molecules

Article
GC-MS Study of the Chemical Components of
Different Aquilaria sinensis (Lour.) Gilgorgans and
Agarwood from Different Asian Countries
Meng-Ru Wang 1 , Wei Li 1 , Sha Luo 1 , Xin Zhao 2 ID
, Chun-Hui Ma 1, * and Shou-Xin Liu 1, *
1 Key Laboratory of Bio-Based Material Science and Technology (Ministry of Education), College of Material
Science and Engineering, Northeast Forestry University, Harbin 150040, China;
nefuwangmengru@163.com (M.-R.W.); liwei19820927@126.com (W.L.); luo.sha.85@163.com (S.L.)
2 Key Laboratory of Pulp and Paper Science & Technology of Ministry of Education/Shandong Province,
Qilu University of Technology, Jinan 250353, China; zhaoxin_zixi@126.com
* Correspondence: mchmchmchmch@163.com (C.-H.M.); liushouxin@126.com (S.-X.L.);
Tel.: +86-451-8219-1204 (C.-H.M.); +86-451-8219-1502 (S.-X.L.)




Received: 10 July 2018; Accepted: 24 August 2018; Published: 28 August 2018


Abstract: As a traditional medicinal herb and valuable natural spice in China, Aquilaria sinensis
(Lour.) Gilg has many significant pharmacological effects. Agarwood is the resinous heartwood
acquired from wounded A. sinensis trees, and is widely used in pharmaceuticals owing to its
excellent medicinal value. In this study, the chemical composition of volatile components and
alcohol extracts from different organs of A. sinensis and agarwoods grown in different regions were
investigated using GC-MS. The results showed that Vietnam agarwood had the highest moisture
content, which was attributed to the local climate, while the fruit and bark of A. sinensis had higher
moisture contents than the other organs. The volatile components of A. sinensis organs included
3-ethyl-5-(2-ethylbutyl)-octadecane, oleic acid 3-(octadecyloxy) propyl ester, and docosanoic acid
1,2,3-propanetriyl ester, while the alcohol extracts of A. sinensis organs contained benzoic acid ethyl
ester, hexadecanoic acid ethyl ester, oleic acid, and n-hexadecanoic acid. Furthermore, the main
active ingredients in agarwood from different habitats were sesquiterpenoids, aromatic species, and
chromone compounds. The role of chromone compound 2-phenylethyl-benzopyran as an elicitor
and the mechanism of agarwood formation were also investigated. Antioxidant tests showed that
essential oils from agarwood and A. sinensis had antioxidant capacities by comparison with butylated
hydroxytoluene and vitamin E. An antibacterial activity test showed that the inhibition effect of the
essential oil was better against Gram-positive bacteria than against Gram-negative bacteria.

Keywords: Aquilaria sinensis (Lour.) Gilg; volatile component; alcohol extracts; GC-MS; antioxidant
capacity; antibacterial activity

1. Introduction
Aquilaria sinensis (Lour.) Gilg (A. sinensis) is a tropical evergreen tree native to China that has been
widely used as traditional medicine in East and Southeast Asia for hundreds of years. The resinous
portion of A. sinensis branches and trunks, known as agarwood, is widely used in traditional medicine
as a digestive, sedative, and antiemetic, and is also used in incense and perfume [1,2]. Agarwood is a
resinous material collected from A. sinensis. In wild forests, only 7–10% of A. sinensis trees can produce
agarwood, and healthy A. sinensis cannot produce agarwood. Agarwood is secreted as a black resin
with an aromatic fragrance only when A. sinensis is stimulated by external stimuli, such as physical or
chemical damage or endophytic bacteria [3,4]. A drastic decline in the number of Aquilaria trees in
natural forests has earned it endangered status. Agarwood resin is widely used in medicine, cosmetics,

Molecules 2018, 23, 2168; doi:10.3390/molecules23092168 www.mdpi.com/journal/molecules

Molecules 2018, 23, 2168 2 of 14

food, and other fields [5]. As a famous traditional Chinese medicine and a highly valuable non-timber
product, the demand for agarwood is much greater than its supply [6]. At the end of last century,
A. sinensis was planted for artificial cultivation in Hainan, Guangdong, and in other regions [7–9].
A. sinensis has been shown to contain chemical components such as terpenoids, flavonoids, lignans,
and steroids [10,11]. Furthermore, an impressive body of literature indicates that the main components
of healthy A. sinensis are fatty alkanes, while the main components of agarwood are sesquiterpenoids
and aromatic species [11–16]. Chemical investigations into agarwood have shown that sesquiterpenes,
2-(2-phenylethyl)-4-H-chromen-4-one derivatives, and aromatic compounds are the main characteristic
chemical constituents [7,17–19].
Existing literature on agarwood is mainly focused on studying the types and structures of
two pharmacologically active components, namely, 2-(2-phenylethyl)chromone derivatives and
sesquiterpenes [15,17,20,21]. However, few studies have compared the contents of these two
compounds in agarwood from different regions. Furthermore, research on A. sinensis has mainly
focused on leaf and seed chemical composition [1,22], while the chemical composition and volatile
contents of different organs of A. sinensis have not been systematically studied. Therefore, herein, the
chemical composition of volatile components and alcohol extracts of different organs of A. sinensis
and agarwood from different regions were investigated by GC-MS. Furthermore, the antioxidant and
antibacterial activities of their essential oils were tested. This study aimed to develop a method
for identifying the chemical composition of agarwood, providing a theoretical basis for further
experiments, and to compare exotic agarwood species and understand the main chemical components
in different samples. The results will provide a basis for selecting the best quality agarwood from
different origins and a foundation for the quality evaluation and mechanism study of agarwood.

