Asian Journal of Biochemical and Pharmaceutical Research Issue 2(Vol.

7) 2017 ISSN: 2231-2560

10.24214/AJBPR/7/2/7075 CODEN (USA): AJBPAD
Research Article

Asian Journal of Biochemical and Pharmaceutical Research

The effect of Kalimantan’s honey propolis toward the quality of mice’s (mus musculus
l.) spermatozoa that exposed by cigarette smoke

Muhammad Eko Andry Setyawan 1, Yusuf Alam Romadhon 2, Retno Sintowati 2, EM Sutrisna3, Oktavian Yoga
Nugraha 1, Fida 'Mushalim Afwan 1

*1Student Of Faculty Of Medicine University Of Muhammadiyah Surakarta

2Lecturer Faculty Of Medicine University Of Muhammadiyah Surakarta
3Dean Of The Faculty Of Medicine University Of Muhammadiyah Surakarta

Received: 06 June 2017; Revised: 24 June 2017; Accepted: 10 July 2017

Abstract: Propolis contains flavonoids that act as antioxidants beneficial in preventing cell damage from
oxidative stress. This study aimed to determine the effect of Kalimantan’s honey propolis toward motility and
viability of male mice spermatozoa (Mus musculus L.) that exposed by cigarette smoke. The study subjects
using 25 male mice Swiss strain were divided randomly into 5 groups. The division of the group consisting of:
normal control (given Aquadest), negative controls (treated by 1 cigarette per day), treatment 1 (treated by one
cigarette + @ 0.1 ml of extract of propolis), treatment 2) treated by 1 cigarette + @ 0 , 2 ml of extract of
propolis), treatment 3 (treated by 1 cigarette + @ 0.4 ml propolis extract). Treatment lasted for 47 days, on day
48 mice in termination, then examined the quality of motility and viability of sperm that. Average results
obtained in mice sperm motility negative controls (13.20%), normal controls (46.34%), treatment 1 (26.56%),
treatment 2 (30.16%), and treatment 3 (33.60 %). The results mean viability of spermatozoa of mice obtained in
the negative control (21.20%), normal controls (31.86%), treatment 1 (48.40%), treatment 2 (48.56%), and
treatment 3 (53.00 %). Test results one way ANOVA there are significant differences in the quality of sperm is
sperm motility and viability of spermatozoa (P <0.05). The Kalimantan’s honey propolis may improve sperm
quality of mice (mus musculus L.) after exposed by cigarette smoke.

Keywords: motility, viability, propolis, flavonoids, antioxidants

INTRODUCTION:
Infertility is a condition that causes a failure of conception in couples who have been married
for more than a year, who do not produce offspring even though they do not follow family planning
programs. The frequency of infertility is about 10-15% of the couples, and about half (40%) of cases
are caused by abnormalities in men.[1]. The etiology for male infertility factors is abnormalities in
sperm motility (SM), viability (SV), and DNA integrity [2].
Some conditions may interfere with spermato- genesis and reduce sperm quality such as drug
therapy, chemotherapy, toxicity, free radicals [3] Exposure to free radicals over a period of time can
lead to decreased fertility by decreasing testicular weight, increased lipid peroxidation, decreased
antioxidants (vitamin C) and Oxidative damage. The common source of free radicals in a man is a
cigarette [4].Smoking is one of the habits that can be bad for health, both for the active smokers
themselves and passive smokers in the surrounding environment [5]. Many potentially cytotoxic
compounds in cigarette smoke, for example, nicotine, carbon monoxide, aldehydes, ketones,
nitrosamines, dihydroxybenzenes [6]. There are three main toxic components contained in cigarette
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smoke, namely carbon monoxide, nicotine, and tar. Several studies on the effects of chemicals from
cigarettes show a disruption to spermatogenesis through increased production of reactive free radicals
or oxygen. Smoking can increase free radicals and reduce antioxidants in semen and can cause DNA
damage through cellular DNA fragmentation and morphological abnormalities (head, neck and tail) of
spermatozoa [7].

