The Effect of Agarwood Leaves Ethanol Extract on Porphyromonas

gingivalis Growth Inhibition and in Vitro Cytotoxicity Assay on

Fibroblast

Vinna Kurniawati Sugiaman

, Henry Yonatan Mandalas

, Ethan Yeshael Tanamal

Nathalia Cahya Calista

and Natallia Pranata

Department of Oral Biology, Faculty of Dentistry, Maranatha Christian University, Jalan Prof. Suria Sumantri, Bandung,

Indonesia

Department of Periodontology, Maranatha Christian University, Bandung, Indonesia

Faculty of Dentistry, Maranatha Christian University, Jalan Prof. Suria Sumantri, Bandung, Indonesia

Keywords: Aquilaria Malaccensis Lamk., Poprhyromonas Gingivalis, Inhibition Assay, Fibroblast, Cytotoxicity.

Abstract: Agarwood leaves (Aquilaria malaccensis Lamk.) have an antibacterial activity that could be used as wound

healing agent. Porphyromonas gingivalis were the main pathogens in periodontitis. This was a laboratory

experimental study post-test only control group design. Agarwood leaves were obtained from Ibun Garden,

Majalaya District, West Java. Sample identified and determined by Biology Research Center, LIPI Indonesia.

Extraction and phytochemical test were conducted at Aretha Medika Utama BBRC Bandung and BPTRO

Bogor. Fibroblast ATCC 3T3 Balb/C obtained and cultured at Aretha Medika Utama BBRC. Cytotoxicity

test was carried out by using MTS Assay method, and the results are adjusted to ISO 10993-5. IC

was

obtained using PROBIT analysis. Inhibition assay was carried out by well-diffusion method and the results

are adjusted to Davis and Stout criteria. Research and P. gingivalis (ATCC 33277) was carried out at

Microbiology Laboratory Faculty of Dentistry, Universitas Padjadjaran. Results were analysed with ANOVA.

The results indicate agarwood leaves had weak inhibitory ability at under concentration of 50% and moderate

inhibition at a concentration of 100%. The cytotoxicity results showed no toxic effect at under concentrations

of 62.5 µg/mL. The IC

at a concentration of 215.54 µg/mL.

1 INTRODUCTION

Periodontal disease is defined as various types of

conditions that affect the supporting structures of the

teeth, including gingiva, alveolar bone, and

periodontal ligament (Kinane et al., 2017).

This

disease is the 11th most common disease in the world

and can be classified into gingivitis and periodontitis

(Nazir, 2017). Periodontitis is an inflammatory

disease in dental support tissue caused by specific

microorganisms resulting in progressive destruction

of the periodontal ligament and alveolar bone by

pocket formation and recession (Newman et al.,

2012).

https://orcid.org/0000-0002-3688-6718

https://orcid.org/0000-0002-9799-3824

https://orcid.org/0000-0002-5634-1942

https://orcid.org/0000-0002-4548-2395

https://orcid.org/0000-0001-7970-1915

Periodontitis has a prevalence of 10.8% or is

experienced by approximately 743 million

individuals in the world and is the sixth highest

prevalence disease according to Global Burden

Disease (Wijaksana, 2019; Séverin, 2018). The

pathophysiology of periodontitis is an imbalance of

microorganisms in the oral cavity that causes chronic

exposure to several pathogenic bacteria periodontitis,

including Porphyromonas gingivalis, Actinobacillus

actinomycetemcomitans, Tannerella forsythia, and

Treponema denticola (How et al., 2016).

P. gingivalis is one of the main etiologic agents in

the development of periodontitis with a prevalence of

92% (How et al., 2016; Liu et al., 2013). P. gingivalis

112

Sugiaman, V., Mandalas, H., Tanamal, E., Calista, N. and Pranata, N.

The Effect of Agarwood Leaves Ethanol Extract on Porphyromonas gingivalis Growth Inhibition and in Vitro Cytotoxicity Assay on Fibroblast.

DOI: 10.5220/0010745500003113

In Proceedings of the 1st International Conference on Emerging Issues in Technology, Engineering and Science (ICE-TES 2021), pages 112-121

ISBN: 978-989-758-601-9

is a gram-negative, anaerobic, saccharolytic, and non-

motile bacteria. These bacteria are shaped like cocci

or rod and belong to the group of black pigmented

bacteria (How et al., 2016). The main habitat of P.

gingivalis is plaque in the subgingival pocket of the

oral cavity. The proportion of P. gingivalis was found

to be higher in the deep periodontal pocket compared

to the shallower periodontal pocket. This is due to the

availability of amino acid fermentation requirements

to produce bacterial energy in the periodontal pocket,

such as low sugar levels, low oxygen levels, rich in

blood and serum protein, and has a stable pH that is

slightly alkaline (How et al., 2016)

P. gingivalis bacteria have several factors that

play a role in the virulence process of human cells,

namely fimbriae, lipopolysaccharide (LPS), capsular

polysaccharide (CPS), hemagglutinin, and gingipain.

In addition, P. gingivalis interacts with body tissues

by adhesion and coaggregation so that these bacteria

can invade the body's epithelial cells. However, the

low biological activity of P. gingivalis, especially its

endotoxicity, causes these bacteria to colonize and

grow in sterile tissue without being detected by the

body (Klein et al., 2012).

The treatment that has been carried out against P.

gingivalis infection still has many drawbacks. The

use of medications such as the antiseptic

Chlorhexidine can cause staining of the teeth and

some other disadvantages. In addition, P. gingivalis

is also known to be resistant to antibiotics, including

amoxicillin, clindamycin, and metronidazole (Gerits

et al., 2017). This has led to research on new and

natural substances in the treatment of periodontitis

due to P. gingivalis infection.

In addition to medicament, periodontal disease

treatment also varies depending on extent of the

affected periodontal tissue and can be performed with

and/or without surgery. Some of the most common

non-surgical procedures are scaling and root planing

and medication either locally or systemically with the

aim of infection control, inhibition of microbial

growth, and restoring the healthy state of periodontal

tissue. The most common surgical procedure is the

periodontal flap, aimed to restore the clinical

attachment of the periodontal ligament. This surgery

involves incisions and requires wound healing as well

as postoperative tissue regeneration (Newman et al.,

2012; Williams et al., 2016; Hudwekar et al., 2019).

