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*

, Tutku Can Acısu

, Nida Badıllı

,Edanur Güler–Ekmen

, Osman Sedat Tanyeri

, Abdullah Toz

Mehmet Çay

, Abdurrauf Yüce

, Mesut Aksakal

Fırat University, Faculty of Veterinary Medicine, Department of Physiology. Elazığ, Türkiye.

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

) 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. ____________________________________________________________

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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

1.84 ± 0.29

122.58 ± 15.47

BS 77.12 ± 18.70

1.91 ± 0.35

133.73 ± 11.05

MTX 137.27 ± 41.11

1.86 ± 0.11

97.25 ± 17.40

MTX+BS 118.71 ± 38.78

2.55 ± 0.18

100.10 ± 29.12

Signicant P<0.001 P<0.001 P<0.01

TABLE II

Boswellia serrata



Groups 







  

Control 75.18 ± 12.38

127.90 ± 50.97

3.01 ± 1.37 2.44 ± 0.68

5.40 ± 1.57

BS 68.66 ± 13.98

127.90 ± 30.22

3.57 ± 0.64 4.90 ± 1.66

8.14 ± 0.87

MTX 29.16 ± 16.97

47.75 ± 21.09

4.11 ± 1.96 5.00 ± 1.5

8.37 ± 2.73

MTX+BS 68.14 ± 23.83

90.25 ± 29.14

3.00 ± 0.94 4.11 ± 1.59

6.80 ± 1.81

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

0,42 ± 0,07

BS 1,43 ± 0,09 0,48 ± 0,03 0,20 ± 0,01 1,30 ± 0,36

0,45 ± 0,09

MTX 1,35 ± 0,12 0,46 ± 0,05 0,18 ± 0,02 0,96 ± 0,30

0,17 ± 0,03

MTX+BS 1,41 ± 0,15 0,49 ± 0,07 0,20 ± 0,03 1,24 ± 0,20

0,24 ± 0,07

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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