SUPPLEMENTARY MATERIAL
Chemical composition and antibacterial activity of the essential oil
from Agathis dammara (Lamb.) Rich fresh leaves
Zhifen Chen, Daohang He, Jingdan Deng, Jiaying Zhu and Qiuping Mao
School of Chemistry and Chemical Engineering, South China University of
Technology, Guangzhou 510640, China
The essential oil of fresh leaves from Agathis dammara (Lamb.) Rich was
extracted using hydro-distillation, and GC-FID and GC-MS were used to
analyse the essential oil. 19 compounds were identified, among which the
major components were Limonene (36.81%), β-Bisabolene (33.43%) and
β-Myrcene (25.48%). In the antibacterial test, disc diffusion method and
micro-well dilution assay proved that the essential oil had significant
antibacterial activities. The inhibition zones against Staphylococcus aureus and
Pseudomonas aeruginosa were 23.7 mm and 23 mm, respectively, which
demonstrated that the inhibition effects were greater than positive control (10
μg/disc Streptomycin). And the lowest MIC value of the essential oil was
found against S. aureus (1.25 mg/mL) and Bacillus subtilis (1.25 mg/mL). This
is the first time to report the antibacterial activities of A. dammara essential oil.
Keywords: Agathis dammara; essential oil; GC/MS; antibacterial activity
Corresponding
author. Email: cehdh@scut.edu.cn
1. Experimental
1.1. Plant material and essential oil isolation
The fresh leaves of A. dammara were collected from Longdong, Tianhe district,
Guangzhou, P.R. China in May 2014. The sample was identified as A. dammara by
Dr. Zezheng Gao and the voucher specimen (No. 247613) was deposited in the
herbarium of South China Botanical Garden, Chinese Academy of Sciences,
Guangdong province, P.R. China.
1.2. Essential oil isolation
The fresh leaves (250 g) of A. dammara were hydrodistillated for 4 h using a
Clevenger apparatus (Zhenzhou Zhongtian experimental instrument CO., LTD,
China). The sample yielded about 0.1% (on a dry mass) of yellowish oil (0.26 g) with
a pleasant smell. The obtained essential oil was dried over anhydrous sodium sulfate
and stored at 4℃ before analysis and bioassay.
1.3. Essential oil analysis
Gas chromatography–flame ionization detector (GC–FID) analyses of the essential oil
were conducted using a Thermo Scientific Focus GC instrument equipped with a
DB-5 column (30m × 0.25 mm i.d. 0.25 μm). Nitrogen was used as the carrier gas at
the constant flow of 1.0 ml/min as well as the split ratio was 1/50. The oven
temperature was raised from 50°C (Hold on 4 min) to 250°C (Hold on 10 min) at a
rate of 5°C/min. The injector and detector (FID) temperatures were kept at 270°C and
270°C, respectively. Semi-quantitative data were obtained from FID area percentages
without the use of correction factors. The retention index of each compound was
calculated under the same temperature-programmed conditions and the same column
for n-alkenes (C7–C30) (Figure S3).
Gas chromatography–mass spectrometry (GC–MS) analysis was carried out
on a SHIMADZU GC–MS instrument (GCMS-QP2010) equipped with a DB-5MS
column (30 m × 0.25 mm i.d.; film thickness 0.25 μm) and temperature programming
and injector temperature as mentioned for GC. Transfer line temperature was 250°C.
Helium was used as the carrier gas at a flow rate of 1.69 ml/min with a split ratio
equal to 1/50. Ion source temperature is 200°C with EI mode and the mass ranges m/z
30-600. Identification of individual compound was made by comparison of their mass
spectra with those of the internal reference mass spectra library (NIST08), or with
authentic compounds and confirmed by comparison of their retention index with
authentic compounds or those reported in the literature (Adams 2007).
1.4. Antibacterial activity
1.4.1. Bacterial strains
The antibacterial activities of the A. dammara essential oil were tested against five
bacterial strains. Two Gram-positive strains (Staphylococcus aureus CMCC 26003
and Bacillus subtilis CMCC 63501) and two Gram-negative strains (Pseudomonas
aeruginosa ACTT 27853 and Escherichia coli CMCC 44825) and Escherichia coli
DH5α.
1.4.2. Disc diffusion method
Inhibition zones were determined according to the disc diffusion method against five
selected bacterial strains (Diao et al. 2013). The bacterial suspension after one night
incubation was adjusted to 108 CFU/ml by MH broth and 100 μL of this suspension
was spread on the MHA solid media plates. Filter paper discs (6 mm in diameter)
were individually impregnated with 10 μL leaves essential oil and then placed on the
incubated plates. The plates were incubated at 37℃for 24 h. The diameter of
inhibition zones were measured with a calliper in mm. Streptomycin (10 μg/disc) was
used as the positive control. All the tests were repeated in triplicate.
1.4.3. Micro-well dilution assay
The minimum inhibition concentration (MIC) of A. dammara essential oil was
determined by the micro-well dilution assay, recommended by the National
Committee for Clinical Laboratory Standards (NCCLS) as described previously
(Haddouchi et al. 2013). Briefly, twofold dilution was carried out in a 96-well plate to
obtain the dilutions of the essential oil with the concentrations ranging from 0.02 to
10 mg/mL, and dimethyl sulfoxide (DMSO) at a final concentration of 2% in each
well was used to dissolve the essential oil. The inocula of the tested strains from
overnight broth culture were added to give a final concentration of 5×105 CFU/mL in
each well. All test samples were prepared in Mueller Hinton Broth. And a negative
control containing DMSO (2%) without essential oil was analysed as well. The MIC
was defined as the lowest concentration of the essential oil at which the bacteria did
not exhibit visible growth after 24 h of incubation at 37℃. The growth of the
micro-organism was indicated by turbidity. All the tests were repeated in triplicate.
1.5. Statistical analysis
Both the antibacterial assays were carried out in triplicate. The data of disc diffusion
method are expressed as means ± SD. One-way analysis of variance and Duncan’s
multiple range tests were carried out to determine significant differences (p＜ 0.05)
between the means.
References
Adams RP. 2007. Identification of essential oil components by gas
chromatography/mass spectroscopy. Carol Stream, IL: Allured Publishing
Corporation.
Diao WR, Hu QP, Feng SS, Li WQ, Xu JG. 2013. Chemical composition and
antibacterial activity of the essential oil from green huajiao (Zanthoxylum
schinifolium) against selected foodborne pathogens. J Agric Food Chem.
61:6044-6049.
Haddouchi F, Chaouche TM, Zaouali Y, Ksouri R, Attou A, Benmansour A. 2013.
Chemical composition and antimicrobial activity of the essential oils from four
Ruta species growing in Algeria. Food Chem. 141:253-258.
Figure S1. The chromatogram of gas chromatography–mass spectroscopy analysis
Figure S2. The chromatogram of gas chromatography analysis
Figure S3. The chromatogram of gas chromatography analysis of C7-C30