The Mammary Histopathology Depiction of Female Mice Induced by

Staphylococcus Aureus Bacteria after Scaevola Taccada Extract

Administration

Nurul Aini Siagian

, Mutiara Dwi Yanti

Andayani Boang Manalu

, Putri Ayu Yessy Ariescha

Firdaus Fahdi

, Prihantono

Midwifery Faculty, Institut Kesehatan Deli Husada Delitua

Pharmacy Faculty, Institut Kesehatan Deli Husada Delitua

Department of Oncology, Medical Faculty of Universitas Hasasanuddin Makassar

Keywords: Histopathology, Scaevola Taccada, Staphylococcus Aureus

Abstract: The public is concerned about chemical-based anti-inflammatory drug usage, so it is an indication to find an

anti-inflammatory drug made from herbal plants, one of them is from the Scaevola taccada (Gaertn.) Roxb.

This research was conducted by finding out the histopathological depiction of female mice (Gaertn.) Roxb.

induced by Staphylococcus aureus bacteria after the administration of Scaevola taccada (Gaertn.) Roxb.

Samples were divided into two groups, namely the control and treatment group. The control group was

given by antibiotic amoxicillin 9.59 mg/kg bb, while the treatment group was given by antibiotic

amoxicillin 9.59 mg/kg bb plus Scaevola taccada extract 400 mg/kg bb, an anti-inflammatory determination

is done by the formation of artificial inflammation or mastitis with the induction of staphylococcus aureus

bacteria in mammary parts of rats.

1 INTRODUCTION

Inflammation is a protective response toward tissue

injury caused by physical trauma, chemicals,

damage, or microbiological substances. Anti-

inflammatory drugs are a class of drugs that have

analgesic (pain relief), antipyretic (heat-reducing),

and anti-inflammatory properties. This drug is

relatively safe if it is used by at the appropriate

dosage. But concerns will arise if it is consumed in

high doses or long term because it will cause side

effects such as liver poisoning, digestive tract

disorders, kidney disorders and others (Sitti Amirah

Rahmawati, Safriani Rahman, 2014).

Staphylococcus aureus is a major pathogen in

humans that causes various clinical infections. At

present, staphylococcus aureus is also used to

diagnose mastitis known as "infectious mastitis"

which is being applied to describe acute case

conditions. Staphylococcus aureus can express a

variety of surface proteins that can play key role in

the infection process because it can promote

bacterial adhesion to host cells and tissues, as well

as obtain important nutrients and avoid immune

responses (Contreras and Rodríguez, 2017)

(Yagdiran et al., 2016) (Habib et al., 2015) (Cells,

2019).

Clinical infection caused by staphylococcus

aureus is likely to have a general or even serious

impact. It can be seen from antimicrobial resistance

increasing even clinical disease is also increasing

due to staphylococcus aureus bacteria (Tong et al.,

2015). Mastitis is an inflammatory disease of the

breast. Staphylococcus aureus is the most dominant

bacterium that is often found by inflamed breasts, in

other studies saying that the mammary glands

sinuses and epithelial cells were damaged due to

staphylococcus aureus bacteria (Chen et al., 2014).

According to WHO 2013, many health cares of

medicinal plants have been used by preventing or

treating certain diseases. Because of the efficacy and

availability and also affordable prices so that the

demand for herbal medicines that use natural

ingredients continues to increase. There are millions

of people in major regions of developing countries

covering from 70 to 80 percent of health care needs

in the world population (Essien et al., 2017).

604

Siagian, N., Yanti, M., Manalu, A., Ariescha, P., Fahdi, F. and Prihantono, .

The Mammary Histopathology Depiction of Female Mice Induced by Staphylococcus Aureus Bacteria after Scaevola Taccada Extract Administration.

DOI: 10.5220/0010015306040610

In Proceedings of the International Conference on Health Informatics and Medical Application Technology (ICHIMAT 2019), pages 604-610

ISBN: 978-989-758-460-2

Scaevola taccada (Geartn.) Roxb. is trusted by

the public for treating digestive, anti-tumor, and

anti-inflammatory problems. The fruit can be used

as juice to overcome menstrual problems and

ringworm. The roots are also commonly used by

people as a treatment for dysentery, syphilis, and

beriberi. The Decoction of the leaves and bark can

prevent tachycardia, this plant can also reduce the

frequency of heartbeats, slow the pulse and stimulate

the heart to contract normally, anti-inflammatory,

anti-fungal, anti-bacterial and cough medicine

(Mejin, 2009) ( Chandran and Arunachalam, 2015)

(Review and Scavola, 2017).

