The Effectiveness of Clerodendrum Paniculatum. L against TNF- α in

Rats Induced by S. Aureus

Mutiara Dwi Yanti

, Prihantono

, Dewi Tiansa Barus

, Putri Ayu Yessy Ariescha

, Kholilah Lubis

Nurul Aini Siagian

, Andayani Boang Manalu

, Tetty Junita Purba

Midwifery Faculty, Institute of Health Science DELI HUSADA, Jl. Besar Delitia No.77, Deli Serdang, Indonesia

Department of Surgery, Faculty of Medicine, Hasanuddin University, Makassar City, South Sulawesi, Indonesia

Midwifery Faculty ,STIKES PRIMA NUSANTARA Bukit Tinggi

Keywords: Effectiveness, Bag Flower Leaf, TNF-α

Abstract: Staphylococus Aureus is one of the mastitis causes which causes inflammation due to elevated levels of

TNF-α. This study aimed to analyze the anti-inflammatory activity of bag flower leaf (Clerodendrum

Paniculatum L) on TNF-α level in rats that is induced by S. Aureus bacteria. This study used post test only

control group design with 15 samples of Strain Sprague Dawley rats divided into 3 groups. K (-) group as

normal control group, K (+) was induced by S. Aureus bacteria and the treatment group was administered by

extract 150mg / kg BB then it is measured TNF-α levels by the enzyme-linked immunosorbent assay

(ELISA) test method. Data were analyzed by using bivariate analysis namely one way ANOVA test. The

results showed that Bag Flower Extract contains secondary metabolites in the form of flavonoids and

tannins. Giving bag flower leaf extract (Clerodendrum Paniculatun L) at 150 mg / kg BB has anti-

inflammatory action by reducing TNF-α levels in rate which is induced by staphylococcus aureus (P <0, 0).

05). It was concluded that the administration of bag flower leaf extract could reduce TNF-α cytokines due to

S. Aureus bacteria. It is expected that future researchers will use human subjects to analyze TNF-α levels so

that later bag flower leaves can be used as complementary therapies in mastitis treatment due to S. aureus

bacteria.

1 INTRODUCTION

Staphylococcus aureus is one of the main

pathogens that are most often isolated from

intramammary infections (IMI) throughout the

world. S. aureus bacterial infections are becoming a

serious problem today due to increased bacterial

resistance to various types of antibiotics (Multi Drug

Resistance / MDR). S. aureus resistant can cause the

spread of other infections and cause other diseases

associated with other infections. Although S. aureus

can cause acute and clinical mastitis with

macroscopic changes in milk, bacterial infections

can develop against chronic and subclinical mastitis,

without macroscopic changes in milk but with high

somatic cell and bacterial counts in the mammary

glands (Green et al., 2012; Chinchali and Kaliwal,

2014)

The occurrence of mastitis begins with an

increase of pressure in the duct (breast milk channel)

due to breast milk stasis. If the milk is not removed

immediately, there is excessive alveoli tension and it

causes the epithelial cells that produce milk become

flat and depressed, so that the connective tissue

permeability will be increase. Some components

(mainly immune proteins and sodium) from plasma

enter to breast milk and subsequently into the tissues

around cells so that it triggers an immune

response(Acosta et al., 2016),

Breast milk stasis, there is an inflammatory

response, and tissue damage facilitate infection.

Breast infections are usually caused by bacteria that

are found in normal skin, namely S. Aureus. Other

bacteria that cause mastitis are Streptococcus beta-

hemolitik (such as Group A or Group B

streptococcus) or Escherichia coli. These bacteria

often originate from the baby's mouth which enters

through the lactiferous duct into secretion lobe,

through cracked nipples to the lymph glands around

the duct (periductal) or through spread of

hematogenous (blood vessel) (Schwartz et al., 2002;

596

Yanti, M., Prihantono, ., Barus, D., Ariescha, P., Lubis, K., Siagian, N., Manalu, A. and Purba, T.

The Effectiveness of Clerodendrum Paniculatum. L against TNF- in Rats Induced by S. Aureus.

