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Chemotherapeutic Potential of Epimedium brevicornum Extract: The cGMP-Specific PDE5 Inhibitor as Anti-Infertility Agent Following Long-Term Administration of Tramadol in Male Rats

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Epimedium brevicornum Maxim (EbM) is a well-known Chinese herb that has been widely used for the treatment of several diseases. The main purpose of this study is to examine the role of Epimedium brevicornum extract in certain andrological parameters in rats as a natural modulator for adverse viewpoints associated with chronic administration of tramadol (TAM). Fifty rats were categorized into five groups. Untreated rats were known as Group I, whereas rats in Groups II and III were administered 2.43 g/kg/day of E. brevicornum extract and 50 mg/kg/day of TAM for 130 consecutive days, respectively. Both of Groups IV and V were administered TAM for 65 successive days, followed by concomitant use of both drugs for another 65 days, with the E. brevicornum extract at doses of 0.81 and 2.43 g/kg/day, respectively. TAM showed an injurious effect on sperm attributes, serum hormones, tissue malondialdehyde, superoxide dismutase, and nitric oxide. Elevation of the apoptotic marker Bax and a reduction of Bcl2 were recorded. Histopathological abnormalities have been reported in rat testicles. Rats treated with E. brevicornum extract with TAM showed an improvement in all the parameters tested. It could be presumed that E. brevicornum extract plus TAM exhibits a promising effect on the enhancement of male anti-infertility effects.
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antibiotics
Article
Chemotherapeutic Potential of
Epimedium brevicornum Extract: The cGMP-Specific
PDE5 Inhibitor as Anti-Infertility Agent Following
Long-Term Administration of Tramadol in Male Rats
Ahmed S. Abdelaziz 1, * , Mohamed A. Kamel 1, Amany I. Ahmed 2, Shimaa I. Shalaby 3,
Salama M. El-darier 4, Amany Magdy Beshbishy 5, Gaber El-Saber Batiha 6, *,
Suliman Y. Alomar 7,* and Dina M. Khodeer 8
1Pharmacology Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44519, Egypt;
makamel@zu.edu.eg
2Biochemistry Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44519, Egypt;
aialsayed@zu.edu.eg
3Physiology Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44519, Egypt;
Siabdallah@zu.edu.eg
4Botany and Microbiology Department, Faculty of Science, Alexandria University, Alexandria 21568, Egypt;
salama.eldarir@alexu.edu.eg
5National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary
Medicine, Nishi 2-13, Inada-cho, Obihiro 080-8555, Hokkaido, Japan; amanimagdi2008@gmail.com
6Department of Pharmacology and Therapeutics, Faculty of Veterinary Medicine, Damanhour University,
Damanhour 22511, Al Beheira, Egypt
7Doping Research Chair, Department of Zoology, College of Science, King Saud University, Riyadh 11495,
Saudi Arabia
8Department of Pharmacology and Toxicology, Faculty of Pharmacy, Suez Canal University, Ismailia 41522,
Egypt; dina_khoudaer@pharm.suez.edu.eg
*Correspondence: asabdelaziz@vet.zu.edu.eg (A.S.A.); dr_gaber_batiha@vetmed.dmu.edu.eg (G.E.-S.B.);
syalomar@ksu.edu.sa (S.Y.A.); Tel.: +20-45-271-6024 (G.E.-S.B.); Fax: +20-45-271-6024 (G.E.-S.B.)
Received: 3 May 2020; Accepted: 5 June 2020; Published: 11 June 2020


Abstract:
Epimedium brevicornum Maxim (EbM) is a well-known Chinese herb that has been widely
used for the treatment of several diseases. The main purpose of this study is to examine the role
of Epimedium brevicornum extract in certain andrological parameters in rats as a natural modulator
for adverse viewpoints associated with chronic administration of tramadol (TAM). Fifty rats were
categorized into five groups. Untreated rats were known as Group I, whereas rats in Groups II
and III were administered 2.43 g/kg/day of E. brevicornum extract and 50 mg/kg/day of TAM for
130 consecutive days, respectively. Both of Groups IV and V were administered TAM for 65 successive
days, followed by concomitant use of both drugs for another 65 days, with the E. brevicornum extract
at doses of 0.81 and 2.43 g/kg/day, respectively. TAM showed an injurious eect on sperm attributes,
serum hormones, tissue malondialdehyde, superoxide dismutase, and nitric oxide. Elevation of
the apoptotic marker Bax and a reduction of Bcl2 were recorded. Histopathological abnormalities
have been reported in rat testicles. Rats treated with E. brevicornum extract with TAM showed
an improvement in all the parameters tested. It could be presumed that E. brevicornum extract plus
TAM exhibits a promising eect on the enhancement of male anti-infertility eects.
Keywords:
Epimedium brevicornum Maxim; tramadol; male fertility; andrological parameters;
nitric oxide
Antibiotics 2020,9, 318; doi:10.3390/antibiotics9060318 www.mdpi.com/journal/antibiotics
Antibiotics 2020,9, 318 2 of 15
1. Introduction
Tramadol hydrochloride (TAM; Figure 1) is classified as a centralized pain reliever agent, used
mainly to treat moderate and severe pain [
1
]. Tramadol was also found to be obtained through opioid
and nonopioid mechanisms through sedative/analgesic action [
2
]. Ecient TAM is a racemic mixture of
two enantiomers containing two well-defined and harmonious mechanisms: the (+) TAM is a
µ
-opioid
selective agonist that inhibits serotonin reuptake to a higher degree and stimulates serotonin eux in
the brain, while the (-) TAM enantiomorph inhibits the reuptake of noradrenaline [
3
]. Considering the
fact of being opiate-related, TAM carries all possible risks from other opiates and shows a wide range
of side eects, including dizziness, headache, drowsiness, vomiting attempts, bowel disease, sweating,
pruritis, and CNS stimulation. These side eects are similar to opioids due to the anity of tramadol
to the micro-opioid receptor [4].
Antibiotics 2020, 9, x FOR PEER REVIEW 2 of 15
1. Introduction
Tramadol hydrochloride (TAM; Figure 1) is classified as a centralized pain reliever agent, used
mainly to treat moderate and severe pain [1]. Tramadol was also found to be obtained through opioid
and nonopioid mechanisms through sedative/analgesic action [2]. Efficient TAM is a racemic mixture
of two enantiomers containing two well-defined and harmonious mechanisms: the (+) TAM is a µ-
opioid selective agonist that inhibits serotonin reuptake to a higher degree and stimulates serotonin
efflux in the brain, while the () TAM enantiomorph inhibits the reuptake of noradrenaline [3].
Considering the fact of being opiate-related, TAM carries all possible risks from other opiates and
shows a wide range of side effects, including dizziness, headache, drowsiness, vomiting attempts,
bowel disease, sweating, pruritis, and CNS stimulation. These side effects are similar to opioids due
to the affinity of tramadol to the micro-opioid receptor [4].
Figure 1. Chemical structure of tramadol hydrochloride.
TAM has been reported to cause respiratory depression and psychological and physiological
addiction [5]. Moreover, long-term administration of TAM results in an increase in oxidative stress
that causes male infertility due to low levels of testosterone and neurodegenerative diseases such as
Alzheimer’s disease [6,7]. There is a disturbing increase in the abuse of TAM, mainly among young
people assuming that it improves sexual excitement and climax without the development of sexual
dysfunctions [8], and small quantities for additional investigations and examinations have been
implanted [9]. There is an urgent need for safer natural antioxidants to minimize the effects of
oxidative and peroxidative damage of TAM on male infertility. Such natural antioxidants can prevent
the issues associated with synthetic drugs, such as various side effects, treatment disappointment,
and drug resistance.