Fundamentally wound healing is a complex

cellular process, focuses on restoring the structure

and function of damaged tissues through 3 (three)

phases, namely the inflammatory phase, proliferation

phase, and remodelling phase. Fibroblasts are

important cells in the wound healing process, derived

from undifferentiated mesenchymal cells. Fibroblasts

produce mucopolysaccharides, amino-glycine, and

proline acids which are the basic ingredients for

linking the edges of the wound. Inflammatory signals

activate the proliferation and maturation of the

fibroblasts, which are responded by collagen

synthesis and cross-bond initiation to form an

extracellular matrix as well as differentiate into

myofibroblast phenotype to facilitate wound closure

(Sugiaman, 2011; Gonzales et al., 2016).

Agarwood (Aquilaria malaccensis Lamk.) is a

plant that grows in the forests of Indonesia. The final

product of agarwood known as gubal contain resin as

a result from mushroom infection in induction

process (Janshen et al., 2017).

Induction process is an

outcome from a long term and complex

microorganism interaction, makes gubal extremely

rare and high in value. Agarwood resin often used as

ingredient in perfume and cosmetic industry (Musir

et al., 2016; Nugraha and Ginting, 2013).

The use of

resin that is too dominant, make the other part of

agarwood often become overlooked and resulted as

waste, especially the leaf (Wangiyana, 2020).

In traditional medicine, agarwood leaves tend to

be used empirically by Indonesians as a treatment for

malaria, diabetes, asthma, abdominal pain

(constipation), and skin care. These potencies

achieved by drinking the leaf’s brew or inhaling the

scent of burned leaves and stems (Wangiyana, 2020;

Syamsul et al., 2020).

Agarwood leaf still can’t reach

its maximum use yet in Indonesia due to the lack of

information about the goods contained (Janshen et al.,

2017).

Agarwood leaf extract as an antibacterial

facilitated by the presence of flavonoids as main

compound that intervene in the destruction of

bacterial cell membranes (Nomer et al., 2019;

Warganegara and Restina, 2016). Along with

alkaloids, flavonoids shall change the protein

structure found on the outer surface of bacteria

namely fimbriae, resulting in the decrease of its

hydrophobic property and inhibits bacterial adhesion

with host cell (Pratiwi et al., 2015).

Furthermore,

there are saponins with active substances to lower the

surface tension of bacterial cell walls. The substance

will bind to the cytoplasmic membrane to destabilize

the bacterial cell membrane, causing cytoplasm

leakage, resulted in bacterial lysis Dennis et al.,

2017).

Phytochemical screening of agarwood leaf

extract shows presence of tannins and

steroids/triterpenoids that invade the bacterial cell

membrane, causing the membrane becomes brittle

and easily destroyed (Sari et al., 2017).

The Effect of Agarwood Leaves Ethanol Extract on Porphyromonas gingivalis Growth Inhibition and in Vitro Cytotoxicity Assay on

Fibroblast

113

Wound healing-wise, agarwood leaf extract

proven to accelerate the inflammatory process, as

well as re-epithelization in mice with DM. Main

compounds that act as mentioned in agarwood are

flavonoids, saponins, and tannins (Suhardiman and

Juanda, 2019; Wahid and Safwan, 2019; Fauzi et al.,

2017).

Flavonoids act in the activation and

proliferation of fibroblasts and induce the production

of collagen fiber in order to accelerate wound healing

process. Along with flavonoids, saponins stimulates

blood vessels proliferation, while tannins act as

homeostat by inhibits the production of prostaglandin

and stimulating vasoconstriction (Suhardiman and

Juanda, 2019; Fauzi et al., 2017; Rahmadhani et al.,

2020).

Research on the comparison of total phenol levels

in agarwood leaf steeping and agarwood leaf ethanol

extract showed that the ethanol extract has higher

flavonoids level, namely 62.9 mg GAE/gram and

28.5 GAE/gram in agarwood leaf steeping. Other

studies showed that there are 8342.82 mg/100 gram

flavonoids contained with antioxidant activity

ranging from 28.50 – 40.30 ppm, classified as very

strong (Nurmiati and Wijayanti, 2018; Komang et al.,

2018; Harahao et al., 2015).

Therefore, agarwood leaf has the potential sources

of antibacterial, antioxidant and anti-inflammatory

that in addition to inhibiting the growth of P.

gingivalis, shall be effective in wound healing

process.

2 METHODS AND MATERIALS

2.1 Agarwood Leaf Extraction

Agarwood leaves was obtained from Ibun Garden,

Majalaya District, West Java and had been identified

by Biology Research Center, Lembaga Ilmu

Pengetahuan Indonesia, Bogor. Extraction was

carried out by maceration method and ethanol as

solvent. For cytotoxicity assay, 5 mg total of extract

was dissolved in 1 mL DMSO 10% and become seven

different extract concentration (500 µg/mL, 250

µg/mL, 125 µg/mL, 62.5 µg/mL, 31.25 µg/mL, 15.63

µg/mL, and 7.81 µg/mL). As for inhibition assay,

serial dilution method used to make working

concentration. 100% extract was dissolved with 10

mL aquadest to 50%, 25%, 12.5%, 6.25%, 3.13%,

1.56% agarwood leaf extract. Both final

concentrations were filtered using 0.22 µm tissue

culture pore syringe resulted in sterile sample.

2.1.1 Phytochemical Test

The qualitative phytochemical assay was carried out

by Farnsworth method. Results showed Agarwood

leaf ethanol extract indeed contain flavonoid,

saponin, tannin, alkaloid, triterpenoid, steroid, and

phenol.

