https://doi.org/10.52973/rcfcv-e34289
Received: 10/07/2023 Accepted: 27/09/2023 Published: 01/01/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34289
ABSTRACT
This study aimed to determine the effect of Boswellia serrata extract
on Methotrexate– induced testicular damage by evaluating antioxidant
system, reproductive organ weights, some spermatological
parametres and serum Testesterone levels. For this purpose, 40
Sprague Dawley rats were divided into 4 groups. 1. Control Group
(n=10): No treatment was given for 10 days. 2. B. Serrata Group (n=10):
B. Serrata was given by gavage at a dose of 500 mg·kg
-1
for 10 days. 3.
Methotrexate Group (n=10): Methotrexate was given intraperitoneally
as a single dose of 20 mg·kg
-1
. 4. Methotrexate + B. Serrata Group
(n=10): After methotrexate was given intraperitoneally as a single
dose of 20 mg·kg
-1
, 500 mg·kg
-1
B. Serrata was given by gavage for
10 days. It was determined that B. Serrata signicantly increased
serum Testosterone levels (P<0.001), testicular GSH levels (P<0.001),
motility of sperm (P<0.001), concentration of sperm (P<0.001), absolute
ventral prostate (P<0.001) and absolute seminal vesicles (P<0.05) organ
weight in Methotrexate + B. Serrata group. The decrease in testicular
MDA levels (P>0.05) and the increase in GSH–Px enzyme activity of
testes (P>0.05) and nal body weight (P>0.05) were not signicant
in Methotrexate + B. Serrata group compared to the Methotrexate
group. In conclusion, the negative effects of Methotrexate on the
male reproductive system can be reduced by administering B.
Serrata extract.
Key words: Boswellia serrata extract; Methotrexate; testicular
injury; sperm; oxidative stress
RESUMEN
El objetivo fue determinar el efecto del extracto de Boswellia
Serrata sobre el daño testicular inducido por metotrexato mediante
la evaluación del sistema antioxidante, el peso de los órganos
reproductores, algunos parámetros espermatológico y los niveles
de testesterona sérica en este estudio. Para ello, 40 ratas Sprague
Dawley se dividieron en 4 grupos. 1. Grupo Control (n=10): No se aplicó
ningún tratamiento durante 10 días. 2. Grupo de B. Serrata (n=10): B.
Serrata se administró a una dosis de 500 mg·kg
-1
por sonda durante
10 días. 3. Grupo de metotrexato (n=10): el metotrexato se administró
por vía intraperitoneal en una dosis única de 20 mg·kg
-1
. 4. Grupo de
metotrexato + B. Serrata (n = 10): el metotrexato se administró por vía
intraperitoneal en una dosis única de 20 mg·kg
-1
, luego se administró
500 mg·kg
-1
de B. Serrata por sonda durante 10 días. Se determinó
que B. Serrata aumentó signicativamente el nivel de testosterona
en suero (P<0,001), el nivel de GSH en los testículos (P<0,001) , la
motilidad de los espermatozoides (P<0,001), la concentración de
espermatozoides (P<0,001), el peso absoluto de los órganos de la
próstata ventral (P<0,001) de las vesículas seminales (P<0,05) en el
grupo de metotrexato + B. Serrata, La disminución en el nivel de MDA
de los testículos (P>0,05) y el aumento en la actividad de la enzima
GSH–Px de los testículos (P>0,05) y el peso corporal nal (P>0,05)
no fueron signicativos en el grupo de metotrexato + B. Serrata en
comparación con el grupo de metotrexato. En conclusión, los efectos
negativos del metotrexato en el sistema reproductivo masculino
pueden reducirse administrando extracto de B. Serrata.