Epimedium is a plant category of the Berberidaceae family, containing 63 plant species. Epimedium
brevicornum Maxim (EbM) is a well-known Chinese herb that is related to the Epimedium genus and
has been widely used for treating several ailments (e.g., osteoporosis, infertility, impotence, amnesia,
senile functional disorders, and cardiovascular diseases) [10]. Chen and Chiu [11] claimed that
intracavernous uptake of EbM extract might lead to a penile erection in rats. Wong et al. [12] defined
the main bioactive components that were extracted from EbM, for instance, epimedin A, B, and C,
and icariin (ICA; Figure 2A–D). ICA is the main flavonoid glycoside obtained from the aeronautical
piece of the plant [13]. Furthermore, ICA shows a cGMP-specific PDE5 inhibitor capable of producing
an orally efficient erectile dysfunction treatment agent [13,14]. Qureshi et al. [15] found that the
administration of 300 to 500 mg/kg/day of E. brevicornum extract is a highly effective therapy for the
treatment of impotency and reduces stress and depression. Furthermore, it was shown to be an
excellent anxiolytic medicine and adaptogen for hormonal disorders. Within the current study, we
assume that E. brevicornum extract might trigger an anti-infertility agent in rats. We tested the
ameliorative effect of two different doses of E. brevicornum under the long-term administration of
TAM.
Figure 1. Chemical structure of tramadol hydrochloride.
TAM has been reported to cause respiratory depression and psychological and physiological
addiction [
4
]. Moreover, long-term administration of TAM results in an increase in oxidative stress
that causes male infertility due to low levels of testosterone and neurodegenerative diseases such
as Alzheimer’s disease [
5
,
6
]. There is a disturbing increase in the abuse of TAM, mainly among
young people assuming that it improves sexual excitement and climax without the development of
sexual dysfunctions [
7
], and small quantities for additional investigations and examinations have
been implanted [
8
]. There is an urgent need for safer natural antioxidants to minimize the eects of
oxidative and peroxidative damage of TAM on male infertility. Such natural antioxidants can prevent
the issues associated with synthetic drugs, such as various side eects, treatment disappointment,
and drug resistance.
Epimedium is a plant category of the Berberidaceae family, containing 63 plant species.
Epimedium brevicornum Maxim (EbM) is a well-known Chinese herb that is related to the Epimedium
genus and has been widely used for treating several ailments (e.g., osteoporosis, infertility, impotence,
amnesia, senile functional disorders, and cardiovascular diseases) [
9
]. Chen and Chiu [
10
] claimed
that intracavernous uptake of EbM extract might lead to a penile erection in rats. Wong et al. [
11
]
defined the main bioactive components that were extracted from EbM, for instance, epimedin A,
B, and C, and icariin (ICA; Figure 2A–D). ICA is the main flavonoid glycoside obtained from the
aeronautical piece of the plant [
12
]. Furthermore, ICA shows a cGMP-specific PDE5 inhibitor capable
of producing an orally ecient erectile dysfunction treatment agent [
12
,
13
]. Qureshi et al. [
14
] found
that the administration of 300 to 500 mg/kg/day of E. brevicornum extract is a highly eective therapy
for the treatment of impotency and reduces stress and depression. Furthermore, it was shown to be
an excellent anxiolytic medicine and adaptogen for hormonal disorders. Within the current study,
we assume that E. brevicornum extract might trigger an anti-infertility agent in rats. We tested the
ameliorative eect of two dierent doses of E. brevicornum under the long-term administration of TAM.
Antibiotics 2020,9, 318 3 of 15
Antibiotics 2020, 9, x FOR PEER REVIEW 3 of 15
Figure 2. Chemical structures of the main bioactive components extracted from Epimedium
brevicornum. (A) epimedin A, (B) epimedin B, (C) epimedin C, and (D) icariin.
2. Materials and Methods
2.1. Chemicals
Epimedium brevicornum extract as a source of icariin was purchased from Naturalin (code: NAT-
091). The Molecular Structure and HPLC analysis chart is available on the company webpage
(https://www.egypt-business.com/product/details/1617-epimedium-extract/35138). Tramadol
hydrochloride 225 mg tablets were kindly given by the Laboratory of Forensic Sciences, Ministry of
Justice (Cairo, Egypt). Its International Union of Pure and Applied Chemistry (IUPAC) name is as
follows: (±) cis-2-{(dimethylamino) methyl}-1-(3-methoxyphenyl) cyclohexan-1-ol hydrochloride. All
other chemicals used in this study were graded analytically.
2.2. Animal Use
Fifty adult male Wister rats of 200 ± 10 gm aged 4 to 5 months were collected from the Animal
Research Unit, Faculty of Veterinary Medicine, Zagazig University, Egypt. The animals were housed
in an environment free of pathogens, with controlled humidity, temperature (22 °C), and in an
alternate 12 h light and dark cycle [16]. Two weeks before the experiment was conducted, the animals
were allowed to acclimatize to the test facility conditions. The methodology was applied to all
creatures dealt with, tested, and authorized by Zagazig University Research Center Institutional
Animal Care and Use Committee (IACUC) under number ZU-IACUC/2/F/32/2018.
2.3. Model Building
Rats were caged into five groups, each of which consisted of ten rats. Group 1 was the control
group that received an oral administration of 0.2 mL saline as the vehicle used for 130 consecutive
days. Groups 2 and 3 were administered 2.43 g/kg/day of E. brevicornum and 50 mg/kg/day of
tramadol for 130 consecutive days, respectively [6,17]. Groups 4 and 5 were received tramadol at 50
mg/kg/day for 65 days, followed by the herb extract at doses of 0.81 g/kg/day and 2.43 g/kg/day for
another 65 days, respectively.
2.4. Sample Collection
At the end of the animal study, all rats were euthanized using an anesthesia system involving
ether for serum and tissue collection [18]. In a BD Vacutainer PST II tube [19], blood samples were
Figure 2.
Chemical structures of the main bioactive components extracted from Epimedium brevicornum.
(A) epimedin A, (B) epimedin B, (C) epimedin C, and (D) icariin.
2. Materials and Methods
2.1. Chemicals
Epimedium brevicornum extract as a source of icariin was purchased from Naturalin (code: NAT-091).
The Molecular Structure and HPLC analysis chart is available on the company webpage (https:
//www.egypt-business.com/product/details/1617-epimedium-extract/35138). Tramadol hydrochloride
225 mg tablets were kindly given by the Laboratory of Forensic Sciences, Ministry of Justice
(Cairo, Egypt). Its International Union of Pure and Applied Chemistry (IUPAC) name is as follows:
(
±
) cis-2-{(dimethylamino) methyl}-1-(3-methoxyphenyl) cyclohexan-1-ol hydrochloride. All other
chemicals used in this study were graded analytically.
2.2. Animal Use
Fifty adult male Wister rats of 200
±
10 gm aged 4 to 5 months were collected from the Animal
Research Unit, Faculty of Veterinary Medicine, Zagazig University, Egypt. The animals were housed in
an environment free of pathogens, with controlled humidity, temperature (22
C), and in an alternate
12 h light and dark cycle [
15
]. Two weeks before the experiment was conducted, the animals were
allowed to acclimatize to the test facility conditions. The methodology was applied to all creatures
dealt with, tested, and authorized by Zagazig University Research Center Institutional Animal Care
and Use Committee (IACUC) under number ZU-IACUC/2/F/32/2018.
