Journal of Experimental Pharmacology
Dovepress
open access to scientific and medical research
ORIGINAL RESEARCH
Journal of Experimental Pharmacology downloaded from https://www.dovepress.com/ by 35.153.143.85 on 16-May-2021
For personal use only.
Open Access Full Text Article
Assessment of Reproductive Toxicity of
Hydroethanolic Root Extracts of Caesalpinia
benthamiana, Sphenocentrum jollyanum, and Paullinia
pinnata
This article was published in the following Dove Press journal:
Journal of Experimental Pharmacology
Mavis Baffoe 1
George Koffuor 1,†
Agyapong BaffourAwuah 1
Lorraine Sallah 2
1
Department of Pharmacology, Faculty of
Pharmacy and Pharmaceutical Sciences,
Kwame Nkrumah University of Science
and Technology, Kumasi, Ghana;
2
Department of Physiology, School of
Medicine and Dentistry, Kwame
Nkrumah University of Science and
Technology, Kumasi, Ghana
†
Prof. George Koffuor passed away on
February 5, 2021.
Correspondence: George Koffuor
Department of Pharmacology, Faculty of
Pharmacy and Pharmaceutical Sciences,
Kwame Nkrumah University of Science
and Technology, Kumasi, Ghana
Tel +233 27-740-0312
Email gkoffuor@yahoo.com
Plain Language Summary
In Ghana, the use of herbal aphrodisiacs by majority of young men is gradually becoming the
order of the day. This study therefore assessed the safety of the use of root extracts of three
common herbal aphrodisiacs: Caesalpinia benthamiana, Sphenocentrum jollyanum, and
Paullinia pinnata. Results revealed that chronic use of these herbals as aphrodisiacs had
223
submit your manuscript | www.dovepress.com
Journal of Experimental Pharmacology 2021:13 223–234
DovePress
© 2021 Baffoe et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.
php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the
work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For
permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php).
http://doi.org/10.2147/JEP.S283557
Powered by TCPDF (www.tcpdf.org)
Purpose: Male sexual dysfunction negatively affects an individual’s quality of life and thus
its of prime public concern, hence the need to boost reproductive abilities in such individuals.
This study assessed the effect of hydroethanolic root extracts of Caesalpinia benthamiana
(CBRE), Sphenocentrum jollyanum (SJRE), and Paullinia pinnata (PPRE), commonly used
as aphrodisiacs in Ghana, using male Sprague-Dawley rats.
Methods: Plasma testosterone, follicle-stimulating hormone, and luteinizing hormone were
assayed in grouped rats treated orally with 1 mL/kg normal saline, 50 mg/kg monosodium
glutamate (MSG), and 100, 300, or 1000 mg/kg CBRE, SJRE, and PPRE, respectively, for
60 days. Epididymis and testis weights were determined. Semen was assessed on spermatozoa count, motility, and morphology. Malonyladehyde formation in lipid-peroxidation assay
and histological examinations were performed to assess pathological changes in testes.
Testicular testosterone was also assayed.
Results: While MSG, CBRE, SJRE, and PPRE treatments did not result in significant
reduction (p>0.05) in plasma testosterone, there was significant reduction (p≤0.05
−0.0001) in plasma luteinizing hormone, and follicle-stimulating hormone. The combined
mean wet weights of epididymides and testes of all treated groups did not vary significantly
(p>0.05) from the control. There was significant reduction (p≤0.0001) in sperm motility and
count, with significant morphological changes (p≤0.05–0.001), ie, bent necks, tails, and
midpieces, and multiple anomalies in the spermatozoa in extract and MSG-treated groups.
There was also significant (p≤0.0001) reduction in testicular testosterone among all treatment
groups.
Conclusion: Hydroethanolic CBRE, SJRE, and PPRE were found to have detrimental
effects on reproductive function with prolonged usage and thus may not be safe to use in
healthy males who intend to reproduce.
Keywords: plasma follicle-stimulating hormone, plasma luteinizing hormone, sperm
anomalies, sperm motility, testicular testosterone, monosodium glutamate, herbal
aphrodisiacs
Baffoe et al
detrimental effects on semen quality and thus could negatively
affect the capability of reproductive-age males to produce offspring, even though they enhance copulation and pleasure.
Journal of Experimental Pharmacology downloaded from https://www.dovepress.com/ by 35.153.143.85 on 16-May-2021
For personal use only.
Introduction
Issues of male sexual dysfunction and reproductive inability are of prime public concern and an important part of an
individual’s quality of life, and thus people continue to
search for substances they can use to heighten their sexual
abilities. In the Ghanaian society, it is often an ignominy
for a man to be identified with sexual weakness; therefore,
sexual vitality is of great necessity to many who eventually use aphrodisiacs.1–3 The introduction of varieties of
herbal aphrodisiacs, driven in part by heavy advertising,
has caused a flutter of public attention4 toward their use
when the individual does not even have sexual
dysfunction.5 There are increasing numbers of people
who may be susceptible to adverse drug effects, should
there be any. The risks of use and abuse of aphrodisiacs
are also high, as these preparations are now sold in supermarkets, fuel stations, market places, transport stations,
commercial centers, and communication centers, among
others, without supervision of any kind.
There are claims that some of these drugs could cause
impotence, kidney failure, and problems with hearing and
sight, among others. Some aphrodisiacs have also been presumed to cause male fertility problems. For example, studies
conducted on Mondia whitei, a herbal aphrodisiac, have
revealed that chronic administration resulted in antispermatogenic and antifertility effects in male albino rats.6 A market
survey in Ghana conducted by the authors revealed
Caesalpinia benthamiana, Sphenocentrum jollyanum, and
Paullinia pinnata to be among herbs popularly recommended
by herbal medical practitioners and used as aphrodisiacs in
Ghana. These plants have also been indicated as such in the
literature.
