Antioxidant Effects of Peppermint (Mentha piperita ...Antioxidant Effects of Peppermint (Mentha piperita) Extract on the Oxidative Balance of Rabbit Spermatozoa . Eva Tvrdá, Natália

Antioxidant Effects of Peppermint (Mentha

piperita) Extract on the Oxidative Balance of

Rabbit Spermatozoa

Eva Tvrdá, Natália Konečná, Katarína Zbyňovská, and Norbert Lukáč Department of Animal Physiology, Slovak University of Agriculture, Nitra, Slovakia

Email: {evina.tvrda, konecna.natali, zbynovska.katarina, norolukac}@gmail.com

Abstract—This study aimed to assess the effects of different

concentrations (0, 1, 5, 10, 50 and 100 µg/mL) of the

peppermint (Mentha piperita) extract on the motility and

oxidative profile of rabbit spermatozoa following 0, 2, 4 and

8h of in vitro culture. Sperm motion was assessed using

Computer aided sperm analysis, while Reactive Oxygen

Species (ROS) production and the total antioxidant capacity

were assessed by chemiluminescence. Protein and lipid

oxidation were evaluated using spectrophotometric assays.

The experiments revealed that low peppermint

concentrations (1-10 µg/mL) exhibited motility-promoting

effects (P<0.05 and P<0.01; 8h). Peppermint concentrations

ranging between 1 and 50 µg/mL led to a significant

preservation of the intracellular oxidative balance of rabbit

spermatozoa (P<0.01; P<0.001; 8h). All selected peppermint

concentrations prevented oxidative degeneration of proteins

and lipids (P<0.05; P<0.01; P<0.001; 8h). Our results

indicate that the peppermint extract could delay oxidative

damage inflicted to spermatozoa by the in vitro

environment.

Index Terms—peppermint, reactive oxygen species,

antioxidants, spermatozoa, rabbits

I. INTRODUCTION

Oxidative Stress (OS), regarded as an imbalance

between the systemic manifestation of Reactive Oxygen

Species (ROS) and a biological system's ability to readily

detoxify the reactive intermediates or to repair the

resulting damage, has become a prominent factor in the

pathogenesis of male sub- or infertility [1], [2]. The

inability to restore the damage induced by ROS along

with the cell membranes rich in polyunsaturated fatty

acids and lack of substantial ROS-scavenging

mechanisms renders spermatozoa to be uniquely prone to

ROS-inflicted damage. Subsequently, a rapid decline of

intracellular ATP causes mitochondrial and axonemal

damage, decreased sperm vitality and increased

morphological defects, all of which contribute to

alterations in the sperm function [3]. OS has become a

significant concern for scientists as ROS-associated

insults may lead to poor fertilization rates and

embryogenesis, pregnancy loss and birth defects [4].

Manuscript received September 10, 2017; revised January 24, 2018.

Recently, a vast variety of medicinal plants have been

suggested to enhance male fertility [5]-[8]. Currently,

medicinal plants are increasingly recognized worldwide

as an alternative source of efficient and cost-effective

biologically active compounds, which could be used as

supplementary remedies to primary health care

management [9], [10].

Peppermint (Mentha piperita L.) is a perennial herb

native to Europe, naturalized in northern America, and

currently cultivated worldwide. Being a hybrid of

spearmint (M. spicata L.) and water mint (M. aquatica

L.), peppermint is best known for its flavoring and

fragrance properties as well as for essential oils extracted

from the leaves used in many food, cosmetic and

pharmaceutical products [11].

The chemical components of peppermint leaves and

extracts vary with plant maturity, variety, geographical

region and processing conditions [11]. The main volatile

components identified in peppermint essential oils are

menthol, menthone, eucalyptol, menthyl acetate,

menthofuran, limonene and carvone [12], [13]. The main

antioxidant activity of mint is closely associated with the

total polyphenolic content of peppermint leaves which is

approximately 19–23% (12% total flavonoids), and

includes eriocitrin, rosmarinic acid, luteolin 7-O-

rutinoside, hesperidin, and smaller quantities of pebrellin,

gardenin B and apigenin [14], [15]. Furthermore, the

salicylic acid content of peppermint candies and tea is

reportedly very high [16].