2.3. Model Building
Rats were caged into five groups, each of which consisted of ten rats. Group 1 was the control
group that received an oral administration of 0.2 mL saline as the vehicle used for 130 consecutive
days. Groups 2 and 3 were administered 2.43 g/kg/day of E. brevicornum and 50 mg/kg/day of tramadol
for 130 consecutive days, respectively [
5
,
16
]. Groups 4 and 5 were received tramadol at 50 mg/kg/day
for 65 days, followed by the herb extract at doses of 0.81 g/kg/day and 2.43 g/kg/day for another
65 days, respectively.
2.4. Sample Collection
At the end of the animal study, all rats were euthanized using an anesthesia system involving
ether for serum and tissue collection [
17
]. In a BD Vacutainer PST II tube [
18
], blood samples
were gathered, left for coagulation at room temperature, and centrifuged at 1200
×
gfor 15 min to
obtain serum. The serum samples were preserved at
20
C to be used for various biochemical
Antibiotics 2020,9, 318 4 of 15
experiments. Each rat pair of testis and epididymis was quickly cut and rinsed with ice-cold saline,
while the testicular tissue was dissected and frozen in liquid nitrogen for further examination. For the
antioxidant measurement, a piece of one testis from each rat was homogenized using a WiseTis HG-15D
homogenizer (Daihan Scientific Co., Seoul, Korea), while the other part of testis was maintained in 10%
neutral buered formalin to be used for histopathological studies.
2.5. Semen Assessment and Sperm Parameters
The caudal portion of the epididymis was precisely isolated from one testis and placed in
a sterilized Petri dish containing 2 mL normal saline where it was macerated; the epididymal contents
obtained were correctly treated as the semen. All the equipment and solutions used were prewarmed
to 37
C before used. A drop of the obtained suspension was then positioned on a clean glass slide and
examined by under a light microscope at a high resolution of
×
400 to assess the individual spermatozoa
motility, whereas the total motile spermatozoa percentage was examined by various microscopical
fields. Eosin-nigrosine-stained epididymal suspensions were prepared for the sperm viability assay to
assess the proportion of live/dead sperm. An improved Neubauer hemocytometer tallying chamber
was used to count the spermatozoa in the semen solution diluted with ordinary saline at a proportion
of 1:4 and a few drops of formalin (40%) [
19
]. The sperm cell count exceeded the all-out spermatozoa
number in four squares
×
2500
×
dilution factor. Eosin–nigrosine-recolored smears were analyzed
under the oil immersion lens to evaluate sperm variation levels from the standard after an arbitrary
analysis of 100 spermatozoa [20].
2.6. Hormonal Analysis
Enzyme-linked immunosorbent assay (ELISA) kits obtained from MyBioSource (San Diego,
CA, USA) were used to examine the plasma luteinizing hormone (LH), testosterone (Tes), estradiol
(E2), and follicle-stimulating hormone (FSH) levels. The absorbance was measured using an ELISA
plate reader (DNM–9602; Beijing Perlong Medical Instrument Ltd., Beijing, China), as instructed by
the manufacturer.
2.7. Assessment of NO and Antioxidant Scavenging Capacity
A suitable homogenate aliquot was ultracentrifuged for 30 min at 10,000
×
gat 4
C and the
supernatant obtained after centrifugation was used for the estimation of NO spectrophotometrically:
catalase (CAT; EC 1.11.1.6), superoxide dismutase (SOD; EC1.15.1.1), and malondialdehyde
(MDA; the lipid peroxidation marker), using Griess reagents, as described by Green et al. [
21
].
Spectrophotometrical determination of the MDA levels [
22
], CAT, and SOD activity at 570 and
480 nm wavelengths, respectively [
23
,
24
], was calculated using a Shimadzu-type spectrophotometer
(UV 120-02). All antioxidant enzyme kits were carried out using Biodiagnostic tools (Biodiagnostic
Company, Dokki, Giza, Egypt), following the fabricator’s stipulations.
2.8. Relative Quantitative RT-PCR Analysis
A detailed description of the protocols used has already been reported [
25
]. Real-time PCR
(RT-PCR) was carried out according to the manufacturer’s instructions using an Mx3005P Real-Time PCR
System (Agilent Stratagene, CA, USA) and 5x HOT FIRE Pol EvaGreen qPCR Mix Plus (Solis BioDyne,
Tartu, Estonia). The primers used are listed in Table 1. The relative expression of each gene normalized
to the housekeeping GAPDH was reported as fold change by 2∆∆CT, relative to control [26].
Antibiotics 2020,9, 318 5 of 15
Table 1. Oligonucleotide sequences for interest and reference genes.
Gene Forward Primer (50–30) Reverse Primer (50–30) Accession No Product Size Ref.
Bax CGAATTGGCGATGA
ACTGGA
CAAACATGTCAGCTGC
CACAC NM_017059.2 109 [25]
Bcl2 GACTGAGTACCTGAACCG
GCATC
CTGAGCAGCGTCTTCAG
AGACA NM_016993.1 135 [25]
GAPDH GGCACAGTCAAGGCTGA
GAATG
ATGGTGGTGAAGACGC
CAGTA NM_017008.4 143 [25]
2.9. The Testes Morphology
The testicles were resolved in formalin (10%) and immersed in paran. Afterwards, 5-
µ
m thick
sections were prepared, stained with hematoxylin–eosin, and tested under an Olympus/3H light
microscope (Olympus, Tokyo, Japan) [25].
2.10. Statistical Analysis
Statistical analysis was carried out using one-way ANOVA accompanied by a posthoc dunken
test, SPSS 16 (SPSS, Chicago, IL, USA). Data were interpreted as mean
±
SEM, and a p-value <0.05 was
considered statistically significant.
3. Results
3.1. Ecacy of Oral Administration of E. brevicornum extract and/or Tramadol on Semen Parameters
In order to detect the harm caused by the male conceptual framework, the sperm parameters
were evaluated in the present investigation (Figure 3A–C). Concerning sperm parameters, sperm
motility and count were remarkably increased, and abnormalities were reduced in the E. brevicornum
group related to the control one, whereas substantial reduction in sperm motility and count and
an improvement in abnormalities were observed in TAM-treated mice with regard to the control one
(
p<0.05
). Regarding the two E. brevicornum-administered groups, a remarkable increase was reported
in the sperm count by 43.7% and 71.8% and motility by 66% and 98%, while reduction by 40% in the
sperm abnormality was detected when compared with the TAM-treated rats.
Antibiotics 2020, 9, x FOR PEER REVIEW 5 of 15
Table 1. Oligonucleotide sequences for interest and reference genes.
Gene Forward Primer (5–3) Reverse Primer (5–3) Accession No Product Size Ref.
Bax CGAATTGGCGATGA
ACTGGA
CAAACATGTCAGCTGC
CACAC NM_017059.2 109 [26]
Bcl2 GACTGAGTACCTGA
ACCGGCATC
CTGAGCAGCGTCTTCAG
AGACA NM_016993.1 135 [26]
GAPDH GGCACAGTCAAGGC
TGAGAATG
ATGGTGGTGAAGACGC
CAGTA NM_017008.4 143 [26]
2.9. The Testes Morphology
The testicles were resolved in formalin (10%) and immersed in paraffin. Afterwards, 5-µm thick
sections were prepared, stained with hematoxylin–eosin, and tested under an Olympus/3H light
microscope (Olympus, Tokyo, Japan) [26].