C. benthamiana and Mezoneuron benthamianum (family
Caesalpiniaceae), locally called akoo boree, are widespread in
West and Central Africa and have been traditionally used in the
management of erectile dysfunction, dysentery, urethral discharge, skin diseases and wounds. These species have antibacterial, vasorelaxation, and antioxidant properties.7,8 S.
jollyanum (family Menispermaceae) is a shrub native to the
tropical forest zones of Sierra Leone, Nigeria, Ghana, Ivory
Coast, and Cameroon.9 It is known locally as aduro kokoo (red
medicine), okramankote (dog’s penis), and krakoo among the
Akan and Asante tribes of Ghana.10 Although the root of this
plant is widely used as a male aphrodisiac, several studies have
224
Powered by TCPDF (www.tcpdf.org)
submit your manuscript | www.dovepress.com
DovePress
Dovepress
indicated that S. jollyanum has antioxidant, antiangiogenic,
anti-inflammatory, antipyretic, antinociceptive, antitumor,
antiviral, laxative, stomachic, and tonic activity.11,12 P. pinnata
(family Sapindaceae) locally known as twantini, is an African
tropical plant whose roots and leaves are used in traditional
medicine for many purposes (wound healing, treatment of
dysentery, arthritis, malaria), especially for erectile
dysfunction.13,14
It is thus required that studies be carried out on these
herbal aphrodisiacs to determine possible adverse effects
associated with their use to get the public and health policy
makers informed to enable them to make decisions that will
help regulate the exposure of individuals to such drugs.15
This study thus assessed the toxicity of hydroethanolic
root extracts of C. benthamiana, S. jollyanum, and P.
pinnata in male Sprague-Dawley rats.
Methods
Choice and Collection of Herbal
Aphrodisiacs
A total of 20 medicinal herb sellers (17 men and three
women) aged 35–60 years in the herbal market section of
the Kumasi Central Market, Kejetia, in the Ashanti region
of Ghana, were interviewed in their local language using
unstructured questionnaires on the patronage of herbal
aphrodisiacs. The sellers gave their knowledge on herbs
reported by users to boost libido, enhance erection, and
increase male virility. From the interactions, information
on local names of plants with aphrodisiac properties, the
plant part used, their form of preparation and usage, and
their mode of activity, among other things, were obtained.
The three most frequently mentioned plants — C.
benthamiana, S. jollyanum, and P. pinnata — were then
selected for this study (Table 1).
Table 1 Eight Frequently Purchased Herbal Aphrodisiacs from
the Kumasi Central Market, in the Ashanti Region of Ghana
Local
Name
Part
Used
Frequency
Caesalpinia benthamiana
Akoo boree
Root
8
Sphenocentrum jollyanum
Kraman koti
Root
8
Paullinia pinnata
Mondia whitei
Twantini
Asaase wham
Root
Root
7
7
Guilandina bonduc
Oware nhyini
Root
6
Chrysophyllum beguei
Khaya senegalensis
Atadwe dua
Mahoghany
Root
Root
5
5
Vitex grandifolia
Dunsikro
Root
4
Journal of Experimental Pharmacology 2021:13
Baffoe et al
Dovepress
Journal of Experimental Pharmacology downloaded from https://www.dovepress.com/ by 35.153.143.85 on 16-May-2021
For personal use only.
Preparation of Hydroethanolic Root Extracts of
Selected Plants
Roots of C. benthamiana, S. jollyanum, and P. pinnata
were sun-dried and crushed into coarse powder using a
hammer-mill (STEDMAN 20×18, USA). The powders (5
kg each) were extracted separately with 70% v:v ethanol
in a soxhlet apparatus for 72 hours. The filtrates obtained
were each concentrated at a low temperature in a vacuum
rotary evaporator (CMRE-10110) under reduced pressure
to obtain a thick mass that was dried in a hot oven
(PT2010) at 40°C for 24 hours. Root-extract yields of
10.2%, 11.7%, and 7.4% were obtained for S. jollyanum,
C. benthamiana, and Paullinia pinnata, respectively.
These extracts were labeled CBRE, SJRE, and PPRE,
respectively, and kept in a desiccator at room temperature
for usey.
Experimental Animals and Husbandry
Male Sprague Dawley rats weighing 150–230 g were
purchased from the Centre for Plant Medicine Research,
Akuapem-Mampong, in the Eastern Region of Ghana and
kept in the Animal Facility of the Department of
Pharmacology, KNUST, Kumasi, Ghana. The animals
were housed in stainless steel cages (36×50×20 cm) with
wood shavings as bedding, maintained under normal
laboratory conditions (24°C–28°C, relative humidity
60%–70%, and ambient light–dark cycle), and given free
access to a solid-pellet diet (Agricare, Kumasi, Ghana) and
water throughout the study. All animals used in this study
were treated in accordance with the US National Institutes
of Health Guidelines for the Care and Use of Laboratory
Animals (Department of Health and Human Services publication 85–23, revised 1985).
Establishing Experimental Doses
To establish favorable doses of the extracts to be used, an
experiment previously described by Irwin16,17 was performed. Normal healthy rats were put into three groups
(n=5) and treated with 50, 500, or 1,000 mg/kg CBRE
daily, observed and assessed for behavior specifically
related to neurotoxicity, central nervous systemstimulation
or depression, autonomic function, and lethality at 30
minutes, 1, 2, 3, and 24 hours, and subsequently daily
for 2 weeks. The same test was done for SJRE and
PPRE. The dose for monosodium glutamate (MSG; the
reference drug for reproductive toxicity in this study) was
selected based on previous research.18
Journal of Experimental Pharmacology 2021:13
Powered by TCPDF (www.tcpdf.org)
Assessing Reproductive Toxicity of SJRE,
PPRE, and CBRE
Experimental Grouping and Dosing of Animals
Experimental animals were put into eleven groups of
seven animals each. Group A (vehicle-treated control)
was administered 1 mL/kg normal saline. Group B (positive control) was treated with 50 mg/kg MSG to induce
oxidative stress in various body organs, including the
testes, while groups C, D, and E were dosed with 100,
300, and 1,000 mg/kg CBRE, respectively. Groups F, G,
and H were treated with 100, 300, and 1,000 mg/kg SJRE,
respectively, and groups I, J, and K 100, 300, and 1,000
mg/kg PPRE, respectively. Treatments were given orally
and daily over a period of 60 days. Blood samples were
taken from animals in each group by cardiac puncture into
well-labeled gel-separation Eppendorf (Vacutainer) tubes
for plasma-hormone assays. The animals were humanely
killed after anesthetization with phenobarbitol (50 mg/kg
intraperitoneally) and testes and epididymides removed for
weight determination, semen analysis, and testicular testosterone assays.