2. Results and Discussion

2.1. Moisture Analysis of A. sinensis Organs and Agarwood from Different Regions
Three precision-weighed (1.00 g) powdered samples of agarwood xylem from different regions
and different A. sinensis organs (including blossom, seeds, peel, leaf, branch, xylem, bark, and root)
were dried to constant weight in an oven at 105 ± 3 ◦ C to calculate their average moisture contents.
Agarwood can resist external attack of the xylem parts and its moisture content is lower than that
of xylem. The main factors influencing the moisture content are ambient humidity and the agarwood
formation mechanism. Compared with other regions, the moisture content of Chinese A. sinensis
agarwood was the lowest, at 7.99 ± 0.13% (Table 1). A. sinensis agarwood is mostly grown in areas
near the Tropic of Cancer, which has a humid tropical and subtropical monsoon climate with high
temperatures and abundant rainfall. A. sinensis agarwood is also commonly grown in mountain
rainforest or semi-evergreen rainforest with moist, porous, and humus thick soil. Agarwood from
Vietnam had the highest moisture content (14.73 ± 0.08%). This was attributed to Vietnam having a
tropical monsoon and tropical rainforest climate, with four distinct seasons in the north, while the
four seasons are divided into dry and rainy seasons in the south owing to the effects of the monsoon.
Furthermore, the moisture contents of Malaysian and Indonesian agarwood were 9.22 ± 0.16% and
11.27 ± 0.31%, respectively. Malaysia is located near the equator and has a tropical rainforest climate
and tropical monsoon climate without obvious seasons, with average temperatures of 26–30 ◦ C and
abundant rainfall. The Indonesian climate is similar to that of Malaysia and typical of a tropical
rainforest climate with abundant rainfall. These fundamental climate factors account for the different
moisture contents of agarwood from different regions. Furthermore, fresh samples recently obtained
from agarwood had a relatively high moisture content. As the weight is not the same as the density,
it is easy to submerge at the place of production. Once separated from the tree, the water supply is
cut off. However, owing to the influence of outside temperature, the moisture stored in the interior
is gradually reduced. Agarwood of good provenance is not likely to sink. The moisture content of
agarwood has a significant influence on its weight, which results in the weight of agarwood changing
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during rainy and dry seasons. The original water content of agarwood is relatively abundant, and its
during rainy and dry seasons. The original water content of agarwood is relatively abundant, and
weight can be reduced under dry conditions and during storage. This is an important reason for
its weight can be reduced under dry conditions and during storage. This is an important reason for
selecting agarwood is the lower moisture content, and the higher quality [23]. Furthermore, testing
selecting agarwood is the lower moisture content, and the higher quality [23]. Furthermore, testing the
the different organs of A. sinensis showed that the root had the lowest moisture content (46.48 ±
different organs of A. sinensis showed that the root had the lowest moisture content (46.48 ± 0.36%).
0.36%). This was attributed to the root comprising the water transport channels of the plant, meaning
This was attributed to the root comprising the water transport channels of the plant, meaning that it
that it does not retain water. The fruit had the highest moisture content, including the blossom, seed,
does not retain water. The fruit had the highest moisture content, including the blossom, seed, and
and peel. Owing to the driving force of transpiration [19], the moisture contents of the blossom, seed,
peel. Owing to the driving force of transpiration [19], the moisture contents of the blossom, seed, and
and peel were 71.98 ± 0.83%, 79.72 ± 1.27%, and 81.20 ± 0.68%, respectively. The moisture content of
peel were 71.98 ± 0.83%, 79.72 ± 1.27%, and 81.20 ± 0.68%, respectively. The moisture content of the
the bark was 70.07 ± 0.23%, while those of the leaves and branches were 63.88 ± 0.66% and 63.23 ±
bark was 70.07 ± 0.23%, while those of the leaves and branches were 63.88 ± 0.66% and 63.23 ± 0.24%,
0.24%, respectively. The moisture content of the xylem was lower, at 51.95 ± 0.03%.
respectively. The moisture content of the xylem was lower, at 51.95 ± 0.03%.
Table 1. Moisture analysis of A. sinensis organs and agarwood from different regions.
Table 1. Moisture analysis of A. sinensis organs and agarwood from different regions.
No. Origin Hydrodistillation Extraction (%) Soxhlet Extraction (%) Moisture Percentage (%)
No.
1 Origin
China Hydrodistillation
NDExtraction (%) Soxhlet2.28
Extraction
± 0.16 (%) Moisture Percentage
7.99 ± 0.13 (%)
21 Malaysia
China NDND 2.28 ±±0.16
21.47 0.90 9.22 ± 0.16
7.99 ± 0.13
32 Indonesia
Malaysia NDND 10.51±± 0.90
21.47 0.36 11.27
9.22 ±± 0.16
0.31
43 Vietnam
Indonesia NDND 11.5±
10.51 ± 0.45
0.36 14.73 ±
11.27 ± 0.08
0.31
54* blossom
Vietnam <0.05
ND 11.5 ±±0.45
21.51 1.05 71.98 ±
14.73 ± 0.83
0.08
65 ** blossom
seed <0.05
<0.1 11.01±± 1.05
21.51 0.43 79.42 ±
71.98 0.83
± 1.27
76 ** seed
peel <0.1
<0.05 25.24±± 0.43
11.01 1.10 81.2 ±±0.68
79.42 1.27
87 ** peel
blade <0.05
<0.05 20.30±± 1.10
25.24 1.33 81.2 ±± 0.68
63.88 0.66
98 ** blade
branch <0.05
ND 3.48±
20.30 1.33
± 0.21 63.23 ±
63.88 0.66
± 0.24
9* branch ND 3.48 ± 0.21 63.23 ± 0.24
10 * xylem ND 2.39 ± 0.28 51.95 ± 0.03
10 * xylem ND 2.39 ± 0.28 51.95 ± 0.03
11 * bark ND 6.85 ± 0.26 70.07 ± 0.23
11 * bark ND 6.85 ± 0.26 70.07 ± 0.23
12 * root ND 3.81 ± 0.26 46.48 ± 0.36
12 * root ND 3.81 ± 0.26 46.48 ± 0.36
“*” Nos. 5–12 represent different organs of A. sinensis (blossom, seeds, peel, leaf, branch, xylem, bark,
“*” Nos. 5–12 represent different organs of A. sinensis (blossom, seeds, peel, leaf, branch, xylem, bark, and root).
andNot
ND: root). ND: Not detected.
detected.