2.2 Cytotoxicity Assay (Viability Test

using MTS Assay)

Fibroblast cell (3T3 Balb/C) ATCC CCL-163 was

obtained from Aretha Medika Utama BBRC Bandung

as collection. Thawing and subculture process was

conducted, and cells were cultured in a complete

medium, contained of 10% FBS (Biowest, S81B-

500), 1% ABAM (Biowest, L0010-100), 1%

Amphotericine B (Biowest, L0009-050), 1% MEM

Vitamins (Biowest, X0556-100), 1% L-Glutamine

(Biowest, X0551-100), 0.2% Nanomycopulitine

(Biowest, LX16-100), 0.1% Gentamicin (Gibco,

15750060) and basal medium DMEM High Glucose

(Biowest, L0103-500).

Cells were harvested and calculated using

hemacytometer after it reached the confluency of

70%, then implanted with 5 x 10

density in a 96 well-

plate. After incubated for 24 h, the old medium was

replaced with 200 µL new medium and 20 µL

agarwood leaf ethanol extract, then proceeded to be

incubated under 37˚C with 5% CO

. Furthermore, 20

µL of MTS reagent was added on each well and was

incubated for 3 h. Absorbance was measured using

spectrophotometer with 490 nm. Cells death was

calculated based on the absorbance and integrated to

standard curve of 3T3 Balb/C.

2.2.1 Statistical Analysis

Results that obtained as data processed with IBM

SPSS 21.0 ver. Normality test was carried out then

proceeded to One-way ANOVA and Tuckey HSD

(Post-Hoc). IC

value was obtained using PROBIT

analysis.

2.3 Inhibitory Assay

P. gingivalis which had been made into a suspension

were taken with a cotton swab and spread into the

blood agar medium. Then a hole is made using a

perforator in the inoculated agar, forming a well.

Furthermore, the wells will be filled with each

treatment, specifically positive control with antiseptic

chlorhexidine solution, negative control with

aquadest, and ethanol extract of agarwood leaves with

ICE-TES 2021 - International Conference on Emerging Issues in Technology, Engineering, and Science

114

various concentrations. The filled media were then

incubated for 24 hours at 37

The same procedure will be repeated three times.

The inhibition zone that formed around the well was

measured using a calliper with units of mm as

research data. Results were further categorized

according to inhibition zone category according to

Davis and Stout.

2.3.1 Statistical Analysis

Results that obtained as data processed with IBM

SPSS 21.0 ver. Normality test was carried out using

Shapiro-Wilk then proceeded to One-way ANOVA

and Dunnett T3 (Post-Hoc).

3 RESULTS AND DISCUSSION

3.1 Results

3.1.1 Cytotoxicity Assay

Phytochemical analysis of agarwood leaf ethanol

extract showed positive result from the presence of

flavonoid, saponin, tannin, alkaloid, triterpenoid, and

phenol as shown in Table 1.

Table 1: Phytochemical Analysis Results.

Compound Results

Flavonoids (+)

Saponins (+)

Tannins (+)

Terpenoids (+)

Triterpenoids/Steroids (+) Triterpenoid

Phenols (+)

Alkaloids (+)

The outcomes of cytotoxicity assay included

mean of absorbance, corrective absorbance, number

of viable cells, percentage of viability, and percentage

of inhibition obtained through calculations and

spectrophotometer measurements.

Parameters used in this study were the percentage

of viability cell and IC

. Cell viability was obtained

and calculated from the number of cells that are still

alive (viable) after treated, while IC

value was

obtained through PROBIT analysis with the aim of

knowing the concentration of agarwood leaf extract

which can inhibit fibroblasts growth by as much as

half the population. Results of cytotoxicity assay

shown in Appendix.

In vitro cytotoxicity assay according to ISO

10993-5: Biological Evaluation of Medical Devices

states that, if the relative cell viability for the extract

concentration of a sample is more than equal to 70%,

then the material must consider as non-toxic

(International Standard Organization, 2009;

International Standard Organization, 2012). Based

on these standards in this study, the concentration of

agarwood leaf extract that did not toxic on 3T3

Balb/C fibroblasts were concentrations of 62.5

µg/mL, 31.25 µg/mL, 15.63 µg/mL and 7.81 µg/mL.

Meanwhile, concentrations of 500 µg/mL, 250

µg/mL, and 125 µg/mL were toxic.

Standard curve of 3T3 Balb/C cells was created

(Figure 1) as a reference for calculating the number

of cells based on the absorbance obtained. The curve

made by regressing the absorbance value and the

number of cells into the standard curve line equation

y = ax + b. The y-axis is the absorbance value, while

the x-axis is the number of cells.

Figure 1: 3T3 Balb/C Standard Curve.

The regression curve shows linear relationship

between the number of cells and the absorbance

value. This could also be seen in observations on well

plates. The darker the color produced on the well

plate, the higher the absorbance value and the number

of viable cells. This curve is then used as a reference

for calculating the number of viable cells in each

treatment.

Furthermore, statistical analysis conducted

towards these data resulting in normality distributed

and homogenous data. Analysis proceeded to

ANOVA and Post-Hoc Tuckey HSD. Results showed

that each treatment significantly affecting the

difference in cell viability by significance (p)<0.5.

The IC50 value obtained was at a concentration of

215.54 µg/mL using PROBIT analysis.

y = 7E-05x + 0,0329

R² = 0,9811

0,0000

0,1000

0,2000

0,3000

0,4000

0,5000

0,6000

0 2000 4000 6000 8000 10000

Absorbance Value

Number of cells

3T3-Balb/C Standard Curve

The Effect of Agarwood Leaves Ethanol Extract on Porphyromonas gingivalis Growth Inhibition and in Vitro Cytotoxicity Assay on

Fibroblast

115

3.1.2 Inhibitory Assay

In this study, 9 treatments were repeated three times

on P. gingivalis. There were 7 concentrations of

agarwood leaf ethanol extract, namely 100%, 50%,

25%, 12.5%, 6.25%, 3.13%, and 1.56%. Other

treatments given were positive control with antiseptic

chlorhexidine solution and negative control with

aquadest.

Parameter used in this study was the diameter of

the inhibition zone produced in each treatment as the

form of a clear zone on agar media. The results of this

inhibition zone measurement will then be interpreted

into the inhibition category according to Davis and

Stout.

