SUPPLEMENTARY MATERIAL
Bioanalytical evaluation of Cinnamomum zeylanicum essential oil
Muhammad Saleem, Haq Nawaz Bhatti*, Muhammad Idrees Jilani and Muhammad Asif Hanif
Department of Chemistry, University of Agriculture Faisalabad Pakistan.
*hnbhatti2005@yahoo.com; haq_nawaz@uaf.edu.pk;0092419200161-3309
Abstract
This manuscript describes the antioxidant activity of essential oil of Cinnamon (Cinnamomum
zeylanicum) bark extracted by supercritical fluid extraction, hydro distillation and steam
distillation. The cinnamon bark essential oil exhibited a wide range of total phenolic contents,
total flavonoid contents, reducing power, inhibition of linoleic acid peroxidation and DPPH
radical-scavenging activity (IC50). Bioactivity of cinnamon essential oil was assayed against
various bacterial strains including Bacillus subtilis, Escherichia coli, Pastrurella multocida and
Straphylococcus aureus) and fungal strains including Aspergillus niger and Aspergillus flavus.
More essential oil yield was obtained using supercritical fluid extraction in comparison to other
methods. The oil extracted by supercritical fluid extraction (SCFE) was dominated by
cinnamaldehyde, limonene, copaene, naphthalene, heptane, bicyclo[4.2.0]octa-1,3,5-triene and 2propenal. Due to the presence of cinnamaldehyde in the essential oil of cinnamon bark it acts as a
good antioxidant and antimicrobial agent.
Keywords: Cinnamon, antibacterial, antifungal, cinnamaldehyde, antioxidants
1. Experimental
1.1. Chemicals and reagents
All chemicals were purchased from Sigma-Aldrich Chemicals USA.
1.2. Collection and extraction of cinnamon sample
Cinnamon (C. zeylanicum) bark was collected from the local market Faisalabad. Samples were
packed in polythene bags and were brought in Analytical laboratory, University of Agriculture,
Faisalabad. The voucher specimen number assigned to Cinnamon was CH0001M. The essential
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oil from cinnamon bark was extracted by hydro-distillation, steam distillation and supercritical
fluid extraction methods. The extracted essential oil was stored in sealed vials at 4°C, until to
further analyses (Wang et al. 2009). Five kilo grams of the C. zeylanicum bark was hydro
distilled for 3 h in Clevenger type apparatus. Five kilo grams of the C. zeylanicum bark was
steam distilled for 3 h in Clevenger type apparatus. Heat was supplied to the heating mantle (120
oC) and the essential oil was extracted for 3 h (until no more essential oil was recovered). The
essential oil was collected and analyzed immediately. Ten kilograms of C. zeylanicum bark was
used to extract the essential oils using supercritical CO2 extraction at 45˚C and 90 bar pressure
for a batch time of 4h.
1.3. Gas chromatographic/mass spectrometry analysis
The components of essential oil extracted from cinnamon bark were analyzed using GC-MS
analysis (Hussain et al. 2013, Lohani et al. 2012). The analysis of the oils was performed using a
gas chromatograph (HP 6890N Network CG System) with a capillary column HP-5 (30 m × 0.32
mm×0.25 µm film thickness) and a flow of 0.5 mL min−1 in 1:20 split ratio mode (split injector;
290 °C). The oven temperature was programmed to increase from 70 °C (held for 5 min) to 250
°C at 3 °C /min, with helium as the carrier gas at an inlet pressure of 3.14 psi. The injections
consisted of 1 L from a 2 mg mL−1 CH2Cl2 solution. The percentage compositions were
determined from electronic integration measurements using flame ionization detection (FID, 250
°C). All samples were analyzed by a gas chromatography coupled to a mass electron impact
detector (GC/MS; HP 6890N) equipped with MSD Productivity Chem. Station Software and a
capillary column HP-5 MS (30 m×0.32 mm×0.25 µm film thickness) under the same
chromatographic conditions as described above: the ion source at 250 °C, electron impact
ionization at 70 eV and acquisition mass range from 10–400 M/Z (3.66 scan/s). The
identification of oil components was accomplished by comparing their GC and GC/MS retention
indices (which were determined relative to the retention times of the series of n alkanes) and
comparing their mass spectral fragmentation patterns with those from the Wiley Library
Software 59943B and literature data (Adams, 2007). The relative proportions of the essential oil
constituents were obtained as percentages by FID peak-area normalization.
1.4. Antioxidant Activity
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Determination of total phenolic contents, total flavonoid contents, reducing power ability and
DPPH scavenging activity was calculated by following method described in literature (AlMaskri et al. 2011, Hanif et al. 2011). The antioxidant activity of sample solution in linoleic acid
system was determined in terms of measurement of % inhibition of peroxidation in linoleic acid
system following a reported method (Iqbal and Bhanger 2006).
1.5. Antimicrobial Activity
The extracts were individually tested against a panel of microorganisms, including four species
of bacteria

Escherichia coli B 10 (E. coli)
[gram negative]

Bacillus subtilis SPS 2 (B. subtilis)
[gram positive]

Pasturella multocida
[gram negative]

Straphylococcus aureus
[gram positive]

Two species of fungi were used

Aspergillus niger (A. niger)

Aspergillus flavus (A. Flavus)
All the tests relating antimicrobial activity were performed in the Biochemistry Lab, University
of Agriculture, Faisalabad. Bacterial strains were cultured overnight at 37 ºC in Nutrient agar
(NA) and fungi isolates were cultured overnight at 30 ºC in potato dextrose agar (PDA). The
essential oil of C. zeylanicum bark extracted by different extraction techniques (supercritical
fluid extraction, hydro distillation and steam distillation) were dissolved to a final concentration
of 30 mg/ml and sterilized by filtration through 0.45 µm Millipore filters. Antimicrobial tests
were then carried out by the disk diffusion method (Liao et al. 2010) using 100 µL of suspension
containing 108 CFU/ml of bacteria and 104 spore/ml of fungi spread on nutrient agar (NA) and
potato dextrose agar (PDA) medium, respectively. The disks (6 mm in diameter) were
impregnated with the 35 mg/ml extracts (350 µg/disk) were placed on the inoculated agar.
Negative controls were prepared using the same solvents employed to dissolve the plant extracts.
Levofloxin (30 mg/disk) for bacterial strains and fluconazole (30 mg/disk) for fungi isolates. The
inoculated plates were incubated at 37 °C for 24 h for clinical bacterial strains and 72 h for fungi
isolates. Plant-associated microorganisms were incubated at 27°C. Antimicrobial activity was
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evaluated by measuring the zone of inhibition against the test organisms in comparison to a
control of negative and reference standards.
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