Plasma-Hormone Assays
Blood samples in the Eppendorf tubes were centrifuged at
2,500 rpm for 5 minutes using a Wisperfuge 1384 (Tamson
Instruments) at 25°C to obtain plasma samples, which
were assayed for testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) with analytical
grade reagents (Syntron Bioresearch) using ELISA.19
Testosterone Assays
A 50 µL measure each of calibrator, control, and test
samples was placed in the corresponding labeled wells
on a microtiter plate (in triplicate) followed by addition
of 100 µL conjugate solution. The solution was properly
mixed and incubated on a microplate shaker running at
approximately 200 rpm for 1 hour at 25–28°C. Each well
was washed properly with 300 µL diluted buffer and the
plate dried by tapping against absorbent paper. A 150 µL
measure of tetramethylbenzidine substrate was added to
each well and incubated for 15 minutes at room temperature before the addition of 50 µL stop solution.
Absorbance in each well was read at 450 nm within 20
minutes after addition of the stop solution using spectrophotometry (LabTech advanced microprocessor UV-vis
single beam 295). A graph of absorbance versus concentration of the calibrator was plotted and used to extrapolate
the actual amount of the hormone in the test samples.
submit your manuscript | www.dovepress.com
DovePress
225
Journal of Experimental Pharmacology downloaded from https://www.dovepress.com/ by 35.153.143.85 on 16-May-2021
For personal use only.
Baffoe et al
Follicle-Stimulating Hormone and Luteinizing Hormone
Assays
Adequately coated microtiter plate wells were selected
to run 0, 25, 50 and 100 mIU/mL for FSH calibrators,
control, and test samples in triplicate. A 50 µL quantity
of each sample was pipetted into the corresponding
coated well with addition of 200 µL of the enzyme–
antibody conjugate solution. The solution was mixed
appropriately and incubated at room temperature for 45
minutes. Wells were then washed with a buffer and
deionized water and decanted. Substrate-chromogen
solutions (100 µL) were added to each well, mixed
thoroughly, and incubated for 15 minutes at room temperature. Shortly after, 100 µL 1 N H2SO4 was added to
each well and appropriately mixed. Absorbance in each
well was read at 450 nm spectrophotometrically. A
graph of absorbance versus concentration of the calibrator was plotted and used to extrapolate the actual
amount of the hormone in the test samples. A similar
procedure was used for LH assays.
Determination of Epididymis and Testis Weight
Epididymides and testes were dissected from treated rats
and weighed using a top-loading balance (Grand G).
Organ weight:body weight ratios were calculated and compared to the control groups.
Semen Analysis
Semen samples from rats in each treatment group were
collected by humanely killing the rat, dismembering the
epididymis, and squeezing its content into 0.5 mL normal
saline. As soon as liquefaction of the semen had occurred
(within 15–30 minutes of collection), sperm count, motility, and abnormalities were assessed.
Estimation of Sperm Count
This was carried out using the modified method of Ekualo
et al.20 Semen samples were mixed thoroughly and drawn
to the 0.5 mark on the pipette. This was then diluted to the
11 mark on the pipette using semen-diluting fluid (sodium
bicarbonate 5 g, formalin 1 mL, and distilled water to 100
mL). A tissue-free aliquot was loaded into a Neubauer
hemocytometer (depth 1/10) and allowed to settle for 2
minutes. Five counts were performed for each sample, and
the mean calculated and taken as the mean count for each
male rat. The sperm count was estimated:
sperm count/mL = n × 40,000
226
Powered by TCPDF (www.tcpdf.org)
submit your manuscript | www.dovepress.com
DovePress
Dovepress
where n = total number of sperm cells in five cells of
the cytometer.
Evaluation of Sperm Motility
Liquefied semen samples from the treatment groups were
placed on a glass slide, covered with a coverslip ringed
with petroleum jelly, and viewed under microscopy
(BX51, China). Assessment was made from a minimum
of five microscopic fields to evaluate sperm motility on at
least 200 spermatozoa for each rat. Sperm motility was
analyzed for progressive motile sperm, unprogressive
motile sperm, and immotile sperm, distinguished by the
movement of the sperm cells.21
Sperm Morphology
A fraction of each of the sperm suspensions was examined
by placing the solution (20:1) on a glass slide, drying, in
air, and fixing by heat. Chloramine solution (1%) was
added for several minutes to remove excess mucus. The
fixate was then washed and dried by blotting on filter
paper. It was stained for 5 minutes (Ziehl–Neelsen carbol
fuchsin two parts, concentrated alcohol solution of eosin
one part, and 95% alcohol one part), washed with water,
and counterstained with Loeffler’s methylene blue for a
few seconds. The slide was then washed, dried, and examined under oil immersion for percentage abnormalities in
every 200 spermatozoa seen on each slide, and five airdried smears were prepared on glass slides for each
sample.22
Testicular Testosterone Assay
Testes of animals of the treatment groups were collected
into glass containers containing 1 g/mL Tris phosphate
buffer, pulverized, and allowed 30 minutes to settle.
Supernatants were then collected into separately labeled
sample tubes and centrifuged (Wisperfuge 1384) at
2,500 rpm for 5 minutes at 25°C to obtain the physiological samples to be assayed for testosterone using
analytical grade reagents (Syntron Bioresearch) using
ELISA.19
Assessment of Pathological Changes in
Testes
MDA Formation in Lipid-Peroxidation Assay
Levels of malonyladehyde (MDA) formation in tissue
were determined as per Tüközkan et al23 with slight modification. To 3 mL reagent solution (3 mL 20% TCA
containing 0.5% thiobarbituric acid [TBA]), 1 mL homogenate was added in a test tube. This was heated at 95°С
Journal of Experimental Pharmacology 2021:13
Dovepress
Journal of Experimental Pharmacology downloaded from https://www.dovepress.com/ by 35.153.143.85 on 16-May-2021
For personal use only.
for 30 minutes, cooled immediately, and centrifuged at
5,000 g for 10 minutes. Absorbance was firstly read at
532 nm and then 600 nm to correct for aspecific absorbance. The molar extinction coefficient of the MDA–TBA
abduct, 155 mM−1 cm−1, was used to estimate the levels
of MDA:
Nmol
MDA
Absorbance at532nm Absorbance at600nm
protein¼
� 106
mg
155 � totalprotein
Histological Examination
Tissue portions from the epididymides and testes were
utilized for histological examinations. Tissue samples
were fixed in 10% buffered formalin (pH 7.2), dehydrated through a series of ethanol solutions, embedded
in paraffin, and routinely processed for histological analysis. Sections of 2 μm thickness were cut and stained
with H&E for examination. The stained tissue samples
were observed through an Olympus microscope (BX51)
and photomicrographs taken with a charge-coupled
camera.
