Activities and mechanisms of Eugenol and esential oil betel leaf (Piper betle, Linn) against some bacterial pathogens
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Eco. Env. & Cons. 25 (3) : 2019; pp. (1461-1465)
Copyright@ EM International
ISSN 0971–765X
Activities and mechanisms of Eugenol and esential oil
betel leaf (Piper betle, Linn) against some bacterial
pathogens
*Muhammad Yanis Musdja1, Ulfa Sadiah1 and Eka Putri1
Department of Pharmacy, State Islamic University, Jakarta, Indonesia
(Received 20 January, 2019; accepted 30 March, 2019)
ABSTRACT
Eugenol is one of the ingredients of essential oils of betel leaf and clove pistil, essential oils of betel leaf and
eugenol are used by health workers and the community against microbes. This aim of this research was to
determine the activity and mechanism of inhibition of the essential oil of betel leaf that was compared to
eugenol against Proteus mirabilis, Proteus vulgaris, Salmonella thypimurium, Shigella flexneri, and Streptococcus
mutans. Determination of Minimum Inhibitory Concentration (MIC) was done by using micro dilution
method. MIC values of essential oil for Proteus mirabilis 9% (v/v), Proteus vulgaris 6% (v/v), Salmonella
thypimurium 5% (v/v), Shigella flexneri 11% (v/v), and Streptococcus mutans more than 17% (v/v), and for
eugenol was obtained MICvalues for Proteus mirabilis 0,4% (v/v), Proteus vulgaris 0,5% (v/v), Salmonella
thypimurium 0,2% (v/v), Shigella flexneri 0,5% (v/v) and Streptococcus mutans more 0,4% (v/v). mechanism
of inhibition of the essential oil of betel leaf and eugenol could cause membrane damage that indicated
with the accurrence of leakage of cellular metabolites. In this cases, could be seen with the increase leaked
nucleic acid and protein from cell of bacteria that indicates an increased absorbance at wavelength of 260
nm and 280 nm by using Ultra Violet-Visible Spectrophotometer. This was reinforced by an increase in
leakage ions Ca ,+and K+,with observed by using Atomic Absorption Spectrophotometre (AAS) moreover
also occurred plasmolysis on bacterial that was observed by using Scanning Electron Microscope (SEM).
Key words : Piper betle, Proteus mirabilis, Proteus vulgaris, Salmonella thypimurium, Shigellaflexneri, Streptococcus mutans,
Antibacteri.
Introduction against bacteria, parasites, viruses and fungi. In this
case if the experts fail to find new drugs to fight in-
Finding new antimicrobial compounds is very ur- fection, then in the next 20-30 years, to treat infec-
gent, because as predicted by WHO, in the near fu- tious diseases will be more difficult. Antimicrobial
ture, we will enter the post-antibiotic era. Because resistance happens when microorganisms, i.e bac-
the antibiotics that exist now, from time to time, teria, fungi, viruses, and parasites change when
have continued to increase resistance to antibiotics they are exposed to Anti-microbial drugs such as
that exist today. Therefore, on the world health day antibiotics, antifungals, antivirals, antimalarials,
in 2011, the WHO has declared combat to antimicro- and anthelmintics. At this ime, new resistance
bial resistance (AMR) worldwide (WHO, 2011) mechanisms are emerging and spreading globally,
Antimicrobial resistance (AMR) has threatened threatening our ability to treat common infectious
the prevention and treatment of infectious diseases diseases, resulting in prolonged illness, disability
*Corresponding author’s email : yanis.musdja @uinjkt.ac.id1462 Eco. Env. & Cons. 25 (3) : 2019
and death. Antimicrobial resistance occurs naturally methods (2006) by using micro-dilution (Supprakul
over time, usually through genetic changes. Antimi- et al., 2006).
