Introduction:
The
use of plants as medicine is believed to be as old as mankind. Till date, a
large part of the world population confides on medicinal plants. Monodora myristica
(MM) is a tropical plant reported to have several medicinal potentials.
Methods:Twenty-eight
adult male Wister rats were separated into four groups of seven rats each.
Group1 received the solvent (1ml) for the extract (control) while group 2,3,
and 4 received 75mg/kgBW, 150mg/kgBW and 300mg/kgBW of extract respectively
for 55days. Five animals from each of the four groups were sacrificed at the
55th day. The weight of rats, testicular, epididymal, prostate,
seminal vesicles were recorded, and sperm parameters (sperm count, motility,
viability, morphology) were assessed on the day of
sacrifice. Blood samples (5ml) were collected and the serum used for hormone
(Luteinizing Hormone (LH), Follicle Stimulating Hormone(FSH), testosterone
and prolactin) assay.
Results:The
two higher doses of the extract treated groups showed a significant
(P<0.05) increase in serum testosterone andFSHas compared to the control.No
significant (P<0.05) difference in LH was observed between extract treated
groups and the control. However, astatistical accepted (P<0.05) decrease
in serum prolactin level was observed in the highest dose (300mg/kgBW) of the
extract treated group as compared to the control. There was significant
(P<0.05) improvement in sperm motility (all treated groups), sperm count
(150mg/kgBW and 300mg/kgBW) and sperm viability (150mg/kgBW and 300mg/kgBW).
Conclusion:
Hydroethanolic
extract of Monodora myristica seed improves testicular functions in
Wistar rats by modulating the neuroendocrine pathway of testicular regulation.
The use of plants as
medicine is believed to be as old as mankind [1]. Records as old as
300 B.C by the Babylonians, Egyptians, Chinese, and Indians described several
plants used in the treatment of different kinds of diseases[6]. Till
date, plants are still used as medicine (traditional medicine) and several
orthodox drugs are derived from medicinal plants[13]. A large part
of the world population, especially in developing countries of the world,
confide on medicinal plants as their source of primary health care, because
herbs are believed to be more affordable, and to have high efficacy and limited
side effects [2,12]. Some of this traditional medicine had been
adequately studied scientifically and shown to be efficacious as traditionally
claimed. However, a great number of them still need proper scientific
evaluation. Plants are known to contained bioactive phytochemicals that can
negatively or positively alter the physiology of organisms [2]. Some
bioactive substances in plants are well known to have toxic effects on vital
organs in animals [14]. Monodora
myristica is a plant that had been identified to contain several bioactive
substances with medicinal potentials. It has as many names as it’s attributed
medicinal potentials. Its common names include; Calabash nutmeg, False nutmeg,
African nutmeg, Jamaican nutmeg [12].Locally it is called Ehuru (Igbo), Ariwo
(Yoruba), Gijuyadanmiya (Hausa), Erhe (Urhobo), Uyengben (Edo), Arigogo
(Ijaw) [2,8] and Obatorr (Abureni).
It is used as culinary spice in preparing several delicacies in Africa and
other countries. Ethnomedicinally, this plant is used in treatment of
arthritis, diarrhea, diabetes, stomach ache,
sickle cell and sexual impotency[2]. Scientifically, it has
antinociceptive, antimalarial, antioxidative, and antimicrobial potentials[2].
However, some reports suggest that this plant has hepatotoxic, and fertility
lowering ability [3,16]. This study aims to identify the effects of
hydroethanolic seed extract of this plant on reproductive functions in male Wistarrats
MATERIALS AND METHOD
Collection and extraction of plant material: Dried Monodora myristica (Calabash Nutmeg) seeds were purchased
from a general market in Choba, Port Harcourt Rivers State. The seed was
identified and authenticated at the department of Crop and Soil Science,
Faculty of Agricultural sciences, Niger Delta University, Wilberforce Island,
Bayelsa State. The seeds were washed, sun-dried, and then dehulled. The
dehulled seeds were grounded to coarse powder using a hand blender (Model:
Corene, A.5 Lander YCIASA).
Extraction
was done as described by Azwanida[5]. Briefly, the grounded seed of MM was macerated in a solvent solution
(30% distilled water plus 70% ethanol) for 72 hours with periodic agitation.
