SUPPLEMENTARY MATERIAL
Chemical constituents and antimicrobial properties of the essential oil and ethanol
extract from the stem of Aglaia odorata Lour.
Nantiya Joycharatad*, Sonesay Thammavongb, Supayang Piyawan Voravuthikunchaicd, Patimaporn
Plodpaie, Watcharapong Mitsuwanc, Surasak Limsuwanad and Sanan Subhadhirasakulaf
a
Faculty of Traditional Thai Medicine, Prince of Songkla University, Songkhla 90112, Thailand; bFaculty
of Pharmacy, University of Health Sciences, Lao PDR 7444; cDepartment of Microbiology, Faculty of
Science, Prince of Songkla University, Songkhla 90112, Thailand; dNatural Products Research Center of
Excellence, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand; eDepartment of
Pest Management, Faculty of Natural Resources, Prince of Songkla University, Songkhla 90112,
Thailand; fFaculty of Pharmaceutical Sciences, Prince of Songkla University, Songkhla 90112, Thailand
*
Corresponding author. Email: nantiya.j@psu.ac.th
Abstract
The stem-derived essential oil of Aglaia odorata Lour was obtained by hydrodistillation
using a Clevenger type system. Gas chromatographymass spectrometry analysis of the oil
revealed the identification of 39 compounds, representing 76.4% of the oil; germacrene D
(20.3%), -humulene (17.1%), -himachalene (12.7%), and -caryophyllene (10.2%) were
the major components. Ar-turmerone (1) and eichlerialactone (2) were isolated from the stem
oil and ethanolic stem extract of this plant species, respectively. Antimicrobial activities of
the oil and ethanol extract were tested against both Gram-positive and Gram-negative
bacterial strains including Bacillus cereus ATCC 11778, Staphylococcus aureus ATCC
25923, Acinetobacter baumannii ATCC 19606, and Escherichia coli ATCC 25922, as well
as three rice fungal pathogens Bipolaris oryzae, Pyricularia oryzae, and Rhizoctonia solani
using broth microdilution method. The oil and 1 exhibited significant antifungal activity
against the three rice pathogens tested whereas 2 exhibited good antibacterial activity against
both of Gram-positive pathogens tested.
Keywords: Aglaia odorata; antimicrobial activity; ar-turmerone; essential oil; rice
pathogens
Experimental
General experimental procedures
GCMS analysis of the oil was carried out using Agilent 7890A GC (Agilent 21 Technologies, USA)
coupled with Pegasus 4D time-of-flight mass spectrometer (LECO Corp, USA). 1D and 2DNMR
spectral data were recorded on FTNMR Bruker Advance 500 MHz. EIMS was performed on MAT 95 XL
mass spectrometer (Thermofinigan, Germany). The following pathogenic microorganisms were used: two
Gram-positive bacteria (Bacillus cereus ATCC 11778 and Staphylococcus aureus ATCC 29213), two
Gram-negative bacteria (Acinetobacter baumannii ATCC 19606 and Escherichia coli ATCC 25922) and
three fungi isolated from rice (Bipolaris oryzae, Pyricularia oryzae, and Rhizoctonia solani). Pathogens
used in this work were obtained both from Department of Microbiology and Natural Products Research
Center of Excellence, Faculty of Science and Department of Pest Management, Faculty of Natural
Resources, Prince of Songkla University, Thailand.
Plant material
The stem of Aglaia odorata Lour was collected from the southern region of Thailand in May 2011.
Botanical identification was performed by Dr. Oratai Neamsuvan, an ethnobotanist at the Faculty of
Traditional Thai Medicine, Prince of Songkla University, where the voucher specimen (NJ0511) is
deposited.
Extraction of the oil and isolation
The stem of A. odorata was dried in an oven at 60 C. The dried stem (800 g) was ground and
hydrodistilled for 8 h using a Clevenger-type apparatus. The essential oil was collected, dried over
anhydrous sodium sulphate, and stored at 4 °C until used. The percentage yield of the oil recovered was
calculated based on the dried weight of plant material. For the compound isolation, the oil (1.5 g) was
applied to column chromatography over silica gel using gradient elution with ethyl acetate
(EtOAc)petroleum ether, and finally washed down with methanol (MeOH). The fractions were then
combined according to their TLC patterns, to give fourteen fractions (IO–XIVO). Fraction VIO (142 mg)
was further purified using a Preparative thin layer chromatography with 3% EtOAc in toluene as the
developing solvent to afford 4.9 mg of ar-turmerone (1) (Figure S1).
Gas chromatographymass spectrometry (GCMS)
The essential oil from A. odorata was analysed using GCMS on Agilent 7890A GC equipped with
Pegasus 4D time-of-flight mass spectrometer. The separation condition was carried out on Rxi-5silMS
capillary column (30 m x 0.25 mm i.d., film thickness 0.25 m). Column temperature was initially kept at
50 C for 2 min, and gradually increased at the rate of 5 C per min to 245 C, at which the temperature
was held for 8 min; split ratio was 1:400; injector temperature was 257 C. Helium was used as carrier
gas at a flow rate of 1 ml/min. The sample of 1 l was injected in the acquisition mode. For MS detection,
an electron ionization system with ionization energy of 70 eV with MS transfer line at temperature of 250
C was used. The scan range was 35334 amu and the scan rate was 0.5 s per scan. The retention indices
(RI) of all the components were determined by co-injection of the sample with a solution containing
C7C36 n-alkanes standards. Identification of the individual components was based on the comparison of
their GC retention indices and mass spectra with those of published data (Adams, 2001) and National
Institute of Standards and Technology mass spectra library (NIST2.0) data of GCMS system.