2.2.Chemical
2.2. ChemicalComposition
CompositionAnalysis
AnalysisofofA.
A.sinensis
sinensis
Samples of
Samples of A.
A. sinensis
sinensis organs,
organs, prepared
prepared according
according to to Section
Section 3.2.2,
3.2.2, were
were tested
tested byby GC-MS
GC-MS to to
determine the yields of volatile components and alcohol extraction. The main composition
determine the yields of volatile components and alcohol extraction. The main composition of the of the
alcohol extracts
alcohol extracts and
and volatile
volatile components
components of
of A.
A. sinensis
sinensis organs
organs are
areshown
shownin inFigures
Figures11andand2.2. The
The
volatilecomponents
volatile componentsand andrelative
relativepercentages
percentageswere
weremeasured
measuredand andare
arelisted
listedininTable
Table2.2.

Figure 1. Chemical composition of alcohol extracts of different A. sinensis organs: (a) root; (b) peel;
Figure 1. Chemical composition of alcohol extracts of different A. sinensis organs: (a) root; (b) peel; (c)
(c) blossom; (d) bark; (e) branch; (f) blade; and (g) seed.
blossom; (d) bark; (e) branch; (f) blade; and (g) seed.
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Figure 2.
Figure 2. Chemical
Chemicalcomposition
compositionofofvolatile
volatilecomponents in in
components different A. A.
different sinensis organs:
sinensis (a) peel;
organs: (b)
(a) peel;
blossom; (c) blade; and (d) seed.
(b) blossom; (c) blade; and (d) seed.

Table 2. Chemical composition analysis of different organs from A. sinensis.

Table 2. Chemical composition analysis of different organs from A. sinensis.
RA%
A. sinensis Molecular Molecular CAS
Main Chemical Components VolatileRA% Alcohol
Organ
A. sinensis Formular Molecular
Weight Number
Molecular CAS Component Extracts
Main Chemical Components Volatile Alcohol
Organ Benzoic acid, 2-hydroxy-, phenylmethyl Formular Weight Number
C14H12O3 228 118-58-1 Component
16.59 Extracts
--
blossom ester
Benzoic acid, n-Hexadecanoic
2-hydroxy-, phenylmethyl C16H32O2 256 57-10-3 -- 15.96
acid C14 H12 O3 228 118-58-1 16.59 –
blossom ester
10-Octadecenoic acid, methyl ester C16 H32CO19H
2 36O2 256 296 57-10-3
13481-95-3 –21.30 15.96
--
seed n-Hexadecanoic acid
Squalene C30H50 410 111-02-4 -- 36.66
10-Octadecenoic acid, methyl
Benzoic acid, 2-hydroxy-, ester
phenylmethyl C19 H36CO 2 12O3
14H
296 228 13481-95-3
118-58-1 21.30
21.50 –--
seed
peel Squalene
ester Benzoic acid, ethyl ester C30 HC 509H10O2 410 150 111-02-4
93-89-0 – -- 36.66
28.28
Benzoic acid, 2-hydroxy-,
Octadecane, phenylmethyl
3-ethyl-5-(2-ethylbutyl)-n- C14 H12 O 3 H54
C26 228 366 118-58-1
55282-12-7 21.50
13.34 –--
peel
blade ester Benzoic acid, ethylacid
Hexadecanoic ester C9 H10CO162H32O2 150 256 93-89-0
57-10-3 – -- 28.28
8.66
4-((1E)-3-Hydroxy-1-propenyl)-2-
Octadecane,
branch C26 HC5410H12O3 366 180 458-35-5
55282-12-7 13.34-- 15.07
–
blade methoxyphenol
3-ethyl-5-(2-ethylbutyl)-n-Hexadecanoic
C16 H32 O2 256 57-10-3 – 8.66
acid 326875-68- --
xylem 8-Naphthol, 1-(benzyloxy)- C17H14O2 250 --
4-((1E)-3-Hydroxy-1-propenyl)-2- 7 18.18
branch 9-Octadecenoic acid, 1,2,3-propanetriyl C10 H12 O3 180 458-35-5 – 15.07
bark methoxyphenol C57H104O6 884 537-39-3 -- 6.74
ester
–
xylem
root 8-Naphthol,
Benzoic1-(benzyloxy)-
acid, ethyl ester C17 H14CO9H2 10O2 250 150 326875-68-7
93-89-0 – -- 20.32
18.18
RA%: Relative area in
9-Octadecenoic acid, 1,2,3-propanetriyl total compounds.
bark C57 H104 O6 884 537-39-3 – 6.74
ester
root Benzoic acid, ethyl ester C9 H10 O2 150 93-89-0 – 20.32
RA%: Relative area in total compounds.
Molecules 2018, 23, 2168 5 of 14

