Antibacterial Activity of Nutmeg Oil

Abstract

Indonesia is one of the largest producer of nutmeg oil (Myristica fragrans). This essential oil has a lot of usefulness for food and pharmaceutical industries, however antibacterial activity of Indonesian nutmeg oil has not been investigated yet. Antibacterial activity Myristica fragrans oil from two areas respectively (Sulawesi and Central Java) were investigated. The essential oils was extracted using water and steam distiller and then its antibacterial activity against pathogenic bacteria (gram-positive bacteria: Staphylococcus aureus, Staphylococcus epidermis, and gram-negative bacteria: Shigella Dysenteriae, Salmonella Typhi) was examined. Resistance pattern was studied by in vitro disc diffusion method using essential oil concentration 20%, 40%, 60%, 80% and 100%. The result showed that the two essential oils inhibited all bacteria. The highest inhibition zone on Central Java nutmeg oil was on 60% concentration of the oil (12.96 16.79, 13.46 and 16.50 mm for S. aureus, S. epidermis, S. dysenteriae, S. typhi respectively), while on Sulawesi nutmeg oil was on 100% concentration (18.84, 16.54, 17.84 and 12.54 mm for S. aureus, S. epidermis, S. dysenteriae, S. typhi respectively).

Full text

ICSAFS Conference Proceedings
2nd International Conference on Sustainable Agriculture and Food Security:
A Comprehensive Approach
Volume 2017
Conference Paper
Antibacterial Activity of Nutmeg Oil
Sarifah Nurjanah, Indira Lanti Putri, and Dwi Pretti Sugiarti
Universitas Padjadjaran, Jl Bandung-Sumedang km 21, Jatinangor, Bandung, Indonesia
Abstract
Indonesia is one of the largest producer of nutmeg oil (Myristica fragrans). This essential
oil has a lot of usefulness for food and pharmaceutical industries, however antibacterial
activity of Indonesian nutmeg oil has not been investigated yet. Antibacterial activity
Myristica fragrans oil from two areas respectively (Sulawesi and Central Java) were
investigated. The essential oils was extracted using water and steam distiller and
then its antibacterial activity against pathogenic bacteria (gram-positive bacteria:
Staphylococcus aureus, Staphylococcus epidermis, and gram-negative bacteria: Shigella
Dysenteriae, Salmonella Typhi) was examined. Resistance pattern was studied by in
vitro disc diffusion method using essential oil concentration 20%, 40%, 60%, 80%
and 100%. The result showed that the two essential oils inhibited all bacteria. The
highest inhibition zone on Central Java nutmeg oil was on 60% concentration of the
oil (12.96 16.79, 13.46 and 16.50 mm for S. aureus, S. epidermis, S. dysenteriae, S. typhi
respectively), while on Sulawesi nutmeg oil was on 100% concentration (18.84, 16.54,
17.84 and 12.54 mm for S. aureus, S. epidermis, S. dysenteriae, S. typhi respectively).
Keywords: Antibacterial activity; Nutmeg oil; Central Java; Sulawesi.
1. Introduction
The Nutmeg tree (Myristica fragrans) was originated from the Banda Island [1], and is
widely distributed all over in Indonesian and throughout the world (India, Grenada and
Malaysia). Maluku (Moluccas), Sulawesi (Celebes), Aceh, Java and Papua are the major
production areas of Indonesian nutmeg [2]. Indonesia produces three quarters of the
total world output and export, while Grenada is in the second rank [3].
Nutmeg mainly used as a spice or extracted to nutmeg oil. Essential oils can be
extracted from both the kernel and mace. Nutmeg refer to the dried kernel, while
mace is a dried scarlet fibrous aril that covers the kernel. Steam and water distillation
or steam distillation is generally used for the extraction oil. The main application used
of nutmeg essential oils is in food flavoring. It has been used as the flavoring agent for
beverage, biscuit, cake, pudding, candy and roasted food such as meat and sausages.
