Gas chromatography – Mass spectrometry

analysis and antibacterial activity of

Cinnamomum burmanii essential oil to
Staphylococcus aureus and Escherichia coli
by gaseous contact
Cite as: AIP Conference Proceedings 1823, 020073 (2017); https://doi.org/10.1063/1.4978146
Published Online: 17 March 2017

Chairunnisa, Hady Anshory Tamhid, and Arde Toga Nugraha

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AIP Conference Proceedings 1823, 020073 (2017); https://doi.org/10.1063/1.4978146 1823, 020073

© 2017 Author(s).
 Gas Chromatography – Mass Spectrometry Analysis and
Antibacterial activity of Cinnamomum burmanii Essential
Oil to Staphylococcus aureus and Escherichia coli by
Gaseous Contact
Chairunnisaa), Hady Anshory Tamhidb), and Arde Toga Nugraha

Dept. of Pharmacy, Islamic University of Indonesia, Yogyakarta 55584, Indonesia

a)
Corresponding author: chairunnisa0924@gmail.com
b)
hadyanshory@uii.ac.id

Abstract. Infectious diseases and antibiotic resistance becomes a problem that must be resolved. Plant based products are
among the alternative agents examined in order to replace conventional antibiotics. Cinnamaldehyde is one of the
compound in cinnamon oil that has antibacterial activity. But the other compounds in cinnamon oil has also the potential
antibacterial activity. The purpose of this study to conduct GC-MS analysis of cinnamon oil and its antibacterial activity
to Staphylococcus aureus and Escherichia coli by gaseous contact. Cinnamomum burmannii was distilled by water-steam
distillation to obtain essential oil. Identification of compounds was analyzed by GC-MS. Antibacterial activity was
observed by gaseous contact method in airtight boxes. The GC-MS analyzed showed that there are four major
compounds of cinnamon oil, trans-cinnamaldehyde (56,10%), 1,8- cineole (16,53%), α-pinene (3,44%) and α –terpineol
(3,05%). The Minimum Inhibitory Dose (MID) of cinnamon oil to E. coli and S. aureus was 12.5 μL/L and 6.26 μL/L
respectively. Gas compounds of cinnamon oil has more effective to gram-positive bacteria than gram-negative bacteria.

INTRODUCTION
Infectious diseases are one of the causes of morbidity and mortality, especially in developing countries [1].
Infectious diseases often caused by pathogenic bacteria. Staphylococcus aureus and Escherichia coli are a few
examples of a pathogenic bacteria that can cause infections such as nosocomial infections [2,3]. The evolving
bacterial strains and the increasing incidence of antibiotic resistance become a big problem for public health [4].
This situation shows the need for the discovery and development of new antibacterial agent. The process of making
new antibiotics that are often take a long time and require a high cost provides an opportunity to develop the
potential of natural material as an antibacterial agent [1]. The advantage of using bioactive compounds of plants are
the compounds do not pollute the environment, leaving no residue, as well as relatively cheaper operational costs
[3].
Aromatherapy is a treatment which harness the power of the scent of essential oils from plants, commonly used
to help maintain health, relieve the stress, as well as stimulate the healing process [5,6,7]. Aromatherapy uses
essential oils as its therapeutic agent. Traditionally, essential oils from plants have been widely used to treat
respiratory tract infections such acute and chronic bronchitis, acute sinusitis, and also colds [8]. Many studies have
shown the effects of essential oils as an antioxidant, antiviral, antifungal, antibacterial, and insecticidal [2,5,6,7]. The
use of essential oils in Indonesia is still limited as holistic therapies to help soothe the mind, while in developed
countries such as Britain, the essential oil has been used as an antiseptic which spread through the air in public
places [5].
Cinnamomum is a genus in the family Lauraceae and there are three known species in Indonesia, they are
Cinnamomum zeylanicum, Cinnamomum cassia, and Cinnamomum burmannii. Cinnamomum burmannii, also

International Conference on Chemistry, Chemical Process and Engineering (IC3PE) 2017

AIP Conf. Proc. 1823, 020073-1–020073-6; doi: 10.1063/1.4978146
Published by AIP Publishing. 978-0-7354-1491-4/$30.00

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known as Indonesian cinnamon, is the most known species in Indonesia. Cinnamon (C. burmannii) often used as a
spice in cooking, has volatile oil as the largest content of the bark with cinnamaldehyde as its major content, and has
the ability to inhibit bacterial growth [9].
There are still a few study which conducted the evaluation of antibacterial effect of Indonesian cinnamon (C.
burmannii) [9]. Most study that has been done mostly using extract and also essential oil in liquid form only [9.10].
Several publications have demonstrated that cinnamon oil has antibacterial effect by effectively inhibiting the
growth of five pathogenic bacteria due to its active compounds, cinnamaldehyde and cinnamic acid [10,11]. The
study of cinnamon oil's antibacterial effect in its vapour state as aromatherapy is still rarely conducted.
The aim of the present study was to investigate the antibacterial effect of cinnamon oil (C. burmannii) in its
vapour phase as aromatherapy against the growth of the Escherichia coli and Staphylococcus aureus using gaseous
contact method. The research benefit includes as an innovation in the use of cinnamon bark (C. burmannii) and its
oil and to provide a new treatment alternative for infections caused by bacteria using natural ingredients.