DOI: 10.5220/0009979605960603

In Proceedings of the International Conference on Health Informatics and Medical Application Technology (ICHIMAT 2019), pages 596-603

ISBN: 978-989-758-460-2

Amir et al., 2007; Indonesian Pediatric Society,

2013)

Tumor Necrosis Factor alpha (TNF α) is a

pro-inflammatory cytokine that is involved in an

inflammatory reaction and it plays a role in the pain

emergence. This was first isolated by Carswell et al

in 1975 toward an effort to identify the Tumor

Necrosis Factors that is responsible for nicrosis from

the Meth A. sarcoma. Most organs appear to be

affected by TNF α, and there are many cytokine

functions which are not fully understood (Sanchez,

Ford and Yancey, 2005; Notebaert et al., 2008; Lai

et al., 2017)

The various roles of TNF-α can be explained

through their effects on endothelial vascular and

endothelial leukocyte interactions. When it is

exposed to TNF-α, endothelial cells will cause

inflammatory reactions by expressing various

adhesion molecules. TNF can also cause

vasodilatation by inducing the expression of



cyclooxygenase 2 (COX-2) and its relationship to

the production of prostacyclin 2 (PGI2). It can

explain erythema and heat which are signs of

inflammation. Other signs of inflammation are

tumors that occur through increased vascular

permeability, vascular through TNF mediators and

liquid passage and transendothian macromolecules

which ultimately cause edema and inflammation

(Nair, M.; Mahajan, 2006; Berthold-Losleben and

Himmerich, 2008; Lai et al., 2017)

Immune response that occurs due to the

invasion of staphylococous aureus bacteria as

antigens is when the entry of s. aureus into the body

will be eliminated by neutrophils and macrophages

as their role in the innate immune system.

Macrophages can also act as Antigen Presenting

Cells (APC). Bacteria will be phagocytosis in

macrophages then recognized by Major

Histocompatibility Complex II (MHC II) then

presented in the form of peptide antigens. Then

MHC II will bind to T lymphocytes. T lymphocytes

have several surface molecules or Cluster of

Differentiation (CD). Peptide agents that have been

presented by MHC II will bind to the T helper

lymphocytes (CD4) in section of T Cell Receptor

(TCR). (Abbas, Licthman and Pillai, 2015; Tong et

al., 2015; Acosta et al., 2016)

Bag flower leaf is one of the plant species

included in the Clerodendrum genus which has a

different species number of 580 species, and it is

spread evenly in Asia, Africa, America and Australia

and it has been used in traditional medicine in Asia

and Africa. India, China, Korea, Thailand, and Japan

are countries that have used several species of this

genus in medical practice (Cucumber, Virus and

Indonesia, 2009; Florence, Joselin and Jeeva, 2012;

India Biodiversity Portal, 2017)

Research on the efficacy of bag flower leaf

which aims to find out the antioxidant and anti-

inflammatory activities of big flower leaf extract

(Cloredendrum Paniculatum L) that is conducted

Anti-inflammatory activity tests on animals, it

showed the antioxidant activity results of bag flower

leaf ethanol is very strong (IC

<50 µg/ml) namely

= 27,73376 µg/ml and it has anti-inflammatory

activity at a dose of 50 mg / kg. Other research on

the anti-inflammatory activity test of Clerodendrum

Paniculatum L. which aims to evaluate the anti-

inflammatory activity of various extracts of

Clerodendrum paniculatum leaf shows the anti-

inflammatory activity in best vitro at a dose level of

200 and 400 mg / kg. Indomethacin at a dose level

of 10 mg / kg is used as a standard reference drug.

Both extracts showed a significant dose reduction (P

<0.001) in edema paw when it is compared to

controls). The study results indicated that petroleum

ether and chloroform extract from bag flower leaf

have anti-inflammatory potential which provides a

scientific basis for the traditional claims of

Clerodendrum Paniculatum Linn leaves as anti-

inflammatory drugs (Joseph, Bindhu and Aleykutty,

2013; Hafiz, Rosidah and Silalahi, 2016a).

This study is aimed to assess the level of

TNF-α which is one of the pro-inflammatory

cytokines that can trigger inflammation in mastitis

caused by S. Aureus bacteria.