2.10. Statistical Analysis
Statistical analysis was carried out using one-way ANOVA accompanied by a posthoc dunken
test, SPSS 16 (SPSS, Chicago, IL, USA). Data were interpreted as mean ± SEM, and a p-value <0.05 was
considered statistically significant.
3. Results
3.1. Efficacy of Oral Administration of E. brevicornum extract and/or Tramadol on Semen Parameters
In order to detect the harm caused by the male conceptual framework, the sperm parameters
were evaluated in the present investigation (Figure 3A–C). Concerning sperm parameters, sperm
motility and count were remarkably increased, and abnormalities were reduced in the E. brevicornum
group related to the control one, whereas substantial reduction in sperm motility and count and an
improvement in abnormalities were observed in TAM-treated mice with regard to the control one (p
< 0.05). Regarding the two E. brevicornum-administered groups, a remarkable increase was reported
in the sperm count by 43.7% and 71.8% and motility by 66% and 98%, while reduction by 40% in the
sperm abnormality was detected when compared with the TAM-treated rats.
Figure 3. The efficacy of oral uptake of E. brevicornum extract and/or tramadol on semen parameters.
Tramadol-administered rats resulted in a remarkable decrease in sperm motility (A) and count (B)
Figure 3.
The ecacy of oral uptake of E. brevicornum extract and/or tramadol on semen parameters.
Tramadol-administered rats resulted in a remarkable decrease in sperm motility (
A
) and count (
B
) and
an improvement in sperm abnormalities (
C
) relative to the control group (* p
0.05). The eect of oral
administration of E. brevicornum extract exerted a remarkable enhancement in sperm count and sperm
motility, while the sperm abnormality associated with tramadol-treated rats was decreased (* p
0.05).
Results are assessed using one-way ANOVA accompanied by Bonferroni’s test.
Antibiotics 2020,9, 318 6 of 15
3.2. Eect of E. brevicornum Extract and/or Tramadol Oral Administration on the Hormonal Assay
As appears in Figure 4A–D, E. brevicornum extract exhibited a significant impact on the sexual
hormones of male rats in comparison with controls. In the interim, substantial decreases in Tes, FSH,
LH, and E2 hormones were observed in the TAM group. In contrast, the herb extract-treated group
showed exceptionally large increases in Tes, FSH, LH, and E2 hormones. The most ameliorative eects
were observed in Group 5.
Antibiotics 2020, 9, x FOR PEER REVIEW 6 of 15
and an improvement in sperm abnormalities (C) relative to the control group (* p 0.05). The effect
of oral administration of E. brevicornum extract exerted a remarkable enhancement in sperm count
and sperm motility, while the sperm abnormality associated with tramadol-treated rats was
decreased (* p 0.05). Results are assessed using one-way ANOVA accompanied by Bonferroni’s test.
3.2. Effect of E. brevicornum Extract and/or Tramadol Oral Administration on the Hormonal Assay
As appears in Figure 4A–D, E. brevicornum extract exhibited a significant impact on the sexual
hormones of male rats in comparison with controls. In the interim, substantial decreases in Tes, FSH,
LH, and E2 hormones were observed in the TAM group. In contrast, the herb extract-treated group
showed exceptionally large increases in Tes, FSH, LH, and E2 hormones. The most ameliorative
effects were observed in Group 5.
Figure 4. The effect of oral uptake of E. brevicornum extract and/or tramadol on hormonal assay.
Results are presented as mean ± SEM and evaluated using one-way ANOVA accompanied by
Bonferroni’s test for multiple comparisons. * indicates significant different at p < 0.05 from the
tramadol-treated group. (A) Effect on testosterone level, (B) effect on FSH level, (C) effect on LH level,
(D) effect on E2 level.
3.3. Effect of E. brevicornum Extract and/or Tramadol Oral Administration on NO and Antioxidant Status
The conceptual framework power of males by TAM administration continues due to oxidative
stress conditions in testicular tissues. The redox system activity has been assessed in the testes along
these lines. In the TAM-treated rats, abnormal changes in oxidant and antioxidant enzyme activities
and a decrease in the antioxidant activity were observed in the reduced SOD activities (p < 0.05) and
increased MDA and nitric oxide (NO) production (p < 0.05), respectively. The two groups subjected
to E. brevicornum were significantly reduced in all irregularities of oxidative stress (p < 0.05) with
regard to TAM-treated rats, as shown in Figure 5A–C.
Figure 4.
The eect of oral uptake of E. brevicornum extract and/or tramadol on hormonal assay. Results
are presented as mean
±
SEM and evaluated using one-way ANOVA accompanied by Bonferroni’s test
for multiple comparisons. * indicates significant dierent at p<0.05 from the tramadol-treated group.
(A) Eect on testosterone level, (B) eect on FSH level, (C) eect on LH level, (D) eect on E2 level.
3.3. Eect of E. brevicornum Extract and/or Tramadol Oral Administration on NO and Antioxidant Status
The conceptual framework power of males by TAM administration continues due to oxidative
stress conditions in testicular tissues. The redox system activity has been assessed in the testes along
these lines. In the TAM-treated rats, abnormal changes in oxidant and antioxidant enzyme activities
and a decrease in the antioxidant activity were observed in the reduced SOD activities (p<0.05) and
increased MDA and nitric oxide (NO) production (p<0.05), respectively. The two groups subjected to
E. brevicornum were significantly reduced in all irregularities of oxidative stress (p<0.05) with regard
to TAM-treated rats, as shown in Figure 5A–C.
Antibiotics 2020,9, 318 7 of 15
Antibiotics 2020, 9, x FOR PEER REVIEW 7 of 15
Figure 5. The effect of oral uptake of E. brevicornum extract and/or tramadol on malondialdehyde
(MDA) (A), superoxide dismutase (SOD) (B), and nitric oxide (NO) (C) activity concentrations in
testes of male rats. Results are represented as mean ± SEM and analyzed using one-way ANOVA
accompanied by Bonferroni’s test for multiple comparisons. * Significant variation in the tramadol-
treated group.
3.4. Expression of Apoptosis-Related Genes in Testicular Tissue
An apoptotic index (Bax) demonstrated a five-fold increase in the TAM-treated group than that
in the saline control one. At the same time, a considerable reduction in the gene expression to half
was observed after E. brevicornum administration when compared to the TAM group (Figure 6).
Tramadol at a dose of 50 mg/kg/day caused an exceptionally huge decrease in the expression of Bcl2
(by 75%) when related to the saline control group, followed by a remarkable rise in groups submitted
to treatment with E. brevicornum alongside TAM.
Figure 6. The effect of oral E. brevicornum extract and/or tramadol administration on the expression
of apoptotic factor Bax and Bcl2 gene in testes of male rats. Tramadol-administered rats showed a
substantial increase in apoptotic index (Bax) levels (by five-fold) relative to the control group (* p
Figure 5.
The eect of oral uptake of E. brevicornum extract and/or tramadol on malondialdehyde (MDA)
(
A
), superoxide dismutase (SOD) (
B
), and nitric oxide (NO) (
C
) activity concentrations in testes of male
rats. Results are represented as mean
±
SEM and analyzed using one-way ANOVA accompanied by
Bonferroni’s test for multiple comparisons. * Significant variation in the tramadol-treated group.