In vitro, mint has exhibited significant antimicrobial

and antiviral activities, strong antioxidant and antitumor

actions, as well as antiallergenic effects. Animal model

studies have reported analgesic and anesthetic behavior of

mint in the central and peripheral nervous system,

immunomodulating and chemopreventive properties.

Furthermore, human studies have emphasized on

beneficial effects of mint extracts on the gastrointestinal

and respiratory tract. Several clinical trials examining the

effects of peppermint oil on irritable bowel syndrome

symptoms have been conducted as well [11]. Our earlier experiments revealed stimulating effects of

the peppermint extract on the functional activity and

mitochondrial metabolism of male gametes [17]. Based

on this body of evidence, the purpose of this in vitro

study was to assess the efficacy of the Mentha piperita

Journal of Advanced Agricultural Technologies Vol. 5, No. 2, June 2018

©2018 Journal of Advanced Agricultural Technologies 117doi: 10.18178/joaat.5.2.117-122

extract on rabbit spermatozoa motility, ROS production

and intracellular oxidative profile during an 8-hour

culture in order to provide information on its possible

antioxidant effects on male reproductive cells.

II. MATERIAL AND METHODS

A. Plant Material

Peppermint (Mentha piperita L.) leaves were obtained

from the Botanical Garden at the Slovak University of

Agriculture in Nitra. After drying, the plant tissues were

crushed, weighed and soaked in ethanol p.a. (96%,

Centralchem, Bratislava, Slovak Republic) during two

weeks at room temperature in the dark. Exposure to

sunlight was avoided to prevent the degradation of active

components. Subsequently the plant extracts were

subjected to evaporation under reduced pressure at 40 °C

to remove any residual ethanol (Stuart RE300DB rotary

evaporator, Bibby Scientific Limited, UK, and vacuum

pump KNF N838.1.2KT.45.18, KNF, Germany). Crude

plant extracts were dissolved in DMSO (Dimethyl

sulfoxide; Sigma-Aldrich, St. Louis, USA) to equal 100.4

mg/mL as a stock solution.

B. Sample Collection and Processing

Ten male rabbits (New Zealand white broiler line;

approx. four months of age; 4.0±0.2 kg of weight) used in

the experiment were obtained from the experimental farm

of the Animal Production Research Centre Nitra,

Slovakia.

Semen samples were collected on a single day (early in

the morning) using an artificial vagina. Immediately after

collection, each sample was diluted in physiological

saline solution (PS) (sodium chloride 0.9% w/v, Bieffe

Medical, Italia), enriched with 5% glucose (Centralchem),

4% bovine serum albumin (BSA; Sigma-Aldrich) and

supplemented with 0 (control group), 1, 5, 10, 50 and

100µg/mL peppermint extract. The samples were

cultured at 37°C. After culture periods of 0, 2, 4 and 8h,

spermatozoa motility and ROS generation were assessed

in each group. Furthermore, an aliquot of each sample

was centrifuged at 800 x g at 25ºC for 10min, the media

were removed and the resulting pellet was sonicated at 28

kHz for 30s on ice using RIPA buffer (Sigma-Aldrich)

with protease inhibitor cocktail for mammalian cell and

tissue extracts (Sigma-Aldrich). Subsequently the

samples were centrifuged at 11,828 x g at 4ºC for 15min

to purify the lysates from the residual cell debris [18].

The resulting supernatants comprising the intracellular

contents were stored at −20ºC for the assessment of the

total antioxidant status, protein and lipid peroxidation.

C. Spermatozoa Motion Analysis

Spermatozoa motility (%; MOT) was assessed using

the computer-aided sperm analysis (CASA, Version 14.0

TOX IVOS II.; Hamilton-Thorne Biosciences, Beverly,

MA, USA). Ten μL of each sample were placed into the

Makler counting chamber (depth 10μm, 37°C; Sefi

Medical Instruments, Haifa, Israel) and immediately

assessed. With the objective to avoid false positive results,

the samples were stained using the IDENT Stain, a DNA-

specific dye based on Hoechst bisbenzimide (Hamilton-

Thorne Biosciences) and analyzed under fluorescent

illumination. Ten microscopic fields were subjected to

each analysis to include at least 300 cells.