2 MATERIALS AND METHODS

2.1 Location and Research Design

This research will be carried out in the

Laboratory of Hasanuddin University Hospital for

ELISA examination and bacterial culture,

Hasanuddin University Bio pharmacy Laboratory

and Hasanuddin University Animal Laboratory. The

research type used is the type of research used true

experimental with post only test control group

design.

2.2 Population and Sample

The population of this study were 15 Strain

Sprague Dawley (Rattus Novergicus) white rats

according to WHO standards and were randomly

selected to avoid bias in the study. The rats that used

in this study were rats weighing between 200-250

The Effectiveness of Clerodendrum Paniculatum. L against TNF- in Rats Induced by S. Aureus

597

grams. Before being given treatment, rats were first

adapted for 7

days in a cage with controlled

temperature and adequate lighting. Rats were fed

with pellets and water on an ad libitum basis. The

use of experimental animals in this study was

approved by the health research ethics commission,

medical faculty of Hasanuddin University, Makassar

(No. 1053 / H4.8.4.5.31 / PP36-KOMETIK / 2017).

2.3 Method of Data Collection

2.3.1 Sampling Criteria

Sampling criteria in this study namely

inclusion criteria: female rat Strain Sprague Dawly

weighing 200-250 grams with age 2-3 months, there

is no anatomical abnormalities. Exclusion criteria:

rats don’t want to eat, rats that are sick during the

adaptation process. The materials needed in this

study were rat maintenance: cages, food containers.

Rat treatment: scales, sterile syringes, feeding tube.

Sample preparation: centrifuge, vortex, shaker,

yellow and blue tip, micropipette for volume 2 µl -

1000 µl.

Female rat Strain Sprague Dawley each

group consisted of 5 rats. Furthermore, rats were

divided into 3 treatment groups, each consisting of

five rats that is consisting of two control groups and

one treatment group so that a total sample of 15 rats

were obtained. To avoid bias factors due to weight

variation, the grouping of samples is done randomly.

To avoid drop outs sample, the sample size which is

used was 6 per sample group so that the number of

rats used was 15 rats for 3 groups.

Blood sampling was taken twice after the

induction of S. aureus bacteria and after treatment in

each group. 1 ml of blood is drawn in centrifuge at a

speed of 5000 rpm for 5 minutes. Then do the

separation of blood with serum. The serum is

suctioned with a 1.0 µL dropper and placed in an

effendorf tube. The serum collected was carried out

by the ELISA method to obtain TNF-α level. Test

reagent for TNF-α examination: TNF-α ELISA Kit

RAT with catalogue number RTA00 .

2.3.2 Extraction Preparation

Bag flower leaves are obtained in the yard of

community houses as much as ± 2 kg of raw leaves,

then cleaned of dirt attached by using running water

then the sample is cut into small pieces, then dried to

contain water content below 10%, after that the bag

flower leaves are sieved with mesh size 40 so that a

smooth simplicia sample is obtained, after it, the

sample is ready to be extracted by maceration

method (Poorter et al., 2012; Hafiz, Rosidah and

Silalahi, 2016b).

Maceration is generally carried out by means

of 10 parts of simplicity put into a vessel, then

poured with 75 parts of the liquid solution, closed

and left for 5 days which is protected from light,

while repeatedly stirring after 5 days the juice is

dispensed, the pulp is squeezed. The residue is

added to solvent sufficiently and cleaned to obtain a

total of 100 parts. The resulting maserat is then

concentrated using a rotary evaporator until a thick

extract is obtained, and then dried by using a water

bath and desiccators (MOH RI, 2000).