According to research conducted (Suthiwong,

Thongsri, and Yenjai, 2016), there is strong

antifungal activation found in the content of

Scaevola taccada (Geartn.) Roxb. Scaevola taccada

(Geartn.) Roxb. has also been used as

dermatological aid in Hawaii. A mixture of root bark

crushed with salt is used by curing skin diseases. In

Indonesia, the roots are used as an antidote when

consuming poisonous fish and crabs. Scaevola

taccada is reported by having chemical constituents

from alkaloids, flavonoids, lipids, terpenoids,

glycosides, and saponins (Mejin, 2009) (Chandran

and Arunachalam, 2015).

Previous research on chemical component

groups found in diethyl ether fraction of Scaevola

taccada (Gaertn.) Roxb. was Flavonoid group with a

wavelength of 239.50 nm and had hydroxy,

aromatic, ketone, alkyl group and supported by spot

spotting after spraying with benedict reagents and

Antimony (III) Chloride reagents. The research

results also support traditional plants used for the

treatment of several diseases and inflammatory

conditions (Rachmat Kosman, 2012).

In this case, the researchers concluded by

utilizing the Scaevola taccada (Gaertn.) Roxb. to

reduce inflammation caused by staphylococcus

aureus bacteria), so it needs to be re-tested whether

there is an effect of the Scaevola taccada (Gaertn.)

Roxb. on inflammation that occurs in mammary

bacteria induced by female mice induced by

staphylococcus aureus bacteria.

2 METHOD

This research was experimental research by using

the post-test only controls group design. The study

was conducted at Biopharmaca Laboratory and

Biology Pharmacy Laboratory of UIN Makassar and

for the adaptation of mice to the end of the treatment

at Hasanuddin University animal laboratory.

Subjects in this study were twelve strains Sprague

Dawley mice with the body-weight of 200-250

grams, divided into control and treatment groups.

The main ingredients were amoxicillin and Scaevola

taccada extract. Other materials were cotton alcohol,

10% formalin buffer solution, and container pots,

injection syringes, tissue, light microscopes, glass

objects, microtomes, water baths, and glass covers.

Mouse cages are plastic tubs covered by wire and

given sawdust and place to eat and drink.

The sample of this study was divided into two

groups. The control group was induced by

staphylococcus aureus (0.2 ml x 108 ml/CFU), and

they are given amoxicillin antibiotics at a dose of

9.59 mg/ml/ 250 gram bb of mice for five days.

While the treatment group induced by

staphylococcus aureus (0.2 ml x 108 ml/CFU), it

was given amoxicillin antibiotics at a dose of 9.59

mg/ml/ 250 gram bb of mice for five days and

Scaevola taccada extracts at dose of 400 mg/ml/kg

bb of mice for five days. Then on the 6

day, the

mice in euthanasia were taken and the mammary

part and examined for processing and making the

histopathological preparations.

The material was the Scaevola taccada plant

obtained from Watang Suppa Village, Suppa

District, Pinrang Regency. They are cleaned from

the dirt attached by using flowing water and then cut

the sample into small pieces. They are dried to

contain water content below 10%. Scaevola taccada

was sieved with a mesh size of 40 so that a smooth

Simplicia sample was obtained. After that, the

sample is ready to be extracted by maceration

method. Extraction by the maceration method used

70% ethanol solvent. First, the sample is moistened

with 70% ethanol until fully submerged for 15

minutes. After that, it is sufficient again to be two

liters with 70% ethanol at the temperature room for

3 x 24 hours while occasionally stirring. Macerate is

then filtered and the pulp is macerated again. The

extract obtained then evaporated by using a rotary

evaporator until it thickens, then dried with the help

of a water bath. The extract obtained is then

evaporated by using a rotary evaporator until it

thickens, and then dried with the help of the water

bath. The resulting viscous extract is inserted into

porcelain vial and weighed the extract weight.

Furthermore, the dose is converted to obtain a dose

of 400 mg/kg bb.