The alcohol extracts of different A. sinensis organs (Figure 1) contained benzoic acid ethyl ester,
hexadecanoic acid ethyl ester, oleic acid, and n-hexadecanoic acid. Among them, benzoic acid was
abundant in the root (20.32%) and peel (28.28%). Compared with the literature [24,25], the RA(%)
value of Benzoic acid, ethyl ester was 14%, while the data in Table 2 is higher than the literature data,
whereas the hexadecanoic acid ethyl ester contents in the branch and seed were similar, at 9.32%
and 9.65%, respectively. As shown in Figure 1, oleic acid was concentrated in the bark (14.11%) and
n-hexadecanoic acid was concentrated in the blossom (15.96%). Because the RA(%) values in other
organs of A. sinensis were not listed in the previous studies, only the chemical structures have been
analyzed, so no comparison could be made. Inorganic compounds are transported upward in the
xylem of plants, while organic matter is transported through the phloem upward and downward, with
preferential transport to the growth centers of the plants [26]. Owing to plant transpiration, water is
transported from the bottom to the top. The organic acid contents are higher in the leaves and blossom
because organic acids are water soluble, while the water solubility of ester organic compounds is poor,
resulting in higher ester contents in the roots and leaves [25].
The main volatile components in the different organs were 3-ethyl-5-(2-ethylbutyl)-octadecane,
oleic acid 3-(octadecyloxy) propyl ester, and docosanoic acid 1,2,3-propanetriyl ester (Figure 2).
These three compounds all have obvious antimicrobial effects and potential bioactivities [27,28].
Therefore, vulnerable external parts of the plant body are rich in these compounds. The
3-ethyl-5-(2-ethylbutyl)-octadecane content was highest in the blades (37.26%), while its contents
in other organs were in the order: blossom (9.41%) > seed (5.34%) > peel (3.38%). The oleic acid
3-(octadecyloxy) propyl ester content was highest in the blade (18.59%), while its contents in the
blossom, peel, and seed were 15.71%, 5.38%, and 4.64%, respectively. Furthermore, the docosanoic
acid 1,2,3-propanetriyl ester content was highest in the blade (6.11%).
The chemical composition and relative percentages of the contents of A. sinensis alcohol extracts,
prepared according to Section 3.2.3, were determined by GC-MS and the results are listed in Table 2.
The results show that the volatile components of the blossom (16.59%), seed (21.30%), peel (21.50%),
and blade (13.34%) contained a high abundance of fatty acids, such as benzoic acid, and 10-octadecenoic
acid methyl ester. Similarly, the alcohol extracts of the blossom (15.96%), seed (36.66%), peel (28.28%),
and blade (8.66%) contained n-hexadecanoic acid and squalene. Squalene has strong oxygen capacity,
antifatigue, and anticardiovascular disease effects [29]. The main chemical constituents in the blade
were myristicin and palmitic acid. In a preliminary study by Wu and coworkers [13], A. sinensis peel
extracts showed obvious antibacterial and antitumor activities. However, the proportion of these
compounds in the branches, xylem, bark, and root was very low.

2.3. Chemical Composition Analysis of Agarwood from Different Regions

Agarwood samples from different geographic regions were prepared according to the procedures
described in Sections 3.2.2 and 3.2.3, and tested using GC-MS. The volatile components and relative
percentages of effective constituents were identified and are listed in Table 3.

Table 3. Chemical composition analysis of agarwood from different regions.

Molecular
Main Chemical Components (RA%) Malaysia China Indonesia Vietnam
Weight
Isoaromadendrene epoxide (C15 H24 O) 220 0.28% 1.14% 0.12% 4.50%
Agarospirol (C15 H26 O) 222 0.11% 0.13% 0.49% 0.66%
β-Guaiene (C15 H24 ) 204 0.08% 0.07% 0.21% 0.21%
Benxylacatone (C10 H12 O) 148 0.21% 0.18% 0.30% 0.15%
2-(2phenylethyl)chromone (C17 H14 O2 ) 1.78% 1.65% 0.07% 0.03%
6-hydroxy-2-[2-(4-methoxyl-phenyl)ethyl]chromone 250
0.56% 0.49% 0.23% 0.21%
(C18 H16 O4 )
6-hydroxy-2-(2-phenylethyl)chromone (C17 H15 O3 ) 0.43% 0.79% 0.77% 0.17%
RA%: Relative area of total compounds.
Molecules 2018, 23, 2168 6 of 14

The chemical composition of agarwood has been studied extensively [30–32]. The chemical
composition of xagarwood from China was the same as that of imported agarwood. Analyzing
Molecules 2018, 23, 6 of 14
the alcohol extracts of agarwood from different regions showed that they contained volatile oils,
sesquiterpenoids,2-(2-phenylethyl)chromone,
sesquiterpenoids, 2-(2-phenylethyl)chromone, fatty fattyacids,
acids,and
andother
othercomponents
components(Figure
(Figure3).
3).Among
Among
them,the
them, themain
mainvolatile
volatileoil
oilcomponents
components in in agarwood
agarwood are are sesquiterpenoids
sesquiterpenoids and
and aromatic
aromatic compounds.
compounds.
2-(2-Phenylethyl)chromone is
2-(2-Phenylethyl)chromone is aa characteristic
characteristic component
component that
that confirms
confirms subfamily
subfamily Agaraceae
Agaraceae asas an
an
independent subfamily,
independent subfamily, while
while itsits precursor
precursor diphenyl
diphenyl pentanone
pentanone isis widely
widely found
found in
in plants
plants of
of the
the
Thymelaeaceaefamily
Thymelaeaceae family[33].
[33].

