SUPPLEMENTARY MATERIAL
Analysis of essential oils from Voacanga africana seeds at different
hydrodistillation extraction stages: Chemical composition, Antioxidant activity
and Antimicrobial activity
Xiong Liu, Dongliang Yang, Jiajia Liu, Na Ren
Department of Pharmaceutical Engineering, College of Chemistry and Chemical
Engineering, Central South University, Changsha 410083, Hunan, P.R. China.
In this study, essential oils from Voacanga africana seeds at different extraction
stages were investigated. In the chemical composition analysis, 27 compounds
representing 86.69-95.03% of the total essential oils were identified and quantified.
The main constituents in essential oils were terpenoids, alcohols and fatty acids
accounting for 15.03-24.36%, 21.57-34.43% and 33.06-57.37%, respectively.
Moreover, the analysis also revealed that essential oils from different extraction stages
possessed different chemical compositions. In the antioxidant evaluation, all analyzed
oils showed similar antioxidant behaviors, and the concentrations of essential oils
providing 50% inhibition of DPPH scavenging activity (IC50) were about 25 mg/mL.
In the antimicrobial experiments, essential oils from different extraction stages
exhibited different antimicrobial activities. The antimicrobial activity of oils was
affected by extraction stages. By controlling extraction stages, it is promising to
obtain essential oils with desired antimicrobial activities.
Keywords: Voacanga africana seeds, essential oils, chemical composition, antioxidant
activity, antimicrobial activity
1. Experimental
1.1.Chemical Reagents and Samples
The reagents and chemicals (analytic grade unless stated otherwise) were
purchased from Sinopharm Chemical Reagent Co., Ltd. and Tianjin Damao Chemical
Reagent Co., Ltd (China). The Voacanga seeds were collected from Ghana, in 2012,

Corresponding author: Tel:+86-0731-88836834, E-mail address: liujj0903@163.net
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and the sample was authenticated by professor Liu at the Central South University. A
voucher specimen (CS-PHA120923) has been deposited in College of Chemistry and
Chemical Engineering of Central South University. The Voacanga seeds were
grounded by a universal grinder. The grounded material was stored at –10 oC in a
domestic refrigerator before the hydrodistillation extraction procedures were
performed.
1.2.Extraction of the essential oils at different extraction stages
In our experiments, the hydrodistillation extraction process was divided into six
stages. The essential oils extracted between 0-60 min, 60-120 min, 120-180 min,
180-240 min, 240-300 min and 300-360 min were named as I, II, III, IV, V and VI,
respectively. As a control, the total essential oil extracted between 0-360 min was also
investigated and was named as VII. All extraction experiments were performed in
triplicate in a Clevenger apparatus with 300 g of powered seeds. The obtained oils
were stored in a freezer for GC-MS analysis and biological test.
1.3.Gas chromatography-mass spectrometry analysis
The chemical composition analysis of the oils was carried out on a gas
chromatograph Shimadzu GC-17A coupled with a mass spectrometer QP 5000 with a
DB-5MS (30 m × 0.25 mm; 0.25 mm film thickness) fused-silica capillary column.
And the analysis was operated under the following conditions: injector temperature,
250 oC; programmed temperature, 60–160 oC (8 oC /min, held for 1 min), 160-200 oC
(3 oC /min, held for 1 min), 200-240 oC (8 oC /min, held for 1 min); carrier gas,
helium, a flow rate of 1.0 mL/min; split ratio, 10:1; EIMS, electron energy, 70 eV.
Based on comparison of the corresponding mass spectra with data from the NIST
mass spectra database and retention indices (RI) relative to the series of C6–C23
alkanes, the constituents in essential oils were identified. Relative contents of the
constituents in the essential oils were assumed to be proportional to the areas under
the corresponding chromatogram peaks (Liu et al. 2009).
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1.4.Antioxidant capacity
The antioxidant activities of the oils from different stages were determined by
the DPPH radical-scavenging assay. One milliliter from a 0.1 mM methanol solution
of the DPPH radical was mixed to 3 mL of essential oils at various concentrations (1.0,
5.0, 10.0, 15.0, 20.0, 25.0 and 30.0 mg/mL). After 30 min in the dark, the absorbance
values were measured at 517 nm using the UV-9600 spectrophotometer (Beijing
Rayleigh Analytical Instrument Co., Ltd, China) and converted into the ability to
scavenge the DPPH• radical (Mohamed et al. 2014). In this paper, ascorbic acid was
used as positive controls (Liu et al. 2013). The ability to scavenge the DPPH• radical
for all essential oils was obtained considering the mean value of triplicate assays.