As shown in appendix and Figure 2,

measurements of inhibition zone diameter showed

that the largest diameter of the inhibition zone was in

group 9, namely chlorhexidine as positive control.

Then followed by a group of 8, namely the ethanol

extract of gaharu leaves with a concentration of

100%. Meanwhile, the smallest diameter of the

inhibition zone was in group 1, namely the negative

control of aquadest.

Figure 2: Mean of inhibition zone diameter.

Note:

1 treatment group for (-) control (aquadest)

2 treatment group for agarwood leaf ethanol extract 1.56%

3 treatment group for agarwood leaf ethanol extract 3.13%

4 treatment group for agarwood leaf ethanol extract 6.25%

5 treatment group for agarwood leaf ethanol extract 12.5%

6 treatment group for agarwood leaf ethanol extract 25%

7 treatment group for agarwood leaf ethanol extract 50%

8 treatment group for agarwood leaf ethanol extract 100%

9 treatment group for (+) control (chlorhexidine)

According to the Davis and Stout inhibition

category, the result will be categorized as weak if the

inhibition zone formed is 5 mm or less. Followed by

moderate inhibitory ability if 5-10 mm inhibition

zone formed. While strong inhibitory ability when

10-20 mm zone is formed. Lastly, inhibitory ability

will be categorized as very strong if more than 20 mm

zone formed (Rastina et al., 2015). Based on these

categories, ethanol extract of agarwood leaf has weak

inhibitory ability at concentrations of 50%, 25%,

12.5%, 6.25%, 3.13%, and 1.56%. While the

concentration of 100% has moderate inhibitory

ability against the growth of P. gingivalis.

Normality test was performed resulting in

normally distributed data by significance of p<0.05.

Data analysis was proceeded to One-way ANOVA

and it was found to support H

, states that agarwood

leaf extract has significance effect on P. gingivalis

growth. Further analysis was carried out by using

Post-Hoc Dunnett T3 method. Results showed that

each concentration of agarwood leaf extract has

different significance effect toward P. gingivalis

growth.

Table 2: Post-Hoc Dunnett T3 Analysis.

Treatment Inhibition Zone

(-) Control 0 ± 0.00

1,56% 2.18 ± 0.08

3,13% 2.35 ± 0.15

6,25% 2.63 ± 0.08

bdc

12,50% 3.23 ± 0.34

cde

25% 3.62 ± 0.10

def

50% 4.30 ± 0.39

100% 6.55 ± 0.23

(+) Control 11.62 ± 0.60

3.2 Discussion

3.2.1 Cytotoxicity Assay

This study results showed that the highest percentage

of cell viability was in the group with concentration

of 7.81 µg/mL, and the lowest was in the group with

concentration of 500 µg/mL. These data indicate that

there is decrease in cell viability which is inversely

proportional to the increase in concentration. The

differences in cell viability at each concentration

could indicate that the cell response towards each

concentration was also different, which is thought to

be due to the variance in the amount of active

compound content at each concentration.

Qualitative phytochemical testing carried out in

this study did not prove the exact amount and

proportion of active compounds contained in

agarwood leaf extract. In addition, the dilution of

agarwood leaf extract is also thought to result in a

decrease in the number of active compounds along

with the smaller the concentration, since the amount

0,00

2,00

4,00

6,00

8,00

10,00

12,00

14,00

123456789

Diameter (mm)

Treatment Group

Mean of Inhibition Zone Diameter

ICE-TES 2021 - International Conference on Emerging Issues in Technology, Engineering, and Science

116

of extract keep reduced during the dilution process.

Referring to several previous studies, the active

compounds that play a role in this research are

saponins, flavonoids, and tannins.

Saponins are derivatives of glycosides that can act

as antioxidant agents capable of neutralizing free

radicals by binding to active oxygen. The lyobipolar

properties possessed by saponins allows their

interaction with cell membranes by reducing the

surface tension of cells (Yildirim and Kutlu, 2015).

Saponins also could increase the expression and

activation of TGF-β, FGF, and VEGF via monocyte

proliferation. Activation of FGF will increase

fibroblast proliferation and stimulate fibronectin

synthesis. Fibronectin synthesis will induce fibroblast

migration. The fibroblasts will then be responsible for

the synthesis of collagen in the extracellular matrix

(Suharto and Etika, 2019; Ardiana et al., 2015).

Signaling performed by FGF plays an important

role in the regulation of cell pluripotency through

interaction with FGFRs. FGF/FGFRs will then be

responsible for cellular processes such as

proliferation, migration, embryonic development,

tissue regeneration, and cell metabolism. This entire

process is mediated by activation of RAS - mytogen-

activates protein kinase (MAPK), Phospholipase C

Gamma, and signal transducers and activators of

transcription (STAT). These various signaling

pathways will also work together with signaling from

other growth factors, such as TGF-β (Mohammadi et

al., 2020).

In molecular level, flavonoids could induce

extracellular signal-regulated kinase (ERK). ERK can

specifically recognize various growth factor receptors

on the cell surface such as FGFRs, KGFR, and EGFR

also significantly activate these rexeptors (Etika et al.,

2017). Arginine, which is a derivative of flavonoids,

has been shown to affect the proliferation of human

gingival fibroblasts and fibroblasts through activation

of amino acid receptors and cyclic-AMP response

element binding (CREB). This activation will then

stimulate the secretion of various growth factors and

also the extracellular matrix (Kurahashi and Fujii,

2015).

Flavonoids contributed in macrophage activation

which will stimulate the synthesis of several growth

factors such as PDGF, FGF, EGF, TGF-β, and TGF-

α. Macrophages together with neutrophils can

synthesize Reactive Oxygen Species (ROS) which is

a chemically reactive molecule, formed due to the

acceptance of electrons by the O

molecule. ROS

produced in the wound healing process is superoxide

radical anion which will then be broken down into

(hydrogen peroxide) and oxygen molecules

through the superoxide dismutase mechanism. The

disintegration of superoxide radical anion aims to

prevent the formation of destructive ROS with high

concentrations such as peroxynirite (ONOO--) or

hydroxyl radical (-OH) Jiang et al., 2018; Ningrum

and Kurniawaty, 2019).