2.3.3 Bacterial Culture

Staphylococcus aureus bacteria obtained

from laboratory of Uhhas Hospital with the type of

staphylococcus aureus bacteria then planted in

BHIB medium and it is incubated for 18-24 hours at

37° C in incubator and then propagated by using

Nutrient Agar (NA) medium which is then re-

incubated for 18-25 hours. After bacterial

incubation, it is done gram staining. Biochemical

tests for S. aureus bacteria were planted in NA by

planting on DNAse agar medium then mannitol salt

agar, then bacitracin and Novobiocin tests were

followed by callatase coagulase test. Then it is

reincubated for 18-24 hours at 37° C. The bacteria

that grew in biochemical test were matched with the

identification table of S. aureus bacteria. To make a

bacterial sample which is injected into rats by

making a suspension in physiological Na Cl solution

of 10 ml mixed with a colony of S. aureus bacterial

which is golden yellow with turbidity level of Mc

Farlan 2 x 10

CFU. The accuracy of Mc Farland

turbidity level is measured by the Densi check tool

(Das, Borah and Ahmed, 2013)

Figure 1 describe the research tools and

materials like : ELISA-Test for TNF-α, Sprague

Dawley Rats, Pagoda Leaves and Staphylococcus

Aureus Bacteria

After getting the concentration of bacterial

cells 10

cell / mL, then centrifuged at a speed of

10,000 rpm for 10 minutes at 25

C. The obtained

pellets were then suspended with 1 mL PBS. The

suspension was then injected into experimental

animals, namely in mammals of rat female Strain

Sprague Dawly, precisely in the lactiferous duct

section with a volume of 100 µL (0.2 ml).

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

598

Figure 1. Research Tools and Materials

2.3.4 Data Analysis

Data analysis begins with test data

normality distribution. To compare TNF-α level in

the group after the induction of S. Aureus bacteria,

and after the intervention in the treatment group was

analyzed by using Paired T-Test. To compare

changes in TNF-α level between the treatment and

control group, statistical analysis used one way

Anova test. The significance limit used in this study

was 5% (p = 0.05).

3 RESULTS

Qualititative phytochemical test of bag flower

leaf extract (Clerodendrum Paniculatum L)

Table 1 shows the phytochemical test results of

secondary metabolite content in bag flower leaf

extract (Clerodendrum Paniculatum L), there was

secondary metabolite compounds include flavonoids

and tannins.

Table 1. Qualitatively analysis of phytochemical content

of sea leafy leaves (Scaevola taccada (Gaertn) Roxb.)

Extracts

Metabolit

Compunds

Analysis result

Alkaloid -

Flavonoid +

Steroid/triterpenoid -

Saponin -

Tanin +

From several studies that conducted

phytochemical testing of Clerodendrum

Paniculatum L plants, there were differences in

interpretation of the results of phytochemical

screening that researchers conducted on several

previous studies. Phytochemical screening results of

bag flower leaf ethanol extract in this study were

identified only 1 from 3 alkaloid solutions used

namely Mayer solution. The difference in

phytochemical screening results in this study can be

caused by several factors, namely the treatment of

the sample and the environmental conditions in

which the bag flower leaves grow (Pidugu and Arun,

2012; Andriani et al., 2017; Wang et al., 2018)

However, differences phytochemical

screening results conducted in this study with

previous studies did not affect the value of the study,

because the compound expected to exist in the

ethanol extract of bag flower leaves that had an

effect on anti-inflammatory activity was the

flavonoid compound. The tannin compound itself in

this study also influences anti-inflammatory activity

and can function as an antibacterial and the

compound does not differ from the results of

previous studies that bag flower leaf extract has

flavonoid compounds (Yadav et al., 2014; Wang et

al., 2018)

3.1 TNF Levels - after Bacterial

Induction and after Intervention

Table 2 shows that in negative control group there

were no differences in mean TNF-α levels on rats

that were not induced with S. Aureus bacteria with a

p value = 0.825. In the positive control group there

was no difference in the mean TNF-α levels after

bacterial induction with p = 0.894. In the treatment

group with a dose of bag flower leaf extract 150 mg

/ Kg BB there was a significant mean difference

between TNF-α levels after bacterial induction and

after administration of the intervention with a p

value = 0.003.