Figure 3. Structural formulas of compounds commonly found in alcohol extracts of agarwood from
Figure 3. Structural formulas of compounds commonly found in alcohol extracts of agarwood from
different regions. (a) isoaromadendrene epoxide, C15 H24 O; (b) agarospirol, C15 H26 O; (c) β-guaiene,
different regions. (a) isoaromadendrene epoxide, C15H24O; (b) agarospirol, C15H26O; (c) β-guaiene,
C15 H24 ; (d) benxylacatone, C10 H12 O; (e) 2-(2-phenylethyl)chromone, C17 H14 O2 ).
C15H24; (d) benxylacatone, C10H12O; (e) 2-(2-phenylethyl)chromone, C17H14O2).

The
Theactive ingredients
active for all for
ingredients agarwood obtained from
all agarwood differentfrom
obtained regions includedregions
different sesquiterpenoids,
included
aromatic species, and
sesquiterpenoids, chromone
aromatic compounds.
species, and chromoneFor example, in Malaysian
compounds. agarwood,
For example, in chromone
Malaysian
compounds accounted compounds
agarwood, chromone for 2.77% ofaccounted
the total content,
for 2.77%while in total
of the Indonesian
content,agarwood, chromone
while in Indonesian
compounds and agarospirol
agarwood, chromone compounds accounted for 0.61% accounted
and agarospirol and 0.49%for of0.61%
the total
and content,
0.49% of respectively. In
the total content,
Vietnamese agarwood, the proportions of agarospirol and 2-(2-phenylethyl)chromone
respectively. In Vietnamese agarwood, the proportions of agarospirol and 2-(2- with its
derivatives were 0.66% and
phenylethyl)chromone with0.41%, respectively.
its derivatives wereThe
0.66%proportion
and 0.41%, of chromone
respectively.compounds was the
The proportion of
highest
chromonein Chinese agarwood
compounds was the(2.93%).
highest in Chinese agarwood (2.93%).
Sesquiterpenoids
Sesquiterpenoidshavehavebeen
beenreported
reportedto tohave
have antineuroinflammatory
antineuroinflammatoryproperties,
properties,while
whilearomatic
aromatic
species
species and chromone
chromone compounds
compoundshave haveshown
shown inhibitory
inhibitory activity
activity toward
toward human
human gastric
gastric cancer
cancer cells
cells [34–36].
[34–36]. According
According to previous
to previous research,
research, thethe defensivereaction
defensive reactionmechanism
mechanism of of agarwood formation
formation
(Figure
(Figure4),4),can
canbe
beinduced
inducedby byphysical
physicalinjury
injury[37,38],
[37,38],chemical
chemicaldamage
damage[39],[39],fungi
fungiinfection
infection [40],
[40], or
or
elicitors,
elicitors,as
asshown
shownin inFigure
Figure44[41,42].
[41,42]. Chromone
Chromone compound
compound2-phenylethyl-benzopyran
2-phenylethyl-benzopyranisisthe theelicitor
elicitor
that
that induces
induces agarwood
agarwood formation.
formation. The The different
different chromone
chromone compound
compound contents
contents among
among agarwood
agarwood
samples provides a scientific basis for the screening of Chinese
samples provides a scientific basis for the screening of Chinese agarwood. agarwood.
Molecules 2018, 23, 2168 7 of 14
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Molecules 2018, 23, x 7 of 14

Figure 4. Defensive reaction mechanism of agarwood formation.

Figure 4.
Figure Defensive reaction
4. Defensive reaction mechanism
mechanism of
of agarwood
agarwood formation.
formation.
2.4. Antioxidant Activity Tests
2.4. Antioxidant Activity Tests
Free Radical
Free Radical Scavenging Activity
Activity Assay of of the Essential
Essential Oil
Free Radical Scavenging
Scavenging Activity AssayAssay of the the Essential Oil
Oil
DPPH
DPPH is is a highly stable nitrogen-centered free radical that cancapture
capture otherfree free radicals, with
DPPH is aa highly
highly stable
stable nitrogen-centered
nitrogen-centeredfree freeradical
radicalthat
thatcan
can captureother other freeradicals,
radicals,with
witha
a strong absorption centeredatat517 517 nm.Freshly
Freshly preparedDPPH DPPH solutionisisaadeep deep purple color
color that
astrong
strongabsorption
absorptioncentered
centered at 517nm. nm. Freshlyprepared
prepared DPPHsolution
solution is a deep purplepurple color that
that
becomes colorless
becomes colorless or pale yellow after reacting with an antioxidant [43]. As shown in Figure 5a, with
with
becomes colorless oror pale
pale yellow
yellow after
after reacting
reacting with
with an
an antioxidant
antioxidant [43]. As shown
[43]. As shown in in Figure
Figure 5a,
5a, with
increasing essential
increasing essential oil concentration,
concentration, the the scavenging
scavenging activity (SC%)(SC%) increased significantly.
significantly. When
increasing essential oil
oil concentration, the scavenging activity
activity (SC%) increased
increased significantly. WhenWhen
the concentration
concentration waswas less
less than
than 60 60 mg/mL,
mg/mL, the scavenging activity increased rapidly, but as
as the
the concentration was less than 60 mg/mL, the scavenging activity increased rapidly, but as the
the the scavenging activity increased rapidly, but the
concentration
concentration waswas increased
was increased further,
increased further, the
further, the scavenging activity increased more slowly. The antioxidant
concentration the scavenging activity increased
scavenging activity increased more
more slowly.
slowly. The
The antioxidant
antioxidant
capacity of
capacity of the essential
essential oil was
was much weakerweaker than thosethose of butylated
butylated hydroxytoluene (BHT) (BHT) and
capacity of the
the essential oil
oil was much
much weaker than than those of of butylated hydroxytoluene
hydroxytoluene (BHT) and and
vitamin E (VE).
vitamin (VE). According to to the literature,
literature, if aa substance
substance has an an SC50 value
value (the
(the concentration
concentration of of
vitamin E E (VE). According
According to the the literature, if if a substance hashas an SC SC5050 value (the concentration of
essential oil
essential oil when
oil when the SC% was 50%) of less than 10 mg/mL, the substance is considered to have an
essential when the
the SC%
SC% was
was 50%)
50%) of of less
less than
than 1010 mg/mL,
mg/mL, the the substance
substance is is considered
considered toto have
have anan
antioxidant capacity
antioxidant capacity [44].
capacity [44]. The DPPH
The DPPH scavenging
DPPH scavenging effect decreased in the order: BHT > VE > HD (HD
antioxidant [44]. The scavenging effect
effect decreased
decreased in in the
the order:
order: BHT
BHT >> VEVE > HD (HD
> HD (HD
represents the
represents the volatile
volatile components
components extracted
extracted by by hydrodistillation).
hydrodistillation).
represents the volatile components extracted by hydrodistillation).