Tannins contributed to the differentiation of

fibroblasts into myofibroblasts along with the

production of an extracellular matrix which important

in wound contraction. But along with this process,

tannins can also control the differentiation and

proliferation of fibroblasts through the expression of

genes involved in extracellular matrix production.

This is performed by induction of Smad2 and Erk

proteins on the TGF-β1 signaling pathways. This

regulation of fibroblast differentiation and

proliferation aims to prevent fibrosis forming in the

wound. The molecular mechanism of the anti-

proliferative effect of fibroblasts by tannins is thought

to be due to the regulation of cyclin gene expression

All these mechanisms suggest that both

differentiation and inhibition of fibroblast

proliferation depend on inhibition of Smad2 and Erk

activation on the TGF-β1 signaling pathways

(Pattarayan et al., 2018).

Toxicity to fibroblasts in this study not only could

be caused by the level of the active compound at each

concentration, mechanism of action, and structure of

the active compound in the agarwood leaf extract. In

vitro, the mechanism of cytotoxicity can be in the

form of cell membrane destruction, prevention of

protein synthesis, binding with irreversible receptors,

prolonged inhibition of polydeoxynucleotide and

enzyme reactions (Aslanturk, 2018). It has also been

shown that toxic agents can induce excessive nitric

oxide production, ROS followed by oxidative stress,

and mitochondrial dysfunction as a result of oxidative

stress. Toxic agents can also potentially release

components that can directly result in DNA damage,

followed by apoptosis (Stammenković-Radak and

Andjelković, 2016; Zhang, 2018; Spindola et al.,

2018).

The determination of IC

value is important in

determining and understanding the pharmacological

and biological characteristics of a chemotherapeutic

agent. The IC

value is a measurement of drug

efficacy indicating the amount of concentration of a

chemotherapeutic agent required to inhibit half the

biological processes. This suggests a description of

the antagonistic potential of chemotherapeutic agents

in a study Aykul and Martinez-Hackert, 2016; He et

al., 2016).

In this study, the IC

value obtained was at a

concentration of 215.54 µg / mL, which indicates that

The Effect of Agarwood Leaves Ethanol Extract on Porphyromonas gingivalis Growth Inhibition and in Vitro Cytotoxicity Assay on

Fibroblast

117

this concentration can inhibit fibroblast proliferation

by up to 50% of the population. These results can be

used as a reference for further research regarding the

potential of agarwood leaf extract as a wound healing

agent by using the IC

value as the minimum

concentration of agarwood leaf extract.

3.2.2 Inhibitory Assay

Based on the mean of inhibition zone measured, it can

be concluded that the diameter of the inhibition zone

is directly proportional towards the concentration of

agarwood leaf extract.

Phytochemical test results showed that agarwood

leaf extract contain several active compounds that can

inhibit the growth of P. gingivalis. The main

compounds contained in the ethanol extract of gaharu

leaves are flavonoids. These compounds contributed

in the destruction of bacterial cells due to damage to

the permeability of their cell membranes (Nomer et

al., 2019; Warganegara and Restina, 2016). The action

mechanism of flavonoids as antimicrobial is divided

into 3, that is nucleic acid synthesis inhibition,

inhibiting the function of bacterial cell membranes,

and inhibiting energy metabolism from amino acids

(Nomer et al., 2019).

Flavonoids can inhibit nucleic acid synthesis in

bacterial cells because of their A and B rings. These

rings contributed in the intercalation process or the

process of hydrogen bonding by accumulating nucleic

acid bases so that the formation process of DNA and

RNA is inhibited (Nomer et al., 2019). These

flavonoid compounds can also cause damage to the

permeability of bacterial cell walls so that bacterial-

toxic substances can enter these bacterial cells. In

addition, flavonoids also form complex compounds

with extracellular proteins that can damage cell

membranes owned by bacteria resulting in leakage of

intracellular compounds. Flavonoids are also able to

inhibit the energy metabolism process of bacteria by

interfering with the macromolecular biosynthesis

process of these bacteria. Due to inhibited metabolic

processes, these molecules cannot develop properly to

meet the needs of these bacteria (Nomer et al., 2019;

Sapara and Waworuntu, 2016).

There are various types of flavonoids, such as

genkwanin, apigenin, and luteolin. Some of these

compounds are a class of flavonoids which are found

in agarwood leaves (Wangiyana, 2020). Genkwanin,

apigenin, and luteolin are known to have antibacterial

activity. Apigenin can affect the cytoplasmic

membrane of bacteria, this compound will interfere

with the metabolic process of bacteria and ultimately

inhibit energy production in bacteria (Xie et al., 2014).

Other active compounds found in agarwood leaf

extract are alkaloids, tannins, saponins, and

triterpenoids/steroids. These compounds are involved

in the destruction of bacterial cell membranes (Sari et

al., 2017). Alkaloids penetrate the bacterial

lipopolysaccharide membrane, causing depolarization

of the cytoplasmic membrane. This compound will

then affect the production of enzymes in bacteria and

cause leakage in the cytoplasm of the bacteria

(Cushnie et al., 2014). Meanwhile, tannins invade the

polypeptides present in the cell walls, these

compounds can denature proteins and eventually lead

to bacterial lysis (Zeniusa et al., 2019).

Saponins would bind water molecules and dissolve

fat so that it could disrupt the surface tension of

bacterial cells and eventually cause cell destruction. In

addition, there are triterpenoids/steroids that can bind

to lipid molecules on the bacterial cell membrane.

These compounds will disrupt the integrity of the

bacterial cell membrane and can change the

morphology of the cell membrane. In the end,

bacterial cells will be fragile and will undergo lysis

(Sari et al., 2017).