Quantification was based on the percentage contribution of each oil components to the total amount
present.
Preparation of ethanol extract and isolation
The plant materials were rinsed thoroughly and cleaned from extraneous matter with tap water, ovendried at 60 C, grounded with an electric-grinder, weighed, and exhaustively macerated with 95% EtOH
at room temperature 3 times. The filtrate was pooled and concentrated under reduced pressure at 45 C in
vacuum rotary evaporator to give the EtOH extract. For the phytochemical isolation, the EtOH extract
was partitioned with various solvents ordered by increasing polarity. Each filtrate was pooled and
evaporated to dryness under reduced pressure at 45 ºC to yield the hexane fraction, CH2Cl2 fraction, and
BuOH fraction. The CH2Cl2 fraction (2.15 g) was applied to CC over silica gel using gradient elution with
acetone–hexane, and finally washed down with MeOH. The fractions were then combined according to
their TLC patterns, to give seven fractions (ID–VIID). Fraction VD (259 mg) was further purified on a
silica gel column with 20% acetone in hexane as the eluent and followed by a Sephadex LH20 column
eluting with CH2Cl2–MeOH (1:1) to afford 4.1 mg of eichlerialactone (2) (Figure S1).
ar-Turmerone (1). yellow oil; EIMS (70 eV, 200 C): m/z = 91, 119, 132, 149, 178, 216; 13C NMR
(125 MHz, CDCl3):  135.5 (s, C-1), 129.1 (d, C-2), 126.6 (d, C-3), 143.7 (s, C-4), 126.6 (d, C-5), 129.1
(d, C-6), 35.2 (d, C-7), 52.7 (t, C-8), 199.9 (s, C-9), 124.1 (d, C-10), 155.1 (s, C-11), 27.6 (q, C-12), 20.7
(q, C-13), 21.9 (q, C-14), 20.9 (q, C-15).
Eichlerialactone (2). Colorless solid; 13C NMR (125 MHz, CDCl3):  24.5 (t, C-1), 28.2 (t, C-2),
178.1 (s, C-3), 147.3 (s, C-4), 41.0 (d, C-5), 31.2 (t, C-6), 33.7 (t, C-7), 40.0 (s, C-8), 49.3 (d, C-9), 39.0
(s, C-10), 21.9 (t, C-11), 25.0 (t, C-12), 43.2 (d, C-13), 50.5 (s, C-14), 31.4 (t, C-15), 26.7 (t, C-16), 50.7
(d, C-17), 15.3 (q, C-18), 20.1 (q, C-19), 89.8 (s, C-20), 22.4 (q, C-21), 29.2 (t, C-22), 34.1 (t, C-23),
176.8 (s, C-24), 113.5 (t, C-28), 23.1 (q, C-29), 16.1 (q, C-30).
Antimicrobial activity
Microbial strains including Bipolaris oryzae, Pyricularia oryzae, Rhizoctonia solani, Bacillus cereus
ATCC 11778, Staphylococcus aureus ATCC 25923, Acinetobacter baumannii ATCC 19606, and
Escherichia coli ATCC 25922 were used in this investigation. Mueller Hinton agar (Difco Laboratories,
USA), Mueller Hinton broth (MHB) (Difco Laboratories, USA), Potato dextrose agar (Difco
Laboratories, USA) and Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, USA)
were used as culture media. Standard drugs including penicillin G and propiconazole were used as
positive controls for Gram-positive bacterial and fungal cultures, respectively. DMSO (1% v/v) was used
as negative control. Antimicrobial activity of essential oil, ethanol extract, and isolated compounds from
A. odorata stem was examined by broth microdilution assay using MHB for bacterial strains (CLSI,
2009) and RPMI 1640 medium for fungal strains (CLSI, 2008). Two fold serial dilutions of the tested
agents were prepared in 96-well microtiter plates in the respective medium to final concentration ranged
from 0.0019 to 2 mg mL-1. The microbial suspensions in the respective test medium were standardized to
McFarland No. 0.5. Twenty microliters of microbial suspension was added to each well of the plate,
resulting in a final inoculums concentration of 5 x 10 5 CFU mL-1. The microbial growth was determined
at 24 h of incubation by spectrophotometric mean at 600 nm. The lowest concentration of the tested
agents required to completely inhibit microbial growth after incubation at 37 C for 24 h for bacteria or at
25 C for 2472 h for fungi was recorded as minimum inhibitory concentration (MIC). After MICs were
recorded, an aliquot (100 µl) from the broth with no growth was pipetted and dropped onto agar plate and
incubated with at 37 °C for 48 h for bacteria or at 25 C for 72 h for fungi. The lowest concentration of
the tested agents required to completely preventing microbial growth was reported as minimum
bactericidal concentration (MBC) for bacteria or minimum fungicidal concentration (MFC) for fungi.