Palabras clave: Extracto de Boswellia serrata; metotrexato; lesión
testicular; semen; estrés oxidativo
Effect of Boswellia serrata extract on Methotrexate induced testicular
damage
Efecto del extracto de Boswellia serrata sobre el daño testicular inducido por Metotrexato
Gözde Arkalı*
1
, Tutku Can Acısu
2
, Nida Badıllı
2
,Edanur Güler–Ekmen
1
, Osman Sedat Tanyeri
1
, Abdullah Toz
1
,
Mehmet Çay
1
, Abdurrauf Yüce
1
, Mesut Aksakal
1
1
Fırat University, Faculty of Veterinary Medicine, Department of Physiology. Elazığ, Türkiye.
2
Fırat University, Faculty of Veterinary Medicine, Department of Reproduction and Articial Insemınation. Elazığ, Türkiye.
*Corresponding Author: garkali@rat.edu.tr
Boswellia serrata extract ameliorate on testicular damage / Arkali et al. ____________________________________________________________
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INTRODUCTION
Methotrexate (MTX) is a drug, which used to treat various neoplasms
such as acute lymphoblastic leukemia, osteosarcoma, non hodgkin
lymphoma and certain types of cancer. Methotrexate, which has
anti–proliferative, anti–inflammatory and immunomodulatory
effects, is used in low doses in inflammatory diseases [1, 2,
3, 4]. Chemotherapeutic drugs affect many systems such as
gastrointestinal, liver, kidney, respiratory and skin [5, 6, 7]. Also
many chemotherapy agents, including MTX, can cause infertility
[8, 9, 10]. In terms of gonadal toxicity of antimetabolites, the most
studied drug in laboratory animals is MTX. Low and medium doses
of MTX were reported to cause oligospermia but not testicular
atrophy in rats (Rattus norvergicus) [11]. In some studies, it has been
determined that MTX causes degeneration by decreasing germinal
epithelial thickness, seminiferous tubule diameter and testicular
weight. Additionally MTX has a lethal effect on all spermatogenic
cells, especially spermatocytes and spermatids, and reduces the size
of sertoli and leydig cells [8, 12]. MTX causes Deoxyribonucleic Acid
(DNA) damage and subsequent apoptosis in germ cells. Additionally,
MTX application causes a decrease in epididymal sperm count and
motility, and an increase abnormal sperm rates [10, 13].
Oxidative stress plays an key role in the pathogenesis of MTX induced
testicular damage [14]. Methotrexate causes an increase in free radicals
due to impairment of antioxidant defenses and differences in the
pro–inflammatory cytokine system in testicular tissue. Oxidative
stress caused by the increase in free radicals causes damage to the
seminiferous tubules and causes a decrease in germ cells [9, 15]. As a
result, testicular dysfunction and fertility problems occur.
Since the toxic effects of medicinal plants are less than chemicals,
medicinal plants are widely used to prevent tissue damage. Boswellia
species (Burseraceae) are one of the most used herbal plants
worldwide. Boswellia serrata is a plant species belonging to the
Burseraceae family. Many pharmacological effects of B. serrata such
as antioxidant, antiinamatory, anticancer, antidiabetic have been
reported [16]. Most of the effects of B. serrata are due to the boswellic
acids its contains [17]. Aqueous extracts of B. serrata, boswellia oil and
methanolic leaf extract’s antioxidant activity is dose dependent [16,
17, 18]. Boswellia species are used all over the world. However, there
is limited literature information on the effect of B. serrata on the male
reproductive system. This aim of this study was to investigate the
effect of B. serrata on MTX–induced testicular damage by examining
the antioxidant effect of B. serrata.
MATERIALS AND METHODS
Animals and experimental design
Firat University Local Animal Use Committees (Elazig, Türkiye)
approved the experimental protocols of this study with protocol
number 2022/18–03. Forty healthy adult male Sprague Dawley
rats were procured (10–12 weeks/250–300 g) and maintained from
Firat University Experimental Research Centre (Elazig, Türkiye).