The mixture was filtered with Whatman No.1 filter paper. The filtrate was then
evaporated to dryness with a Rotary Evaporator at 800c, yielding dry
oily brown coloured extract which was weighed, stored in a closed container and
refrigerated until needed.
Animal Models: Male Wistarrats bred at the animal
house, Faculty of Basic Medical Sciences, University of Port Harcourt used for
this study were kept in wooden cages with adequate ventilation, ambient temperature
of 250c and natural light and dark cycles, and were allowed to
acclimatized for 10 days. Feeds (TopFeeds, Super Delux Animal Feeds, Nigeria)
and water were provided ad libitum. Each animal was weighed at the
commencement of the study. Animals were handled with care and with strict
compliance with NIH guidelines for care and use of laboratory animals[7].
Ethical approval for this study was given by the University of Port Harcourt
research ethics committee.
Study Design: Twenty male wistar rats weighing
180±20g) were selected into four groups of 5 rats each. Group 1(normal control)
received distilled water. Group 2, 3 and 4 received 75mg/kg, 150mg/kg and
300mg/kg of rat respectively. Treatments were done
through oral gavage (9am-10am daily) for 54 days. Five rats from each group
were weighed, then sacrificed, under chloroform anaesthesia, on study day 55,
after overnight fast.
Sample collection:Four milliliters (4mls) of blood was
collected from each sacrificed rat through cardiac puncture and transferred
into a plane sample bottle to clot. Each sample was centrifuged at 2000rpm for
15minutes, serum separated and stored in a refrigerator until needed for
hormonal assay. Thereafter, the serum was analyzed for Luteinizing hormone,
Follicle Stimulating hormone, prolactin and testosterone levels by the enzyme
linked immunoassay (ELISA) technique; using analytical grade reagents
[4,10].
The
right testis was separated, epididymis excised and both organs weighed.
Epididymal semen was minced for sperm analysis.
Assessment of sperm count and motility: The organ weights for
epididymis and testes were recorded for each rat. The sperm motility and count
were determined. Sperm count was conducted according to the method of Dills et
al.Briefly, the right testis was
cut into very fine pieces using a scalpel and homogenized for 20 min in 50 mL
of STM solution containing 0.9 NaCl, 0.05% Triton X-100 and 0.01% merthiolate.
Four separate haemocytometer slides were made and the testis sperms were
counted under light microscopy with the use of a manual counter. The left
testis was also taken and minced by a sharp blade and immersed in 1 mL of
physiological saline and the solution was kept in 37°C. After gentle mixing, a
drop of the solution was taken on Neubaur chambers counting and then each was
assessed for sperm motility. This was done by counting motile and non-motile
sperm in different fields and was expressed as a percentage. All the solutions
and instruments that were used in this experiment were kept in an incubator at
37°C.
Assessment of sperm viability and morphology:
A
drop of sperm suspension was smeared on a microscopic slide, fixed with 95%
ethanol and stained with Nigrosin-Eosin. The stained slide was air dried for
12h and viewed with a light microscope at ×400 objective. Blue stained cells
were counted as live cells and purple stained cells were counted as dead cells.
At least, 200 cells were count in each slide and percentage of live cells
calculated. A portion of the sperm suspension was smeared on a slide, fixed
with 95% ethanol and stained with Nigrosin-Eosin. The slides were examined with
a light microscope with ×400 objective. Sperm cells with oval head and one
third covered with acrosome, short middle piece and a slender single tail were
considered normal. Two hundred spermatozoa were viewed and percentage of
abnormal cells were recorded [21].
Assessment
of Serum LH, FSH, Testosterone and Prolactin: The blood samples
were centrifuged at 2000rpm for 15min to obtain the serum sample which was
analyzed for testosterone, prolactin, follicle stimulating hormone (FSH) and
luteinizing hormone (LH) level using enzyme linked immunoassay (ELISA)
technique; using analytical grade reagents [9,10,11] and absorbency
was read at 450nm. A graph of absorbance versus concentration was plotted to
read off the actual amount of each hormone in test samples[5].
Assessment
of Testicular tissue oxidative stress biomarkers (SOD, MDA) and Total Protein: A quantity of 0.4 ml
of the sample was mixed with 1.6 ml of Tris-KCl buffer to which 0.5 ml of 30%
TCA was added. Then 0.5 ml of 0.75% TBA was added and placed in a water bath
for 45 minutes at 80oC. This was then cooled in ice and centrifuged
at 3000 g. The upper potion in the container was collected and absorbance
measured against a reference blank of distilled water at 532nm. Lipid
peroxidation in unit/mg protein was computed with a molar extinction
coefficient of 1.56 x 105 M-1Cm-1 [22].