D. Reactive Oxygen Species (ROS) Production

ROS production in each fraction was assessed by the

chemiluminescence assay using luminol (5-amino-2, 3-

dihydro-1, 4-phthalazinedione; Sigma-Aldrich) as the

probe [19]. The test samples consisted of luminol (10μL,

5mmol/L) and 400μL of control or experimental sample.

Negative controls were prepared by replacing the sperm

suspension with 400μL of each culture medium. Positive

controls included 400μL of each medium, 10μL luminol

and 50μL hydrogen peroxide (H2O2; 30%; 8.8M; Sigma-

Aldrich). Chemiluminescence was measured on 48-well

plates in 15 1 min-cycles using the Glomax Multi+

Combined Spectro-Fluoro Luminometer (Promega

Corporation, Madison, WI, USA) [20]. The results are

expressed as relative light units (RLU)/s/106 sperm.

E. Total Antioxidant Capacity (TAC)

An improved enhanced chemiluminescence

antioxidant assay using horseradish peroxidase conjugate

and luminol was used to study the total antioxidant

capacity of the sample. 5-100μmol/L Trolox (6-hydroxy-

2,5,7,8-tetramethylchroman-2-carboxylic acid; Sigma-

Aldrich) was used as the standard, while a signal reagent

consisting of 0.1mol/L Tris-HCl (Sigma-Aldrich),

12mol/L H2O2 (Sigma-Aldrich), 41.8mmol/L 4-

iodophenol (Sigma-Aldrich) and 282.2mmol/L luminol

(Sigma-Aldrich) was used to induce the reaction.

Chemiluminescence was measured on 96-well plates in

10 cycles of 1 min using the Glomax Multi+ Combined

Spectro-Fluoro Luminometer (Promega Corporation).

The results are expressed as μmol Trolox Eq./g protein

[20].

F. Protein Oxidation

Carbonyl group quantification was performed through

the traditional 2,4-dinitrophenylhydrazine (DNPH)

method. Briefly, 1mL of the pretreated sample solution

was added to 1mL of DNPH (10 mM in 2 NHCl; Sigma-

Aldrich), mixed, and incubated for 1 h in the dark at room

temperature. After the addition of 1 mL of trichloroacetic

acid (20% w/v; Sigma-Aldrich) the mixture was

incubated at 4°C for 10min before centrifugation at

11,828 x g for 15min. The supernatant was discarded

without disturbing the pellet that was subsequently

washed three times with 1mL of ethanol/ethyl acetate

(1/1; v/v) to remove free DNPH reagent. The sample

pellet was resuspended in 1mL of 6M guanidine-HCl

(Sigma-Aldrich) before absorbance measurement at 360

nm. The molar absorption coefficient of 22,000 1/M.cm

was used to quantify the concentration of protein

carbonyls groups. Protein carbonyls are expressed as

nmol/mg protein [20], [21].

G. Lipid Peroxidation

Lipid peroxidation expressed through malondialdehyde

(MDA) production was assessed with the help of the


©2018 Journal of Advanced Agricultural Technologies 118

TBARS assay, modified for a 96-well plate and ELISA

reader. Each sample was treated with 5% sodium dodecyl

sulfate (Sigma-Aldrich), and subjected to 0.53%

thiobarbituric acid (TBA; Sigma-Aldrich) dissolved in

20% acetic acid adjusted with NaOH (Centralchem) to

pH 3.5, and subsequently boiled at 90–100°C for 1h.

Following boiling, the samples were placed on ice for

10min and centrifuged at 1,750 x g for 10min.

Supernatant was used to measure the end-product

resulting from the reaction of MDA and TBA under high

temperature and acidic conditions at 530–540nm with the

help of the Multiskan FC microplate photometer (Thermo

Fisher Scientific Inc.) [18]. MDA concentration is

expressed as μmol/g protein.

H. Protein Quantification

Protein concentration was quantified using the DiaSys

Total Protein (DiaSys, Holzheim, Germany) commercial

kit and the semi-automated clinical chemistry

photometric analyzer Microlab 300 (Merck, Darmstadt,

Germany). The measurement is based on the Biuret

method, according to which copper sulfate reacts with

proteins to form a violet blue color complex in alkaline

solution, and the intensity of the color is directly

proportional to the protein concentration when measured

at 540 nm.

I. Statistical Analysis

Statistical analysis was carried out using the GraphPad

Prism program (version 3.02 for Windows; GraphPad

Software, La Jolla California USA, www.graphpad.com).