1.5.Microbial strains and growth conditions
In the experiments, six pathogenic bacteria: Bacillus subtilis, Staphylococcus
aureus, S. epidermidis (gram-positive), Proteus vulgaris, Pseudomonas aeruginosa,
Escherichia coli (gram-negative) and pathogenic fungi: Candida albicans were used
as test organisms. All microbial strains were provided by XiangYa School of
Medicine, Central South University. The microbial strains were maintained at -80 oC
in 25% (v/v) glycerol with the appropriate medium. Bacteria were cultured in
Mueller-Hinton agar culture medium and incubated in biochemical incubator at 37 oC.
Fungi were cultured in potato dextrose agar culture medium and incubated in diurnal
growth incubator at 28 oC. The microbial strains were subcultured three times. One
loopful of the third subcultured cells was transferred from solid medium to liquid
medium, then shaken for 24 h. The cultures were diluted to 106-107 CFU/mL by
phosphate-buffered saline (PBS).
1.6.Minimum inhibitory concentration (MIC)
In this study, the broth dilution method was applied to determine the minimum
inhibitory concentration (MIC) (Carson et al. 1995, Pessoa et al. 2002, Senatore et al.
2013). Briefly, the Tween 80 was used to enhance the solubility of essential oils in the
Mueller-Hinton broth (5 %). The concentrations of essential oils were diluted with
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Mueller-Hinton broth to 1024, 512, 256, 128, 64, 32 and 16 μg/mL. The
Mueller-Hinton broths introduced with Tween 80 (5 %) were used as a negative
control. The lowest concentration of essential oils that prevented any visible bacterial
growth after 24 h of incubation at 37 oC was the MIC value. In this paper,
levofloxacin and fluconazole were used as positive controls. All assays were
performed in triplicate.
1.7.Statistical analysis
All tests were performed in triplicate. The results were expressed as mean value
± SD. Statistical comparison was performed via a one-way analysis of variance
(one-way ANOVA) followed by Tukey test by using GraphPad Prism 5 software.
Difference at p < 0.05 was considered to be statistically significant.
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References
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susceptibility of Escherichia coli and Staphylococcus aureus to the essential oil of Melaleuca
alternifolia (tea tree oil). Microbios.82:181-185.
Liu JJ, Yang DL, Zhang Y, Yuan Y, Cao FX, Zhao JM, Peng XB. 2009. Chemical component and
antimicrobial activity of volatile oil of Calycopteris floribunda J Cent South Univ
Technol.16:0931-0935.
Liu X, Yang D-L, Liu J-J, Xu K, Wu G-H. 2013. Modeling of supercritical fluid extraction of
flavonoids from Calycopteris floribunda leaves. Chem Pap.68:316-323.
Mohamed AA, Ali SI, El-Baz FK, Hegazy AK, Kord MA. 2014. Chemical composition of essential oil
and in vitro antioxidant and antimicrobial activities of crude extracts of Commiphora myrrha
resin. Ind Crop Prod.57:10-16.
Pessoa LM, Morais SM, Bevilaqua CML. 2002. Anthelmintic activity of essential oil of Ocimum
gratissimum Linn. and eugenol against Haemonchus contortus. Vet Parasitol.109:59-63.
Senatore F, Oliviero F, Scandolera E, Taglialatela-Scafati O, Roscigno G, Zaccardelli M, De Falco E.
2013. Chemical composition, antimicrobial and antioxidant activities of anethole-rich oil from
leaves of selected varieties of fennel [Foeniculum vulgare Mill. ssp. vulgare var. azoricum
(Mill.) Thell]. Fitoterapia. 90:214-219.
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Table S1. Chemical compositon of the essential oils at different extraction stages. nd
= not detected; tr = traces.