MATERIALS AND METHODS

Plant Materials
Dried cinnamon bark (Cinnamomum burmannii) was obtained from CV. Merapi Farma Herbal, Yogyakarta,
Indonesia on May 2016. The cinnamon bark then observed for its macroscopic and microscopic characteristics.

Isolation of the Essential Oils

Essential oils were obtained by steam and water distillation method. 1 kg of dried cinnamon bark were distilled
for 4 hours. The essential oils yielded were kept and sealed in a glass vial for further analysis.

Identification of Components by GC-MS

The GC-MS analysis of cinnamon EO analysis was carried out on Shimadzu QP2010 SE system. The operating
conditions were Helium as a carrier gas, 0.75 ml/min of flow rate, 250oC injector temperature, 250oC detector
temperature, 0.1 μL sample volumel; 36.1 kPa pressure, and Willey library data.

Bacterial Strains
Escherichia coli and Staphylococcus aureus were used as the respectative of Gram-negative and Gram-positive
bacteria. For antibacterial assay, Mueller-Hinton Broth (MHB) and Mueller-Hinton Agar (MHA) were used for
culturing the bacteria. Stock cultures of E. coli and S.aureus were grown in MHB at 37oC for 24 h before the test.

Antibacterial Assays by Gaseous Contact

Antibacterial activity of cinnamol oil was assayed using gaseous contact method [8]. Airtight boxes (1.3 L) were
prepared, the inside of the boxes were covered with aluminium foil to avoid contamination by essential oil vapour,
and the upper edges of the airtight boxes was affixed by wax to prevent air leaks. Petri dishes with MHA (20 mL
each) were inoculated with 10 μL of each bacterial strains (108 CFU/mL) by spread plate method. Five
concentrations of cinnamon oil were prepared by dissolving cinnamon oil with DMSO: 25; 12.5; 6.25; 3.13; and
1.56% v/v. Part of the solution (260 μL) was soaked on filter paper (9 cm in diameter) using micropipette and placed
in the airtight box by sticking it on one side of inner walls of the box. The airtight boxes then sealed with adhesive
tape to prevent air leaking. Control box was prepared by inserting a filter paper soaked by DMSO alone. Analyses
were carried out in triplicate. The boxes were incubated at 37 oC for 24 h and the Minimal Inhibitory Dose (MID)
was recorded. MID was the minimal inhibitory dose per unit space required to suppress the growth of
microorganisms in closed system. MID values were expressed as weight per unit volume [8].

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 RESULTS AND DISCUSSION

Plant Material Characteristics

Macroscopic characteristics of dried cinnamon bark were slightly rolled shape, thick, the outer surface was
brown-reddish brown, and the inner surface was dark brown-blackish brown. There were pale wavy striped
lengthwise and white lichens on the outer surface. Dried cinnamon bark is shown in Fig. 1. Figure 2 shows the
microscopic characteristics of cinnamon bark. Microscopic characteristics observed were including schlerenchyme
fiber, volatile oil glands, periderm, and calcium oxalate crystals. These characteristics were in accordance with the
characteristics of Cinnamomum burmannii mentioned in Materia Medika Indonesia Volume I [12].

FIGURE 1. Dried cinnamon bark (Cinnamomum burmannii)

(a) (b) (c)

(d) (e) (f)

FIGURE 2. Microscopic characteristics of Cinnamomum burmannii bark a. calcium oxalate crystals; b. periderm; c. volatile oil
gland on periderm; d. volatile oil gland; e. schlerenchyme fiber on parenchyma; f. schlerenchyme fiber

Isolation of Essential Oil

Isolation of essential oil from dried cinnamon bark was done by water-steam distillation. This method was
chosen because essential oils are volatile substances which do not stand a heating to high temperatures. Water-vapor
distillation method will allow the essential oil, which has a low vapor pressure and low boiling point, to be distilled
properly and minimizes the risk of damage due to the hydrolysis reaction. Water-steam distillation is a better method

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for isolating volatile oil compared to water distillation because the decomposition of oil are smaller and can avoid
the hydrolysis reaction and polymerization of some compounds due to the influence of boiling water [13].
The volume of cinnamon oil obtained from steam and water distillation process was 2.5 mL and the yield was
0.25% v/w. The quality of essential oil is affected by the type of distillation method, duration of distillation, and
steam pressure, while material sizes and distillation methods affect to the yield of essential oil produced [13, 14].