Effect of Extracts on Plasma Reproductive
Hormones
Treatments with extracts generally did not result in significant reduction (p>0.05) in plasma testosterone, except
for groups treated with 100 mg/kg PPRE, which caused a
reduction (p≤0.05). The various treatments, however,
resulted in significant reductions (p≤0.05–0.0001) in
plasma LH at the various doses, except for the 300 and
1,000 mg/kg CBRE treatments. CBRE, SJRE, and PPRE
treatments showed significant reductions in serum FSH
(p≤0.05–0.0001) (Figure 1).
Statistical Analysis
All data collected on sperm and hormonal assays are
presented as means ±SEM and subjected to one-way
ANOVA using GraphPad Prism 6.0 (GraphPad Software,
San Diego, CA, USA). Statistical significance between
treatments and control was established using Dunnett’s
multiple-comparison post hoc test using a 95% confidence
limit. p≤0.05 represented significant variation between
variables.
Results
Choosing the Herbal Aphrodisiacs
C. benthamiana, S. jollyanum, and P. pinnata were
selected for this study based on their frequency of mention
in the questionnaire as has having been used successfully
by the populace in enhancing libido, inducing erection,
and improving male virility (Table 1).
Roots of C. benthamiana, S. jollyanum, and P. pinnata
were bought from the market and authenticated by Dr GH
Sam, a lecturer and botanist of the Department of Herbal
Medicine, KNUST, Kumasi, Ghana. Samples of these
plant parts with voucher numbers KNUST/HM1/2017/
L011, KNUST/HM1/2017/L012, and KNUST/HM1/
2017/L013, respectively, have been deposited at the herbarium of the Department of Herbal Medicine, KNUST,
Kumasi, Ghana.
Journal of Experimental Pharmacology 2021:13
Powered by TCPDF (www.tcpdf.org)
Baffoe et al
Figure 1 Plasma testosterone, luteinizing hormone (LH), and follicle-stimulating
hormone (FSH) concentrations in rats treated with normal saline (NS), monosodium glutamate (MSG), and hydroethanolic root-bark extracts of Caesalpinia
benthamiana (CBRE), Sphenocentrum jollyanum (SJRE), and Paullinia pinnata
(PPRE) for 60 days. Data presented as group means ± SEM, n=7. Significant
differences between treatment and control: ns, p>0.05; *p≤0.05; **p≤0.01;
***p≤0.001; ****p≤0.0001 (one-way ANOVA followed by Dunnett’s multiplecomparison test).
submit your manuscript | www.dovepress.com
DovePress
227
Baffoe et al
Dovepress
Epididymis and Testicular Weight
Journal of Experimental Pharmacology downloaded from https://www.dovepress.com/ by 35.153.143.85 on 16-May-2021
For personal use only.
The combined mean wet weights of left epididymides and
left testes of the treated groups did not vary significantly
(p>0.05, Table 2).
Semen Analysis
Sperm Count
There were significant reductions (p≤0.05–0.0001) in
sperm count in all treatment groups compared to the vehicle-treated group (Figure 2).
Sperm Motility
There was a significant reduction (p≤0.0001) in sperm
motility in the extract-treated animals compared to the
vehicle-treated groups (Figure 3).
Figure 2 Sperm counts of rats treated with normal saline (NS), monosodium glutamate
(MSG), and various doses of hydroethanolic root-bark extracts of Caesalpinia benthamiana
(CBRE), Sphenocentrum jollyanum (SJRE), and Paullinia pinnata (PPRE) for 60 days. Data
presented as means ± SEM, n=7. Significant differences between treatment and control:
*p≤0.05; ***p≤0.001; ****p≤0.0001 (one-way ANOVA followed by Dunnett’s multiplecomparison test).
Sperm Morphology
There were varied significant morphological changes
(p≤0.05–0.001) in spermatozoa of animals in all treatment
groups compared to vehicle-treated groups. The dominant
changes occurring in animals treated with CBRE were
bent tails and midpieces. PPRE-treated animals showed
dominant bent necks, tails, and midpieces. SJRE-treated
animals had significant levels of all the deformities
recorded. Treatments showed significant levels (p>0.05)
of multiple anomalies and headless tails (Figure 4).
Assessment of Pathological Changes
Lipid Peroxidation
The effect of each of the extracts on lipid peroxidation of
testes of treated rats was measured as the amount of MDA
(nmol/mg of tissue protein) formed. There were significant
(p≤0.001) increases in MDA concentrations in MSG-,
Figure 3 Percentage sperm motility of rats treated with normal saline (NS),
monosodium glutamate (MSG), and various doses of hydroethanolic root-bark
extracts of Caesalpinia benthamiana (CBRE), Sphenocentrum jollyanum (SJRE), and
Paullinia pinnata (PPRE) for 60 days. Data presented as means ± SEM, n=7. Significant
differences between treatment and control: *p≤0.05; ***p≤0.001; ****p≤0.0001
(one-way ANOVA followed by Dunnett’s multiple-comparison test).
1,000 mg/kg CBRE–, 300 mg/kg SJRE–, and 1,000 mg/
kg PPRE–treated rats. MDA concentration was highest in
rats treated with 1,000 mg/kg CBRE (Figure 5).