Table 2. Difference in mean TNF-α level before induction

of S. Aureus bacteria, after induction of S. Aureus bacteria

and after administration of Bag Flower Leaf Extract

Group

After

Induction

Average ±

After

Treatment

Average ±

Value

Control (-

)

251,3 ±

8,3*

249 ± 20,2*

P =

0,825

Control

(+)

305,5 ±

22,9

303,3 ±

35,6

P =

0,894

EDP 150

mg/Kg

319 ±

11,1

247,0 ±

25,9

P =

0,003

The Effectiveness of Clerodendrum Paniculatum. L against TNF- in Rats Induced by S. Aureus

599

Table 3. Difference in mean TNF-α level after

administration of Bag Flower Leaf Extract

Table 3 shows that the statistical test results obtained

p value <0.008 so that there are significant

differences in average TNF-α levels in the negative

control group, positive control group, and EDP150

mg / Kg BB group after treatment. From the results

of continued tests (post hoc) in table 4 shows the

average comparison of TNF-α levels after giving

treatment in the negative control group with a

positive control group significantly different with a p

value = 0.022. In the negative control and EDP

treatment group 150 mg / kg BB there was no

significant difference in the average TNF-α level

after administration of the treatment with a p value>

0.05. In the positive control group and the EDP150

mg / Kg BB group, there was a significant difference

in the mean TNF-α levels after administration of

treatment with p = 0.016.

Table 4. Continued Test (Post Hoc) Differences in TNF-α

Levels after Giving Bag Flower Leaf Extracts

Group Control

(-)

Control

(+)

EDP150

mg/KgB

Control (-) - 0,022* 1,000

Control (+) - - 0,016*

EDP150

mg/KgBB

- -

* Significantly different groups

4 DISCUSSION

This study showed that there were

differences in the mean TNF-α level in rats between

groups that were not induced by S. Aureus bacteria

and those that were induced by S. Aureus. The

statistical results in this study showed that the

administration of bag flower leaf extract to rats at a

dose of 150 mg / Kg BB was able to reduce levels of

TNF-α in rat after being significantly induced by S.

aureus bacteria.

This study was in line with several previous

studies, namely mice infected with Staphylococus

aureus, and then there was an increase in TNF-α

production compared to mice that were not induced

by staphylococcus aureus. (Mufidah & Rifa'i., 2015;

Pereyra et al., 2017)

S. Aureus contains llipoteicoic acid which

is found on the surface of bacteria which is

recognized as toll-like receptors II (TLR II) which

will further stimulate IL-12 production so as to

stimulate INF- γ. This bacterium can also activate

the adaptive immune response through superantigen

induction. The interaction between superantigens

and cells can lead to greater stimulation of T cells

compared to other antigens. TNF-α cytokines and

IL-1 have immunostimulatory activity and work

synergistically with IFN-γ to enhance immune and

inflammatory reactions. However, if the levels of

cytokines are at high concentrations it can cause

epithelial cell damage and cause toxic shock (Liang

and Ji, 2007; Krakaeuer, 2011; Phuneerub et al.,

2015; Tong et al., 2015).

In the phytochemical test results on bag

flower leaf (Clerodendrum Paniculatum L), it is

found secondary metabolite compounds in the form

of flavonoids and tannins. The existence of

secondary metabolites is an important factor through

its mechanism of a bacterium. Flavonoids are the

largest group of phenol compounds. Flavonoid

compounds are good reducing compounds that

inhibit oxidation reactions both enzymes and non-

enzymes. Its mechanism as an antibacterial is to

form complexes with extracellular and dissolved

proteins and with microbial walls, flavonoids also

play a direct role by interfering with the function of

microorganism cells and inhibiting microbial cell

cycles, denaturating bacterial cell proteins and

damaging cell membranes that can result in lysis of

bacterial cells (Parubak, 2013; Nanda, Bora and

Tiwari, 2016; India Biodiversity Portal, 2017).

The work mechanism of tannin as an

antibacterial is to inhibit the reverse transcriptase

and DNA topoisomerase enzymes so that bacterial

cells cannot be formed. Tannin has antibacterial

activity related to its ability to inhibit and kill

bacterial growth by reacting toward cell membranes,

inactivation of essential enzymes in bacteria and

destruction of functions and genetic material and

interfering with the transport of proteins in the inner

layers of cells. Tannins also have targets on cell wall

polypeptides so that cell wall formation is less than

perfect. This causes the bacterial cell become lysis

due to osmotic and physical pressure so that the

bacterial cell will die. Tannin acts as an antibacterial

because it can form of protein complexes and

hydrovobic interactions, if the hydrogen bond

between tannin and protein enzymes contained in the

protein is likely to be denatured so that bacterial

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

600

metabolism is disrupted (Daglia, 2012; Yadav et al.,

2014; Phuneerub et al., 2015).

