Evaluation of Antibacterial and Antioxidant Effects of Mix Essential

Oil for Oral Health Care

Juniarti

1,2,3

, Moch Abdussalam

, Indah Permata Yuda

and Indra Kusuma

2,4,5

Biochemistry Department, Faculty of Medicine, YARSI University, Jakarta, Indonesia

Magister of Biomedical Science, Graduate School, YARSI University

Herbal Research Center, YARSI University, Jakarta, Indonesia

Physiology Department, Faculty of Medicine, YARSI University, Jakarta, Indonesia

Stem Cell Research Center, YARSI University, Jakarta, Indonesia

Keywords: Mix essential oil, antioxidant, antibacterial

Abstract: Essential oil have some antioxidant and antimicrobial properties. The aim of this study was to determine the

chemical compounds, antioxidant and antimicrobial activities of essential oil. The analysis of the mix essential

oil was carried out using gas chromatography mass Spectrometry. The antioxidant activity of the essential oil

was also evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Antimicrobial

properties of the essential oil were assessed against Enterococcus faecalis, Streptococcus mutans,

Streptococcus sanguinis using the disk diffusion method. Free radical scavenging potentials showed values

for IC

in 194 µg/ml for mix essential oil, which are close to the natural antioxidant (ascorbic acid) with an

of 2.98 µg/mL. The major of mix essential oil were α-pinene (24.54%), D-limonen (18.00%), cis-1-

methyl-4-(1-methylethenyi)-l-cyclohexane (14.95%), 3-carene (8.92%), L-menthone (8.26) and β-pinene

(5.72%).

1 INTRODUCTION

In the 21st century, multidrug resistant antibiotic is

widely recognized as a serious threat to global health

(Martelli and Giacomini, 2018.) According to World

Health Organization (WHO) data in 2017, the most

dangerous multidrug-resistant to which new

antibiotics should be highly discovered (World

Health Organization, 2017). The discovery of new

antibiotics agents was mainly from natural product

(Jackson et al., 2018). Natural products have been a

source of medicinal agents and traditional medicine

system that have been used for thousands of years in

many countries (Dias et al., 2012; Newan and Cragg,

2016). Natural antimicrobial and antioxidant agents

can be obtained from different sources including

plants, bacteria, algae animals, and fungi, but there

has been an increased interest in plant-based active

compound as an alternative to the common antibiotics

(Rossiter et al., 2017; Helal et al., 2019).

Essential oil of many plant special have become

popular in recent years. Essential oils are volatile

natural mixtures extracted from different plant parts

(seeds, flowers, buds, leaves, twigs, bark, herbs,

wood and roots), and are composed of terpenoid

structures with broad activities (Seow et al., 2014).

Plant essential oils are also well-known to be the rich

sources of bioactive compounds. They are use as

alternative medicines, particulary as anti-

inflammatoty, antimicrobial, analgesic,

antipasmodic, anthelmintic, antipruritic and many

other theraperutic (Bakkali et al., 2008; Jaradat et al.,

2017). Nowadays, essential oils are used broadly in

preservatives in food and beverages industry,

cosmetics and pharmaceutical products (Seow et al.,

2014; Bakkali et al., 2008). Research on the use and

efficacy of essential oils significantly contribute to

the disclosure of their therapeutic properties, so that

they are frequently prescribed, even if their chemical

constituents are not always completely knowns.

Therefore, in this study the antimicrobial and

antioxidant activities of essential oils are the subjects

of particular interest. Evaluation of antioxidant

properties and antimicrobial activity against different

oral bacteria.

Juniarti, ., Abdussalam, M., Permata Yuda, I. and Kusuma, I.

Evaluation of Antibacterial and Antioxidant Effects of Mix Essential Oil for Oral Health Care.

DOI: 10.5220/0009957801190122

In Proceedings of the 2nd International Conference of Essential Oils (ICEO 2019), pages 119-122

ISBN: 978-989-758-456-5

119

2 MATERIAL AND METHODS

2.1 Material

Essential oil was provided by from WA Japan, Co

(Saitama-shi, Saitama-ken, Japan), which was dried

with anhydrous sodium sulphate and stored in vial at

4°C before use. Ascorbic acid, methanol (Merck,

German), 2,2-diphenyl-1-picrylhydrazyl (DPPH),

chlorhexidine (were purchased from Sigma Aldrich)

as positive control, and anaerobic jar (for anaerobic

condition) for antibacterial assay.