3.4. Expression of Apoptosis-Related Genes in Testicular Tissue
An apoptotic index (Bax) demonstrated a five-fold increase in the TAM-treated group than that in
the saline control one. At the same time, a considerable reduction in the gene expression to half was
observed after E. brevicornum administration when compared to the TAM group (Figure 6). Tramadol at
a dose of 50 mg/kg/day caused an exceptionally huge decrease in the expression of Bcl2 (by 75%) when
related to the saline control group, followed by a remarkable rise in groups submitted to treatment
with E. brevicornum alongside TAM.
Antibiotics 2020, 9, x FOR PEER REVIEW 7 of 15
Figure 5. The effect of oral uptake of E. brevicornum extract and/or tramadol on malondialdehyde
(MDA) (A), superoxide dismutase (SOD) (B), and nitric oxide (NO) (C) activity concentrations in
testes of male rats. Results are represented as mean ± SEM and analyzed using one-way ANOVA
accompanied by Bonferroni’s test for multiple comparisons. * Significant variation in the tramadol-
treated group.
3.4. Expression of Apoptosis-Related Genes in Testicular Tissue
An apoptotic index (Bax) demonstrated a five-fold increase in the TAM-treated group than that
in the saline control one. At the same time, a considerable reduction in the gene expression to half
was observed after E. brevicornum administration when compared to the TAM group (Figure 6).
Tramadol at a dose of 50 mg/kg/day caused an exceptionally huge decrease in the expression of Bcl2
(by 75%) when related to the saline control group, followed by a remarkable rise in groups submitted
to treatment with E. brevicornum alongside TAM.
Figure 6. The effect of oral E. brevicornum extract and/or tramadol administration on the expression
of apoptotic factor Bax and Bcl2 gene in testes of male rats. Tramadol-administered rats showed a
substantial increase in apoptotic index (Bax) levels (by five-fold) relative to the control group (* p
Figure 6.
The eect of oral E. brevicornum extract and/or tramadol administration on the expression
of apoptotic factor Bax and Bcl2 gene in testes of male rats. Tramadol-administered rats showed
a substantial increase in apoptotic index (Bax) levels (by five-fold) relative to the control group
(* p0.05)
. The eect of oral administration of E. brevicornum extract exhibited a marked decrease in
gene expression when compared to tramadol-treated rats (* p
0.05). Tramadol at a dose of 50 mg/kg/day
caused a significant reduction in expression of Bcl2 (by 75%) when related to the control animals,
followed by a substantial increase in E. brevicornum extract-treated groups. Results are presented as
mean ±SEM and evaluated using one-way ANOVA accompanied by Bonferroni’s test.
Antibiotics 2020,9, 318 8 of 15
3.5. Eect of Oral E. brevicornum Extract and/or Tramadol Administration on Testicular Histopathology
Histopathological evaluation was carried out in all experimental groups, and the results are
shown in Figure 7A–E. A typical histological structure of the seminiferous tubules in testis tissue was
observed under light photomicrography, in sections prepared from control and E. brevicornum-treated
groups (2.43 g/kg/day for 130 days). Necrosis and degeneration of the seminiferous tubule, (sloughing
of the stratified seminiferous epithelium), spermatogenic cells and Sertoli cells, necrosis of some
spermatogenic cells (especially spermatogonia, spermatocytes), and spermatids with a pyknotic
nucleus appeared in animals receiving TAM 50 mg/kg/day for 130 consecutive days. Reasonable
normalization of the testicular tissue, with a sustained normal histological structure of most of the
seminiferous tubules, a moderate number of spermatozoa, and mild edema in the intertubular space,
were observed in the tubular lumen of rats receiving both TAM and E. brevicornum in dierent doses,
with a noticeable improvement in the higher proportion of E. brevicornum.
Antibiotics 2020, 9, x FOR PEER REVIEW 8 of 15
0.05). The effect of oral administration of E. brevicornum extract exhibited a marked decrease in gene
expression when compared to tramadol-treated rats (* p 0.05). Tramadol at a dose of 50 mg/kg/day
caused a significant reduction in expression of Bcl2 (by 75%) when related to the control animals,
followed by a substantial increase in E. brevicornum extract-treated groups. Results are presented as
mean ± SEM and evaluated using one-way ANOVA accompanied by Bonferroni’s test.
3.5. Effect of Oral E. brevicornum Extract and/or Tramadol Administration on Testicular Histopathology
Histopathological evaluation was carried out in all experimental groups, and the results are
shown in Figure 7A–E. A typical histological structure of the seminiferous tubules in testis tissue was
observed under light photomicrography, in sections prepared from control and E. brevicornum-
treated groups (2.43 g/kg/day for 130 days). Necrosis and degeneration of the seminiferous tubule,
(sloughing of the stratified seminiferous epithelium), spermatogenic cells and Sertoli cells, necrosis
of some spermatogenic cells (especially spermatogonia, spermatocytes), and spermatids with a
pyknotic nucleus appeared in animals receiving TAM 50 mg/kg/day for 130 consecutive days.
Reasonable normalization of the testicular tissue, with a sustained normal histological structure of
most of the seminiferous tubules, a moderate number of spermatozoa, and mild edema in the
intertubular space, were observed in the tubular lumen of rats receiving both TAM and E. brevicornum
in different doses, with a noticeable improvement in the higher proportion of E. brevicornum.
Figure 7. Plate (A): photomicrograph of the rat testis in the control rats shows normal, intact
seminiferous tubules with normal lining epithelium and stratified seminiferous epithelium with
normal interstitial connective tissue. Plate (B): photomicrograph of the rat testis in Group 2, which
received E. brevicornum extract at a dose of 2.43 g/kg/day, show normal testicular parenchyma. Plate
(C): photomicrographs of the testis in Group 3, which received tramadol at doses of 50 mg/kg/day
and oral daily plate C, show necrosis, degeneration of the seminiferous tubule, sloughing of the
stratified seminiferous epithelium, spermatogenic cells, and Sertoli cells, indicated with black arrows.
Plate (D): photomicrographs of the rat testis of Group 4, which received tramadol at 50 mg/kg/day
for 65 days, followed by TAM and E. brevicornum extract at a dose of 0.81 g/kg/day for another 65
days, show slight destruction and sloughing of spermatogenic cells, with slight hyperplasia of Leydig
cells and slightly hyaline degeneration of the interstitial CT. Plate (E): photomicrographs of the testis
in Group 5, which received tramadol at 50 mg/kg/day for 65 days, followed by tramadol and E.
brevicornum extract at a dose of 2.43 g/kg/day for another 65 days.
4. Discussion
Tramadol (TAM) is an atypical, midway-acting manufactured pain relief used to get rid of
moderate to severe agony, with antinociceptive impacts that are interceded by a mixture of mu-
opioid agonist impacts and inhibition of serotonin and norepinephrine reuptake [2]. In addition to
A B C
D E
Figure 7.
Plate (
A
): photomicrograph of the rat testis in the control rats shows normal, intact seminiferous
tubules with normal lining epithelium and stratified seminiferous epithelium with normal interstitial
connective tissue. Plate (
B
): photomicrograph of the rat testis in Group 2, which received E. brevicornum
extract at a dose of 2.43 g/kg/day, show normal testicular parenchyma. Plate (
C
): photomicrographs of
the testis in Group 3, which received tramadol at doses of 50 mg/kg/day and oral daily plate C, show
necrosis, degeneration of the seminiferous tubule, sloughing of the stratified seminiferous epithelium,
spermatogenic cells, and Sertoli cells, indicated with black arrows. Plate (
D
): photomicrographs of
the rat testis of Group 4, which received tramadol at 50 mg/kg/day for 65 days, followed by TAM
and E. brevicornum extract at a dose of 0.81 g/kg/day for another 65 days, show slight destruction
and sloughing of spermatogenic cells, with slight hyperplasia of Leydig cells and slightly hyaline
degeneration of the interstitial CT. Plate (
E
): photomicrographs of the testis in Group 5, which received
tramadol at 50 mg/kg/day for 65 days, followed by tramadol and E. brevicornum extract at a dose of
2.43 g/kg/day for another 65 days.