crobial resistant-microbes are found in people, Analysis of Proteins and Nucleic Acids was de-
animals, food, and the environment (in water, soil termined based on Carson et al., methods (2002)
and air). Antibiotic resistance is present in every from suspension of test bacteria that had been incu-
country, especially against, resistance in tuberculo- bated for 24 hours in Mueller-Hinton Broth Medium
sis (TB), malaria. HIV, influenza etc (WHO, 2018, (MHB) by using UV-VIS spectrophotometer
Cowan, 1999). (Carson et al., 2002; Glasel, 1995)
Therefore it is very necessary to look for new an- Analysis of leakage of metals were measured in
tibacterials to prevent and treat infectious diseases, the form ions Ca2+ and K+ based Carson et al., meth-
especially those sourced from natural products. In ods (2002) by using Atomic Absorption Spectrum
the essential oil of betel leaf, there are many (Carson et al., 2002)
phenol compounds which are strongly suspected to Analysis of changes in bacterial cell morphology
have efficacy against microbes. Because of the large was done based on Carlson methods (2002) by us-
number of chemical compounds, where the possi- ing Scanning Electron Microscopy (SEM) (Carson
bility of work is synergistic. It is likely that mi- et al. methods, 2002; Cox et al., 2001)
crobes are more difficult to become resistant to
betel leaf essential oil. Eugenol has long been used Results and Discussion
as a disinfectant, especially by dentists. Eugenol can
be made synthetically or can also be obtained by iso- The result of the identification was carried out by
lating from betel leaves, or cloves. (Coralie Pavesi et the Biology Research Center, Indonesian Institute of
al., 2018; Abd El-Baky et al., 2016; Hamed et al., Sciences, Bogor, Indonesia, showed that the plants
2012). used was true betel leaves (Piper betle Linn).
Usually natural products have smaller side ef- Essential oil of betel leaf was isolated in a fresh
fects than synthetic chemical compounds or single condition by using steam distillation methods
chemical compounds (Cowan, 1999). However, to which was clear yellowish in color with a distinctive
compare the advantages and disadvantages be- odor such as betel nut with a yield value of 0.14% (v
tween eugenol and betel leaf essential oil as antibac- / w).
terial, other studies are needed, such as safety and Essential oil was analyzed by using GC-MS and
side effects, types of bacteria that are more sensitive obtained results in the form of chromatograms.
and the prices of these two compounds, etc. Chromatogram results showed the chemical compo-
nents of essential oil of betel leaf has 57 components
Materials and Methods with major components (> 1%) as many as 18 com-
ponents and minor components (MUSDJA ET AL 1463
As shown in Figure 1. Table 2. The absorbance values of nucleic acid com-
Synthetic Eugenol was obtained from the Indone- pounds (260 nm) of essential oils and eugenot
sian Institute of Sciences, after analysis by using against Shigella flexneri with UV-Vis.
GCMS, showed that the compound was Eugenol Treatment Absorbance Absorbance
with very high purity or high quality. As shown in betel oil eugenol
Figure 2.
Normal Control 0.283 0.283
The test results of the zone of inhibition of vola- 1 MIC 1.018 1.446
tile oil and eugenol, each with a concentration of 2 MIC 1.366 1.842
15% against several gram (+) pathogenic bacteria
are shown in Table 1.
Table 3. The absorbance values of protein (280 nm) of es-
Antibacterial activity test results with concentra- sential oils and eugenot against Shigella flexneri
tion 15% of both essential oil of betel leaf & eugenol with UV-Vis.
against some bacteria.
Determination of MIC value for essential oils of Treatment Absorbance Absorbance
betel oil eugenol
betel leaf was startted from a concentration of 4% to
11% (v / v), except for Sreptococcus mutans was Normal Control 0,260 0,260
started from a concentration of 4-17% (v / v) and for 1 MIC 1,351 1,552
eugenol was started from a concentration of 0.1% - 2 MIC 1,630 1,709
0 , 8% (v / v). This concentration determination was
based on 100% bacterial growth inhibition by essen- Table 4. Measurement of Ca 2+ ion levels of Shigella
tial oils of betel leaf and eugenol at the lowest con- flexneri by using Atomic Absorption Spectro-
centration. As shown in Table 2 photometry (AAS).