In addition cigarettes also reduce infertility by decreasing testosterone levels. This will
interfere with the process of spermatogenesis because spermatogenesis runs under the influence of
testosterone. So at the maturation stage of spermatids into mature spermatozoa, interference may occur
that can affect the normal morphology [8]. We have done various researches on traditional medicine
that has many benefits in balancing the body's defense system, one of them is propolis [9].Propolis has
the benefit of being anti-bacterial [10], antifungal (Hasanah 2012), antiviral [12]. Propolis contains
50% resin, 30% wax, 10% essential fat, 5% pollen, and 5% organic components. Resins consist of
alkaloids, phenols, amino acids and 12 different types of flavonoids, including chrysene in them.
Propolis also contains complete vitamins, including vitamin A, vitamin B1, vitamin B2, vitamin B6,
vitamin C, vitamin E and vitamin D (Nugroho 2016)
Flavonoids serve as exogenous antioxidants that have been proven useful in preventing cell
damage due to oxidative stress. The mechanism of action of flavonoids as antioxidants can be directly
or indirectly. Flavonoids as antioxidants directly are donated hydrogen ions to neutralize the toxic
effects of free radicals. Flavonoids as antioxidants indirectly by increasing the expression of
endogenous antioxidant genes through several mechanisms. One of the mechanisms to increase the
expression of antioxidant gene is through activation (Nrf2) so that there is an increase in genes that
play a role in the synthesis of endogenous antioxidant enzymes such as SOD gene (Sumardika & Jawi
2012). Flavonoid performance in the body is also able to bind to estrogen alpha receptors (REα) in the
testes and epididymis which can replace estrogenic function and work together with testosterone for
spermatozoa maturation [15]
This study aimed to determine the effect of Kalimantan’s honey propolis toward motility and
viability of male mice (Mus musculus L.) spermatozoa that exposed by cigarette smoke.

MATERIALS AND METHODS:

Research Design: This research is laboratory experimental with complete randomized design (post-
complete randomized design) posttest only with control group desaign. This research has met the
declaration of Helsinki 1975 and national health research ethics Department of Health of the Republic
of Indonesia in 2004, with number 658/A.2.KEPK-FKUMS/IV/2017.
Place and Time of Research: Was performed in the anatomy pathology laboratory of the Faculty of
Medicine, Muhammadiyah University of Surakarta in September 2017. The subjects used in this study
were male webster males aged 2-3 months with weight ± 20-30 gr.
Ethanol Propolis Extract: The method of extraction used in this research is the maceration method.
[16] The first step in the extraction of ethanol propolis is rough propolis weighed 500 grams, then
dissolved in 1000 mL of ethanol 70% and fed into Erlenmeyer flask. Erlenmeyer flask that has
contained propolis extract wrapped in dark plastic then incubated over the orbital shaker for 7 days.
After incubation for 7 days, Erlenmeyer flask containing propolis extract was put into the microwave
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for 30 min at 50 ° C. The ethanol extract of propolis is then filtered and placed on a steam plate heated
at a temperature of about 40 ° C. [16]
Calculation of Dosage and Treatment: This study used 25 male mice Swiss strain were divided into
5 groups, each group consisting of 5 mice were selected by random sampling. Division of the group
consists of: normal control (given Aquadest), negative control (given 1 cigarette every day), treatment
1, treatment 2, and treatment 3.
The EEP treatment dose was devided into 3 group: treatment 1 was treated by a cigarette + 0.1 ml of
propolis extract, treatment 2 was treated by 1 cigarette + 0.2 ml of propolis extract, treatment 3 was
treated by 1 cigarette + 0.4 ml extract Propolis.
Treatment of exposure to cigarette smoke is done after the animals try to experience acclimatization
for one week [18]. Each treatment of exposure to cigarette smoke done in the cage treatment, then just
given the extract of propolis. This treatment is done every day for 47 days starting at 16:00 pm until
complete. On day 47, all mice were sacrificed by dislocation of vertebrae cervicalis. [19]. Cauda
epididymis is separated by cutting the proximal portion of the epididymis corpus and the distal part of
the vas deferens. The cauda epididymis was then fed into a petri dish containing 1 ml of 0.9% NaCl,
the proximal portion of the cauda was cut slightly with scissors and the cauda was gently pressed until
the epididymic fluid secretion came out and suspended with Nacl 0.9%. [20] The process is carried out
several times then stirred homogeneously. Suspension of spermatozoa from cauda epididymis is used
for spermatozoa quality observation which includes motility and viability.
Sperm Motility Test: Homogeneous samples were collected as much as 10 μl. [19] Subsequently
placed on a hemocytometer, Spermatozoa motility examination was performed using a 400 ×
magnification microscope [21]. Observations were made on 100 spermatozoa and repeated 3 times for
one test animal. [22]. The motility of spermatozoa can be known by counting the number of moving
spermatozoa from 100 spermatozoa [19].
Sperm Viability Test: Spermatozoa solution was taken as much as 10 μl using a micro pipette and
dripped on the hemocytometer. The sample is then dropped with a 10 μl Eosin-Y solution and covered
with a cover glass. The viability of the sperm was calculated using a microscope with 400x
magnification [21]. Percentage of live spermatozoa counts of 100 spermatozoa for each replication.
[19]. For sperm viability assessments, spermatozoa with red or dark red heads are considered dead
while spermatozoa with white heads or pink heads are considered alive. The viability parameter is
collected and recorded as a percentage of viability. [23]

RESULTS
Research on the effect of Kalimantan’s honey propolis toward sperm quality of mice (mus
musculus L.) that exposed by cigarette smoke can be seen the following results:

1. Motility: The mean of the research showed that motility of spermatozoa mice on negative control
(13,20%), normal control (46,34%), treatment 1 (26,56%), treatment 2 (30,16%), and treatment 3
(33, 60%).
2. Viability: The mean of the research were obtained viability of spermatozoa mice on negative
control (21,20%), normal control (31,86%), treatment 1 (48,40%), treatment 2 (48,56%), and
treatment 3 (53, 00%).