Figure 5. Results of (a) DPPH free radical scavenging activity and (b) ferric ion reducing antioxidant
Figure
Figure 5. Results
Results of
of (a)
(a) DPPH
DPPH free radical scavenging
free radical scavenging activity
activity and
and (b)
(b) ferric
ferric ion reducing antioxidant
ion reducing antioxidant
power 5.
(FRAP) assay.
power (FRAP) assay.
power (FRAP) assay.
The antioxidant activity of the sample was determined using the ferric ion reducing antioxidant
The antioxidant activity of the sample was determined using the ferric ion reducing antioxidant
power (FRAP) assay established by Benzie and Strain [45,46]. The standard curve equation obtained
power (FRAP) assay established by Benzie and Strain [45,46]. The standard curve equation obtained
by the test (y = 0.002x + 0.1049, R2 = 0.999) was used to determine the reducing ability of the extract.
by the test (y = 0.002x + 0.1049, R2 = 0.999) was used to determine the reducing ability of the extract.
The reduction capacity of the sample was expressed as the FRAP value, with a higher FRAP value
The reduction capacity of the sample was expressed as the FRAP value, with a higher FRAP value
Molecules 2018, 23, 2168 8 of 14

The antioxidant activity of the sample was determined using the ferric ion reducing antioxidant
power (FRAP) assay established by Benzie and Strain [45,46]. The standard curve equation obtained
by the test (y = 0.002x + 0.1049, R2 = 0.999) was used to determine the reducing ability of the extract.
The reduction capacity of the sample was expressed as the FRAP value, with a higher FRAP value
representing a better reduction capability. As shown in Figure 5b, the ferric reducing power increased
with increasing concentration, and the antioxidant capacities were in the order: BHT > VE > HD.

2.5. Antibacterial Properties

In recent years, plant essential oils have received much attention in bacteriostatic compound
research owing to their remarkable bacteriostatic activities and unique advantages of reduced side
effects and residual toxicity compared with synthetic chemical bacteriostatic agents. In general,
Gram-positive bacteria are more sensitive to plant essential oils than Gram-negative bacteria because
the outside of the cell wall of Gram-negative bacteria contains a lipopolysaccharide layer that prevents
hydrophobic compounds from entering the cells, which reduces the bacteriostatic effect [47]. As shown
in Table 4, with increasing essential oil concentration, the Bacillus subtilis (B. subtilis) and Staphylococcus
aureus (S. aureus) inhibition ratios became stronger, at 60.80 ± 3.82% and 64.46 ± 3.01%, respectively.
Meanwhile, the inhibitory effect on Escherichia coli (E. coli) was weaker, at 57.97 ± 3.44%. The inhibition
rates were in the following order: S. aureus > B. subtilis > E. coli.

Table 4. Inhibition activities of essential oils on E. coli, B. subtilis, and S. aureus.

E. coli B. subtilis S. aureus

Concentration(mg/mL) Average OD Inhibition Average OD Inhibition Average OD Inhibition
(cm) Ratio (%) (cm) Ratio (%) (cm) Ratio (%)
0 2.03 0 2.05 0 2.04 0
0.2 2.15 5.58 ± 0.22 2.44 15.98 ± 0.32 2.43 16.05 ± 0.33
0.4 2.34 13.25 ± 0.43 2.72 24.63 ± 0.82 2.77 26.35 ± 0.81
0.6 2.56 20.70 ± 1.07 3.12 34.29 ± 1.30 3.36 39.29 ± 1.21
0.8 2.84 28.52 ± 1.31 3.57 42.58 ± 1.42 3.76 45.74 ± 1.24
1.0 3.09 34.30 ± 1.52 3.89 47.30 ± 1.33 4.01 49.13 ± 1.65
1.2 3.53 42.49 ± 1.88 4.39 53.30 ± 1.82 4.48 54.46 ± 1.75
1.4 3.81 46.72 ± 2.07 4.66 56.01 ± 2.31 4.82 57.68 ± 1.97
1.6 4.07 50.12 ± 2.44 4.97 58.75 ± 2.82 5.30 61.51 ± 2.65
1.8 4.40 53.86 ± 2.47 5.12 59.96 ± 2.34 5.54 63.18 ± 2.57
2.0 4.83 57.97 ± 3.44 5.23 60.80 ± 3.82 5.74 64.46 ± 3.01

3. Materials and Methods

3.1. Materials

3.1.1. Apparatus
GC-MS analysis of the essential oils was conducted on an Agilent 6890N-5973 insert gas
chromatograph (Agilent Technologies, Palo Alto, CA, USA) using an HP-5MS5% phenyl methyl
siloxane capillary column (30 mm × 0.25 mm × 0.25 µm) and equipped with an Agilent 6890N-5973
mass selective detector in electron impact mode.