Then, staphylococcus aureus cultured was

planted in BHIB medium and incubated for 18-24

hours at 37° C in an incubator. Then the bacteria

were planted on NA (Nutrient Agar) medium and re-

incubated for 18-24 hours at 37° C. After bacterial

The Mammary Histopathology Depiction of Female Mice Induced by Staphylococcus Aureus Bacteria after Scaevola Taccada Extract

Administration

605

incubation, do gram staining. Biochemical tests were

carried out by NA colonies for staphylococcus

aureus by planting on DNAse agar medium and then

mannitol salt agar, then doing bacitracin and

Novobiocin tests followed by catalase coagulase

test. Then it was re-incubated for 18-24 hours with a

temperature of 37° C. The bacteria that grew on

biochemical tests were matched with the

identification table of staphylococcus aureus

bacteria. To make bacterial samples that were

injected into mice by making suspension in a

physiological NaCl solution as much as 10 ml mixed

with golden yellow S. aureus bacterial colony with

turbidity level of Mc Farlan 2 x 108 CFU. The

accuracy of the Mc Farland turbidity level is

measured by the Densi check tool.

Adaptationofanimal

Scaevolataccada Extractsacevolataccada

Histopathologicaltestmikroskopik Givingextracts

Figure 1. Research Scheme

3 RESULT AND DISCUSSION

The identification results of alkaloid compounds,

flavonoids, steroids/triterpenoid, saponins and

tannins groups in sea leaves (Scaevola taccada

(Gaertn.) Roxb. extracts showed positive results (+)

which means that there are alkaloid compounds,

flavonoids, steroids/triterpenoid, saponins and

tannins in Scaevola taccada (Gaertn.) Roxb).

extracts.

Flavonoid compounds are found in almost all

parts of plants including the outer bark, leaves, fruit,

and roots. Flavonoids are also natural compounds

that have potential as antioxidants that can

counteract free radicals that play a role in the

generation of degenerative diseases through the

mechanism of damage to the immune system, lipid

oxidation, and protein (Aminah, Tomayahu, and

Abidin, 2017).

Scaevola taccada plant used in this study is only

part of the leaf. The Scaevola taccada leaf can be

used as an anti-inflammatory because it contains

several compounds. One of them is the flavonoid

compound.

In this study, the Scaevola taccada leaves were

obtained from the coast of Watang Suppa Village,

South Sulawesi. Scaevola taccada leaves must be

cleaned of dirt that attaches to the leaves so as not to

interfere with the extraction process. Next, the

Scaevola taccada leaves cut into small pieces to

facilitate the drying process. The dried leaves are

extracted.

Extraction is a technique used to extract

compounds in the plant. The extraction technique

used in this study was the maceration technique

because the maceration technique is the simplest

extraction technique that is the easiest to do.

Maceration is done by using three times

recapitulation for 70% ethanol sailor for 24 hours

after the extraction process of liquid extract is

evaporated using a rotavator to obtain thick green

and dark extracts.

The subjects were 12 female Sprague Dawley

mice that were adapted by seven days at Hasanuddin

University animal laboratory, which met the

inclusion criteria. The results of the

histopathological study of female mammary mice

induced by bacterium Staphylococcus aureus after

Scaevola taccada (Gaertn.) Roxb. extract

administration by comparing with the control group

were only given antibiotics.

According to figure 3, it can be seen in the group

that only has not been given antibiotics. There are

many inflammatory cells in milk glands and

connective tissue, while after being given

amoxicillin, the inflammation antibiotics appear to

be reduced but still appear in some cells. However,

In Figure 4, inflammation cells can be seen before

antibiotic amoxicillin administration and Scaevola

taccada extracts, there are many inflammation cells

in milk glands and connective tissue after antibiotic

ICHIMAT 2019 - International Conference on Health Informatics and Medical Application Technology

606

treatment and extra Scaevola taccada inflammation

cells are reduced by only visible inflammation cells

in the connective tissue section.

Pre Post

Figure 2: Microscopic features in mammary mice control

group

Pre Post

Figure 3: Microscopic features in the mammary mice

treatment group

Figure 4: Inflammation Number Diagram

Table 1 shows that the average number of

inflammation in the control group was 54,000 while

in the treatment group was 91,600 with a mean

difference of 37.6. It meant that the treatment group,

namely the group was given by the Scaevola taccada

extract (Gaertn.) Roxb. was more influential in

reducing inflammation cells than the control group

that was only given amoxicillin.