2.2 GC-MS Analysis Conditions

The analysis of the mix essential oil was performed

using Agilent 19091S-433, Equipped with HP-5 MS

capillary column (30 m x 0.25 mm, i.d., 0.25 μm film

thckness) and a HP 5972 mass selective detector. For

GC-MS detection an electron ionization with

ionization energy of 70 eV was used. Helium was the

carrier gas at a flow of 20 mL/min. Injector and MS

transfer line temperatures were set at 150 and 250

respectively. Column temperature was initially kept

C for 3 min, then gradually increased to 325

C at

3°C/min rate. 2 μL of sample were injected manually

and split mode.

2.3 Antimicrobial Screening

The antibacterial activity against Enterococcus

faecalis ATCC 29212, Streptococcus mutans ATCC

25175, S. sanguinis ATCC 10556 was detected using

disk diffusion method. The Kirby-Bauer disk

diffusion susceptibility test was used to determine the

sensitivity or resistance of bacteria to essential oil.

Bacteria was inoculated to nutrient broth (NB),

incubated at 37°C for 24 hours. Inoculum was diluted

by using physiological solution (NaCl 0.9%) to match

0.5 Mc Farland standard. A paper disk was dropped

50-μl essential oil in certain concentration and put the

disk in Mueller Hinton agar plate content bacteria

inside. The plates were incubated at 37°C for 24

hours. Chlorhexidine was used as a positive control.

Inhibition area diameter (IAD) was recorded as

sensitivity by measured the clear zone of growth

inhibition on agar surface around the disk.

2.4 Antioxidant Activity

The antioxidant activity of essential oil was

determined using the 2,2-diphenyl-1-picrylhydrazyl

(DPPH) radical scavenging activity. The DPPH

method was employed to evaluate the antioxidant

activities of essential oil radical-scavenging activity

as described by Panda (Panda, 2012). Briefly, 1 mL

of the extract at varying concentrations (25–200

ߤg/mL) was stirred together with 1 mL of DPPH in

methanol (0.3 mM) and 1 mL of methanol. The

mixture solution was incubated in dark room for 30

minutes and then the absorbance was measured using

spectrophotometer at wavelength 517 nm. The

percentage of DPPH inhibition was calculated using

the following equation: % inhibition = [(ܣDPPH−

ܣܵܣDPPH)] × 100, (1) where ܣDPPH is the

absorbance of DPPH without a sample and AS is the

absorbance of DPPH with a sample or the standard.

DPPH scavenging activity was presented as the

concentration of a sample required to decrease DPPH

absorbance by 50% (IC

). This value can be

determined by plotting the absorbance (the

percentage of inhibition of DPPH radicals) against the

concentration of DPPH and fitting the slope of the

linear regression.

3 RESULT AND DISCUSSION

The chemical composition of mix essential oil

was analysed by employing GC-MS, leading to

comparison of the relative retention time and the

mass spectra of mix oil component with data

library as shown in Table.1.

Table 1: Chemical composition for mix essential oil

No RT

(min)

Component

Composition

(%)

1 2.113 α-pinene 24.54

2 2.353 β-pinene 5.72

3 2.498 α-phellandrene 1.56

4 2.669 D-limonen 18.00

5 2.891 γ-terpinene 1.69

6 3.250 3-carene 8.92

7 3.926 L-menthone 8.26

8 4.063 1-menthone 3.14

9 4.191 cis-1-methyl-4-

(1-

methylethenyi)c

yclohexane

14.95

10 5.234 D-carvone 4.21

11 5.952 4-methyl-1-(1-

methylethyl)cyc

lohexene

1.34

12 7.294 Eugenol 1.93

13 8.380 Caryophyllene 1.91

major component (> 1%)

ICEO 2019 - 2nd International Conference of Essential Oil Indonesia

120

Structure analysis resulted in the identification of

thirteen compound representing 96.8% of the mix oil.

The main component were cyclic monoterpenes and

sesquiterpene. The result of bioassay showed that mix

essential oil exhibit antimicrobial activity against

Enterococcus faecalis, Streptococcus mutans, S.

sanguinis using the disk diffusion method shown as

in Table 2 and Figure. 1. Pinene compounds (α-

pinene and β-pinene) have oral antibacterial

bioactivity. Mercier (2009) reported that α-pinenes is

the largest contribution of active fractions against

gram-negative bacteria that seek jaw infections,

pardontitis or periodontitis (Mercie et al., 2009). D-

limonene compound, the main component of citrus

essential oil has activity against the bacteria

Porphylomonas gingivalis with a significant

inhibition in the range of 0.33-1.00 mg/mL (Mizrahi

et al., 2006). Mint leaves (Mentha piperita), the main

component is the L-menthone compound has

antibacterial activity against Aggregatibacter

actinomycetemcomitans, periodontal disease bacteria

(Karicheri and Antony, 2016).