Polycarbonate cages (Tecniplast Laboratory Animal Equipment,
Italy) were used to the animals kepting (a 12 h day night cycle and
temperature of 24 ± 3°C). Standard commercial pellet food and fresh
drinking water were given to animals as ad libitum.
The rats were divided into 4 groups, after a one–week adaptation
period. 1. Control Group (n=10): No treatment was applied for 10 days.
2. B. serrata Group (n=10): B. serrata was given at 500 mg·kg
-1
dose
[19] by gavage for 10 days. 3. Methotrexate Group (n=10): Methotrexate
was given by intraperitoneally as a single dose of 20 mg·kg
-1
[9]. 4.
Methotrexate + B. serrata Group (n=10): Methotrexate was given by
intraperitoneally as a single dose of 20 mg·kg
-1
[9], then 500 mg·kg
-1
B. serrata [19] was given by gavage for 10 days.
ELISA analysis
The blood samples were taken into serum tubes and centrifuged
(Nüve NF800R, Türkiye) (3220 G/10 min), the serums were separated.
Serum Testosterone levels of all animals were measured using rat
specic enzyme linked immunosorbent assay (ELISA) kits (sunred
201–11–0260) according to the manufacturer’s recommendations.
The standard curve was used to determined the concentration of
Testosterone hormone.
Analysis of malondialdehyde and antioxidant enzyme activity
Testicular tissue samples were weighed and then homogenized with
a mechanical homogenization device (IKA Ultra–Turrax T25) using
Tris Buffer (dilution ratio: 1/10, g/v) while maintaining their coldness.
The homogenates were centrifuged at 3220 G for 60 min and the
supernatant was separated [20]. Malondialdehyde (MDA), Glutathione
(GSH) levels, Glutathione peroxidase (GSH–Px) enzyme activities and
total protein content were measured from the supernatant. Total
protein content was determined according to Lowry method [21] by
using spectrophotometre (Shimadzu, UV–1700 PharmaSpec, Kyoto
Japan). The method described by Placer et al. [22] was used for MDA
analysis using spectrophotometer. The method described by Sedlak
and Lindsay [23] was used for GSH analysis using spectrophotometer.
The method described by Lawrence and Burk [24] was used for GSH–
Px enzyme analysis using spectrophotometer.
Analysis of spermatological parameters
Reproductive organs (testes, ventral prostate, seminal vesicles and
cauda epididymis) were removed and weighed (HR–250AZ,Türkiye).
Epididymal sperm concentration was determined using the
hemocytometer method. The right epididymis was minced in 1 mL
of 0,09% NaCl and incubated at room temperature for 4 h. After
incubation semen was drawn up to the 0.5 line of the pipette (red
blood cell count pipette) and 2% eosin solution was drawn up to the 101
line of the pipette. The diluted sperm suspension was transferred to
Thoma lame counting chambers (the volume is 0,1 mm
3
) and counted
at 400 (10×40) magnication with using a light microscope (Nikon
eclipse Ci–L, Japan). The result was expressed in million/right cauda
epididymis. Freshly isolated left cauda epididymal tissue was used for
analysis motility of sperm. Percent motility of sperm was assessed
using a light microscope (heated stage, 37°C) (Nikon eclipse Ci–L,
Japan). For determining abnormal sperm (morphological) percentage,
Tris buffer spermatozoa suspension was mixed with eosin nigrosine
stain (1.67% eosin, 10% nigrosine and 0.1 M sodium citrate) and
peripheral smear slides were prepared, and examined under a light
microscope at 400 (10×40) magnication. A total of 200 spermatozoa
were examined per slide and the abnormality rates of spermatozoa
(total, tail and head) were expressed as a percentage [25, 26].
Statistical analysis
It was determined by Shapiro Wilk normality analysis whether the
values obtained as a result of the study showed normal distribution.