4. Discussion
Tramadol (TAM) is an atypical, midway-acting manufactured pain relief used to get rid of moderate
to severe agony, with antinociceptive impacts that are interceded by a mixture of mu-opioid agonist
impacts and inhibition of serotonin and norepinephrine reuptake [
2
]. In addition to cytotoxic drugs,
dierent drugs can aect human fertility through various components. By modifying the hormones or
nonhormonal systems of the hypothalamic–pituitary–gonadal axis, medicines can legitimately lead to
sexual interruption and impairment of spermatogenesis and a change in epididymal growth. Previous
reports documented the adverse eects of some eective drugs (e.g., sulfasalazine, testosterone,
Antibiotics 2020,9, 318 9 of 15
anabolic steroids, opioids, cyproterone acetic acid derivation, tramadol, sartan, and GnRH analogs) [
27
].
E. brevicornum Maxim (EbM) is a Chinese herb that possesses many pharmacological activities, including
hormonal enhancement, immunological capacity regulation, antiosteoporosis, antitumor, antioxidation,
antimaturity, and antiatherosclerosis. Moreover, over 2000 years, it has been used as an antirheumatic
natural tonic and aphrodisiac in China, Japan, and Korea [9].
In this research, we present the risks of long-term use of TAM over 130 consecutive days. We tested
its eects on male fertility by evaluating some andrological parameters on Wister male rats, namely,
sperm motility, count, livability and abnormalities, serum testosterone, LH and FSH hormones,
and serum estradiol (E2). These parameters are indicators that determine male ability to create feasible
spermatozoa, some tissue-scavenging ability, and an apoptosis index (Bcl-2 and Bax gene expression).
Additionally, we figured out the possible improvements in the aforementioned parameters using
dierent doses E. brevicornum extract combined with TAM through the 65 days of treatment to detect
their therapeutic eect.
The hypothalamus assumes a vital function in the maintenance of spermatogenesis by controlling
FSH and LH secretion from the pituitary organ through GnRH [
28
]. At the same time, testosterone is
integrated into the regulation of GnRH synthesis feedback and released through a long-circle input
system [
29
]. Male maturity is dependent mainly on serum testosterone, LH and FSH concentrations,
sperm count, and sperm quality. The regulated male sex hormone levels indicate the male reproductive
brokenness. In the current study, Figure 4A reveals that blood testosterone levels delivered mainly in
the testicles were substantially diminished (p
0.05) in the TAM-treated group compared to the control
group. Testosterone is a significant androgen that plays a key role in sexual development, behavior,
spermatogenesis, separation, and the maintenance of the adornment sex organs [
30
]. The synthesis
and release of androgens are dependent on the pituitary gonadotropins, which are LH and FSH. Both
LH and FSH are essential for testicular capacity and spermatogenesis. LH is the fundamental tropic
controller of Leydig cells; without it, the androgen generation cannot be imagined [31].
Our findings show that the TAM-administered group had a marked decrease in serum
concentrations of LH in the central pituitary organ (p
0.05). Serum FSH levels showed a substantial
decrease in the TAM-administered group in contrast to the control group. Our findings of a reduction
rather than an increase in circling LH in the TAM-treated group indicate that the drug was acting
on the hypothalamus axis by suppressing LH release by the pituitary gland. Moreover, estradiol
(E2), which has shown a controlled decrease in TAM-treated rats, is the overwhelming controller of
FSH discharge in human males [
32
]. Estradiol is generated by the aromatase activity in the Leydig
cells of the mammalian testis, as well as in certain germ and Sertoli cells of immature warm-blooded
animals [
33
]. Raven et al. [
34
] suggested that the levels of peripheral E2 directly reflect the inhibitory
tone used by gonadotropin-release estrogens and are significant determinants of testosterone and
LH and FSH levels. E2 prevents the release of gonadotropin in men by acting on the pituitary and
hypothalamus. However, the mean plasma E2 level expected to restore LH, FSH, and testosterone
to the standard levels was not substantially the same as the mean E2 level. E. brevicornum extract at
a dose of 2.43 g/kg/day for 130 successive days stimulated the most impressive values, in contrast
with testosterone, LH, FSH, and E2 control rats, showing improvement in male fertility, which was
confirmed by sperm motility, abnormalities, and count when related to control rats, where a marked
rise in sperm count and motility could be seen, with a marked decrease in abnormalities. Groups 4
and 5 received TAM at 50 mg/kg/day for 65 days and TAM and E. brevicornum extract at doses of
0.81 g/kg/day and 2.43 g/kg/day for 65 days, respectively, resulting in remarkable improvements in the
previously mentioned parameters compared to the TAM-treated group.
In folk medicine, E. brevicornum extract has been documented in traditional Chinese medicine
for treating erectile dysfunction as it has testosterone mimetic activity and therapeutic potential in
hypoandrogenism management [
35
]. Shindel et al. [
13
] reported the beneficial eect of E. brevicornum
on erectile function after cavernous nerve damage. They revealed that rats treated with a low-dose of
E. brevicornum resulted in a remarkable increase in the intracavernous pressure (ICP) to mean arterial
Antibiotics 2020,9, 318 10 of 15
pressure (MAP) ratio and area under the curve (AUC) to MAP ratio in comparison to control animals.
They concluded that E. brevicornum might have neurotrophic eects on penile tissue in addition to
its known phosphodiesterase type 5 (PDE-5)-inhibiting eects. Interestingly, high blood testosterone
levels in high-dose E. brevicornum-treated rats show significant degradation when compared to control
or low-dose E. brevicornum-treated rats. At the same time, no improvement in the serum LH has been
documented in all treated groups.
Irregular morphology of spermatozoa was increased in TAM-treated groups, whereas this
irregular morphology was improved in E. brevicornum-treated groups compared to control. The sperm
characteristics shown in Figure 3showed a significant reduction (p
0.05) in sperm count, motility,
and percent of viability in TAM oral administration (50 mg/kg/day b.wt.) for 130 consecutive days
in comparison to healthy animals. Our results are compatible with those reported by Nna and
Osim [
36
], who indicated a significant increase in spermatozoa with abnormal morphology in all TAM
(20 mg/kg/day b.wt.)-treated groups, with regard to the control animals. TAM was impressively capable
of implementing its malignant activities in the production of the testis as well as the function and,
thus, the regulation of serum hormonal levels that regulate the male richness [
27
,
36
,
37
]. Furthermore,
Abdel-Hamid et al. [
7
] suggested that TAM may exhibit a beneficial eect on premature ejaculation
therapy. Rats treated with E. brevicornum at a dose of 2.43 g/kg/day for 130 consecutive days elicited
a remarkable increase in sperm motility and count, with a marked decrease in sperm abnormalities
compared to the control or TAM-treated groups. Nantia et al. [
38
] reviewed the eect of E. brevicornum
extract on mammalian fertility and documented that the herb extract improved sperm parameters,
as well as the treatment of libido dysfunction, sexual asthenia, and erection. In addition, another study
documented that the oral administration of lipid-based E. brevicornum extract improved the erectile
function of aged rats [39].