Table 2 Organ Weight to Body Weight Ratio of Sprague-Dawley Rats Treated with Normal Saline (NS), Monosodium Glutamate (MSG), and
various Concentrations of Hydroethanolic Root-Bark Extracts of C. benthamiana (CBRE), Sphenocentrum jollyanum (SJRE), and Paullinia pinnata
(PPRE)
Weight of Testes
Testes: Body Weight Ratio
Weight of Epididymides
Epididymis: Body Weight Ratio
2 mL/kg NS
50 mg/kg MSG
1.725±0.14
1.530±0.03ns
0.0063±0
0.0062±0ns
0.495±0.05
0.345±0.07ns
0.0015±0.0002
0.0017±0.0001ns
100 mg/kg CBRE
1.288±0.32ns
0.0073±0
300 mg/kg CBRE
1,000 mg/kg CBRE
1.445±0.10ns
1.428±0.06ns
0.0069±0ns
0.0064±0ns
0.450±0.05ns
0.460±0.02ns
0.0019±0.0003ns
0.0019±0ns
100 mg/kg SJRE
1.478±0.03ns
0.0071±0ns
0.412±0.01ns
0.0020±0.0001ns
300 mg/kg SJRE
1,000 mg/kg SJRE
1.693±0.13ns
1.683±0.15ns
0.0065±0ns
0.0069±0ns
0.527±0.02ns
0.046±0.02ns
0.0017±0.0001ns
0.0021±0.0001ns
100 mg/kg PPRE
1.713±0.04ns
0.0074±0
0.465±0.02ns
0.0021±0.0001ns
300 mg/kg PPRE
1,000 mg/kg PPRE
1.688±0.08ns
1.305±0.11ns
0.0068±0ns
0.0077±0ns
0.497±0.01ns
0.407±0.05ns
0.0018±0.0002ns
0.0023±0ns
ns
ns
0.3575±0.08
ns
0.0019±0.0003ns
Notes: Results presented as means ± SEM, n=7. Treatment versus control: nsp>0.05 (one-way ANOVA using Dunnett’s multiple-comparisons test).
228
Powered by TCPDF (www.tcpdf.org)
submit your manuscript | www.dovepress.com
DovePress
Journal of Experimental Pharmacology 2021:13
Baffoe et al
Journal of Experimental Pharmacology downloaded from https://www.dovepress.com/ by 35.153.143.85 on 16-May-2021
For personal use only.
Dovepress
Figure 4 Sperm morphology at the end of 60 days of treatment with normal saline (NS), monosodium glutamate (MSG), and various concentrations of the hydroethanolic
root-bark extracts of Caesalpinia benthamiana (CBRE), Sphenocentrum jollyanum (SJRE), and Paullinia pinnata (PPRE). Values are means ± SEM, n=7. ns, p>0.05; *p≤0.05;
**p≤0.01, ***p≤0.001; ****p≤0.0001 (one-way ANOVA followed by Dunnett’s multiple-comparison test).
Journal of Experimental Pharmacology 2021:13
Powered by TCPDF (www.tcpdf.org)
submit your manuscript | www.dovepress.com
DovePress
229
Baffoe et al
Dovepress
Journal of Experimental Pharmacology downloaded from https://www.dovepress.com/ by 35.153.143.85 on 16-May-2021
For personal use only.
embedded in connective tissue. MSG treatment resulted
in necrosis of connective tissue. High doses (1,000 mg/kg)
of extracts resulted in disorganization of epididymal cells,
and although there were greatly convoluted tubules
embedded in connective tissue, there was very scanty
spermatozoa (Figure 8, A–E).
Discussion
Figure 5 Malondialdehyde concentration indicative of lipid peroxidation of testes
after treatment with normal saline (NS), monosodium glutamate (MSG), and various concentrations of hydroethanolic root-bark extracts of Caesalpinia benthamiana
(CBRE), Sphenocentrum jollyanum (SJRE), and Paullinia pinnata (PPRE) for 60 days.
Data presented as group means ± SEM, n=7. Significant differences between treatments and control: ns, p>0.05; *p≤0.05, ***p≤0.001 (one-way ANOVA using
Dunnett’s multiple-comparison test).
Effect of Extracts on Testicular
Testosterone
There were significant (p≤0.0001) reductions in testicular
testosterone for all treatments compared to the normal
saline–treated animals (Figure 6).
Histological Studies
Histological assessment of control and drug-treated rats
indicated that testes of normal ratsshowed well-layered
seminiferous tubules with different stages of spermatogenic cells. Treatment with MSG and high doses (1,000
mg/kg) of CBRE, SJRE, PPRE caused mild–severe
degrees of seminiferous tubule atrophy, reduction in interstitial Leydig cells, and destruction (Figure 7, A–E).
Histological assessment of epididymides showed normal epididymides with greatly convoluted tubules
Figure 6 Testicular testosterone concentration inf rats treated with normal saline
(NS), monosodium glutamate (MSG), and various concentrations of hydroethanolic
root-bark extracts of Caesalpinia benthamiana (CBRE), Sphenocentrum jollyanum
(SJRE), and Paullinia pinnata (PPRE) for 60 days. Data presented as means ± SEM,
n=7. Significant differences between treatments and control: ***p≤0.001;
****p≤0.0001 (one-way ANOVA followed by Dunnett’s multiple-comparison test).
230
Powered by TCPDF (www.tcpdf.org)
submit your manuscript | www.dovepress.com
DovePress
Effects of hydroethanolic root extracts of C. benthamiana,
S. jollyanun, P. pinnata on reproductive function were
studied in male SpragueDawley rats. In a survey, these
herbs were believed to be effective in increasing sexual
desire, inducing erection, and enhancing male fertility, a
finding similar to other research.24 All these plants have
been studied for their traditional use as aphrodisiacs.25,26
The roots of these plants have been used frequently in
polyherbal preparations to achieve maximum results.27
Oral administration of the C. benthamiana, P. pinnata,
and S. jollyanun for 60 days resulted in a significant
(p≤0.05) dose-dependent reduction in sperm count and
motility and had a detrimental effect on sperm morphology,
possibly due to oxidative stress. It has been demonstrated
that administration of high concentrations of MSG, induces
oxidative stress in different organs, including the testes.18,28
It has already been established that oxidative stress within
the testes can impair their functionality, particularly with
respect to the quality of spermatozoa produced.29 Oxidation
of glucose and fructose provides the energy required for
spermatozoa motility. The reduction in sperm motility seen
in this study is evidence that the extracts used may be
inhibiting the uncoupling reaction of oxidative
phosphorylation30 and hence rendering spermatozoa immotile. The susceptibility of sperm cells to oxidative damage is
a result of their enrichment with unsaturated fatty acids
within sperm plasma membrane.31 Sanocka and Kurpisz,
200432 revealed that increased lipid peroxidation leads to
oxidative damage to sperm DNA, modification of membrane function, impairment of mobility, and a possiblly
significant effect on spermatogenesis.29 Lipid peroxidation
is one of the major processes in oxidative damage,33 and
this was assessed in this study by the measurement of TBAreactive substances, particularly MDA. Polyunsaturated
fatty–acid degradation is measured by the amount of
MDA formed and is thus the common indicator of polyunsaturated fatty–acid oxidative damage.34
The effects of CBRE, SJRE, and PPRE, and MSG on
sperm count, motility, and morphology could be related to an
increased production of free radicals and hence oxidative
Journal of Experimental Pharmacology 2021:13
Journal of Experimental Pharmacology downloaded from https://www.dovepress.com/ by 35.153.143.85 on 16-May-2021
For personal use only.