Previous research states that agarwood leaf extract

produced moderate to strong inhibition of

Staphylococcus aureus bacteria, a gram-positive

bacterium (Liana, 2014). While research conducted

upon gram-negative bacteria showed that the

inhibition power of agarwood leaf extract was weaker

when compared to gram-positive bacteria. This is

caused by 3 layers of the wall owned by gram-negative

bacteria. Those are the outer lipoprotein layer, the

middle lipopolysaccharide layer, and the outer

peptidoglycan layer (Septiani et al., 2017).

In this study, the inhibitory ability produced

towards P. gingivalis was weak to moderate. This

bacterium is an encapsulated gram-negative which

makes it more resistant to antibacterial activity

compared to other bacteria. This is supported by other

research which states that agarwood leaf extract has

weak to moderate inhibitory power against several

other encapsulated gram-negative bacteria such as

Escherichia coli, Klebsiella pneumoniae and Vibrio

mimicus (Hendra et al., 2016; Begum, 2016; Jihadi et

al., 2020).

In this study, the diffusion method used was agar

well diffusion method which is widely used to

evaluate the antimicrobial activity of extracts derived

from plants. In this method, a hole with a diameter of

8 mm is made which will then be filled with agarwood

leaf extract. The agar well diffusion method is used

with the aim that the results obtained directly

reflecting the agarwood leaf extract inhibitory ability.

This is ought due to the diffusion process is better and

ICE-TES 2021 - International Conference on Emerging Issues in Technology, Engineering, and Science

118

the volume of material placed is more than the agar

disk diffusion method Balouiri et al., 2016).

There are several factors that can affect the

diameter of the bacterial growth inhibition zone. The

first factor is the temperature used at the time of

incubation. If during the incubation process the agar

media are stacked, there is a possibility that there is a

temperature difference between the plates. In addition,

the thickness of the agar also affects the diameter of

the inhibition zone. The best agar thickness is 4 mm.

More than that, the diffusion process of the extract will

be slower (Zeinusa et al., 2019). The dilution of the

extract could also affect the diffusion ability. Higher

concentration means decreasing solubility or thicker

extract. Therefore, the extract diffusion process with

higher concentrations will be slower.

Another factor that can affect the diameter of

inhibition zone is the turbidity of the bacterial

suspension. In this study, the measurement of the

turbidity level was only visually performed by

comparison with the 0.5 Mc Farland solution. If the

bacterial suspension is too turbid, the resulting

inhibition zone diameter will be smaller and vice

versa.

The phytochemical test carried out in this study

was a qualitative test. Hence, it is difficult to

determine with certainty the number of compounds

contained in agarwood leaf extract which can inhibit

P. gingivalis growth. In addition, the solvent used in

diluting the ethanol extract of gaharu leaves was

distilled water (aquadest) which was also used as a

negative control in this study. It was found that this

treatment did not produce a clear zone on the agar

medium, meaning that distilled water confirmed had

no inhibitory ability and did not affect P. gingivalis

growth.

In this study, the concentration of agarwood leaf

extract was made in percent units makes the range of

concentrations that could be made as a treatment was

less than optimal. Making the concentration in units of

PPM (parts per million) might be better because this

method refers more to the unit of concentration. This

is a way to measure the concentration of a substance

that is both very low and high, which 1 ppm is

equivalent to 1 milligram per liter, or the concentration

in percent is 0.0001%.

4 CONCLUSIONS

This study showed that there was a cytotoxicity effect

of the agarwood leaf extract towards fibroblasts in

vitro at concentrations of 500 µg/mL, 250 µg/mL, and

125 µg/mL, and there was no cytotoxicity effect of

agarwood leaf extract towards fibroblasts in vitro at

concentrations of 62.50 µg/mL, 31.25 µg/mL, 15.63

µg/mL, and 7.81 µg/mL. Agarwood leaf extract also

had effect towards P. gingivalis growth with the

maximum inhibition at a concentration of 100%

which is classified into the moderate inhibition

category.

ACKNOWLEDGEMENTS

We thank Faculty of Dentistry of Maranatha Christian

University for the technical support during the

research. This research received grant from

Maranatha Christian University.

REFERENCES

Kinane DF, Stathopoulou PG, Papapanou PN. (2017).

Periodontal diseases. Nat Rev Dis Prim. 3(1), 1–14.

Nazir MA. (2017). Prevalence of periodontal disease, its

association with systemic diseases and prevention. Int J

Health Sci (Qassim). 11(2), 72–80.

Newman MG, Takei HH, Klokkevold PR, Carranza FA.

(2012). Carranza’s clinical periodontology, Saunders

Elsevier. St. Louis, 12

ed.

Wijaksana Evan IK. (2019). Periodontal chart dan

periodontal risk assessment sebagai bahan evaluasi dan

edukasi pasien dengan penyakit periodontal. J Kesehat.

Gigi. 6(3), 19–25.

Séverin T. (2018). Periodontal health and disease: A

practical guide to reduce the global burden of

periodontal disease, FDI World Dental Federation.

Switzerland.

How KY, Song KP, Chan KG. (2016). Porphyromonas

gingivalis: An overview of periodontopathic pathogen

below the gum line. Front. Microbiol. 7(1), 1–14.

Liu Y, Zhang Y, Wang L, Guo Y, Xiao S. (2013).

Prevalence of porphyromonas gingivalis four rag locus

genotypes in patients of orthodontic gingivitis and

periodontitis. PLoS One. 8(4), 1–6.

Klein BA, Tenorio EL, Lazinski DW, et al. (2012).

Identification of essential genes of the periodontal

pathogen porphyromonas gingivalis. BMC Genomics.

13(8), 1–17.

Gerits E, Verstraeten N, Michiels J. (2017). New

approaches to combat porphyromonas gingivalis

biofilms. J. Oral Microbiol. 9(1), 1–11.

Williams AJ, Wang Z, Taylor SF. (2016). Prevention and

treatment of periodontal diseases in primary care-dental

clinical guidance. Scott. Med. J. 22(5), 1–57.

Hudwekar AD, Beldar A, Murkute S, Lendhey SS, Thamke

M. (2019). Aloe vera on wound healing after

periodontal flap surgery in chronic periodontitis

patient: A randomized control trial. J. Oral Res. Rev.

11(7), 72-76.

