The Potency of Antifertility Effect of Stem Bark Extract of Mangrove

(Avicennia Marina) on Male White Rats (Rattus Novergicus)

Nikeherpianti Lolok, Nurlena Ikawati, and Fatmasari Siharis

STIKES Mandala Waluya Kendari Jl. Jend. AH. Nasution, Kambu, Kota Kendari, Sulawesi Tenggara,

Keywords: Male contraception, antifertility, stem barkof mangrove Avicennia marina.

Abstract: The issue of Indonesian population density is one of must be faced problems. Stem bark of mangrove

Avicennia marina is one of many plants can be used as contraception. Recently reported, in many countries

this Avicennia marina used as antifertility nevertheless there are no focus research of the topic proven. The

object of this study is to determine the effect of stem bark extract of mangrove Avicennia marina on

number, motility and viability of spermatozoa of male white rats and also to assess the effect of certain

treatment group of some fractions compared to control CMC-Na. This study is experimentally designed in

laboratorium. Stem bark of Avicennia marina was extracted by soxhlet extraction with methanol solvent,

while animal testing were classified to 4 group (each group for 6 rats randomly). The subject were treated

along 34 days everyday, the group 1: 1% CMC-Na as a control; Group 2: low dose methanol suspension

extract of 1.25%; Group 3: medium dose methanol suspension extract of 2.5%; and Group 4: high dose

methanol suspension extract of 5%.Motility test was conducted by putting a drop of sperm on improved

neubauer counting chamber and observed by Olympus BX53F microscope (400x magnifying). Then the

data analyzed by ANOVA (p value 95%) prior to post hoc test by Duncan (SPSS 20).Motility index of each

group 5%, 2.5%, 1.25% and controlwere35%, 47%, 67% and 79% respectively. While, for viability index in

group 5%, 2.5%,1.25% and control were 62%, 76%, 73% and 88% respectively.The treatment of stem bark

extract of mangrove Avicennia marina in male white rats had significantly related to motility and viability

index of male rats sperm (p <0.005). This results means that stem bark extract of mangrove Avicennia

marina showed antifertility effect in male rats.

1 INTRODUCTION

The problem of Indonesian population density is one

of many problems that still appearing and not

solved. The raising of population number in every

year is still alarming because not balanced with the

increasing of prosperity. Based on population census

in 2010, the number of population raised to

237,641,326 compared to 2000 that only

206,264,597 (BPS,2012). The other problem is

Indonesian population growth rate increase 1.38%

from 2010-2015 (BPS, 2017).

The growth of population does not only impact

to the citizen prosperity in agricultural field but also

in job opportunities, education, health, and

residences. Therefore, government concerns to

promote family planning program to help a person

managing birth distance with many methods of

contraception offered by government for women and

men (satriyasa,et all,2017).

Family planning program held by government

does not work optimally because the participation of

men in the program still in low level compared to

women. This situation occurred because there are

still no safe and comfort family planning tools for

men. Based on the data, the participation of

Indonesian men joining family planning program

still in 5.5% comparing to other countries like

Pakistan of 10.9%; Nepal of 18% and Bangladesh

reached to level of 19% (Askrening, 2017).

Therefore, it needs to be discovered the men

contraception that safe for long term use

(Wiryawanet al, 2017).

The use of herbal remedies as traditional

medicines in Indonesia has been developing since a

few decades. Many kinds of plants can be used as

herbal remedies to make contraception effect. WHO

has formed a group of study to maintain the

regulation of men fertility with explorating materials

Lolok, N., Ikawati, N. and Siharis, F.

The Potency of Antifertility Effect of Stem Bark Extract of Mangrove (Avicennia Marina) on Male White Rats (Rattus Novergicus).

DOI: 10.5220/0009587701410146

In Proceedings of the 6th International Conference on Advanced Molecular Bioscience and Biomedical Engineering (ICAMBBE 2019) - Bio-Prospecting Natural Biological Compounds for

Seeds Vaccine and Drug Discovery, pages 141-146

ISBN: 978-989-758-483-1

141

or compounds as antifertility agent that safe,

effective, and acceptable (Febrianti, 2016).