Descriptive statistical characteristics (mean, standard

error) were evaluated at first. One-way ANOVA was

used for specific statistical evaluations. Dunnett test was

used as a follow-up test to ANOVA, based on a

comparison of every mean to a control mean, and

computing a confidence interval for the difference

between the two means. The level of significance was set

at P<0.05; P<0.01 and P<0.001.

III. RESULTS AND DISCUSSION

Recently, plant extracts have attracted widespread

scientific interest due to their broad range of

antimicrobial, anti-cancer, anti-inflammatory or

antioxidant properties [9], [11], [15], [22], [23].

Different in vitro studies have reported that mint

extracts are well absorbed and tolerated, and no distinct

toxicity was reported [24]-[26]. On the other hand, in vivo

studies on the impact of the Mentha extracts on male

fertility suggest its potentially toxic effects on testicular

function [27]. Due to the existing controversy on the

exact in vitro behavior of peppermint on male gametes,

we focused on the in vitro impact of Mentha extracts on

the functional and antioxidant competence of rabbit

spermatozoa.

The CASA assessment showed a continuous decrease

of spermatozoa motility in all groups over the course of

the in vitro culture (Table I). The initial (0h) as well as

short-term (2h) MOT was higher in the experimental

groups supplemented particularly with low Mentha

extract concentrations (5 and 10µg/mL) when compared

to the control group, which became statistically

significant at time 2h (P<0.05 in case of 10µg/mL;

P<0.01 with respect to 5µg/mL). Moreover, 100µg/mL

peppermint extract caused an instant decrease of the

spermatozoa MOT. After 2h, the decline of spermatozoa

motion became significant when administering

100µg/mL Mentha (P<0.05). At the end of the

experiment (8h), MOT was significantly decreased with

respect to a concentration range of 50-100µg/mL Mentha

(P<0.001 in case of 100µg/mL and P<0.01 in relation to

50 µg/mL extract). In the meantime, spermatozoa motion

was significantly improved following exposure to a

concentration range of 1-10µg/mL peppermint extract

(P<0.001 with respect to 5 and 10µg/mL; P<0.01 in case

of 1µg/mL peppermint).

TABLE I. SPERMATOZOA MOTILITY (%) IN THE ABSENCE (CTRL) OR

PRESENCE OF THE PEPPERMINT EXTRACT IN DIFFERENT TIME PERIODS

Time 0h 2h 4h 8h

Ctrl 59.232.55 47.332.12 35.331.65 22.671.35

100

µg/mL 37.331.70 34.331.65* 15.671.60*** 1.550.56***

50

µg/mL 65.001.52 43.502.18 25.402.00* 14.601.44*

10

µg/mL 60.000.57 58.333.05* 47.601.18* 43.431.09**

5

µg/mL 64.670.66 63.331.11** 57.671.37** 48.222.13**

1

µg/mL 57.601.73 54.352.08 44.221.52 42.371.12*

Mean±SEM; *** (P<0.001); ** (P<0.01); *(P<0.05)

It has been previously stated that M. piperita contains a

variety of flavonoids, such as isoflavones, flavanones,

flavonols and dihydrochalcon [14] all of which have been

extensively studied for their potential roles on

spermatogenesis or in vitro sperm survival. Improved

spermatozoa motility and mitochondrial activity after

flavonoid administration were recorded in different

studies on fresh as well as frozen goat, mouse and human

semen [28]-[30].

With respect to pure mint extracts, currently available

studies were focused primarily on the anti-androgenic

effects of Mentha species in in vivo studies. Akdogan et

al. [31] focused on the effects of peppermint and

spearmint teas on the total testosterone, Luteinizing

Hormone (LH) and Follicle-Stimulating Hormone (FSH)

levels as well as testicular histological features in rats.

The animals were randomly divided into four groups. The

control group was given commercial drinking water,

while the experimental groups were administered with

20g/L peppermint tea, 20g/L and 40g/L spearmint tea

respectively. The study revealed that following exposure

to mint tea, FSH and LH levels increased while the total

testosterone levels decreased in comparison to the control

group. Histological assessment revealed an increase of

the mean seminiferous tubular diameter. The only

significant histological changes detected following M.

piperita treatment were segmental maturation arrest in the

seminiferous tubules, although the effects of M. spicata



extended from maturation arrest to diffuse germ cell

aplasia in relation to the dose.