Dovepress
Figure 7 Representative photomicrographs of histological assessment of testes of Sprague-Dawley rats after 60 days of treatment. (A) Normal saline–treated rats showing welllayered seminiferous tubules with different stages of spermatogenic cells; (B) 50 mg/kg monosodium glutamate (MSG)-treated rats showing severe atrophy in some seminiferous
tubules and reduction in interstitial Leydig cells; (C) 1,000 mg/kg SJRE–treated rats showing mild atrophy of seminiferous tubules; (D) 1,000 mg/kg CBRE–treated rats showing
severe atrophy in some seminiferous tubules; (E) 1,000 mg/kg PPRE–treated rats showing different degrees of atrophy (transverse section, 200×, H&E stain).
Abbreviations: NT, normal tubule; DT, degenerated tubule; AT, atrophied tubule.
stress in rat reproductive organs. Excessive glutamate metabolism, as in chronic MSG intake, decreased levels of major
antioxidant enzymes, and increased lipid peroxidation, can
be a source of reactive oxygen species.35,36 It is possible that
CBRE, SJRE, and PPRE, and MSG lead to the excessive
production of free radicals and that endogenous antioxidants
are insufficient to meet the demand. The increase in freeradical production could lead to sperm-membrane dysfunction, sperm-DNA damage, and impaired sperm movement.
The increases in abnormal sperm cells are consistent with
findings of testicular damage.
There was no significant difference in testicular and
epididymal weight between the normal and extract-treated rats. As reported by Franca and Russell,37 testicular
weight or size generally establishes the normalcy of
testes, and thus changes that may be drug-induced can
be assessed. The weight or size of an organ correlates
partly with the presence of functional units, and thus all
functional units were likely to be present after the
60 days of extract treatments, but probably with
impaired functionality.
Serum testosterone, FSH, and LH levels were lower in
extract-treated rats. The mechanism of action of the
Journal of Experimental Pharmacology 2021:13
Powered by TCPDF (www.tcpdf.org)
Baffoe et al
antispermatogenic effect of the various extracts could be
due to interference in anterior pituitary function and a
direct effect on testes. The primary effects of FSH and
androgen appear to be similar in rodents, primates, and
other mammals, and thus the effect exerted by the various
treatments on hormones can be extrapolated to occur also
in humans.
The lowered serum-hormone levels would affect
spermatogenesis and hence semen quality. It has been
established that germ-cell proliferation and survival
depends profoundly on gonadotropin-dependent
mechanisms.38,39 FSH exerts its biological effects via
G protein–coupled receptors found in the testes.
Evidence for the critical role of the LH in initiating
and maintaining spermatogenesis has been obtained
from several animal models and experimental
approaches.28 Even though FSH, LH, and testosterone
have separate roles in the process of spermatogenesis,
they also act in a cooperative way to promote quantitative spermatogenesis, likely through the modulation of
postreceptor events within Sertoli cells.38,39 Studies
have suggested that testosterone together with FSH
promotes spermatogenesis by promoting adhesion of
submit your manuscript | www.dovepress.com
DovePress
231
Journal of Experimental Pharmacology downloaded from https://www.dovepress.com/ by 35.153.143.85 on 16-May-2021
For personal use only.
Baffoe et al
Figure 8 Representative photomicrographs of histological assessment of the epididymides of Sprague-Dawley rats after 60 days of treatment. (A) Normal saline–treated
rats showing well-layered seminiferous tubules with different stages of spermatogenic cells; (B) MSG-treated rats showing necrosis in connective tissue; (C) 1,000 mg/kg
SJRE–treated rats showing disorganization of cells and epididymitis; (D) 1,000 mg/kg CBRE–treated rats showing greatly convoluted tubules, but with very scanty
spermatozoa; (E) 1,000 mg/kg PPRE–treated rats showing greatly convoluted tubules, but with scanty spermatozoa (transverse section, 200×, H&E stain).
round spermatids to Sertoli cells.40,41 The combined
effect of FSH and testosterone is mainly demonstrated
in spermiation.42 The maturation cessation, which may
have been caused by the extracts, could be related to
testosterone inhibition, thus leading to the termination
of spermatogenesis.
Histology of the testes and epididymides of treated rats
revealed changes in the histoarchitecture and decrease in
basal lamina, as well as degenerative changes in germ
cells, suggestive of mild, moderate, and severe oligospermia with some degree of testicular atrophy from the lowest
to the highest treatment doses. The relationship between
production of fertile sperm cells and histological integrity
of testes and epididymides is well established.43
Histopathological changes can cause spermatogenic arrest,
edema, hypospermia, and decreased basal lamina, likely to
be a result of oxidative damage to testicular membrane and
tissue.44
Conclusion
Hydroethanolic root extracts of C. benthamiana, S. jollyanum, and P. pinnata were found to have detrimental effects
on reproductive function on prolonged usage. Chronic use
232
Powered by TCPDF (www.tcpdf.org)
Dovepress
submit your manuscript | www.dovepress.com
DovePress
of these may thus lead to male infertility and hence would
not be safe in healthy males who want to reproduce.
Abbreviations
CBRE, Caesalpinia benthamiana root extract; FSH, folliclestimulating hormone; LH, luteinizing hormone; MDA, malondialdehyde; MSG, monosodium glutamate; PPRE,
Paullinia pinnata root extract; SJRE, Sphenocentrum jollyanum root extractTBA, thiobarbituric acid.
Data-Sharing Statement
All data generated or analyzed during this study are
included in this published article. The data sets used and/
or analyzed are available from the corresponding author on
reasonable request.
Ethics Approval
The Committee on Animal Research, Publication, and
Ethics (CARPE) of the Department of Pharmacology,
Faculty of Pharmacy and Pharmaceutical Sciences,
KNUST, approved this study from an ethical point of
view (FPPS/PCOL/007/2016).
Journal of Experimental Pharmacology 2021:13
Dovepress
Consent for Publication
No images, videos, or recordings requiring consent for
publication were used in this manuscript.