Table 1: Histopathological examination results of mice

induced by Staphylococcus Aureus who were given by

treatment

Group Mean±SD

Mean

Deviation

P-

value

Control

(Amoxicil

lin)

54.000 ±

11.916

37.6

0.001

Treatment

(Amoxicil

lin+

Scaevola

Taccada)

91.600 ±

6.804

0.000

Inflammation is a localized protective response

caused by tissue damage or injury that functions to

destroy the agent and causes injury. It is

characterized by pain, heat, redness, swelling, and

loss of function. Inflammation is divided into two

namely acute inflammation that lasts quickly or

briefly, and chronic inflammation that occurs

repeatedly (Willianto and Wijayahadi, 2016).

The inflammation mechanism in the body which

is commonly called inflammation is an immune

response that maintains tissue homeostasis in the

body and allows the body to survive during the

process of injury or infection. Inflammation is also

normal and healthy body processes that are usually

produced by several disorders or various diseases

from the body's localized response to the general

response. So it can be concluded that inflammation

is the body's first reaction when injury and infection

occur in a person's body. Inflammation can be

treated with various types of anti-inflammatory

agents that are effective and safe to help treat or

reduce inflammation which is called agents (Ganesh

et al., 2014).

The occurrence of mastitis begins with an

increase in pressure in the duct (ASI channel) due to

stasis of ASI. If the milk is not removed

immediately, there is excessive alveoli tension and

causes the epithelial cells that produce milk to

become flat and depressed so that the connective

tissue permeability increases. Some components

(mainly immune proteins and sodium) from plasma

enter the breast milk and subsequently into the

The Mammary Histopathology Depiction of Female Mice Induced by Staphylococcus Aureus Bacteria after Scaevola Taccada Extract

Administration

607

tissues around cells so that it triggers an immune

response. Untreated ASI stasis results in an

inflammatory response and tissue damage making it

easier for breast infections (IDAI, 2013, Tristandi &

Nariyah 2019).

There are several ways the entry of germs into

the breast is through the lactiferous ducts into the

secretion lobes, through cracked nipples to the

lymph glands around the duct (periductal) or through

hematogenous (blood vessel) spread. The most

frequent organisms are Staphylococcus aureus,

Escherichia coli, and Streptococcus. (IDAI, 2013).

Mastitis treatment and prevention depend on

antibiotics usage. So, the abuse of antibiotics can

lead to antibiotic resistance. So it is necessary to

develop treatment strategies with natural ingredients

that have been widely used by various countries in

the world, which can reduce the risk of drug usage

which is made by chemicals (Cheng et al., 2019).

In another study stated that the treatment of

mastitis both infectious and non-infectious with

antibiotics can provide rapid resolution.

Erythromycin is considered the drug of choice

because it has high efficacy, low cost, and has a low

risk of inducing bacterial resistance. Handling of

mastitis using antibiotic therapy with breast

cleansing is more effective for relieving symptoms

quickly compared to only giving antibiotic therapy

(Kamal et al, 2012, Jahanfar et al, 2013).

Antibiotics have often used the treatment of

mastitis. The World Health Organization also says

the use of antibiotics is too excessive. But the

correct use of antibiotics is also a wise treatment of

mastitis. Ask for non-pharmacological assistance in

mastitis such as giving a hot compress to the breast

to help relieve swelling and pain, Allow breast milk

to reduce swelling (Yu Z et al., 2018).

Scaevola taccada (Geartn.) Roxb. is a species of

plant that lives in coastal shrubs scattered throughout

the coastal regions of the Pacific Ocean and Hindia

(Ando et al., 2014). In general, Scaevola taccada

(Geartn.) Roxb. has been used by the community as

traditional medicine, among others, it is used for the

treatment of digestive problems, anti-tumor, anti-

inflammatory, menstrual complaints, ringworm,

dysentery, syphilis, beriberi and others (Chandran

and Arunachalam, 2015).

Phytochemical screening is conducted to

determine the content of any secondary metabolites

contained in these plants. From the results of

phytochemical screening, it can be concluded that

what compounds can provide anti-inflammatory

activity. As screening results of phytochemicals that

have been carried out at UIN Makasar

pharmaceutical biology laboratory toward Scaevola

taccada (Geartn.) Roxb. plant, there are flavonoid

compounds, alkaloids, saponins, terpenoids, and

tannins. It is in line with previous research that the

content of Scaevola taccada (Geartn.) Roxb. is an

alkaloid, flavonoid, Scaevola, and saponin (Rachmat

Kosman, 2012) (Chandran and Arunachalam, 2015).