One of plant that predicted as contraception is

stem bark of mangrove Avicennia marina. In many

contries, stem bark of this kind of mangrove is

empirically used as antifertility agent, but there are

no study reported focus on this. Avicennia marina

has many kind of secondary metabolites such as

terpenoid,steroid, naphthalene, flavonoid, glucoside

iridoid, phenyl propanoid glycoside and diterpenoid

glucoside (Esau et all., 2015). Flavonoid is one of

compounds that have antifertility effect. The

preview research showed that flavonoid contained in

black tea had antifertility effect on male rats

(Delfita, 2014).Therefore, the authors conducted this

research to determine the potencial of antifertility

effect of Avicennia marinaon male white rats

(Rattusnovergicus) especially to count the number,

motility, and viability of spermatozoa of male white

rats. Also to assess the effect of certain treatment

group of some fractions compared to control CMC-

Na. Then, this study hopefully can be reference of

the next researcher in developing safe and comfort

contraception.

2 LITERATURE REVIEW

Avicennia marinais known as white mangrove

(avicenniaceae). Forming shrubs or trees with a

height of 10 meters, and 14 meters in the tropics, it

is living in areas with high salinity and coastal

protection. And it has been reported that it can

tolerate extreme weather and harsh winds (Nayaket

all , 2014).

Avicennia marina is commonly used as an

antioxidant, antitumor, anti-inflammatory,

antimicrobial, antiaging, renal anticholine,

antiartheroscelerosis, and antituberculin. But it is

mentioned that Avicennia marina extract is more

effective as an antibacterial compared to antifungal.

This is related to the chemical content in the form of

alkaloids, flavonoids, tannins and glycosides

(Danata, 2014). In addition to above compounds

there are also other bioactive compounds such as

similar components, fatty acids, heterocyclic

oxygen, proanthocyanidins, quinones, stilbenes,

terpenoids and triterpenoid saponins (behbahaniet all

, 2012).

White rats are rodents and are often used as

experimental animals or used for research because

rats are animals that represent the class of mammals,

which humans are also from the mammal group so

that homogenity, organ completeness, nutritional

requirements, biochemical metabolism, reproductive

system, respiration, blood circulation, genes and

excretions resembling humans. Male white rats

generally have a weight of 450 - 520 g and females

250 - 300 g. Daily need for food is 5-10 g / 100 g

body weight and drink 10 ml / 100 g body weight.

Normal cholesterol levels range between 40-130 mg

/ dl and blood glucose levels 50-135 mg / dl

(Wolfensohn& Lloyd, 2013). In contrast to other

mammals, white rats have longer spermatozoa

around 150-100 nm. The morphology of the rat

sperm head is shaped like the same hook as most

rodents (Wuwunganet all, 2017).

Contraception is a tool or a way that is conducted

to inhibit the normal process of ovulation,

fertilization or implantation. The decision to use

contraception is influenced by the partner, health

and method of contraception. (Novita et all , 2016)

The method of contraception in men that is

currently available is limited to condoms, vasectomy

and hormone injections, but the contraceptives

above cause many side effects and do not

completely prevent pregnancy so that it is less

accepted by the public, therefore it is necessary to

discover and develop new contraceptive alternatives

that prevent fertilization, safely, effective, minimal

side effects, and does not reduce the potential for sex

and libido (Priastini, 2014).

Antifertility contraception is one of family

planning program effort to prevent conception after

marriage. Compounds such as alkaloids, flavonoids,

terpenoids, steroids, saponins and tannins are found

in peel extracts of durian which is proven to have

activity as male antifertility (setyowatiet all, 2015).

Herbal antifertility for men needs to be

prioritized because it has several ways to suppress

fertilization by suppressing spermatogenesis and

decreasing sperm motility so that it fails to enter the

cervix temporarily (Satriyasa, 2017).

3 METHODS

3.1 Design of Study

This is an experimental design research with one

factor (difference level of stem bark extract of

mangrove) completely randomized design and aimed

to determine the antifertility potency of stem bark

extract of Avicennia marina on male white rats.

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3.2 Instruments and Materials

3.2.1 Instruments

Instruments used in this study were blender, siever

(mesh 40), electric scale (OHAUS), microscope

Olympus BX53F, Neubauer counting chamber,

rotary evaporator, glasswares, soxhlet instruments,

spuit injection, hematocryte pipette, and animal feed

equipments.

3.2.2 Materials

Materials used in this study were stem bark of

Avicennia marina, methanol, 1% CMC-Na

suspension, aquadest, alcohol 70%, dye, giemsa,

0.9% saline solution, chloroform, and animal feed.

3.3 Sample Preparation

Stem bark of mangrove Avicennia marina were

collected from Langkema, West Kabaena, Bombana

regency, South East Sulawesi. The bark used must

come from a tree with a trunk diameter of about 10-

20 cm.