Interestingly, this is the first study to demonstrate the ecacy of the E. brevicornum–TAM
combination treatment on male infertility. Animals receiving TAM at 50 mg/kg/day for 65 days, followed
by TAM and E. brevicornum at doses of 0.81 and 2.43 g/kg/day for 65 days, respectively, had a marked
increase in sperm characteristics compared to TAM-treated animals (Figure 3),
and an improvement
was documented in the rodents receiving E. brevicornum at a dose of 2.43 g/kg/day. Tramadol
administration at a dose of 50 mg/kg/day b.wt. for 130 consecutive days led to a substantial reduction
in SOD activity and a remarkable rise in MDA levels when compared to the control group. MDA is
the main reactive aldehyde that is known to have a toxic eect on cells [
40
]. All of these defensive
antioxidant enzymes can work together and, therefore, protect against oxidative injury or damage
caused by free radicals. These results are consistent with previous reports that show that TAM causes
oxidative anxiety in testicular tissue [
37
,
41
,
42
]. In addition, TAM has a narcotic pain-relieving eect,
which is usually recommended for moderate to severe pain at the usual dose of up to 200 mg/day,
as indicated by Costa et al. [
41
]. TAM is also reported as one of the synthetic opioids that have
significant cellular impacts through the expansion of lipid peroxidation that can be used as an indicator
of cell injury caused by reactive oxygen species (ROS) [
43
]. However, the findings documented by
Atici et al. [
44
] showed that morphine and TAM treatment aorded an elevated MDA level, suggesting
an increased level of lipid peroxidation. In addition, reduced levels of glutathione (GSH) were observed
in isolated rat hepatic cells incubated with dierent concentrations of opioids that resulted in cell
necrosis and decreased GSH levels, as well as CAT, SOD, and glutathione peroxidase (GPx) activity.
Recently, Nna and Osim [
36
] confirmed that SOD, CAT, GSH, and GPx were completely reduced,
while MDA was increased mainly in all TAM (20 mg/kg/day b.wt.)-treated groups when compared
with control.
Additionally, TAM administration at 50 mg/kg/day b.wt. for 130 consecutive days led to
a remarkable rise in the NO levels in contrast with dierent groups. These data were supported
by those obtained by Ahmed and Kurkar. [
37
], who revealed that TAM substantially enhanced
nitric oxide testicular levels and lipid peroxidation when compared to the control group. Similarly,
immunohistochemical studies demonstrated that TAM enhanced endothelial nitric oxide synthase
Antibiotics 2020,9, 318 11 of 15
(eNOS) expression in testicular tissues. E. brevicornum administration, at a dose of 2.43 g/kg/day
for 130 successive days, resulted in a detectable increase in SOD activity and a marked decrease in
both MDA and NO levels when compared to either control or TAM-treated groups. Zhao et al. [
45
]
evaluated the
in vitro
antioxidant activity using a DPPH assay. The assay showed that phenolic
compounds (e.g., p-hydroxybenzoic acid, gallic acid, vanillic acid, ferulic acid, rutin, caeic acid,
catechin, quercetin, and benzoic acid) isolated from EbM exhibit potent antioxidant activity. Moreover,
they documented that E. brevicornum pretreatment for 24 h markedly enhanced cisplatin-actuated
oxidative stress by diminishing the MDA and ROS levels, while increasing GSH levels in HEK-293
cells, as well as decreasing nuclear translocation and nuclear factor-kappa B (NF-
κ
B) phosphorylation,
followed by reduced iNOS, interleukin-1
β
(IL-1
β
), and tumor necrosis factor-
α
(TNF-
α
) secretions.
It also decreased cellular apoptosis by reducing Bax and cleaved caspase-3/9 levels and enhancing the
antiapoptotic protein Bcl-2 in the cells [46].
Nitric oxide (NO) is a reactive radical particle formed by oxidizing l-arginine guanidino nitrogen
by NO synthase (NOS), and it is essential for the host defense strategy against several pathogens,
such as parasites, fungi, viruses, and bacteria [
47
]. However, too much NO production can lead to the
development of inflammatory disorders such as autoimmune disease and rheumatoid arthritis [
48
].
Although the exact mechanisms for controlling the anti-inflammatory activity of E. brevicornum
have not yet been understood, Yuk et al. [
49
] showed that E. brevicornum water extract suppresses
lipopolysaccharide-stimulated (LPS) proinflammatory mediator production, such as IL-10, IL-3, IL-12,
IL-17, and NO in RAW264.7 rodent mononuclear phagocyte systems. These findings demonstrate that E.
brevicornum possesses anti-inflammatory eects and may change macrophage-mediated inflammatory
activation. Notably, Shindel et al. [
13
] reported that both Western blot and immunohistochemistry
assays showed substantially higher positivity for nNOS expression and calponin in penile tissues
of all E. brevicornum-treated animals. Histopathological assessment of testicular slices related to
control animals revealed typical, unblemished seminiferous tubules with normal lining epithelium
and stratified seminiferous epithelium with typical interstitial connective tissue. In comparison,
TAM (50 mg/kg/day b.wt.)-treated rats demonstrated necrosis and seminiferous tubules degeneration,
sloughing of the stratified seminiferous epithelium, spermatogenic cells, and Sertoli cells. There was
exceptional necrosis of some spermatogenic cells, spermatogonia, spermatocytes, and spermatids that
appeared with a pyknotic nucleus. Concerning the seminiferous tubules, focal disorder, irregularity,
increased spaces between the tubules, and detached basement membrane of some tubules, as well as
shrunken seminiferous tubules, were also detected.
Elnaga et al. [
50
] could not agree more as they noticed spermatogenic population depletion in
most tubules, focal disorganization, anomaly, expanded spaces between the tubules, and detached
basement membrane of some tubules, as well as shrunken seminiferous tubules. They documented
that spermatogenic cells of animals that received 40 mg/kg/day TAM daily for 6 successive weeks were
replaced by vacuoles, whereas other seminiferous tubules lack sperms and spermatids. Moreover, they
detected darkly stained nuclei of spermatogonia and spermatocytes. They also observed vacuolated
homogenous acidophilic material of the interstitium, Pyknotic nuclei, binucleated cells, vacuolated
spermatogonia, and spermatocytes, as well as deeply stained nuclei of Leydig cells. Ghoneim et al. [
51
]
revealed that TAM treatment for 4 weeks showed distinct histological changes. The seminiferous
tubules existed in abnormal shape, broadly isolated from each other, and some tubules had ruptured
without spermatozoa. Numerous vacuoles were observed between the multinucleated giant cells,
apoptotic cells, and spermatogenic cells. Several inflammatory cells were shown in the intertubular
tissues. In TAM (40, 80, 120, and 160 mg)-treated groups, severe testicular-diused degradation,
with multiple spermatocytes and spermatid giant cell formation, unaccompanied by spermatogenesis,
was detected. Spermatocytes were mainly necrotic in lesser dosed samples with a recovery attempt,
and inexhaustible cores have been shown by the regenerated cells. Micro spermatozoa and dystrophic
calcification were found inside the lumen of seminiferous tubules. Histopathology injuries were
aected by the overhaul dose of TAM before testicular tissue has been calcificated [52].