(A) Serum Testosterone levels of groups (PP
 
______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34289
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As a result of Shapiro Wilk normality analysis, group means normally
distributed data were determined by Anova one–way analysis of
variance, and differences between groups were determined by Duncan
test. Signicance level was accepted as P<0.05. SPSS package
program (IBM SPSS Version 22.0) was used for statistical evaluations.
Data were given as Mean ± Standard Deviation (x̄ ± SD).
RESULTS AND DISCUSSIONS
Methotrexate is the most usually used anticancer drug due to its
signicant benets. However, it also has side effects on all organs
of the body. Due to the high mitotic activity of the testicular germinal
epithelium, testicular germinal epithelium is very sensitive to damage
caused by cytotoxic drugs [27]. In this study the effects of B. serrata
on testicular damage caused by MTX were investigated by evaluating
body weight, serum Testosterone levels, oxidative stress parameters
and spermatological parameters.
In this study, the nal body weight and serum Testosterone levels
of rats were significantly reduced in the Methotrexate groups
(P<0.001) compared to the control group. However, compared to
the Methotrexate group while there was no signicant difference
in nal body weight in the Methotrexate + B. serrata group (P>0.05),
a signicant increase in serum Testosterone level was determined
(P<0.001) (FIG 1). Also there was no difference between the control
group and the B. serrata group (P>0.05) in nal body weight and
Testosterone levels (P>0.05). Testicular MDA levels increased (P<0.001)
signicantly, testicular GSH–Px enzyme activities reduced (P<0.01)
signicantly and there was no signicant difference in GSH levels
(P>0.05) in Methotrexate group compared to the control group.
Compared to the Methotrexate group, while there was no signicant
difference in GSH–Px enzyme activities and malondialdehyde levels
(P>0.05) in the Methotrexate + B. serrata group, a signicant increased
in GSH level (P<0.001) was determined (TABLE I).
When the effect of B. serrata on spermatological parameters and
reproductive organs weight were examined, it was observed that
sperm motility (P<0.001), sperm concentration (P<0.001), absolute
seminal vesicles organ weight (P<0.05) and absolute ventral prostate
organ weight (P<0.001) signicantly decreased in the Methotrexate
group. When abnormal spermatozoon rates were examined, ıt was
observed that tail and total abnormal spermatozoon rate (P<0.01)
signicantly increased in B. serrata and Methotrexate groups compared
to the control group. However compared to the Methotrexate group
while there was no signicant difference in tail and total abnormal
spermatozoon rate (P>0.05) in the Methotrexate + B. serrata group, a
signicant increase in sperm motility (P<0.001), sperm concentration
(P<0.001), absolute seminal vesicles organ weight (P<0.05) and TABLES
II and III). The study showed that MTX causes testicular damage by
increasing oxidative stress and MTX causes deterioration in sperm
parametres and Testosteron levels. This is similar to several study on
MTX–induced testicular damage [9, 28, 29, 30]
Al–Yahya et al. [31] reported that the up to 1000 mg·kg
-1
boswellic
acid was safe in rats. Singh et al. [19] stated that no side effects
were observed at the 500 mg·kg
-1
dose of B. serrata. Sami et al. [32]
declared that a dose of 500 mg·kg
-1
B. serrata was more ameliorative
than the lowest dose. Therefore, in this study, B. serrata dose was
chosen as 500 mg·kg
-1
. Nusier et al. reported that doses of B. serrata
differed in their effects on the rat reproductive system [33].
Some studies [34, 35] have reported that MTX administration
causes a decrease in body weight. Padmanabhan et al. reported
that MTX administration (10 and 20 mg·kg
-1
, once a week, 10 weeks)
resulted in a reduction in nal body weight [34]. Armağan et al. [35]
reported that a 20 mg·kg
-1
single dose of MTX administered on the
rst day caused a decrease in nal body weight. This study is parallel
to many studies on MTX [8, 33, 34, 35]. However, the increase in nal
body weight was not statistically signicant in the Methotrexate +
B. serrata group compared to the MTX group (FİG. 1). Singh et al.
explained that the 500 mg·kg
-1
dose group had no effect on the body
weights of rats. This study is similar to the study by Singh et al. [19].