In beverage industry the oils are used for soft drink of cola-type, beer, whisky and
wine [4–6]. The adding aroma of nutmeg essential oils on these products linked to
How to cite this article:Sarifah Nurjanah, Indira Lanti Putri, and Dwi Pretti Sugiarti, (2017), “Antibacterial Activity of Nutmeg Oil” in 2nd International
Conference on Sustainable Agriculture and Food Security: A Comprehensive Approach, KnE Life Sciences, pages 563–569. DOI 10.18502/kls.v2i6.1076 Page 563
Corresponding Author:
Sarifah Nurjanah
sarifah@unpad.ac.id
Received: 28 July 2017
Accepted: 14 September 2017
Published: 23 November 2017
Publishing services provided
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article is distributed under the
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spicy aroma. Nutmeg essential oils also used in pharmacy due to the antithrombotic,
anti-dysentery, anti-inflammatory, rheumatism and narcotic activities [7].
Morover, nutmeg oil reveals their antibacterial activity. A number of report have
been published about these activities. The oils can be effective against gram pos-
itive and gram negative bacteria: Escherichia coli,Aeromonas hydrophila,Salmonella
choleraesuis,Pseudomonas aeruginosa,Staphylococcus aureus,Listeria monocytogenes,
Listeria innocua [8] and others: Acinebacter calcoacetica,Alcaligenes faecalis,Bacillus
subtilis,Benecka natriegens,Brevibacterium linens,Brocothrix thermosphacta,Citrobacter
fruendii,Enterobacter aerogenes,Erwinia carotovora,Flavobacterium suaveolens,Kleb-
siella pneumonia,Micrococcus luteus,Moraxella sp., Proteus vulgaris,Serratia marcescens
and Yersinia enterocolitica [9].
However, research on antibacterial activity of nutmeg oil in Indonesia has not been
reported widely. Therefore, this research aimed to examine the antibacterial activity of
nutmeg oil from Sulawesi and Central Java against Staphylococcus aureus, Staphylococ-
cus epidermis, Shigella dysenteriae and Salmonella typhii. Among the major production
area in Indonesia, Sulawesi is the biggest. Moreover nutmeg oil from this area has
been renowned since a long time ago as an export commodity. While Central Java is a
new area for nutmeg oil production.
2. Materials and Method
2.1. Materials
Nutmeg oils from the kernel were obtained from distillers in Sulawesi and Central
Java (2 samples for each), wich are distilled using water and steam distillation. S.
aureus, S. epidermis, S. dysenteria and S. typhii were obtained from the Laboratory of
Microbiology, Faculty of Pharmacy, Padjadjaran University.
2.2. Methods
Antibacterial activity of nutmeg oils (zone of inhibition). The nutmeg oils was pre-
pared into solutions with concentration of 20%, 40%, 60%, 80% and 100% (v/v)
by diluting with ethanol. S. aureus, S. epidermis, S. dysenteria and S. typhii suspension
were prepared with cell content of 3 ×108CFU/ml (absorption in spectrometry was
compared with the Mc Farland scale 1 for each microorganism). S. aureus, S. epidermis,
S. dysenteria and S. typhii were cultured on Beef Extract Peptone Agar (NA) medium.
With the aid of moist sterile swab the suspensions were spread on plates of Mueller
Hilton Agar (MHA). Paper disc of 0.5 cm was soaked in oil solution and left to dry
for about 7 minute. Paper disc was placed on MHA and incubated at 37∘C for 48 h.
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After incubation, the inhibition zones were measured to determine the antibacterial
activity. The experiment was conducted at three replications. Diameter of inhibition
was calculated by subtracting the diameter of bacterial activity with the diameter of
paper disc. The resulting diameters of the inhibition zones were compared with control
disc without nutmeg oils.