Constituents
α-Pinene
3-Ethylpyridine
Ethylhexanol
1,3-Diethylbenzene
Naphthalene
α-Terpineol
α-Longipinene
(-)-β-Elemene
α-Cedrene
β-Cedrene
Thujopsene
β-Farnesene
Germacrene D
Acoradiene
Allo-Aromadendrene
2-Tridecanone
Eudesma-4(14),11-diene
epi-α-Selinene
β-Himachalene
(+)-.delta.-Cadinene
β-Sesquiphellandrene
8β-H-Cedran-8-ol
Cedrol
n-Hexadecanoic acid
9,12-Octadecadienoic acid (Z,Z)9-Octadecenoic acid, (E)Octadecanoic acid
Terpenoids
Alcohols
Fatty acids
Other constituents
Total identifed
a
RTa)
(min)
RIb)
5.07
5.50
6.69
7.1
9.77
9.87
13.17
13.22
13.75
13.92
14.08
14.21
14.58
14.62
14.72
15.05
15.11
15.23
15.39
15.61
15.70
17.86
18.13
27.26
31.60
31.76
32.24
937
966
1032
1059
1172
1193
1352
1370
1414
1421
1427
1458
1464
1465
1467
1483
1484
1505
1519
1523
1527
1600
1612
1974
2119
2143
2173
I
nd
nd
7.5
0.67
2.92
1.67
tr
1.05
7.9
2.23
0.74
0.29
nd
0.32
tr
0.22
0.87
0.70
0.35
0.52
0.50
24.93
0.33
27.25
2.25
8.55
1.42
18.39
34.43
39.47
0.89
93.18
II
0.21
1.46
4.3
0.59
2.22
2.11
0.22
1.23
9.02
2.61
0.83
0.32
nd
0.64
0.69
0.26
0.90
0.65
0.27
0.58
0.55
24.33
tr
26.72
3.12
8.94
1.10
20.94
30.74
39.88
2.31
93.87
Content (%)
III
IV
V
nd
nd
nd
0.93
tr
nd
1.87
0.94
tr
0.35
tr
tr
0.56
tr
tr
1.15
0.42
0.51
0.22
tr
0.32
1.29
0.9
1.48
9.66
7.01
11.98
2.76
2.02
3.41
0.87
0.64
1.04
0.35
0.27
0.45
tr
0.28
tr
0.68
0.24
0.84
0.75
0.75
1.23
0.29
0.27
tr
0.93
0.77
0.89
0.64
0.60
0.68
0.29
0.32
0.53
0.61
0.6
0.76
0.60
0.63
0.75
26.67 19.91 29.03
tr
0.30
tr
28.23 35.51 23.45
2.76
3.74
1.86
10.98 16.26 9.26
1.60
1.86
1.76
20.21 15.03 24.36
29.68 21.57 29.54
43.57 57.37 36.33
1.57
0.27
tr
95.03 94.24 90.23
Identificationc)
VI
nd
nd
tr
tr
tr
tr
0.34
1.4
11.75
3.37
1.15
0.51
0.39
tr
tr
tr
1.39
0.97
0.55
0.75
0.76
30.3
tr
22.05
1.34
8.03
1.64
23.33
30.3
33.06
tr
86.69
VII
nd
nd
tr
tr
0.73
0.95
0.26
1.47
12.11
3.4
1.06
0.40
tr
0.82
0.94
tr
0.95
tr
tr
0.68
tr
25.25
tr
25.29
1.68
9.49
1.98
22.82
26.2
38.44
tr
87.46
) Retention time on a DB-5MS column; b) Retention Index on a DB-5MS column; c)
Identification method: RI, retention indices matching with literature data; MS, mass
spectra matching with data from the NIST mass spectra database.
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MS
MS
RI, MS
MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
MS
MS
RI, MS
Table S2. The IC50 values of essential oils from different extraction stages.
Essential oils
IC50 ± SD (mg/mL)
26.23 ± 1.54b
I
24.10 ± 0.61ab
II
26.05 ± 1.34b
III
26.34 ± 1.13b
IV
22.48 ± 0.62a
V
23.64 ± 0.56ab
VI
23.86 ± 0.93ab
VII
Means with different superscript letters (a-c), within same column are significantly different at P
﹤0.05.
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Table S3. Minimum inhibitory concentration (MIC) of oils from Voacanga seeds at
different extraction stages.
Essential oils
MIC (μg/mL)
B. subtilis
I
128
II
128
III
128
IV
256
V
>1024
VI
>1024
VII
>1024
Levofloxacin 1.25
Fluconazole
S. aureus
>1024
128
64
128
>1024
256
256
0.75
S. epidermidis
512
128
128
64
512
256
512
1.25
P. vulgaris
128
128
64
256
256
256
256
0.75
P. aeruginosa
>1024
>1024
>1024
128
64
>1024
256
2.5
E. coli
256
512
>1024
>1024
>1024
>1024
>1024
0.75
C. albicans
256
>1024
512
256
256
128
64
0.5
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