Superoxide dismutase was analyzed by mixing 0.03 ml of the sample and 4.0 ml of
50 M Na2CO3 buffer. Then, 0.03 ml stock solution of epinephrine was added to
the sample-buffer mixture before taking the absorbance reading at 480 nm. A
blank solution containing all reagents only was used for background correction[22].
Assessment of Total Protein in Testicular Tissue and
Serum:
The testis was homogenate with KOH buffer. The homogenate mixture or blood
sample was centrifuged at 3000 rpm for 15 minutes. Five milliliter of ice cold
water was added to 2.0mls to the collected supernant to make up 7ml. A standard
bovine albumin protein solution of 0.3 mg was prepared in the same manner. Five
ml of KOH solution was added to each tube, mixed and kept to stand at ambient
temperature for 15 minutes. Then, 0.05 ml of dilute Folin-ciocalteu reagent was
added to each tube and mixed immediately to a blue color mixture. The
absorbance was read spectroscopically at 750 nm against a blank reagent. The
amount of protein in each sample was determined from a standard curve[22].
Histological Evaluation: The testis was fixed
in 10% formaldehyde and was dehydrated by increasing percentage hydroethanol to
absolute ethanol. The cube shaped testicular tissue was later cleared of
alcohol by impregnating it with xylene. The xylene infused tissue was later
impregnated with hot paraffin wax and left to cool. The wax embedded tissues
were sectioned with a microtome (Rotary) into 3µm thick tissues. The sectioned
tissues were removed of the wax, and later rehydrated with decreasing
percentage of hydroethanol. The hydrated tissues mounted on a slide and stained
with Nigrosin-Eosin [15]. The stained tissues were air dried for
three days. Dried slides were viewed with a camera aided microscope (Olympus,
CX31RTSF) and pictures taken at ×400 objective.
Statistical Analysis: Data were presented
as mean and standard error of mean (mean ± SM) and then subjected to analysis
of variance (ANOVA) using Statistical Package for Social sciences (SPSS)
version 23.0. Multiple comparisons of the test group mean and control was
compared with 2-sided Dennett t-test. Significant means were separated using
the least significant difference at 5% probability level.
RESULTS
Effects of M.
myristica seed extract on body weight and organs weight: The results on the
effect of the extract on body weight (g) and reproductive organs weight (g) of
the rats is in table 1. It shows that the body weight increased in all the test
groups. However, the percentage increase in weight in all the test groups: 75mg/kg
(19.46%), 150mg/kg (25.92%) and 300mg/kg (14.95%) were all significantly
(p<0.05) lower than that of rats in the control group (30.88%); implying a
significant (p<0.05) reduction in relative weight (g) of all the rats in the
test groups when compared with the weight(g) of rats in the control group.
Effects of M.
myristica seed extract on male reproductive hormones: The result (table 2)
shows a significant (p<0.05) reduction in serum Luteinizing hormone (LH) at
the low treatment dose 75mg/kg body weight, while at the higher treated doses-
150mg/kg body weight and 300mg/kg body weight, there was no difference
(P>0.05). Follicle Stimulating Hormone (FSH) and Testosterone, on the other
hand, significantly (p<0.05) increased in groups treated with higher doses
of the extract (150mg/kg and 300mg/kg body weight) respectively, compared with
the control group. It was further observed that Prolactin decreased dose
dependently in all the test groups: 75mg/kg body weight (P>0.05), 150mg/kg
body weight (P>0.05) and 300mg/kg body weight (P<0.05) respectively, when
compared with the control group.
Effects of M.
myristica seed extract on sperm parameters: The results (table 3)
showed significant (p<0.05) increase in sperm count and in percentage motile
spermatozoa respectively, in all the extract treated groups, when compared with
the control group. Also, sperm viability increased in all the test groups,
being significant (p<0.05) in the groups treated with 150mg/kg and 300mg/kg
respectively, compared with the control group. However, no change in sperm
morphology was observed in animals in all the test groups, when compared with
the control group animals.