In a different study, Sharma and Jacob [27] studied

possible contraceptive effects of Japanese mint and its

possible reversibility. The study revealed that the oral

administration of the methanolic mint extract led to a

fertility inhibition in mice, while maintaining their

normal sexual behavior. With an increase in the treatment

duration, a corresponding decrease in the mean weight of

testes and accessory glands was observed. Sperm count,

motility and viability were also decreased. Furthermore,

spermatozoa with coiled tails were frequently observed in

microscopic smears. Nevertheless, all the recorded effects

returned to a normal state within 30 days following

completion of the 60-day treatment. At the same time, the

methanolic mint extract had no impact on the body

weight, blood cell count, packed cell volume, hemoglobin,

blood or serum biochemistry of the animals.

Oxidative Stress (OS) has become one of the leading

causes related to the loss of viable spermatozoa in ex vivo

conditions. ROS overgeneration is nowadays accepted as

a notable side effect of in vitro processing and handling

protocols of semen, leading to major disruptions in the

cellular oxidative metabolism. The resulting OS may

subsequently lead to irreversible alterations of membrane

structures via LPO, as well as oxidative degradation of

proteins or DNA, followed by apoptotic activation [1],

[4]. As such, we focused on the potential in vitro

antioxidant activities of the mint extract in rabbit

spermatozoa.

The luminometric analysis revealed no instant effects

of the peppermint extract on the ROS production by

rabbit spermatozoa (Table II). Further experiments

following 2h, 4h and 8h revealed a mild pro-oxidant

impact of 100 µg/mL peppermint extract on rabbit male

gametes, although without any significance. On the other

hand, administration of Mentha extract concentrations

lower than 50 µg/mL led to a significant reduction of

ROS overgeneration by male reproductive cells (P<0.05

in case of 2h, P<0.01 with respect to 4h, P<0.001 in

relation to 8h; Table II).

TABLE II. REACTIVE OXYGEN SPECIES (ROS) PRODUCTION

(RLU/SEC/106 SPERM) IN THE ABSENCE (CTRL) OR PRESENCE OF THE

PEPPERMINT EXTRACT IN DIFFERENT TIME PERIODS

Time 0h 2h 4h 8h

Ctrl 2.12±0.72 4.55±0.89 9.33±1.17 14.46±1.59

100

µg/mL 2.80±0.93 5.55±0.99 9.90±1.13 15.99±1.78

50

µg/mL 2.10±0.49 3.87±0.81* 7.66±1.31** 12.00±1.27***

10

µg/mL 1.85±0.52 3.29±0.75* 6.44±1.17** 11.88±1.24***

5

µg/mL 2.05±0.68 3.77±0.62* 6.77±1.50** 11.99±1.36***

1

µg/mL 2.10±0.57 4.12±0.65 7.19±1.23** 11.99±1.24***

Mean±SEM; *** (P<0.001); ** (P<0.01); *(P<0.05)

The TAC assessment revealed a significant increase of

this capacity in the experimental samples subjected to 5-

100μg/mL peppermint (P<0.01) after 2 hours, probably

due to the enrichment of the intracellular milleu by the

antioxidant molecules present in mint. This rapid increase

was followed by a slow decrease of the antioxidant

capacity however TAC was still significantly higher in

the samples exposed to 5-100 μg/mL peppermint after 4h

as well as 8h (P<0.01; Table III).

TABLE III. TOTAL ANTIOXIDANT CAPACITY (TAC; EQ. µMOL

TROLOX/G PROT.) IN THE ABSENCE (CTRL) OR PRESENCE OF THE

PEPPERMINT EXTRACT IN DIFFERENT TIME PERIODS

Time 0h 2h 4h 8h

Ctrl 4.17±0.38 3.98±0.23 3.00±0.22 2.12±0.17

100

µg/mL 4.20±0.38 5.43±0.45** 5.04±0.22** 3.35±0.22**

50

µg/mL 4.17±0.24 5.44±0.67** 5.43±0.29** 4.76±0.30**

10

µg/mL 4.20±0.29 5.04±0.65** 4.90±0.39** 3.50±0.50**

5

µg/mL 4.11±0.42 4.34±0.60** 4.12±0.20** 3.20±0.18**

1

µg/mL 4.16±0.29 4.10±0.62 3.32±0.18 2.27±0.15

Mean±SEM; *** (P<0.001); ** (P<0.01); *(P<0.05)