Each assay was performed in triplicate.
Table S1. Composition of the stem essential oil of Aglaia odorata Lour.
Compounds
Benzaldehyde
6-Methyl-5-hepten-2-one
β-Pinene
Camphor
Borneol
Bezoic acid, 2-(acetyloxy)-, methyl ester
(E,E)-2,4-Decadienal
α-Ylangene
α-Copaene
β-Cubebene
β-Elemene
Longifolene
-Caryophyllene
-Elemene
trans-α-Bergamotene
(E)-β-Farnesene
-Himachalene
-Humulene
Acoradiene
γ-Gurjunene
ar-Curcumene
Germacrene D
-Zingiberene
β-Bisabolene
-Cadinene
δ-Amorphene
trans-Calamenene
Spathulenol
Viridiflorol
Humulene epoxide II
Khusimone
γ-Eudesmol
α-Cadinol
ar-Turmerone
RIa
954
972
979
1142
1160
1187
1310
1367
1379
1382
1400
1405
1417
1427
1442
1447
1456
1462
1467
1472
1481
1486
1493
1502
1515
1518
1530
1578
1593
1596
1599
1632
1639
1660
Peak area (%)
tb
t
t
t
t
t
t
1.8
t
0.7
0.1
0.5
10.2
0.8
1.3
0.1
12.7
17.1
4.5
0.1
0.3
20.3
t
2.1
0.2
0.5
t
0.1
0.1
0.1
0.1
0.1
0.1
2.3
Identification
MS, RI
MS
MS, RI
MS, RI
MS, RI
MS
MS
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI,
1D& 2D NMR
MS, RI
MS, RI
MS, RI
MS
MS
α-Bisabolol
1671
0.1
Vitiselinenol
1729
0.1
Cyclohexadecanolide
1934
0.1
1,3-Bis-(2-cyclopropyl,2-methylcyclopropyl)-but-2-en-1-one
1981
t
9,12,15-Octadecatrienoic acid methyl ester
2269
0.1
Monoterpene hydrocarbons
t
Sesquiterpene hydrocarbons
73.1
Oxygenated sesquiterpenes
3.1
Others
0.2
Total amount identified (%)
76.4
Essential oil yield (% )
0.07
a
Retention indices relative to C7C36 n-alkanes on a Rxi-5silMS capillary column. bTrace ( 0.1%).
Table S2. Antimicrobial activity of essential oil (EO), ethanol extract (EE), ar-turmerone (1), and
eichlerialactone (2) from Aglaia odorata stem.
Pathogenic microorganisms
Gram-positive bacteria
Bacillus cereus ATCC 11778
Staphylococcus aureus ATCC 25923
Gram-negative bacteria
Acenetobacter baumannii ATCC 19606
Escherichia coli ATCC 25922
Rice fungi
Bipolaris oryzae
Pyricularia oryzae
Rhizoctonia solani
MICa (MBCb or MFCc )
(mg mL-1)
1
2
EO
EE
Controls
2.0
(2.0)
2.0
(2.0)
2.0
(2.0)
2.0
(2.0)
0.25
(0.25)
0.25
(0.25)
0.25
(0.25)
0.5
(1.0)
0.0002
(0.0002)
0.0002
(0.0002)
2.0
(2.0)
2.0
(2.0)
2.0
(2.0)
2.0
(2.0)
Nd
Nd

Nd
Nd

0.50
(1.0)
0.0625
(0.25)
0.25
(0.25)
2.0
(2.0)
1.0
(2.0)
2.0
(>2.0)
0.25
(>0.25)
0.0625
(0.25)
0.125
(0.25)
1.0
(2.0)
0.25
(0.25)
1.0
(>2.0)
0.0002
(0.0002)
0.0002
(0.0002)
0.0019
(0.0019)
Note: Controls used are already mentioned in experimental section. aMinimum inhibitory concentration;
b
Minimum bactericidal concentration; cMinimum fungicidal concentration. Nd: Not determined; : No
inhibitory effect (1% DMSO).
O
O
H
HOOC
O
1
2
Figure S1. Antimicrobial compounds isolated from the stem of Aglaia odorata Lour.
References
Adams, R.P. (2001). Identification of essential oil components by Gas Chromatography/Mass
Spectroscopy. Allured Publishing Corporation, Carol Stream, IL.
Clinical and Laboratory Standards Institute (CLSI). (2008). Reference Method for Broth Dilution
Antifungal Susceptibility Testing of Filamentous Fungi, Approved Standard, 2 nd ed. CLSI
Document M38-A2, Clinical and Laboratory Standards Institute, Wayne, Pennsylvania.
Clinical and Laboratory Standards Institute (CLSI). (2009). Methods for Dilution Antimicrobial
Susceptibility Tests for Bacteria That Grow Aerobically, Approved Standard, 8 th ed. CLSI
Document M07-A8, Clinical and Laboratory Standards Institute, Wayne, Pennsylvania.