0.05. Group I: Normal control placed on a basal diet; Group II: Positive
control placed on a basal diet/L-NAME/Viagra (5 mg/kg/day); Group III: ED-induced group placed on a basal
diet/L-NAME (40 mg/kg/day); Group IV: Processed TN supplemented diet (20%)/L-NAME; Group V: Raw
TN supplemented diet (20%)/L-NAME (20%); Group VI: Processed WN supplemented diet (20%)/L-NAME;
Group VII: Raw WN supplemented diet (20%)/L-NAME.

4. Discussion
This study’s hypothesis was accepted owing to the observed results. The pathophys-
iology of several disorders, including strokes, venous thrombosis, arterial thrombosis,
and myocardial infarction, has been linked to platelet dysfunction [39]. The release of
vasoconstrictor substances and reactive oxygen species by platelet and leucocyte adhe-
sion has been demonstrated to have a negative impact on erection, which may play a
role in the progression of ED [40]. In hypertensive patients, the aetiopathogenesis of ED
is complex, influenced by a range of psychological and biological factors. Among the
Nutrients 2021, 13, 3529 9 of 12

organic causes are diabetic neuropathy, oxidative stress, dyslipidaemia, arterial hyper-
tension, endothelial dysfunction, hypogonadism, and pharmaceutical side effects [41,42].
Platelets are also thought to have a function in atherothrombosis pathogenesis [43]. In
comparison to L-NAME-treated and normal control rats, results obtained from the activity
of E-NTPDase demonstrated a modulatory influence of TN and WN on platelet activity.
Hypertension, according to Lunkes et al. [44], is a contributing factor for thrombus devel-
opment. E-NTPDase activity in blood platelets has been found to change in a variety of
physiological and pathological situations, implying that it is involved in thromboregula-
tion [45,46]. As a result, it is believed that both TN and WN contain polyphenolics [9,31],
which aid in thromboregulation. Our findings, which revealed that L-NAME increased
adenosine deaminase activity, while TN and WN reversed this effect when compared to the
control, also demonstrated the influence of adenosine. This suggests that ADA’s decrease
with TN and WN supplementation will improve adenosine bioavailability, which is consis-
tent with studies showing that adenosine prevented platelet aggregation by activating the
A2A receptor on platelets and caused vasodilation in smooth muscle cells via the A2B re-
ceptor [47,48]. Nitric oxide is a vasodilator that inhibits platelet activity by increasing cyclic
guanosine monophosphate (cGMP) levels and thereby restricting vasoconstriction [49,50].
Nitric oxide may affect platelet responses by stimulating soluble guanylate cyclase, which
catalyses the production of the secondary messenger cGMP [51]. Platelet cGMP levels
rise, causing a change in intracellular Ca2+ mobilization that favours smooth muscle relax-
ation [52–54]. Both adenosine and NO have been found to be powerful vasodilators and
have been linked to erectile dysfunction [9,13]. We determined that L-NAME induced a
drop in ADA activity across groups, which is consistent with what we found in our lab.
TN and WN, on the other hand, were able to counteract the effects of L-NAME. Thousands
of research papers on testosterone and its biological consequences have been published
lately, with more than a third of them published in the last decade. One important factor
fuelling this growing scientific interest is testosterone’s impact on human health, particu-
larly in men. Not only is it a basic regulator of male reproduction and the maturation of
external genital features [55], but testosterone has also been related to men’s well-being
and overall health [56]. According to our findings in this experiment, L-NAME induced
a reduction in serum testosterone levels, which agrees with the findings of numerous re-
ports in which low testosterone levels were linked to diabetes [57,58], Alzheimer’s [59,60],
atherosclerosis [61,62], cancer [63], osteoporosis [64,65], and infertility [66,67], which are
common characteristics of aging disorders/diseases. However, no toxicological effect of
the supplemented diet was observed in the experimented rats. The mechanism by which
testosterone secretion increases without parallel increase in the secretion of LH is still
unknown. Therefore, further efforts are ongoing to determine if there is a direct effect of
TN and WN on Leydig cells which bypasses the hypothalamic-pituitary (HP) axis.

5. Conclusions
According to our findings, TN and WN improved testosterone levels and modulated
the activities of ectonucleotidases in platelets of L-NAME-stressed rats as a model of
hypertension. As a result, testosterone boosting and the modulation of the E-NTPDase as
well as ADA in the pathogenesis of hypertension may represent mechanisms by which
TN and WN contribute to erectile function. However, the mechanism of action by which
testosterone secretion increased without a corresponding increase in the secretion of LH is
still being investigated. The potential attributes of these nuts can be said to be provided
by the presence of bioactive components (polyphenolics) in TN and WN which has been
documented from our laboratory [9,32].
Nutrients 2021, 13, 3529 10 of 12

Author Contributions: A.A.O.: conceptualization, methodology, funding acquisition, data cura-

tion, writing—original draft preparation. G.O.: conceptualization, visualization, and investiga-
tion. M.R.C.S.: supervision and writing—reviewing and editing. V.M.M.: validation and writing—
reviewing and editing. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by CAPES-PrInt project number- 88881310287/2018-01.
Institutional Review Board Statement: The rats were used in accordance with the rules of the Na-
tional Council for the Control of Animal Experiments (CONCEA) as well as international standards.
All experimental protocols were approved by the Animal Ethics Committee of the Federal University
of Santa Maria, Brazil (Project Ethical Approved number: CEUA 7019030816).
Informed Consent Statement: Not applicable.
Acknowledgments: Ayodeji Augustine Olabiyi (FR number: 3240286543), wish to thank the Con-
selho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Fundação de Amparo à
Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Fundação Coordenação de Aperfeiçoamento
de Pessoal de Nvel Superior (CAPES-PrInt project number-88881310287/2018-01), and the Academy
of Sciences for the Developing World (TWAS) for funding this research.
Conflicts of Interest: There is no conflict of interest among the authors.

Abbreviations

ATP adenosine triphosphate

ADP adenosine diphosphate
AMP adenosine monophosphate
ADA adenosine deaminase
E-NTPdase ectonucleotidases
ED erectile dysfunction
L-NAME Nω-nitro-L-arginine methyl ester hydrochloride
NOS nitric oxide synthase
SIL sildenafil
TN tiger nut
WN walnut

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