Table 2. Antimicrobial activity from mix essential oil

Concen

trations

(%)

Inhibition Zone (mm)

E. faecalis S. mutans S. sanguinis

CHX

CHX EO CHX EO

2.0 18.4 - 28.0 - 13,9 -

12.5 NA - - - - -

25.0 - 8.8 - - - -

50.0 - 9.8 - 8.0 - -

100.0 - 11.8 - 9.5 - 7.8

Chlorhexidine

Essential Oils

Figure 1: Antibacterial efficacy of essential oils compared

to a chlorhexidine

Free radical scavenging activity was measured

with DPPH methods. Employing the DPPH methods

the reult show in Table 3, antioxidant activity (IC

194.90 ± 1.36 μg/mL) for the essential oils studied,

was lower efficient than ascorbic acid (IC

2.98 ±

0.06 μg/mL). The absence of antioxidant activity

observed for the essential oils in the DPPH reduction

can be explained by the reality that they are not

capable of donating a proton and the low solubility

provided by them in the reaction medium of the assay,

because this test utilizes methanol as solvent.

Otherwise, ascorbic acid have the ability to donate the

hydrogen atoms to DPPH reagent, can also describe

this low inhibition concentration oxidizing activity

(

Gharred

et al., 2019;

Umaru

et al., 2019). Therefore,

the reality that the essential oils of this study do not

show significant antioxidant activity can be

explained, since both oils are composed of

monoterpene and sesquiterpene compound.

Table 3. Antioxidant activity of essential oils

Sample Calibration

equation

(μg/mL)

Essential

oils

0,2752x -

4,0685

0,9984 194.90 ±

1.36

Ascorbic

acid

14,05x + 9,016 0,9950 2.98 ± 0.06

value IC

±SD, n: 3

4 CONCLUSIONS

The major of mix essential oil were monoterpene and

sesquiterpene such as α-Pinene, D-Limonen, cis-1-

methyl-4-(1-methylethenyi)-l-cyclohexane, 3-carene,

L-menthone and β-pinene. Antimicrobial properties

of the essential oil were give less active assessed

against Enterococcus faecalis, Streptococcus mutans,

Streptococcus sanguinis using the disk diffusion

method. Free radical scavenging potentials showed

values moderate activity for IC

in 194.90 ± 1.36

μg/mL for mix essential oil, which are close to the

natural antioxidant (ascorbic acid) with IC

of 2.98 ±

0.06 μg/mL.

REFERENCES

Martelli G and Giacomini D 2018 Eur. J. Med. Chem. 58

91-105

World Health Organization 2017. Geneva, Switzerland

Jackson N, Czaplewski L, Piddock L J V 2018 J.

Antimicrob. Chemother. 73 1452-59

Dias D A, Urban S and Roessner 2012 Metabolites 2 303-

Newan D J and Cragg G M 2016 J. Nat. Prod 79 629-61

Evaluation of Antibacterial and Antioxidant Effects of Mix Essential Oil for Oral Health Care

121

Rossiter S E, Fletcher M H and Wuest W M 2017 Chem.

Rev. 117 12415-74

Helal I M, Bessoumy A E, Bataineh E A, Joseph M R P,

Rajagopalan P, Chandramoorthy H C and Ahmed S B

H 2019 Hindawi 2019 1-9.

Seow YX, Yeo CR, Chung HL and Yuk HG 2014. Critical

Rev. in Food Sci. and Nutrition 54 625-44.

Bakkali F, Averbeck S, Averbeck D and Idaomar M 2008

Food and Chemical Toxicology 46 446-75.

Jaradat N, Adwan L, K’aibni S, Zaid A N, Shatya M J Y,

Shraim N and Assali M 2017 BioMed Re.s Intern. 2017

1-9

Panda S K 2012 Antioxidant Enzym (Egypt: IntechOpen) p

381-400

Mercie B, Prost J and Prost M 2009 Int J Occup Med

Environ. Health 22 331-42

Mizrahi B, Shapira L, Domb A J and Houri Haddad Y 2006

J. Periodontol 77 963-8

Karicheri R and Antony B 2016 European Journal of

Pharma. and Med. Res. 3 577-81

Gharred N, Dbeibia A, Falconieri D, Hammami S, Piras A

and Dridi S D 2019 J Ess. Oil Res. 2019 1-8

Umaru I J, Badruddin F A and Umaru H A 2019 Bio. Res.

Int 2019 1-8.

ICEO 2019 - 2nd International Conference of Essential Oil Indonesia

122