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Spermatozoa Motility : Based on the results of data analysis showed that there was a significant
improvement of spermatozoa motility percentage on the mice given the extract of natural honey
propolis typical of Borneo and exposure to cigarette smoke compared with the negative control which
only given exposure to cigarette smoke. The data were tested for normality to know the distribution of
data evenly using Saphiro-Wilk test (n = 21 samples). The spermatozoa motility data tested
homogeneity with Levene test with result p = 0,342 (p> 0,05). It means that the data was homogenous.
The following test was one way anova. One way Anova test results showed that p = 0,008 (p <0,05), It
means that there were significant differences among groups. To determine which groups differed
significantly, a Tukey test was performed (table 1).
The Viability Of Spermatozoa : Based on the results of data analysis showed that there was a
significant improvement of spermatozoa motility percentage on the mice given the extract of natural
honey propolis typical of Kalimantan and exposure to cigarette smoke compared with the negative
control which only given exposure to cigarette smoke. The data were tested for normality to know the
distribution of data evenly using Saphiro-Wilk test (n = 21 samples). The spermatozoa motility data
tested homogeneity with Levene Test with result p = 0,113 (p> 0,05). It means that the data was
homogenous. The following test was one way anova. One way Anova test results showed that p =
0.001 (p <0.05). It means that there were significant differences among groups. To determine which
groups differed significantly, a Tukey test was performed (table 1).

DISCUSSION
Spermatozoa Motility :Based on the results of research and data analysis, male mice were given
exposure to cigarette smoke showed lower concentrations compared with mice given distilled (normal
control) and given a propolis extract natural honey typical of Kalimantan and exposure to cigarette
smoke (P1, P2, and P3). This shows a decrease in spermatozoa concentration in the group of mice
given exposure to cigarette smoke and improved spermatozoa concentration in the group of mice given
propolis extract and exposure to cigarette smoke.
The decline in sperm quality resulting oxidative stress caused by the increase in ROS (Reactive
Oxygen Species) from cigarette smoke. The decrease in the number of spermatozoa also occurs as a
result of chemical substances in cigarette smoke such as nicotine, tar, carbon dioxide so that it has the
potential to lead to increased free radical production [24]which will lead to DNA damage and
ultimately apoptosis spermatozoa. So it will be a decline in sperm quality [20]. Propolis natural honey
is proven to improve the quality of spermatozoa, this is because in Propolis contain 50% resin, 30%
wax, 10% essential fat, 5% pollen, and 5% organic components. Propolis also contains complete
vitamins, including vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin E and vitamin
D (Nugroho 2016). Besides propolis also contains flavonoid which has the most powerful antioxidant
activity against oxidants and free radicals (radical H2O2, O2 ● -, OH ●). [25]
This is supported by Luthfi's research stating that vitamin content in aphrodisiac material also
positively affects fertility improvement. It is supported by Hardiyono and Soekanto that proves
vitamin content in royal jelly can increase the weight of the testes which means that there is an
increase in fertility after the provision of royal jelly. One ingredient that compares chrysene and
various vitamins is propolis which is a product of bees origin [13]

CONCLUSION
The Kalimantan’s honey propolis may improve sperm quality (motility and viability) of mice (mus
musculus L.) that exposed by cigarette smoke.

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Table 1. Tukey Test with Post Hoc Test on motility test

Variable Negative controls Normal control Treatment 1 Treatment 2 Treatment 3
Negative controls - , 007 * , 078 , 047 * , 017 *
Normal control , 007 * - , 809 , 927 , 999
Treatment 1 , 078 , 809 - , 999 , 927
Treatment 2 , 047 * , 927 , 999 - , 983
Treatment 3 , 017 * , 999 , 927 , 983 -

Table 2. Tukey Test with Post Hoc Test on viability test

Variable Negative controls Normal control Treatment 1 Treatment 2 Treatment 3
Negative controls - , 991 , 023 * , 026 * , 009 *
Normal control , 991 - , 036 * , 044 * , 014 *
Treatment 1 , 023 * , 039 * - 1,000 , 988
Treatment 2 , 026 * , 044 * 1,000 - , 981
Treatment 3 , 009 * , 014 * , 988 , 981 -
Signs * = show significantly different results (p <0.05)

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Corresponding Author: Muhammad Eko Andry Setyawan, Student of Faculty Of Medicine University Of
Muhammadiyah Surakarta, INDONESIA

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