Antibiotics 2020,9, 318 12 of 15
The current study examines the eects of chronic administration of TAM (50 mg/kg/day b.wt.)
and E. brevicornum at a dose of 2.43 g/kg/day for 130 consecutive days and TAM at 50 mg/kg/day for
65 days, followed by concurrent usage of TAM and E. brevicornum at doses of 0.81 and 2.43 g/kg/day
for 65 days, respectively. E. brevicornum, at a dose of 2.43 g/kg/day, illustrated normal testicular
parenchyma, as shown in the control group. Tramadol treatment followed by concomitant use of TAM
and E. brevicornum, at a dose of 0.81 g/kg/day, showed that testicular parenchyma appeared intact but
with a slight pathological lesion within the epithelium of the seminar tubular epithelium. Furthermore,
there were some destruction and sloughing of spermatogenic cells. Tramadol administration, followed
by concurrent use of TAM and higher doses of E. brevicornum, showed that the testicular formation of
parenchyma appeared to be homogeneous and healthy, apparently normal, with a typical arrangement
of stratified seminiferous epithelium, and spermatogenic and Sertoli cells. Likewise, Leydig cells are
claimed to be healthy and intact, without any pathological lesions.
5. Conclusions
In conclusion, the present investigation reveals the significant potential role of E. brevicornum
extract in alleviating the negative impacts of tramadol in male Wister rats by regulating antioxidant
functioning and hormonal and apoptotic markers. E. brevicornum extract application prevented
oxidative damage to tramadol by upregulating the NO level and antioxidant-functioning, with
a subsequent decline in the oxidant status. As a result, E. brevicornum extract may cause a protective
action and enhancement of testicular tissue function, increasing sperm count and motility in the rat
model of male infertility. The present study suggests that the supplementation of E. brevicornum extract
may improve spermatogenesis by reducing oxidative stress in the luteinizing hormone-releasing
hormone agonist-induced rat model of male infertility. Therefore, E. brevicornum extract application
can be exploited to improve anti-infertility agents by its active involvement in key regulatory functions.
Author Contributions:
Conceptualization, A.S.A. and M.A.K.; methodology, A.I.A., S.I.S., D.M.K., S.Y.A.,
and A.S.A.; software, D.M.K., M.A.K., and S.I.S.; validation, D.M.K., M.A.K., S.Y.A., and A.I.A.; formal analysis
A.S.A., M.A.K., and G.E.-S.B.; investigation, G.E.-S.B., M.A.K., S.M.E.-d., A.S.A., and D.M.K.; data curation,
A.S.A. and D.M.K.; writing—original draft preparation, A.S.A., D.M.K., and S.M.E.-d.; writing—review and
editing, D.M.K., A.S.A., A.I.A., S.Y.A., S.M.E.-d., and G.E.-S.B.; visualization, A.S.A., M.A.K., D.M.K., S.I.S., A.M.B.,
S.M.E.-d., A.I.A., and M.A.K.; supervision, A.S.A., D.M.K., G.E.-S.B., S.M.E.-d., A.M.B., S.Y.A., and S.I.S.; funding
acquisition, M.A.K., A.I.A., A.M.B., S.I.S., S.Y.A., and S.Y.A. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Acknowledgments:
The authors are grateful to the Deanship of Scientific Research, King Saud University,
for funding through the Vice Deanship of Scientific Research Chairs.
Conflicts of Interest: The authors declare no conflict of interest.
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article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
... Presently, antioxidant supplementation is one of the most common methods of male infertility treatment. TCMs with antioxidant functions, such as Epimedium, is increasingly being used to ameliorate male infertility caused by high levels of OS [45][46][47][48][49][50]. Nevertheless, due to the complexity of TCM components, most traditional pharmacological studies have focused mainly on the beneficial effect of a single component, such as flavonoids and icariin in YYH. ...
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Oxidative stress (OS) is one of the primary factors leading to male infertility. Oral administration of antioxidants has thus far been found to significantly improve the quality of human sperm. Therefore, antioxidant treatment has become the consensus among international experts on male infertility. In this study, peroxisomal biogenesis factor 3 ( Pex3 )-knockout (KO, −/−) mice were used as a model to compare the efficacy of three types of traditional Chinese medicine (TCM) granules (Epimedium [YYH], Cuscuta [TSZ], and Rhodiola [HJT]) for male reproductive function rescue. YYH was revealed to be the best and exerted a rescue effect on Pex3 −/− mice with spermatogenesis defects. In addition, YYH prominently reduced ROS levels in the testes, inhibited DNA oxidative damage in spermatogenic cells, promoted the proliferation of spermatogenic cells, and inhibited apoptosis in Pex3 −/− male mice. Furthermore, the mechanism by which YYH ameliorated dyszoospermia was confirmed via the establishment of cyclin-dependent kinase inhibitor 2 A (P16Ink4a)-KO mice. Specifically, Pex3 −/− mice produced elevated amounts of ROS, which damaged germ cell DNA and further activated the signaling pathway of the cell senescence regulatory protein P16-CDK6, resulting in cell cycle arrest and eventually contributing to spermatogenesis dysfunction. YYH supplementation partially corrected the associated phenotype in gene KO mice by affecting P16 expression levels, thus improving the reproductive outcome to a certain extent.
... FIP administration caused oxidative damages in the testicular tissues, as demonstrated by increased levels of MDA and NO tissue with reduced activity of antioxidant enzymes such as GPx and SOD [32]. Exposure to xenobiotics including pesticides results in increased production of ROS with a recurrent cellular damage of proteins, DNA, and lipids [33,34]. Furthermore, ROS could influence the NO's bioavailability from its biosynthesis to complete removal [35]. ...
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... FIP administration caused oxidative damages in the testicular tissues, as demonstrated by increased levels of MDA and NO tissue with reduced activity of antioxidant enzymes such as GPx and SOD [32]. Exposure to xenobiotics including pesticides results in increased production of ROS with a recurrent cellular damage of proteins, DNA, and lipids [33,34]. Furthermore, ROS could influence the NO's bioavailability from its biosynthesis to complete removal [35]. ...
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Tramadol is widely used as a pain reliever; stimulant and it may delay ejaculation. But chronic tramadol users have adverse side effects. Aim: To clarify the worst effect of tramadol hydrochloride on male fertility and its role in neurodegeneration disorders due to oxidative stress in spite of indeed its use as ejaculation delaying. Materials and methods: This experimental study was performed upon 30 adult wistar rats. They were purchased from the National Research Centre Cairo, Egypt. These rats were divided into three groups (n=10), Group I, rats received vehicle and served as normal control. Group II, rats received low dose of tramadol (20 mg/Kg body weight/ day). Group III, rats received high dose of tramadol (50 mg/Kg body weight/day) for 12 weeks. Results: The results illustrated that tramadol in both low and high doses ingestion lead to weight loss, elevated Nitric Oxide Synthase (NOS) expression and plasma peroxidation value (MDA) but reduced Glutathione (GSH) and Superoxide Dismutase (SOD) in plasma were significantly decreased. Furthermore, plasma Monoamine Oxidase (MAO) was increased leading to serotonin (5-HT) and dopamine decrease in brain tissues proteins (p˂0.001). Plasma Testosterone level, sperm content and sperm vitality were significantly decreased (p˂0.001). Furthermore, hematoxylin and eosin (H&E) stain of the brain tissues showed few purkinje cells and the cells of granular layer were widely separated in the rats of low dose of tramadol group, but there was shrunken purkinje cells, some cells had pykinotic nuclei and majority of the cells of granular layer were non-nucleated, few of them had pykinotic nuclei in the rats of high dose of tramadol when compared to normal brain tissues of normal control rats. Conclusion: Long term tramadol administration developed oxidative stress, neurodegenerative disorders and male infertility.
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