Oxidative stress is an important factor in male infertility [36].
Therefore, studies suggest that testicular MTX toxicity can be prevented
or reduced by the use of antioxidant agents (enzymatic and non–
enzymatic) in studies. Different doses of MTX cause oxidative stress,
and can have harmful effects on the testicular tissue [8, 34, 37]. In
this study, the high MDA levels and low GSH–Px enzyme activities in
the MTX group are similar to many studies investigating MTX induced
Boswellia serrata extract ameliorate on testicular damage / Arkali et al. ____________________________________________________________
4 of 6
oxidative stress in testicular tissue [9, 37, 38, 39]. MTX causes cells to
become sensitive to ROS and reduces the antioxidant enzyme system
effectiveness in the cells. In this study, the decrease in MDA levels
and increase GSH–Px enzyme activity level in the Methotrexate + B.
serrata group wasn’t signicant compared to the Methotrexate group
(TABLE I). But increased GSH levels in testicular tissue were statistically
signicant in Methotrexate + B. serrata group (TABLE I). Doaee et al.
suggest that B. serrata resin extract plays a role as an antiinammatory
and antioxidant agent, protecting nigrostriatal dopaminergic neurons
and ameliorating motor disorders in rat with Parkinson's disease [40].
Tohamy et al. reported that B. serrata showed a protective effect against
testicular damage caused by Fipronil poisoning on the male rat model
for 60 days [41]. In this study, signicant difference in antioxidant
properties of B. serrata was determined only in GSH levels (P<0.001)
(TABLE I). Gupta et al. in a study, which they examined the relationship
between the antioxidant, antiproliferative and antimicrobial activities
of topographically collected B. serrata oleo gum resin and the boswellic
acid concentration, showed that the extracts antioxidant activities
could be because of the percentage of boswellic acids (BAs) [ 42].
MTX induced oxidative stress caused testicular damage and
reduced Testosterone levels by directly effect spermatogenic, sertoli
and leydig cells and therefore reduced Testosterone levels [43]. The
sperm is highly susceptible to damage caused by oxidative stress.
This leads to loss of sperm motility and reduced sperm count. In this
study, the concentration of sperm, motility of sperm (TABLE II) and
Testosterone concentration of serum (FIG. 1A) was decreased in the
Methotrexate group. The concomitant administration of B. serrata with
MTX signicantly increased motility of sperm, concentration of sperm
and Testosterone levels of serum as shown in FIG. 1A and TABLE II.
These results are parallel to the study of Tohamy et al. [41], in
which they reported the protective effect of BA on motility of sperm,
concentration of sperm and Testosterone level of serum. In this study the
tail and total abnormal spermatozoon rate (P<0.01) increased in B. serrata,
Methotrexate and Methotrexate + B. serrata groups (TABLE II) compared
to the control group. The decrease in the abnormal spermatozoa rate
was not statistically signicant in the Methotrexate + B. serrata group
(P>0.05) (TABLE II). However, while B. serrata reduced this adverse effect,
the B. serrata group had the highest in the rate of anormal spermatozoon
compared to the control group (P<0.01) (TABLE II). In the study by Tohamy
et al. [41] in which they invigestigated the effect of boswellic acid on
Fipronil induced male reproductive toxicity, it was observed that the
rate of coiled tail and bent head sperm in the boswellic acid group at a
dose of 500 mg·kg
-1
was higher than the control group.