GC-flame ionization detection analysis. The nutmeg oil was analyzed using GC 2010
Shimadzu machine with a ZB-1MS fused m𝜇silica capillary column (30m ×0.25mm
×0.25μm film thickness). The injector and detector temperature was 280 and 230∘C,
respectively. The oven temperature was programmed to rise from 60∘C to 290∘C in 29
min at rate of 8∘C/min. The carrier gas was hydrogen with a flowrate of 1.31 mL/min.
The percentage of constituents were based on electronic integration of peak area
without the use of response factor correction.
GC-MS Analysis. The analysis of nutmeg oil was performed on Shimadzu QP 2010
Ultra with a ZB-5MS fused silica capillary column (30m ×0.25mm ×0.25μm film thick-
ness). The GC operating conditions were the same as described above. The detector
was operated in EI mode with a mass scan range from m/z 35 to 500 at 0.7 kV. The
components were identified by using The National Institute of Standards and Technol-
ogy (NIST 3.0) and WILEY 275 libraries provided with controlling the GC –MS system
and mass spectra with published data.
3. Result and Discussion
3.1. Antibacterial activity
The antibacterial activity of Sulawesi nutmeg oil on both gram positive (S. aureus
and S. epidermis) and gram negative (S. dysenteriae and S. typhii) were represented
in Table 1, while antibacterial activity of Central Java nutmeg oil were represented
in Table 2. The results from the disc diffusion method indicated that Sulawesi and
Central Java nutmeg oils had inhibitory effect against all bacteria tested in middle to
strong area. Sulawesi Myristica fragrans oil had smaller antibacterial activity average
better than Central Java Myristica fragrans oil. However, the increasing concentration
of oils did not reveal increasing of inhibition diameter. It is more influenced by the
cell wall of each type of bacteria, gram-positive bacteria have a thicker peptidoglycan
than gram-negative bacteria. In addition, the gram-negative bacteria had proteins and
lipoproteins layer that serves as a protective. The mechanism of inhibition against
the bacteria include damage cell membranes, inhibits protein synthesis, and specific
enzymes disrupt cell membranes and cell biological functions [10].
Phenolic compound in essential oil appears to contribute on most antibacterial
activity, while other constituens contributes little to this activity [11]. However, the
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T 1: Antibacterial activity units of Sulawesi Myristica fragrans oil.
Bacteria 0% (control) 20% 40% 60% 80% 100%
Stapphylococcus aureus 0 11.59 14.29 12.96 12.75 18.84
Stapphylococcus epidermi. 0 9.63 10.79 11.46 11.88 16.54
Shigella dysenteriae 0 10.04 12.17 13.04 15.59 17.84
Salmonella typhi 0 9.54 10.75 10.38 10.79 12.54
T 2: Antibacterial activity units of Central Java Myristica fragrans oil.
Bacteria 0% (control) 20% 40% 60% 80% 100%
Stapphylococcus aureus 0 12.16 12.54 12.96 9.37 8.35
Stapphylococcus epidermis 0 9.54 13.62 16.79 9.91 9.29
Shigella dysenteriae 0 13.46 14.62 13.46 12.71 14.21
Salmonella typhi 0 12.46 12.75 16.50 10.04 8.71
antibacterial activity of nutmeg oils was primarily caused by pinene-� component [12].
This constituent presents in both nutmeg oils (Sulawesi dan Central Java). Although -�
pinene component is higher in Central Java nutmeg oils (Table 3), it did not show higher
antibacterial activity. The differences of antibacterial activity in the same component
allegedly influenced by many other active components found in the nutmeg oils. It
has been identified 45 components were identified in Sulawesi nutmeg oil and 39
components were identified in Central Java nutmeg oil. Each component can synergize
to produce antibacterial activity. However, further research needs to be done on the
interaction between the active component in nutmeg oils.
3.2. Composition of nutmeg oils
Chemical composition of Sulawesi and Central Java nutmeg oils with GC-MS analysis
was presented in Table 3. In total, 45 components were identified in Sulawesi nutmeg
oil and 39 components were identified in Central Java.