Protein leak analysis at a wavelength of 260 nm Treatment Iion Ca2+ betel Ion Ca2+ eugenol
and nucleic acid at a wavelength of 280 nm was car- (Concentration) oil (ppm) (ppm)
ried out using a UV / VIS spectrophotometer. In
Normal control 4,21 4,21
this experiment, Shigella flexneri was used as a com-
1 MIC 27 31
parison with no essential oils and eugenol. In this 2 MIC 32 35
case a dose of 1MIC (11%) and 2 MIC (22%) was
used for essential oils and a dose of 1MIC (0.5%)
Table 5. Measurement of K+ ion of Shigella flexneri by
and 2MIC(1%) for eugenol. As shown in Table 3.
using Atomic Absorption Spectrophotometri
Analysis of Ca2+ and K+ ion leakage were carried
(AAS)
out by using Atomic Absorption Spectrophotometry
(AAS). In this experiment, Shigella flexneri was used Perlakuan Ion K+ betel Ion K+ eugenol
as a comparison without giving essential oils and (Konsentrasi) oil (ppm) (ppm)
eugenol. In this case a dose of 1MIC (11%) and Normal control 52,3 52,3
2MIC (22%) were used for essential oils and a dose 1 MIC 61 78
of 1MIC (0.5%) and 2MIC (1%) for eugenol, as 2 MIC 86 90
shown in Table 5
Morphological changes in Shigella flexneri cells shown in Figure 2.b and 2.c for essential oils and 2.d
was caused by betel oils and eugenol at a dose of 1 and 2.e for eugenol.
MIC and 2 MIC was observed by using SEM, as In this case there was a difference between nor-
Table 1. Antibacterial activity test results with concentration 15% of both essential oil of betel leaf & eugenol against
some bacteria.
No. Bacteria Test sample % (v/v) “ Zone of inhibition
1. Proteus mirabillis 15% Oil of betel leaf 15% Eugenol 3 mm4 mm
2. Proteus vulgaris 15% Oil of betel leaf 15% Eugenol 3 mm7 mm
3. Salmonella thypemerium 15% Oil of betel leaf 15% Eugenol 5 mm6 mm
4. Shigella flexneri 15% Oil of betel leaf 15% Eugenol 5 mm8 mm
5. Streptococcus mutans 15% Oil of betel leaf 15% Eugenol 1 mm10 mm1464 Eco. Env. & Cons. 25 (3) : 2019
mal control cells and cells given the betel oils and wall become destroyed (Figure 2d). Whereas with
eugenol, as shown in Fig. 2a. eugenol treatment at 2 MIC dose on the surface of
In cells of Shigella flexneri the dose of betel oil of perforated bacterial cells and morphology of bacte-
1MIC could be seen on bacterial cells with cell walls rial cells becomes irregular because plasmolysis oc-
that were not intact (Figure 2.b), whereas in betel oil curs in bacterial cells, as shown in Figure 2.e.
with a dose of 2 MIC can be seen bacterial cells have According to Brock (1973): Substances that pro-
holes and are destroyed and for eugenol 1 MIC cell duce larger inhibitory zones are not necessarily
(a) Complete cell surface (b)
(c) (d)
(e)
Fig. 2. Changes in cell morphology was observed by using SEM. Type: Shigella flexneri normal cells (a), Shigella flexneri
cells with 1MIC betel oil treatment (b) and Shigella flexneri cells with 2 KHM betel oil treatment (c). While Shi-
gella flexneri cells with 1MIC eugenol treatment (d) and Shigella flexneri cells with 2 MIC eugenol treatment (e).
(15,000x magnification)MUSDJA ET AL 1465
more active than substances that produce smaller sian Institute of Sciences (LIPI), Bogor, Indonesia for
inhibitory zones (Brock, 1973). The size of the inhibi- their technical assistance in this study
tory zone is influenced by sensitivity, media culture,
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