TABLE IV. PROTEIN OXIDATION EXPRESSED AS THE CONCENTRATION

OF PROTEIN OXIDATION EXPRESSED AS PROTEIN CARBONYLS [NMOL

PC/MG PROT] ASSESSED IN RABBIT SPERMATOZOA IN THE ABSENCE

(CTRL) OR PRESENCE OF THE PEPPERMINT EXTRACT IN DIFFERENT TIME

PERIODS

Time 0h 2h 4h 8h

Ctrl 1.20±0.13 2.65±0.21 4.43±0.33 7.71±0.99

100

µg/mL 1.16±0.12 1.77±0.18* 3.25±0.20** 5.55±0.50**

50

µg/mL 1.10±0.10 1.70±0.15*** 3.20±0.21** 5.05±0.40***

10

µg/mL 1.10±0.11 1.69±0.17*** 3.45±0.23** 5.51±0.35***

5

µg/mL 1.13±0.10 1.80±0.21* 4.11±0.21* 5.81±0.67**

1

µg/mL 1.10±0.12 1.80±0.19* 4.40±0.14 6.31±0.72*

Mean±SEM; *** (P<0.001); ** (P<0.01); *(P<0.05)

TABLE V. LIPID PEROXIDATION EXPRESSED AS MALONDIADEHYDE

CONTENT [µMOL MDA/G PROT] ASSESSED IN RABBIT SPERMATOZOA

IN THE ABSENCE (CTRL) OR PRESENCE OF THE PEPPERMINT EXTRACT IN

DIFFERENT TIME PERIODS

Time 0h 2h 4h 8h

Ctrl 0.35±0.05 0.95±0.04 2.20±0.10 6.18±0.22

100

µg/mL 0.31±0.03 0.52±0.03*** 1.15±0.15*** 3.40±0.30***

50

µg/mL 0.26±0.03 0.39±0.03*** 0.85±0.10*** 2.66±0.18***

10

µg/mL 0.28±0.03 0.40±0.03*** 1.22±0.10*** 3.58±0.21***

5

µg/mL 0.33±0.03 0.55±0.05** 1.29±0.14** 4.23±0.20***

1

µg/mL 0.33±0.05 0.55±0.04** 1.59±0.11** 4.90±0.22**

Mean±SEM; *** (P<0.001); ** (P<0.01); *(P<0.05)

To evaluate the ability of Mentha to provide protection

against oxidative insults to proteins and lipids, we

focused to quantify the amount of protein carbonyls as

well as MDA as crucial end-products of protein and lipid

oxidation respectively, following exposure of male

reproductive cells to the peppermint extract. Both

examinations revealed that all chosen concentrations of



Mentha exhibited protective effects on the protein and

lipid molecules early on to the in vitro culture (2h; Tables

IV and V), and maintained these beneficial effects

throughout later assessment times resulting into a

significantly lower occurrence (P<0.05; P<0.01; P<0.001)

of both protein carbonyls (Table IV) and MDA (Table V)

in all experimental groups when compared to the control.

Numerous reports have emphasized on the fact that

plant extracts possess significant antioxidant activities

[11], [13]. The antioxidant ability could be attributed to

the exceptionally high content of phenolic compounds,

particularly flavonoids with potent ROS-scavenging

activities. Thus, mint extracts could be a promising

natural source of antioxidants, possibly used in nutritional

or pharmaceutical industry for the prevention of ROS-

mediated diseases.

Previous studies on male reproductive performance

have shown that biologically active compounds

frequently found in Mentha were able to significantly

decrease LPO, restore glutathione synthesis and catalase

activity, associated with normal spermatogenesis and

sperm viability [32]. In a different study [33], polyphenol

administration led to a significantly increased TAC,

superoxide dismutase levels, as well as sperm percentage,

viability, motility, accompanied by a decrease of MDA in

rats, hence suggesting that flavonoids could be effective

in enhancing healthy semen parameters.