The Effect of Agarwood Leaves Ethanol Extract on Porphyromonas gingivalis Growth Inhibition and in Vitro Cytotoxicity Assay on

Fibroblast

119

Sugiaman VK. (2011). Peningkatan penyembuhan luka di

mukosa oral melalui pemberian aloe vera (Linn.) secara

topikal. Maranatha J Med Heal. 11(1), 70–9.

Janshen YR, Sidharta BR, Swasti R. (2017). Aktivitas

antibakteri ekstrak daun gaharu (Aquilaria malaccensis

Lamk.) terhadap pseudomonas aeruginosa dan

staphylococcus aureus. J. Biotechnol. Biosci. 53(9), 1–

16.

Musir A, Winarti W, Siregar SHP. (2016). Phytochemical

screening and toxicity test BSLT of 70% ethanol extract

of gaharu leaves (Aquilaria beccariana Tiegh.).

Pharmacol. Sci. Res. 13, 34–9.

Nugraha R, Ginting H. (2013). Uji aktivitas antioksidan

ekstrak etanol daun gaharu (Aquilaria malaccensis

Lamk) berdasarkan umur pohon. Peronema. For. Sci. J.

6(3), 1–9.

Wangiyana IGAS. (2020). Medicinal effect review of

agarwood leaves from aquilaria and gyrinops genera. J.

Silva. Samalas. 3(1), 36–9.

Syamsul ES, Amanda NA, Lestari D. (2020). Perbandingan

ekstrak lamur aquilaria malaccensis dengan metode

maserasi dan refluks. J. Ris. Kefarmasian. Indones.

2(2), 97–104.

Nomer NMGR, Duniaji AS, Nocianitri KA. (2019).

Kandungan senyawa flavanoid dan antosianin ekstrak

kayu secang (Caesalpinia sappan L.) serta aktivitas

antibakteri terhadap vibrio cholerae. J. Ilmu dan

Teknol. Pangan. 8(2), 216-225.

Warganegara E, Restina D. (2016) Getah jarak (Jatropha

curcas L.) sebagai penghambat pertumbuhan bakteri

streptococcus mutans pada karies gigi. Med. J.

Lampung Univ. 5(3), 64–5.

Pratiwi EW, Praharani D, Mahdiyah Y. (2015) Daya

hambat ekstrak daun pepaya (Carica papaya L.)

terhadap adhesi bakteri porphyromonas gingivalis pada

neutrofil. 3(2), 193–8.

Dennis, Nurliza C, Savitri W. (2017). Antibacterial effect

of ethanol extract of the avocado seed (Persea

Americana Mill.) as an alternative root canal irrigants

against porphyromonas gingivalis (in vitro). Int. J.

Appl. Dent. Sci. 3(1), 90-92.

Sari R, Muhani M, Fajriaty I. (2017). Uji aktivitas

antibakteri ekstrak etanol daun gaharu (Aquilaria

microcarpa Baill.) terhadap bakteri staphylococcus

aureus dan proteus mirabilis. Pharmacol. Sci. Res.

4(3), 143–54.

Suhardiman A, Juanda D. (2019). Pengembangan obat

herbal fraksi daun gaharu (aquilaria malaccensis Lam)

dalam bentuk gel untuk penyembuhan luka bakar. J.

Sains dan Teknol. Farm. Indones. 3(1), 16–24.

Wahid AR, Safwan S. (2019). Efek antioksidan ekstrak

etanol daun gaharu (Aquilaria malaccensis L.) pada

tikus jantan galur sprague dawley yang diinduksi

paracetamol (kajian aktivitas enzim katalase, SGOT

dan SGPT). Pharmauho. J. Farm. Sains, dan Kesehat.

4(2), 22-4.

Fauzi R, Cahaya N, Hidayaturrahmah. (2017). Pengaruh

pemberian ekstrak etanol daun gaharu terhadap waktu

perdarahan pada tikus putih jantan galur wistar. J. Ilm.

Ibnu Sina. 2(2), 169–73.

Rahmadhani N, Yudaniayanti IS, Saputro AL, Triakoso N,

Wibawati PA, Yudhana A. (2020) Efektivitas krim

ekstrak buah naga merah (Hylocereus polyrhizus)

dalam meningkatkan jumlah sel fibroblas luka bakar

derajat II pada tikus putih (Rattus norvegicus). J. Med.

Vet. 3(1), 65–72.

Nurmiati N, Wijayanti E. (2018). Perbandingan kadar

fenolik total antara seduhan daun gaharu dan kombucha

daun gaharu. JC-T. 2(1), 6–10.

Komang N, Septiani A, Oka IM, Parwata A, Bawa A.

(2018). Penentuan kadar total fenol, kadar total

flavonoid dan skrining fitokimia ekstrak etanol daun

gaharu. J. Mat. Sains, dan Pembelajarannya. 12(1), 78–

87.

10993-5 I. (2009). Biological evaluation of medical devices

- Part 5: Test for in vitro cytotoxicity. Int. Stand. Organ.

3(5), 17–8.

10993-12 I. (2012). Biological evaluation of medical

devices - Part 12: Sample preparation and reference

materials. Int. Stand. Organ. 4(12), 7–8.

Rastina, Sudarwanto M, Wientarsih I. (2015). Aktivitas

antibakteri ekstrak etanol daun kari terhadap

staphylococcus aureus, escherichia coli, dan

pseudomonas sp. J. Kedokt. Hewan. 9(2), 185–188.

Yildirim I, Kutlu T. (2015). Anticancer agents: saponin and

tannin. Int. J. Biol. Chem. 9(6), 333–5.

Suharto I, Etika AN. (2019). Pengaruh ekstrak jahe

(Zingiber officinale roscoe) terhadap kepadatan serabut

kolagen luka insisi. J. Ilmu Kesehat. 7(1), 32–4.

Ardiana T, Rizkia Putri Kusuma A, Dian Firdausy M.

(2015). Efektivitas pemberian gel binahong (Anredera

cordifolia) 5% terhadap jumlah sel fibroblast pada soket

pasca pencabutan gigi marmut (Cavia cobaya).