Traditional medicinal plants have enormous

potential as the development of antimicrobial drugs.

Many studies have described the antibacterial

properties of traditional medicinal plant extracts

isolated from traditional medicinal plants (Ilanko

and Cock, 2019).

The Scaevola taccada plant is a large shrub,

reaching 3-4 meters long which is typical in coastal

areas where it grows very close to a splash of open

seawater, usually on sandy soil or graveled soils.

The leaves widen upwards containing water

measuring 20 cm long and surrounded by bunches of

edges. The color is yellowish-green and shiny, the

edges are curved and the leaf surface is waxy. It

looks attractive with a yellowish-green color. The

fruits and flowers are white. Scaevola taccada

blooms throughout the year and the flowers have a

pleasant shape so they are called pleasure flowers or

beak flowers. The fruit floats in the sea and is spread

by ocean waves, this shrub becomes a new

pioneering plant on the sand edge of tropical waters

(Whiffin, et al. 2010, Chandran, et al. 2013).

Figure 5: The Scaevola Taccada Plant

One of Scaevola taccada (Geartn.) Roxb. extract

content is a flavonoid compound. Various

nutraceutical applications, pharmaceuticals,

cosmetic medicines use flavonoids as indispensable

components. This is due to the anti-inflammatory,

ICHIMAT 2019 - International Conference on Health Informatics and Medical Application Technology

608

antimutagenic and anti-carcinogenic properties

contained therein to modulate the function of the

cellular enzyme kuci (Panche, Diwan, and Chandra,

2016).

Products produced from arachidonic acid

metabolism are one of the inflammation causes.

Through phospholipase, cells have been activated by

mechanical, chemical, or physical stimuli which as a

way to release arachidonic acid. Arachidonic acid is

also an unsaturated fatty acid with 20 carbon atoms.

There are two main pathways in the metabolic

process of arachidonic acid, namely cyclooxygenase

by synthesizing prostaglandins as well as

thromboxane and lipoxygenase which synthesize

leukotriene and lipoxin (Arfan, 2016).

In particular, the flavonoid content is also able to

stop the formation and release substances that cause

inflammation due to allergic reactions. The

compounds included in the flavonoid group have

different effects on inflammation. Anti-

inflammatory mechanism carried out by flavonoids

can go through several pathways, namely inhibiting

the activity of COX enzyme and lipoxygenase

directly which causes inhibition of prostaglandin and

leukotriene biosynthesis which is the final product of

COX and lipoxygenase pathways (Nijveldt RJ, Nood

EV, Hoorn DEV, Boelens PG, Norren KV, 2001)

(Panche, Diwan and Chandra, 2016).

Previous research that said that the anti-

inflammatory effect of Scaevola taccada (Geartn.)

Roxb. extract was evaluated using various methods

by using mice experimental animals. The results

support traditional Scaevola taccada (Geartn.) Roxb.

plant usage in several inflammatory conditions

(Mejin, 2009) (Chandran and Arunachalam, 2015)

(Rachmat Kosman, 2012) (Rahmawati et al., 2014)

(Umrah, 2018).

Complementary therapy of saponin compounds

can modulate the immune system that mediated by

cell systems to increase antibody production.

Saponin does not only has a stimulating effect on

certain components of immunity but also it affects

several non-specific immune reactions such as

inflammation, as well as tannin compounds that

function as antibacterial (Iqbal et al., 2007).

Thus the presence of complementary therapy of

Scaevola taccada (Gaertn.) Roxb. extract) can help

heal inflammation caused by staphylococcus aureus

bacteria at a dose of 400 mg/kg bb.

4 CONCLUSIONS

Based on the results and discussion, it can be

concluded that the administration of amoxicillin and

Scaevola taccada (Gaertn.) Roxb. extracts are better

able to reduce inflammation cells in female

mammary mammals induced by staphylococcus

aureus at dose of 400 mg/kg bb compared to groups

that are only given amoxicillin with mean deviation

of 37.6 /kg.

ACKNOWLEDGEMENTS

This research was supported by Health Institute of

Deli Husada Deli Tua, Health Institute of Medistra

Lubuk Pakam, Sembiring Hospital Foundation, and

Grand Medistra Hospital Foundation. Indonesia

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