Table 1. Effects of M. myristica seed extract on body weight
and organs weight
Parameters
Control
75mg/kgBW
150mg/kgBW
300mg/kgBW
Initial Body Weight. (g)
154.00±2.44
162.00±2.00
178.00±2.00
180.00±6.32
Final Body Weight. (g)
201.80±7.68
193.44±3.53
224.22±5.80
206.76±6.42
Change in Body Weight (%)
30.88
19.46*
25.97
14.95*
Testis Wt.(g)
1.12±0.07
1.00±0.05
1.44±0.06*
1.26±0.04*
Testiculo-somatic index
0.023
0.032*
0.031*
0.048*
N=5,
values are presented as Mean ± SEM and values with asterisk are significant at
P < 0.05.
Table 2. Effects of M. myristica seed extract on male
reproductive hormones
Groups
LH
(mlU/ul)
FSH
(mlU/ul)
Prolactin
(ng/ul)
Testosterone
(ng/ul)
Control
3.76±0.21
2.60±0.21
5.87±2.05
2.45±0.75
75mg/kgBW
3.19±0.12
2.57±0.32
5.39±1.62
3.03±0.45
150mg/kgBW
3.49±0.41
3.23±0.19*
5.46±1.76
4.44±0.50*
300mg/kgBW
3.23±0.28
5.12±0.10*
2.97±1.10*
3.83±0.23*
N=5,
values are presented as Mean ± SEM and values with asterisk are significant at
P < 0.05.
Table 3. Effects of M. myristica seed extract on sperm
parameters.
Groups
Sperm count
(106
cells/ml)
Sperm Motility
(%)
Sperm Viability (%)
Normal Sperm
Morphology (%)
Control
57.50±2.02
51.25±2.39
55.00±2.04
93.75±1.25
75mg/kgBW
65.20±0.35
72.00±2.55*
60.00±4.18
82.00±1.22
150mg/kgBW
70.20±0.97*
81.00±1.00*
93.00±1.22*
93.00±1.22
300mg/kgBW
68.40±2.50*
67.00±3.39*
72.00±1.22*
91.00±1.87
N=5,
values are presented as Mean ± SEM and values with asterisk are significant at
P < 0.05.
DISCUSSION
The study results showed a significant increase in serum testosterone and
follicle stimulating hormone (FSH) level in some0f the extract treated groups.
Also, a notable improvement was observed in the measured sperm parameters
(sperm count, sperm motility, sperm viability, and sperm morphology) in the
extract treated groups when compared with the control group. This improvement
in sperm parameters is positively correlated to the sustained rise in plasma
level of testosterone. Testosterone is the androgen that stimulate and modulate
spermatogenesis. Testosterone is less water soluble and requires androgen
binding protein (ABP) to effectively stimulation the division and
specialization of spermatogonia cells into spermatozoa[20]. The facultative rise in FSH due to increase demand in ABP should have
resulted to the physiological feedback inhibition by inhibin, but this never
happen in the extract treated groups. This suggest that the extract may have a
way of intrinsically preventing the actions of inhibin or increasing sertoli
cells tolerance to seminiferous tubules luminal increase in spermatozoa [17,19].
However, serum luteinizing hormone (LH) and
prolactin were significantly reduced when compared with control group. This
might be due to feedback inhibition by testosterone and sterones in the extract
at the hypothalamus and pituitary gland[18]. Logically, the
concurrent reduction in LH would have subsequently caused decrease in serum
testosterone level. Contrarily, all extract treated groups maintain a
significant increase in testosterone level. This may suggest the extract to
have the inherent ability to induce testosterone synthesis via a mechanism
different from the hypothalamic pituitary gonadal axis [19].The extract induced significant increase in
testiculo-somatic index suggest that Monodora
myristica seeds extract improve testicular functions by either altering
external hormonal influence, or the testicular cells niche surrounding the convoluting
seminiferous tubules. This is evidence in improve sperm parameters, antioxidant
mechanism, and testicular histology.
CONCLUSION
Hydroethanolic crude extract
of M. myristica seed enhanced Wistar rats’
testicular functions by directly or indirectly facilitating the synthesis and
secretion of FSH and testosterone.
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Cite this Article:Adienbo, OM; Azosibe, P; Karibi, A (2024). Neuroendocrine modulation of Testicular Functions by Seed
Extract of Monodora myristica
(Calabash Nutmeg) in Male Experimental Animal Model.Greener Journal of Medical Sciences, 14(1):
37-41.