Journal of Experimental Pharmacology downloaded from https://www.dovepress.com/ by 35.153.143.85 on 16-May-2021
For personal use only.
Acknowledgments
The authors wish to express their gratitude to Dr George
Sam of the Department of Herbal Medicine, KNUST,
Kumasi, Ghana and Dr Sampene of the Department of
Pathology, KATH, Kumasi for their technical assistance.
Author Contributions
Mavis Sersah Baffoe, Baffour Awuah Agyapong,
George Asumeng, and Lorraine Sallah conceived the
concept and design of this study. Mavis Sersah Baffoe
and Baffour Awuah Agyapong did the literature search,
experimental studies, and data acquisition. All authors
contributed to data analysis, drafting or revising the
article, have agreed on the journal to which the article
will be submitted, gave final approval to the version to
be published, and agree to be accountable for all aspects
of the work.
Funding
The authors did not obtain funding from any institution or
organization. Funding was provided solely by the
researchers.
Disclosure
The authors report no conflicts of interest for this work.
The project from which this article was written was a
graduate research work carried out in the Department of
Pharmacology, KNUST, Kumasi, Ghana with which the
authors are affiliated.
References
1. Amidu N, Owiredu WK, Woode E, et al. Incidence of sexual dysfunction: a prospective survey in Ghanaian females. Reprod Biol
Endocrinol. 2010;8(1):106. doi:10.1186/1477-7827-8-106
2. Goldstein I. The mutually reinforcing triad of depressive symptoms,
cardiovascular disease, and erectile dysfunction. Am J Card. 2000;86
(2):41–45. doi:10.1016/S0002-9149(00)00892-4
3. Nyarko-Sampson E, Dabone K, Brenya E. Perception and patronage of
aphrodisiacs among male students in university of Cape Coast: implications for counselling in higher educational institutions. Zimbabwe J
Educ Res. 2017;29(1).
4. Braun M, Klotz T, Mathers M, et al. ‘Viagra® effect’– influence of
mass media on patient behavior. Urol Int. 2001;66(3):145–148.
doi:10.1159/000056594
5. Makwana S, Solanki M, Raloti S, Dikshit R. Evaluation of recreational use of aphrodisiac drugs and its consequences: an online
questionnaire based study. Int J Res Med Sci. 2013;2(1):51–59.
Journal of Experimental Pharmacology 2021:13
Powered by TCPDF (www.tcpdf.org)
Baffoe et al
6. Watcho P, Kamtchouing P, Sokeng S, et al. Reversible antispermatogenic and antifertility activities of Mondia whitei L. in male albino
rat. Phytother Res. 2001;15(1):26–29. doi:10.1002/1099-1573(200
102)15:1<26::aid-ptr679>3.0.co;2-n
7. Dickson RA, Annan K, Komlaga G. Pharmacognostic standardization
of the leaves and root bark of Caesalpinia benthamiana. Pharmacogn
J. 2011;3(24):31–34. doi:10.5530/pj.2011.24.6
8. Zamblé A, Martin-Nizard F, Sahpaz S, et al. Vasoactivity, antioxidant
and aphrodisiac properties of Caesalpinia benthamiana roots. J
Ethnopharmacol. 2007;116(1):112–119. doi:10.1016/j.jep.2007.11.
016
9. Nia R, Paper DH, Essien EE. Evaluation of the anti-oxidant and antiangiogenic effects of Sphenocentrum jollyanum Pierre. Afr J Biomed
Res. 2004;7:129–132.
10. Amidu N An evaluation of the central and sexual behavioral effects
and toxicity of the root extract of Sphenocentrum jollyanum Pierre
(Menispermaceae) [Dissertation] Kumasi: department of molecular
medicine, Kwame Nkrumah University of Science & Technology
Ghana; 2008.
11. Oke JM, Hamburger MO. Screening of some Nigeria medicinal plant
for antioxidant activity using 22-Diphenyl-picryl-hydrazyl radical.
Afr J Biomed Res. 2002;5:77–79.
12. Olorunnisola SO, Olumi de Samuel Fadahunsi SO, Adegbola P. A
review on ethno-medicinal and pharmacological activities of
Sphenocentrum jollyanum Pierre. Medicines (Basel). 2017;4(3):50.
doi:10.3390/medicines4030050
13. Zamblé A, Carpentier M, Kandoussi A. Paullinia pinnata extracts
rich in polyphenols promote vascular relaxation via endotheliumdependent mechanisms. J Cardiovasc Pharmacol. 2006;47(4):599–
608. doi:10.1097/01.fjc.0000211734.53798.1d
14. Jimoh FO, Sofidiya MO, Afolayan AJ. Antioxidant properties of the
methanol extracts from the leaves of Paullinia pinnata. J Med Food.
2007;10(4):707–711. doi:10.1089/jmf.2006.253
15. Mensah MLK, Komlaga G, Forkuo AD, Firempong C, Anning AK,
Dickson RA Toxicity and safety implications of herbal medicines
used in Africa. In:Philip F, editor. Herbal Medicine. Builders,
IntechOpen;2019. doi: 10.5772/intechopen.72437.
16. Irwin S. Comprehensive observational assessment: ia. A systematic,
quantitative procedure for assessing the behavioral and physiologic
state of the mouse. Psychopharmacologia. 1968;13(3):222–257.
doi:10.1007/BF00401402
17. Roux S, Sablé E, Porsolt RD. Primary observation (irwin) test in
rodents for assessing acute toxicity of a test agent and its
effects on behavior and physiological function. Curr Protoc
Pharmacol. 2005;Chapter 10:10. doi:10.1002/0471141755.
ph1010s27
18. Hamza RZ, Al-Harbi MS. Monosodium glutamate induced testicular
toxicity and the possible ameliorative role of vitamin E or selenium
in male rats. Toxicol Rep. 2014;1:1037–1045. doi:10.1016/j.
toxrep.2014.10.002
19. Ekaluo U, Ikpeme E, Udensi O, et al. Effect of aqueous leaf extract of
neem (Azadirachta indica) on the hormonal milieu of male rats. Int J
Curr Res. 2010;4:1–3.
20. Ekaluo U, Ikpeme E, Udokpoh A. Sperm head abnormality and
mutagenic effects of aspirin, paracetamol and caffeine containing
analgesics in rats. Inet J Toxicol. 2009;7(1):1–9.