The stem bark were separated from the

impurities then washed and dried. Samples were

dried premises n aerated for 2-4 days and powdered.

The sampleextracted by the soxhletation method

with methanol as a solvent. The soxhletation tool

was installed, then the 250- gram of dried sample

was wrapped in filter paper, tied with yarn, put into

a round bottom flask on the soxhlet, with a solvent

volume of 1000 mL (1: 4). Soxhletation carried out

at a temperature of 70°C until the cycle droplets

were no longer colored. The liquid extract obtained

then concentrated using a rotary evaporator at 40°C

until viscous methanol extract was obtained.

3.4 Treatment of Subjects

The experimental animals used in this study were

Wistar strain male rats that were 8 weeks old and

weighed between 150-200 g and placed in a cage.

The animals were adapted in the experimental cage

one week prior to be treated. The state of the cage is

maintained at a temperature of 28-32C and a dark-

light cycle of 12 hours each. Experimental animals

were fed standard diet pellets and ad libitum

drinking water (Sornalakshmi, TresinaSoris,

Paulpriya, PackiaLincy, & Mohan, nd; Suresha et

al., 2012). The ethical clearance approval was issued

by Commission on ethical clearancefor preclinical

research Integrated Research and Testing

Laboratory.

3.5 Antifertility Test

For animal testing, the test was divided into 4 groups

with each group consisting of 6 rats randomly

selected. Rats were weighed and given identification

on the tail. Then the treatment was given every day

for 34 days with the following procedures:

Group 1 : 1% CMC-Na Control

Group 2 : low dose of methanol extract

1.25% suspension

Group 3 : medium dose of methanol extract

2.5% suspension

Group 4 : high dose of methanol extracts 5%

suspension

Furthermore, on day 35 animal surgery and

retrieval of sperm in the cauda epididymis obtained

by sucking the sperm from the cauda epididmis with

0.5 hematocryte pipette up to the mark then diluted

with normal saline (dilution 200 times). This

suspension is used to see the number, motility and

viability of spermatozoa.

Observation of motility was conducted by

dripping sperm preparations in the counting

chamber, then observed under an Olympus BX53F

microscope with magnification 400 times. Motility

index of spermatozoa in this study was counted by

calculating the percentage of progressive categories

which straight forward and fast in 100 spermatozoa.

The dyes were used to observe spermatozoa

morphological at the same magnification. Viability

index of sperm was observed by observing

suspension droplets added by one drop of giemsa

added with alcohol on glass objects and observed

under a microscope, live sperm were stated to be

counted from 100 sperm in percent.

3.6 Data Analysis

The analysis of this study used SPSS 20 software.

The method used was the analysis of variation

(ANOVA) with 95% confidence level and continued

with post hoc using the Duncan test.

The Potency of Antifertility Effect of Stem Bark Extract of Mangrove (Avicennia Marina) on Male White Rats (Rattus Novergicus)

143

4 RESULT AND DISCUSSION

4.1 Result

Sample

Motility

0 1 2 3

1 5 % 4

100 35 %

2 2.5 % 2

100 76 %

3 1.5 % 1

100 73 %

4 CMC-

8 1

100 88 %

NO Sample Σ colored Uncolored

Percentage

1 5 % Σ 32 68 62 %

2 2.5 % Σ 24 76 76 %

3 1.5 % Σ 22 73 73 %

4 CMC-Na Σ 12 88 88 %

NO Treatment

Sperm

(million/

mL)

Motiliy

(%)

Viability

(%)

1 5 % 1,400 35 62 %

2 2.5 % 2,140 47 76 %

3 1.5 % 1,440 67 73 %

4 CMC-Na 1,900 79 88 %

5 DISCUSSION

Based on ANOVA and post hoc table with Duncan

test, it was shown that the administration of stem

bark extract ( Avicennia marina ) caused a decrease

in the number of spermatozoa motility and viability

in the male white rat epididymis (Rattusnovergicus).

The number of rat spermatozoa (Rattusnovergicus)

in the treatment of 5% stem bark extract of

Avicennia marina was significant (p ≤ 0.5)

compared to the control.On the other hand, the 1.25

% and 2.5% stem bark extract of Avicennia marina

were not significantly different. The average number

of white rat spermatozoa in the control was 1,900

million/mL; 1.25% was 1,400 million/ml; 2.5% was

2,140 million/ml and 5% was 1,440 million/ml.