3.1.2. Plant Material

Agarwood from Vietnam, Indonesia, and Malaysia was purchased from SanKeshu Medicinal
Materials Market, Harbin, Heilongjiang Province, China, and authenticated using scanning electron
microscopy (Quanta 200, FEI, Eindhoven, The Netherlands) by Yongzhi Cui, Northeast Forestry
University. A. sinensis (diameter, 8.5 cm) was obtained from Guangdong. Different parts were
separated and stored at −20 ◦ C, and then underwent water distillation or Soxhlet extraction for 48 h
after smashing. Climatic information about agarwood from different regions is shown in Table 5.
Molecules 2018, 23, 2168 9 of 14

Table 5. Climatic information of agarwood from different regions.

Origin Geographical Location Information

Guangdong, China is located between latitude 20◦ 130 ~25◦ 310 and
China longitude 109◦ 390 ~117◦ 190 ; subtropical monsoon climate, annual
average precipitation is 1300~2500 mm.
Malaysia is located between 1~7◦ north latitude and 97–120◦ east
longitude, tropical rainforest climate and tropical monsoon climate, no
Malaysia
obvious four seasons, average temperature is 26~30 ◦ C, abundant
rainfall.
Indonesia is located between 12◦ S–7◦ N and 96◦ E~140◦ E, tropical
Indonesia rainforest climate, annual average temperature is 25~27 ◦ C, no four
seasons, abundant precipitation, annual precipitation is 1600~2200 mm.
Vietnam is located at 8◦ 300 ~23◦ 220 north latitude and 102◦ 100 ~109◦ 300
Vietnam east longitude, tropical monsoon climate, average annual rainfall is
1500~2000 mm.

3.1.3. Bacterial Strains

E. coli, B. subtilis, and S. aureus were obtained from the Food Microbiology Laboratory of Northeast
Forestry University, Harbin, China. Bacterial strains were kept on an agar slant nutrient medium at
4 ◦ C until use.

3.1.4. Preparation of Culture Medium

A beef paste peptone culture medium was used in this experiment. This culture medium was
prepared from beef paste (3.0 g), peptone (1.0 g), NaCl (0.5 g), agar (2.0 g), and water (1000 mL), and
adjusted to pH 7.0–7.2 using 1 mol/L NaOH.

3.2. Methods

3.2.1. Moisture Detection Method

Precisely weighed powdered samples of agarwood from different regions and different A. sinensis
organs (1.00 g) were dried to a constant weight in an oven at 105 ± 3 ◦ C to calculate the moisture
content. Samples were measured in triplicate and average values were recorded.

3.2.2. Hydrodistillation (HD) Method for Extracting Volatile Oils

Powdered samples of different A. sinensis organs (200.0 g) were added into a round-bottom flask
with distilled water (1800 mL), and subjected to water distillation for 6 h using an electric heater
(450 W). The essential oil was extracted with diethyl ether, dried with anhydrous sodium sulfate, and
stored under cryopreservation conditions at −20 ◦ C for further analysis.

3.2.3. Soxhlet Extraction Method for Preparation of Alcohol Extracts

Powdered samples of different A. sinensis organs (10.00 g) were put in filter paper bag and
subjected to ethanol Soxhlet extraction for 8 h. After evaporating the ethanol, the extracts were stored
at −20 ◦ C.

3.2.4. GC-MS Analysis Condition

GC was performed using the following conditions: manual injection, 1 µL; injector temperature,
270 ◦ C; carrier gas (He) flow rate, 1 mL/min; programmed oven temperature, 60 ◦ C held for 5 min,
ramped from 60 to 120 ◦ C at a rate of 4 ◦ C/min, held for 5 min, ramped from 120 to 170 ◦ C at
a rate of 3 ◦ C/min, held for 2 min, and ramped from 170 to 280 ◦ C at a rate of 10 ◦ C/min. The
Molecules 2018, 23, 2168 10 of 14

detector temperature was 280 ◦ C. MS was performed under the following conditions: Scan range,
15–500 amu; scan-TIC mode; injection port temperature, 250 ◦ C; column oven temperature, 60 ◦ C; ion
source temperature, 230 ◦ C; carrier gas (N2 ) flow rate, 1.6 mL/min; EI voltage, 70 eV; quadrupole rod
temperature, 150 ◦ C; quality scan range, m/z 40–400. The chemical compositions of essential oils were
identified by direct comparison of their mass spectra in the NIST11 Mass Spectral Library.