Bag flower leaf extract has active compounds

namely flavonoids and tannins that can function as

anti-inflammatory mediators that can reduce the

release of proinflammatory cytokines in rats induced

by S. Aureus bacteria. From previous studies, bag

flower leaves that contain active compounds

flavonoids, tannins and steroids can reduce

inflammation that occurs in mice which is induced

carrageen (Hafiz, Rosidah and Silalahi, 2016a).

Other studies on the methanol extract of

mahogany with flavonoid active substances have

also been shown to reduce TNF-α levels in mice

induced by MLD-STZ. A similar study was carried

out by using purple leaf extract (Gratophyllum

pictum L.) with active flavonoid content, it has

proven to reduce TNF-α level and NO in mice which

is infected by S. Aureus bacteria(Suryani, Endang H

and Aulanni’am, 2013; Tjahjani, Kristina and

Endang Sri Lestari2, 2015; Bond, Morris and

Nassar, 2017)

In this study, the treatment group with a

dose of 150 mg / kg BW had a TNF-α value are

below TNF-α level in the negative control group or

it was not significantly different from the negative

control group where p value> 0.005 so that the

administration of bag flower leaf extract with a dose

of 150 mg / kg body weight can reduce TNF-α levels

in mice with normal conditions that are not induced

by S. Aureus bacteria.

The content of secondary metabolites found

in bag flower leaves (Clerodendrum Paniculatum L)

which is derived from bag flower leaf extract is

thought to act as an anti-inflammatory caused by S.

aureus bacteria. The main bioactive component of

phenols in flavonoids is quercetin. The mechanism

of bag flower leaf extract as an anti-inflammatory

can cause a decrease in TNF-α levels through

inhibition of Nuclear Factor kappa B (NF-kB). NF-

kB becomes active due to a stimulus from the

Reactive Oxygen Synthase (ROS) agent which

causes endothelial dysfunction, pathogen exposure,

DNA damage and physical stress. NF-κB has the

functions in controlling the genes expression of

cytokines and proinflammatory chemokins TNF-α,

IL-1β. The decrease in NF-kB activation is

influenced by inhibitory effect of monocytes on p56

Protein Tyrosin Kinase (PTK) enzyme, which causes

PTK to be inactive. Inactivated PTK causes NF-κB

transcription factors to remain bound to NF-κB

inhibitors so that it cannot trigger transcription and

translation of TNF-α proinflammatory cytokines

secreted by macrophages, thereby reducing TNF-α

levels (Nair, M.; Mahajan, 2006; Fassihi and Sabet,

2008; Rathee et al., 2009)

The dynamics of level changes in TNF-α

between positive control and treatment group

whereas in the treatment group after administration

of bag flower leaf extract, it is decreased TNF-α

levels after returning to the TNF-α level value equal

to the negative control group. It showed that the

content of compounds found in bag flower leaves

can reduce the inflammatory effect by suppressing

the release of TNF-α which is caused by S.Aureus

bacteria.

5 CONCLUSIONS

This research shows that bag flower leaf extract

contains secondary metabolite compounds including

flavonoids and tannins which can reduce TNF-α

level which are inflammatory mediators due to S.

Aureus bacteria. Bag flower leaf extract with a dose

of 150 mg / Kg BB is an effective dose to reduce

TNF-α level in rats induced by S. Aureus bacteria.

The administration of bag flower leaf extract can be

a solution in the treatment of complementary

therapies. It is necessary to evaluate the quantitative

test of bag flower leaf to determine the activity of

secondary metabolites and the percentage of

compound content in grams.

ACKNOWLEDGEMENT

This research was supported by Institut Kesehatan

Delihusada Delitua, Institut Kesehatan Medistra

Lubuk Pakam, Sembiring Hospital Foundation, and

Grand Med Hospital Foundation, Indonesia

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