T 3: Composition of Sulawesi and Central Java Nutmeg Oils.
No Retention Time Chemical component Sulawesi (%) Central Java (%)
1 3.844 alpha.-Thujene 0.81 2.1
2 3.976 1R-.alpha.-Pinene 4.24 16.54
3 4.155 Beta-Fenchene 0.03
4 4.180 Camphene 0.5 0.87
5 4.532 Sabinene 9 18.82
6 4.603 Beta- Pinene 5.31 13.82
7 4.702 Beta -Pinene 3.75 3.52
8 4.959 Phellandrene 1.38 1.49
9 5.055 Carene 1.16 2.96
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Table 3: Continued.
No Retention Time Chemical component Sulawesi (%) Central Java (%)
10 5.146 alpha-Terpinene 4.23 2.59
11 5.272 Cymene 2.14 1.63
12 5.352 Limonene 10.31 8.41
13 5.592 beta-Ocimene 0.1 0.09
14 5.822 gamma-Terpinene 5.4 3.18
15 5.962 Dimethylsiloxane pentamer 0.14
16 6.240 trans Sabinene hydrate 0.41 0.43
17 6.317 alpha-Terpinolene 2.78 2.43
18 6.409 Adamantanol 0.14
19 6.485 Linalool 0.82
20 6.949 Terpeniol 0.13
21 7.145 Mentha-tiene 0.05
22 7.910 Therpinen-ol 7.99 2.8
23 8.286 alpha-Terpineol 0.88 0.8
24 9.099 Linalyl acetate 0.06 0.08
25 9.707 Borneol acetate 0.46 0.25
26 9.786 Isosafrole 3.68 2.17
27 9.919 Terpinene acetate 0.23
28 9.980 Anisole 0.82 0.39
29 10.733 alpha Terpinenyl acetate 0.81 0.41
30 10.770 alpha Cubebene 0.42 0.17
31 10.984 Eugenol 0.42 0.34
32 11.235 Copaene 1.95 1.16
33 11.457 Germacrene 0.14
34 11.648 Methyl isoeugenol 8.5 0.19
35 11.902 Cyclohexene 0.11
36 11.983 Caryophyllen 0.12 0.23
37 12.150 Norpinene 0.3 0.21
38 12.418 Isoeugenol 0.37
39 12.403 Farnesol 0.12
40 12.872 Alpha Curcumene 0.27 0.17
41 12.943 Germacrene 0.22 0.09
42 13.060 Zingibere 0.25 0.11
43 13.106 Cis-methyl isoeugenol 2.04
44 13.200 alpha-Farnesene 0.46 0.11
45 13.267 beta-Bisabolene 0.32 0.13
46 13.586 Myristicine 13.73 9.32
47 13.720 Cadina diene 0.03
48 13.780 Alpha humulene 0.02
49 13.971 Elemicin 3.81 0.39
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The differences of component caused by differences in species of nutmeg. The main
components for both nutmeg oils were alpha pinene, beta pinene, sabinene, limonene
and myristicin. This findings were likely to be similar with other previous studies [4, 13–
16]. The highest component of Sulawesi nutmeg oil was myristicine (13.73%), while in
Central Java nutmeg oil, the highest component was sabinene (18.82%).
4. Conclusion
The study showed that Sulawesi and Central Java nutmeg oils were effective against
bacteria (S. aureus, S epidermis, S. dysenteriae and S. typhi). The highest inhibition zone
on Central Java nutmeg oil was on 60% concentration of the oil (12.96 16.79, 13.46
and 16.50 mm for S. aureus, S. epidermis, S. dysenteriae, S. typhi respectively), while
on Sulawesi nutmeg oil was on 100% concentration (18.84, 16.54, 17.84 and 12.54 mm
for S. aureus, S. epidermis, S. dysenteriae, S. typhi respectively). Therefore nutmeg oils
can be considered as a potential natural antibacterial product. The main components
of the nutmeg oils were sabinene, myristicin, pinene and limonene.
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