3.2.5. Antioxidant Capacity

DPPH Radical Scavenging Activity Assay of Volatile Oils (DPPH)

DPPH (3.5 mg) was made up to 100 mL with methanol in a brown 100-mL volumetric flask.
Essential oil–methanol solution (1.0 mL) was mixed with DPPH–methanol solution (2.0 mL), and the
mixture was shaken (100 rpm) and allowed to stand at room temperature for 30 min. The absorbance
was measured at 517 nm using a TU-1900 UV spectrophotometer (Persee, Beijing, China) with methanol
as the blank. The DPPH anion radical scavenging activity of the mixture was expressed as the SC%,
which was calculated using the following equation:

A0 − A
SC% = × 100% (1)
A0

where A0 is the concentration of the antioxidant components in the A. sinensis essential oil extract
before the reaction with free radicals, and A is the concentration of the antioxidant components in
the A. sinensis essential oil extract after the reaction with free radicals. The oil concentration that
provides 50% inhibition (SC50 ) was calculated from the plot of percentage inhibition of different
oil concentrations. Experiments were performed in triplicate. BHT and VE were used as positive
controls [48].

Ferric Ion Reducing Antioxidant Power (FRAP) of the Volatile Oil

Acetate buffer (300 mmol/L, pH 3.6), FeCl3 ·6H2 O solution (20 mmol/L), and TPTZ
(2,4,6-tripyridyl-S-triazine) solution (10 mmol/L) were mixed in a 10:1:1 (v/v/v) ratio to obtain
the TPTZ working solution. Sample solution (0.3 mL) was added to TPTZ working solution (2.7 mL)
preheated to 37 ◦ C. The mixtures were shaken for 10 min and measured at 593 nm using a TU-1900 UV
spectrophotometer (Persee). Before the experiment, the initial absorbance of the reagents (3 mL) and
the acetate buffer (3 mL) were measured at 593 nm and used as blanks. Experiments were conducted in
triplicate. The corresponding Trolox mass fraction obtained from the standard curve of the absorbance
after the reaction is defined as the FRAP value (mg Trolox/g extract) [49].

3.2.6. Antibacterial Activity

The bacteriostatic activity of the essential oils was tested by measuring bacteriostatic circles
using the filter paper method. Qualitative filter paper was cut into small round pieces (diameter,
1.5 cm) using a puncher, and autoclaved before use. E. coli, B. subtilis, and S. aureus were cultured in
nutrient agar medium at 37 ◦ C for 24 h. Under sterile conditions, the corresponding culture medium
(20–25 mL) was poured into the sterilized culture dish and the solid plate was prepared after cooling
and solidifying. The prepared bacterial suspension (200 µL) was added to the corresponding solid
plate medium and uniformly coated. The filter paper sheet was attached to the bacteria-bearing plate.
Essential oil (5 µL) was added to each piece of filter paper. The essential oil diffused from the center to
the periphery of the bacteria-bearing tablet, gradually forming a concentration gradient that inhibited
the growth of the tested strains and formed a transparent bacteriostatic circle. The bacteriostatic
Molecules 2018, 23, 2168 11 of 14

activity of the essential oils can be preliminarily determined according to the OD of the bacteriostatic
ring [50]. The inhibition ratios were calculated as follows:

Rt − Ro
Inhibition ratio (%) = × 100% (2)
Rt

where Rt is the average OD of the inhibition zone, and Ro is the average OD of the blank sample.
Experiments were performed in triplicate.

4. Conclusions
Agarwood is widely used in traditional Chinese medicine, incense, and perfumes. A. sinensis
is the only certified source of agarwood products listed in the China Pharmacopoeia. The moisture
content of agarwood from different regions, including China, Indonesia, Malaysia, and Vietnam, was
investigated, with Chinese agarwood found to have the lowest moisture content and Vietnamese
agarwood the highest. This result was attributed to Vietnam being located just south of the Tropic
of Cancer, with a tropical monsoon climate and high humidity. Among the parts of A. sinensis, the
moisture content of the root was the lowest, while that of the fruit was the highest. According to
chemical composition analysis of A. sinensis, the seeds had the highest volatile component and alcohol
extract contents. Squalene, fatty acids, and highly unsaturated fatty acids were abundant. The chemical
constituents of agarwood from different regions were also analyzed. The results showed that all species
contained active chemical compounds, such as sesquiterpenoids, aromatic species, and chromone
compounds. The proportion of chromone compounds was highest in Chinese agarwood. Chromone
compound 2-phenylethyl-benzopyran is a type of elicitor that induced agarwood formation. The
antioxidant capacity of essential oils extracted by HD were determined and compared with traditional
antioxidants. The DPPH scavenging effect decreased in the order BHT > VE > HD, while the FRAP test
results decreased in the order BHT > VE > HD. In the antibacterial ability test of the essential oils, the
essential oil concentration increased and inhibition rate decreased from S. aureus to E. coli. The present
study identified the chemical compositions of agarwood and provides a basis for selecting the best
quality agarwood from different origins. This study also provides a new approach to the identification
of agarwood from domestic and exotic species.

Author Contributions: M.-R.W., X.Z. and S.L. conducted experiments; W.L. and C.-H.M. analyzed data and
prepared the Tables and Figures; M.-R.W., W.L. and C.-H.M. designed the research study and prepared this
manuscript; S.-X.L. contributed chemical reagents and analytical tools.
Funding: This work was financially supported by the Fundamental Research Funds for the Central Universities
(2572017ET02), the postdoctoral scientific research developmental fund of Heilongjiang Province, 2016 (Grant No.
LBH-Q16001), the Research Start-up Funding for Introducing Talents in Northeast Forestry University (Grant
No. YQ2015-02), the National Natural Science Foundation of China (Grant Nos. 31500467, 31570567), the Natural
Science Foundation of Heilongjiang Province for Young Scholar (Grant No. QC2015034), and the Foundation of
Key Laboratory of Pulp and Paper Science and Technology of the Ministry of Education/Shandong Province of
China (No. KF201709).
Acknowledgments: We thank Simon Partridge, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac),
for editing the English text of a draft of this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are available from the authors.

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