In spite of the fact that mint contains a broad variety of

biologically active compound with promising antioxidant

effects, Kumar et al. [34] conducted a study examining

the pro- or antioxidant role of mint within its suggested

anti-androgenic activity. The study has revealed that the

aqueous extract of this plant induced OS in the

hypothalamic region and exhibited anti-androgenic

activities in rats. Administration of the extract decreased

activities of detoxifying enzymes such as superoxide

dismutase, catalase, glutathione peroxidase and

glutathione reductase in the hypothalamus of rats. RT-

PCR and Western blot analysis revealed a decreased

expression of selected steroidogenic enzymes,

cytochrome P450scc, cytochrome P450C17, 3beta-

hydroxysteroid dehydrogenase, 17beta-hydroxysteroid

dehydrogenase and related proteins such as steroidogenic

acute regulatory protein, androgen receptor and scavenger

receptor class B-1 as well as testicular 3beta-

hydroxysteroid dehydrogenase and 17beta-

hydroxysteroid dehydrogenase. Histopathological

assessment revealed a decreased sperm density in the

cauda epididymis and degeneration of the ductus deferens. Our data highly emphasize on the need to further

examine the exact impact the biomolecules present in

mint extracts have individually or collectively on the in

vitro sperm survival and oxidative balance. We may

suggest that high concentrations of the Mentha extract

may stimulate the activity of the mitochondrial

respiratory chain of complex II, thus significantly

increasing the risk of ROS overproduction. At the same

time, high ROS concentrations often lead to

mitochondrial dysfunction and rupture, resulting in an

increased ROS release into the surrounding environment.

Lastly, the decreasing mitochondrial viability measured

by the MTT test was mirroring the increasing superoxide

production detected by the NBT assay in our earlier study

[17], based on which we may assume that a significant

amount of ROS could be leaking from dysfunctional

mitochondria.

IV. CONCLUSIONS

Our results, although preliminary, provide evidence for

the dose-dependent in vitro biological activity and

scavenger activity of the Mentha piperita extract against

oxidative tension induced in rabbit spermatozoa. The

design of semen extenders offering a better protection to

male gametes from oxidative damage and improve their

energy requirements is more than necessary. Peppermint

extracts, in small amounts, could be used as a ROS

scavenging and a metabolic promoting supplement,

especially in common andrology protocols including in

vitro fertilization, artificial insemination or spermatozoa

cryopreservation. At the same time, we may emphasize

on the importance to examine specific effects the

biomolecules present in Mentha piperita may have

individually or collectively on the in vitro sperm vitality

and oxidative profile.

ACKNOWLEDGMENT

This study was supported by the European Community

Project no. 26220220180: Building Research Centre

“AgroBioTech”, by the Scientific Grant Agency of the

Ministry of Education of the Slovak Republic and of the

Slovak Academy of Sciences VEGA Project no.

1/0039/16, and by the Slovak Research and Development

Agency Grant no. APVV-15-0544.

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Eva Tvrdá was born in Nitra, on August 4th, 1984. She gained her

Mater title in Biotechnologies, followed by a PhD in Molecular Biology

at the Slovak University of Agriculture in Nitra, Slovakia. She works as a senior research scientist at the Department of Animal

Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra. At the same time, she is the head of

the Laboratory for Andrology and Molecular Toxicology at the

AgroBioTech Research Center. She has published over 100 articles and 6 book chapters on the topics of male reproduction, reproductive

toxicology, oxidative balance and antioxidants. Dr. Eva Tvrdá is a former Sciex MNSch and Fulbright fellow, member

for the European Federation of Animal Science and the Association for

Applied Animal Andrology.

Natália Konečná is a Master student at the Faculty of Agrobiology and

Food Resources, Slovak University of Agriculture in Nitra. Her research

focuses on the effects of plant extracts on male reproduction.

Katarína Zbyňovská is a junior research scientist at the Department of

Animal Physiology, Faculty of Biotechnology and Food Sciences,

Slovak University of Agriculture in Nitra. She is interested in oxidative and antioxidant profiles of biological systems.

Norbert Lukáč is a full professor at the Department of Animal

Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra. His research is focused on the

behavior of reproductive cells and tissues in health and disease.



Documents

Antioxidant Effects of Peppermint (Mentha piperita ...Antioxidant Effects of Peppermint (Mentha piperita) Extract on the Oxidative Balance of Rabbit Spermatozoa . Eva Tvrdá, Natália