Chemical Compounds of Cinnamoin Oil

A total of 27 compunds were identified in cinnamon oil by GC-MS analysis as shown in table 1. It presented the
compunds with their retention time and concentration (%). The dominant compounds were trans-cinnamaldehyde
(56.10%), 1,8-cineol (16.53%), and alpha-pinene (3.44%). Trans-cinnamaldehyde was the most dominant volatile
compound of the essential oil. Precious reports of compounds identified in cinnamon oil also stated that trans-
cinnamaldehyde was the most dominant volatile compound in cinnamon oil [14, 15]. Trans-cinnamaldehyde is the
main constituent of essential oil compounds derived from the genus Cinnamommum [16]. The different growth
environment, local climate, harvest time, harvest treatment and post-harvest treatment, as well as age of the plant
can affect the compounds of essential oils [17]. Although in the same species, a variation of the compounds can be
found in cinnamon plant which grows in a different places [16].
TABLE 1. Compounds identified in Cinnamomum burmannii essential oil
No. R.Time (min) Area (%) Names of the Compound
1. 4.379 0.16 alpha-thujene
2. 4.505 3.44 alpha-pinene
3. 4.723 1.31 camphene
4. 4.852 0.72 benzaldehyde
5. 5.014 0.21 sabinene
6. 5.099 1.43 beta-pinene
7. 5.610 0.27 alpha-terpinene
8. 5.724 0.41 p-cymene
9. 5.798 1.12 bornylene
10. 5.865 16.53 1,8-cineole
11. 6.218 0.41 gamma-terpinene
12. 6.777 1.87 linalool
13. 7.814 1.23 benzenepropanal
14. 8.080 2.27 terpinen-4-ol
15. 8.271 3.05 alpha-terpineol
16. 8.713 0.35 cis-cinnamaldehyde
17. 9.595 56.10 trans-cinnamaldehyde
18. 9.701 1.18 bornyl acetate
19. 11.049 1.14 alpha-copaene
20. 11.523 0.22 farnesol
21. 11.717 0.93 beta-caryophyllene
22. 11.881 2.86 cinnamyl acetate
23. 11.984 0.36 coumarin
24. 12.183 0.25 alpha-humulene
25. 12.726 0.47 alpha-muurolene
26. 13.023 0.51 delta-cadinene
27. 16.027 1.19 benzyl benzoate
Total 100

Antibacterial Assay by Gaseous Contact

Cinnamon oil has demonstrated growth inhibition of E. coli on concentration 6.25% and S. aureus on
concentration 3.13%. Table 2 and Table 3 shows the MID values of cinnamon oil obtained under closed condition.
These results indicate the lowest dose (MID value) of cinnamon oil that can inhibit the growth of E. coli and S.

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aureus were 12.5 μL/L air and 6.26 μL/L air, respectively. The lower MID value, the antibacterial activity is
stronger [8]. Cinnamon oil requires a lower MID to inhibit the growth of S. aureus than E. coli, that shows it has
higher activity for Gram-positive bacteria than Gram-negative bacteria.
TABLE 2. MID value of cinnamon oil against E. coli
MID (μL/L air)
Replication
50 25 12.5 6.26 3.12
1 - - - - +
2 - - - + +
3 - - - + +
Note: (+): growth observed; (-): no growth observed
TABLE 3. MID value of cinnamon oil against S. aureus
MID (μL/L air)
Replication
50 25 12.5 6.26 3.12
1 - - - - +
2 - - - - -
3 - - - - +
Note: (+): growth observed; (-): no growth observed

Several researches stated that antibacterial effect of cinnamon oil is more active against Gram-positive bacteria
than Gram-negative bacteria [9,18,19]. Gram-negative bacteria showed a higher resistance to essential oils as
compared to Gram-positive bacteria. The mechanism of action of antibacterial agents depends on the type of
microorganisms, cell wall structure, and the structure of the outer membrane [9]. E. coli is a Gram-negative bacteria
which have a thick layer of lipopolysaccharide outer membrane surrounding the cell wall. The hydrophillic surface
of Gram-negative outer membrane which is rich in lipopolysaccharide molecules creates a barrier for many
antibiotic molecules so they are more resistant to hydrophobic compounds in the essential oil compared to Gram-
positive bacteria [9, 20]. S. aureus is a Gram-positive bacteria that have a single layer structure of peptidoglycan
which making it easier for antibacterial compounds to destroy the cell wall and cytoplasmic membrane, causing cell
leakage and cytoplasm discharge [20]. Essential oils can affect the cell envelope structure because the major
components of essential oils that act as an antibacterial agents can penetrate the cell wall and damage the
cytoplasmic membrane [20]. Cinnamon oil may affect the plasma membrane, interfering its integrity and causing
changes in the morphology of the cell wall membrane, damaging cell permeability, causing cytoplasm leakage, and
result in bacteria death [20].

CONCLUSION
Gas compounds of Cinnamomum burmannii essential oil (cinnamon oil) showed the ability to inhibit the growth
of E. coli and S. aureus. The Minimum Inhibitory Dose (MID) of cinnamon oil to E. coli and S. aureus was 12.5
μL/L air and 6.26 μL/L air, respectively. Gas compounds of cinnamon oil was more effective to gram-positive
bacteria than gram-negative bacteria.

ACKNOWLEDGMENTS
The authors gratefully thank the Pharmaceutical Laboratory, Department of Pharmacy in Islamic University of
Indonesia for providing the facilities to carry out the work.

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