Additionally, MTX caused a decrease in ventral prostate weight
(P<0.001) and seminal vesicles weights (P<0.05). However, in this study,
the decrease in testes, epididymis, cauda epididymis organ weights
as a result of MTX application was not found to be signicant (P>0.05)
(TABLE III). There are different results regarding male reproductive
organ weights in studies conducted with a dose of 20mg·kg
-1
Methotrexate. Yüncü et al. reported that intraperitoneal MTX caused
a decrease in testicular weight [43]. Aslankoc et al. reported that
intraperitoneal MTX did not affect testes weight, but MTX caused a
decrease in epididiymis organ weights [44]. Güvenç et al. reported that
intraperitoneal MTX did not affect weights of testes, epididiymis, and
cauda epididymis organs, but MTX administration led to a decrease in
absolute ventral prostate weight and seminal vesicles weight [9]. This
study is similar to the study of Güvenç et al. [9]. In this study; B. serrata
showed a positive effect on the MTX induced reduction in seminal
vesicles (P<0.05) and ventral prostate weight (P<0.001), (TABLEIII).
This study is similar to the study of Tohamy et al. [41].
TABLE I


Groups

(nmol·g
-1

GSH
(nmol·g
-1


(IU·gr prot
-1
)
Control 72.64 ± 10.02
b
1.84 ± 0.29
b
122.58 ± 15.47
a
BS 77.12 ± 18.70
b
1.91 ± 0.35
b
133.73 ± 11.05
a
MTX 137.27 ± 41.11
a
1.86 ± 0.11
b
97.25 ± 17.40
b
MTX+BS 118.71 ± 38.78
a
2.55 ± 0.18
a
100.10 ± 29.12
b
Signicant P<0.001 P<0.001 P<0.01
TABLE II
Boswellia serrata

Groups 



  
Control 75.18 ± 12.38
a
127.90 ± 50.97
a
3.01 ± 1.37 2.44 ± 0.68
b
5.40 ± 1.57
b
BS 68.66 ± 13.98
a
127.90 ± 30.22
a
3.57 ± 0.64 4.90 ± 1.66
a
8.14 ± 0.87
a
MTX 29.16 ± 16.97
b
47.75 ± 21.09
c
4.11 ± 1.96 5.00 ± 1.5
a
8.37 ± 2.73
a
MTX+BS 68.14 ± 23.83
a
90.25 ± 29.14
b
3.00 ± 0.94 4.11 ± 1.59
a
6.80 ± 1.81
ab
Signicant P<0.001 P<0.001 P>0.05 P<0.01 P<0.01
TABLE III
Boswellia serrata 


Groups
Testes









Control 1,36 ± 0,15 0,49 ± 0,02 0,20 ± 0,03 1,28 ± 0,16
a
0,42 ± 0,07
a
BS 1,43 ± 0,09 0,48 ± 0,03 0,20 ± 0,01 1,30 ± 0,36
a
0,45 ± 0,09
a
MTX 1,35 ± 0,12 0,46 ± 0,05 0,18 ± 0,02 0,96 ± 0,30
b
0,17 ± 0,03
c
MTX+BS 1,41 ± 0,15 0,49 ± 0,07 0,20 ± 0,03 1,24 ± 0,20
a
0,24 ± 0,07
b
Signicant P>0.05 P>0.05 P>0.05 P<0.05 P<0.001
______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34289
5 of 6
CONCLUSION
In conclusion, signicant improvement was determined in motility
of sperm, count of sperm, Testosterone level of serum and GSH level
of testes. However no signicant difference was detected in abnormal
spermatozoon rate, MDA level and GSH–Px enzyme activity after B.
serrata. Different doses of B. serrata should be investigated through
different mechanisms in MTX induced testicular damage. At the same
time, it was thought that B. serrata application should be studied at
different times.
Limitations of the study
We were not able evaluate the amount of boswellic acids in B.
serrata extract and histology of the testis due to the budget constraint
and lack of equipment.
Conict of interest
The authors declare no conicts of interest.
ACKNOWLEDGEMENTS
This present study was funded by the Scientic Research Projects
Coordination Unit of Firat University (Project No: VF.23.12).
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