The spermatozoa motility of subjects in the

treatment of 5% sample was significantly different

(p ≤ 0.5) with control and other groups of treatment.

The mean motility of rat spermatozoa in control was

79%; 1.25% was 67%; 2.5% was 47% and 5% was

35%.

Viability index of rat spermatozoa in the

treatment of 5% sample was significantly different

(p ≤ 0.5) with control and other groups, nevertheless

the viability index for group treatment between

1.25% and 2.5% sample were significantly different.

The average viability of white rat spermatozoa in

control, group 2, 3, and 4 were 88%, 73%, 76% and

62% respectively. The average number, motility and

viability index of spermatozoa in the control and

each treatment can be seen in table 3.

Based on the results of the study it is known that

the administration of sample1ml/100g BW adult

male rats orally causes a decrease in the number of

spermatozoa in cauda epidedimis, motility and

viability of spermatozoa. The higher dose of sample

was given, the lower amount of motility and

viability of rat spermatozoa (Rattusnovergicus).

In this study it was known that the administration

of sampleaffected the number of spermatozoa in

cauda epidedimis. The number of spermatozoa in the

group 3 (5% of sample suspension) treatment was

significantly different from other groups (p ≤

0.05%). The decrease in the number of spermatozoa

was probably caused by a decrease in the number of

leydingcells by ROS thereby reducing

intratestheticteston levels so that it affected

spermatogenesis. In other words, ROS causes a

decrease in the number of hormone-producing

leyding cells that play a role in spermatogenesis, so

that spermatogenesis is disturbed/stopped, as a result

the number of spermatozoa is reduced. This result is

in line with the research of Nayantara et al.,(2008: 3)

which showed a decrease in the number of

spermatozoa due to increased ROS in rats

(Rattusnovergicus) treated with monosodium

glutamate. The decrease in the number of

spermatozoa is also caused by the death of

spermatozoa cells due to ROS, especially hydroxyl

radicals causing lipid peroxidation.

Based on the results above, it is known that the

administration of stem bark extracts of Avicennia

marina 1ml/100g BW in adult male rats orally

causes a decrease in spermatozoa motility. Motility

index of spermatozoa between treatments were

significantly difference (p≤ 0.05%). Observed

spermatozoa motility index were divided into four

categories: fast moving, slow moving, localized

moving and stationary moving spermatozoa. In this

study, the stem bark extract of Avicennia marina

decreased the spermatozoa motility of rats. This is

because mammalian sperm are rich in unsaturated

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fatty acids in the plasma membrane so that they are

very vulnerable to ROS attack (Tre mallen, 2008:

244).

Spermatozoa motility is produced by a long tail.

Tail movements are carried out by energy produced

by mitochondria which are concentrated in the

middle of the sperm (Shewood, 2004: 520). In this

experiment, observation on living and dead sperm

cells was carried out by an indication of

Eiosinnigrosin. Live sperm cells will be bright white

(transparent) while the dead ones will be red. Died

sperm cells occurred due to the direct contact with

air that lead to sperm oxidation. Long term storage

of sperm cells causes a decrease in sperm motility

due to remaining cell metabolism namely lactic acid

which causes acid condition of the medium and can

be toxic to spermatozoa which ultimately causes

sperm death (Sugiarti et al., 2004). To assess the

viability index of spermatozoa, Eiosinnigrosin

staining was used. According to WHO standards,

this staining technique provides valid result with a

review of the motility obtained data. This technique

of staining eiosinnigrosin is a simple technique. In

this case the eosin dye will be absorbed by the dead

spermatozoa so that they will turn red or pink due to

increased cell wall permeability when the

spermatozoa die. While nigrosin will color the

background of spermatozoa (Septiyani, 2012).

The result also showed that the suspension of

samples affected to the viability index of

spermatozoa. The higher dose of the extract was

given, the lower viability of rat spermatozoa was

obtained. This is caused by flavonoids/ antioxidants

that contain excessive doses of sample cause an

increase in antioxidants in the body. It is also caused

by the ability of flavonoids/antioxidants to form

ROS, both of which will damage the spermatozoa

plasma membrane.

6 CONCLUSION

From the results above, can be concluded that stem

bark extract of mangrove Avicennia marina had

potential activity as antifertility on male rats proven

by decreasing of epidedimis weight, spermatozoa

number, motility and viability of rats. The best dose

as antifertility is 5%.

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