21. World Health Organization. WHO Laboratory Manual for the
Examination of Human Semen. 5th ed. Geneva-Switzland: WHO
Press; 2010.
22. Ekaluo UB, Udokpoh AE, Udofia UU, Ajang RO. Comparative
toxicity of five commonly used analgesics on sperm count and
sperm head abnormalities. Glob J Pure Appl Sci. 2005;11(1):81–84.
doi:10.4314/gjpas.v11i1.16465
23. Tüközkan N, Erdamar H, Seven I. Measurement of total malondialdehyde
in plasma and tissues by high-performance liquid chromatography and
thiobarbituric acid assay. Firat Med J. 2006;11(2):088–092.
submit your manuscript | www.dovepress.com
DovePress
233
Journal of Experimental Pharmacology downloaded from https://www.dovepress.com/ by 35.153.143.85 on 16-May-2021
For personal use only.
Baffoe et al
24. Bella AJ, Shamloul R. Traditional plant aphrodisiacs and male sexual
dysfunction. Phytother Res. 2014;28(6):831–835. doi:10.1002/
ptr.5074
25. Mathur M, Sundaramoorthy S. Plants with aphrodisiac potentials–
The knowledge and the gaps. In: Indian Medicinal Plants. (eds.)
Trivedi P.C. Jaipur, India: Aavishkar Publisher; 2009;1–3.
26. Iwu MM. Handbook of African Medicinal Plants. 2nd ed. Boca
Raton: CRC press; 2014.
27. West E, Krychman M. Natural aphrodisiacs—a review of selected sexual enhancers. Sex Med Rev. 2015;3(4):279–288. doi:10.1002/smrj.62
28. Voja P, Dusica P, Gordana K, et al. Effect of monosodium glutamate
on oxidative stress and apoptosis in rat thymus. Mol Cell Biochem.
2007;303(1–2):161–166. doi:10.1007/s11010-007-9469-7
29. Guerriero G, Trocchia S, Abdel-Gawad FK, Ciarcia G. Roles of
reactive oxygen species in the spermatogenesis regulation. Front
Endocrinol. 2014;5:56. doi:10.3389/fendo.2014.00056
30. Abu A, Uchendu C. Antispermatogenic effects of aqueous hydroethanolic extract of Hymenocardia acida stem bark in Wistar rats. J
Med Plant Res. 2010;4(23):2495–2502. doi:10.5897/JMPR10.407
31. Krzyściak W, Monika M, Bąk E, et al. Sperm antioxidant biomarkers
and their correlation with clinical condition and lifestyle with regard
to male reproductive potential. J Clin Med. 2020;9(6):1785.
doi:10.3390/jcm9061785
32. Sanocka D, Kurpisz M. Reactive oxygen species and sperm cells.
Reprod Biol Endocrinol. 2004;2(1):12. doi:10.1186/1477-7827-2-12
33. Ognjanović B, Marković S, Pavlović S, Žikić R, Štajn A. Effect of chronic
cadmium exposure on antioxidant defense system in some tissues of rats:
protective effect of selenium. Physiol Res. 2008;57(3):403–411.
34. GrottoI D, MariaI LS, ValentiniI J, et al. Importance of the lipid
peroxidation biomarkers and methodological aspects for malondialdehyde quantification. Quím Nova. 2009;32:1. doi:10.1590/S010040422009000100032
35. Paul MV, Abhilash M, Varghese MV, Alex M, Nair RH. Protective
effects of alpha-tocopherol against oxidative stress related to nephrotoxicity by monosodium glutamate in rats. Toxicol Mech Methods.
2012;22(8):625–630. doi:10.3109/15376516.2012.714008
Dovepress
36. Thomas M, Sujatha KS, George S. Protective effect of Piper longum
Linn. on monosodium glutamate induced oxidative stress in rats.
Indian J Exp Biol. 2009;47(3):186–192.
37. França L, Russell L. The testis of domestic animals. Male Reprod.
1998;197:219.
38. Meeker JD, Godfrey-Bailey L, Hauser R. Relationships between
serum hormone levels and semen quality among men from an infertility clinic. J Androl. 2006;28(3):397–406. doi:10.2164/jandrol.106.
001545
39. Ruwanpura SM, McLachlan RI, Meachem SJ. Hormonal regulation
of male germ cell development. J Endocrinol. 2010;205(2):117–131.
doi:10.1677/JOE-10-0025
40. Matthiesson KL, McLachlan RI, O’donnell L, et al. The relative roles
of follicle-stimulating hormone and luteinizing hormone in maintaining spermatogonial maturation and spermiation in normal men. J Clin
Endocrinol Metab. 2006;91(10):3962–3969. doi:10.1210/jc.20061145
41. Sluka P, O’Donnell L, Bartles J, Stanton PG. FSH regulates the
formation of adherens junctions and ectoplasmic specialisations
between rat Sertoli cells in vitro and in vivo. J Endocrinol.
2006;189(2):381–395. doi:10.1677/joe.1.06634
42. Saito K, O’donnell L, McLachlan RI, Robertson DM. Spermiation
failure is a major contributor to early spermatogenic suppression
caused by hormone withdrawal in adult rats. Endocrinology.
2000;141(8):2779–2785. doi:10.1210/endo.141.8.7628
43. Hafez ESE, Hafez B, eds. Reproduction in Farm Animals. 7th ed.
Philadelphia: Wiley-Blackwell; 2013.
44. Ding WX, Shen HM, Ong CN. Critical role of reactive oxygen
species and mitochondrial permeability transition in microcystininduced rapid apoptosis in rat hepatocytes. Hepatology. 2000;32
(3):547–555. doi:10.1053/jhep.2000.16183
Dovepress
Journal of Experimental Pharmacology
Publish your work in this journal
The Journal of Experimental Pharmacology is an international, peerreviewed, open access journal publishing original research, reports,
reviews and commentaries on all areas of laboratory and experimental pharmacology. The manuscript management system is completely
online and includes a very quick and fair peer-review system. Visit
http://www.dovepress.com/testimonials.php to read real quotes from
published authors.
Submit your manuscript here: https://www.dovepress.com/journal-of-experimental-pharmacology-journal
234
Powered by TCPDF (www.tcpdf.org)
submit your manuscript | www.dovepress.com
DovePress
Journal of Experimental Pharmacology 2021:13