ODONTO Dent. J. 2(1), 64-70.

Mohammadi M, Quan M, Zhang JS, Li X. (2020). FGF

signaling pathway: A key regulator of stem cell

pluripotency. Front. Cell Dev. Biol. 8(12), 1–3.

Etika A, Nurrahayu K, Suhato I. (2017). Pengaruh ekstrak

jahe (Zingiber officinale roscoe) terhadap jumlah sel

fibroblas pada tikus (Rattus Norvegicus). J. Nurs. Care

Biomol. 2(1), 12–3.

Kurahashi T, Fujii J. (2015). Roles of antioxidative

enzymes in wound healing. J. Dev. Biol. 3(2), 57–61.

Jiang X, Liu L, Qiao L, Zhang B, Wang X, Han Y, et al.

(2018). Dracorhodin perchlorate regulates fibroblast

proliferation to promote rat’s wound healing. J.

Pharmacol. Sci. 136(2), 66–72.

Ningrum AP, Kurniawaty E. (2019). The role of

mesenchymal stem cells in repairing lung parenchymal

damage. J. Major. 8(1), 201–4.

Pattarayan D, Sivanantham A, Bethunaickan R,

Palanichamy R, Rajasekaran S. (2018). Tannic acid

modulates fibroblast proliferation and differentiation in

response to pro-fibrotic stimuli. J. Cell Biochem.

119(8), 6732–42.

Aslanturk OS. (2018). In vitro cytotoxicity and cell viability

assays: Principles, advantages, and disadvantages,

Licens IntechOpen J. London 1

edition.

Zhang Y. (2018). Cell toxicity mechanism and biomarker.

Clin. Transl. Med. 7(1), 1–4.

ICE-TES 2021 - International Conference on Emerging Issues in Technology, Engineering, and Science

120

Spindola DG, Hinsberger A, Antunes VM de S, Michelin

LFG, Bincoletto C, Oliveira CR. (2018). In vitro

cytotoxicity of chemical preservatives on human

fibroblast cells. Brazilian J. Pharm. Sci. 54(13), 5–6.

Aykul S, Martinez-Hackert E. (2016). Determination of

half-maximal inhibitory concentration using biosensor-

based protein interaction analysis. Anal. Biochem.

508(16), 97–8.

Sapara TU, Waworuntu O. (2016). Efektivitas antibakteri

ekstrak daun pacar air (Impatiens balsamina L.)

terhadap pertumbuhan porphyromonas gingivalis.

Pharmacon. 5(4), 10–7.

Cushnie TPT, Cushnie B, Lamb AJ. Alkaloids: An

Overview of Their Antibacterial, Antibiotic-Enhancing

and Antivirulence Activities. Int J Antimicrob Agents.

2014;44(5):377–86.

Zeniusa P, Ramadhian MR, Nasution SH, Karima N.

(2019). Uji daya hambat ekstrak etanol teh hijau

terhadap escherichia coli secara in vitro. Majority. 8(2),

136–43.

Liana Y. (2014). Uji aktivitas antibakteri ekstrak daun

gaharu (Aquilarria malaccensis) terhadap

staphylococcus aureus. Nurs J. Indones. 11(7), 96-100.

Septiani, Dewi EN, Wijayanti I. (2017). Aktivitas

antibakteri ekstrak lamun (Cymodocea rotundata)

terhadap bakteri staphylococcus aureus dan escherichia

coli. J. Fish. Sci. Technol. 13(1), 1-4

Hendra H, Moeljopawiro S, Nuringtyas TR. (2016).

Antioxidant and antibacterial activities of agarwood

(Aquilaria malaccensis Lamk.) leaves. AIP Conf Proc.

1755(140004), 1–9.

Begum Y. (2016). Study on agarwood (Aquilaria

malaccensis) to evaluate antibacterial and antioxidant

activities of n-hexane, chloroform and ethyl acetate

extracts. Pharma. Tutor. 4(2), 47–50.

Jihadi NIM, Hashim YZHY, Rahim NA, Kamal KM, Noor

NM, Sani MSA, et al. (2020). Antibacterial activity of

ethanolic leaf extract of aquilaria malaccensis against

multidrug-resistant gram-negative pathogen. Food Res.

4(6), 1962–8.

Balouiri M, Sadiki M, Ibnsouda SK. (2016). Methods for in

vitro evaluating antimicrobial activity: A review. J.

Pharm. Anal. 6(2), 71–9.

APPENDIX

Cytotoxicity Assay

Treatment Results

XA XC Number of Cells %Viability %Inhibition

Cell Control 1,7049 1,2395 17238 100,00 0,00

DMSO 10% 1,6521 1,2337 17154 99,51 0,49

500 µg/mL 0,8455 0,4081 5360 31,10 68,90

250

µg

/mL 1,0178 0,6374 8636 50,10 49,90

125 µg/mL 1,0430 0,6675 9065 52,59 47,41

62.5

µg

/mL 1,4046 1,0277 14211 82,44 17,56

31.3 µg/mL 1,5046 1,1200 15530 90,09 9,91

15.6

µg

/mL 1,6080 1,2064 16765 97,26 2,74

7.81 µg/mL 1,9290 1,5217 21269 123,39 -23,39

XA : Mean of absorbance

XK : Mean of corrective absorbance

ALE : Agarwood leaf extract

Inhibitory Assay

Treatment

Inhibition Zone

Mean

1 2 3

- Control 0,00 0,00 0,00 0,00

1,56% 2,20 2,10 2,25 2,18

3,13% 2,35 2,20 2,50 2,35

6,25% 2,65 2,55 2,70 2,63

12,50% 2,85 3,50 3,35 3,23

25% 3,50 3,70 3,65 3,62

50% 3,85 4,50 4,55 4,30

100% 6,50 6,35 6,80 6,55

+ Control 10,95 12,10 11,80 11,62

The Effect of Agarwood Leaves Ethanol Extract on Porphyromonas gingivalis Growth Inhibition and in Vitro Cytotoxicity Assay on

Fibroblast

121