Journal of Ethnopharmacology 253 (2020) 112652

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Metabolomics analysis to evaluate the antibacterial activity of the essential T

oil from the leaves of Cinnamomum camphora (Linn.) Presl
Jiali Chena, Cailin Tanga, Rongfei Zhanga, Shaoxia Yea, Zhimin Zhaoa, Yuquan Huangb,
Xinjun Xua, Wenjian Lana, Depo Yanga,∗
a
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, 510006, Guangdong Province, China
b
China Resources Sanjiu Medical & Pharmaceutical Co.,Ltd., Shenzhen, 518110, Guangdong Province, China

A R T I C LE I N FO A B S T R A C T

Keywords: Ethnopharmacology relevance: Cinnamomum camphora (Linn.) Presl (C. camphora) is one of the oldest herbal
Essential oil medicines used as a traditional medicine, owning a wide range of biological functions including anti-bacterial,
Cinnamomum camphora (Linn.) Presl anti-oxidative, anti-fungal, anti-inflammatory, insecticidal and repellent activities.
Antibacterial Objective: The aim of this study was to investigate the antibacterial activity and mechanism of action of the
MRSA
essential oil (EO) from C. camphora.
Metabolomics
Materials and methods: The EO was isolated from the leaves of C. camphora by hydrodistillation, and the che-
TCA cycle
mical compositions of the EO were analyzed by gas chromatography-mass spectrometry (GC-MS). The minimum
inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) values of the EO were esti-
mated by the microbroth dilution method. Growth curve was investigated by turbidimetry. Apoptosis was
measured by flow cytometry. Morphological change of bacteria was observed by field emission scanning electron
microscopy and transmission electron microscopy. The integrity of cell membrane was evaluated by NanoDrop
and BCA Protein Assay Kit. The methicillin-resistant Staphylococcus aureus (MRSA) metabolic profile in the
presence of the EO was explored by GC-MS-based metabolomics. Isocitrate dehydrogenase (ICDH), α-ketoglu-
tarate dehydrogenase (α-KGDH), succinic dehydrogenase (SDH) and malic dehydrogenase (MDH) activities were
detected by commercial kits.
Results: The main components of the EO from the leaves of C. camphora were identified to be linalool (26.6%),
eucalyptol (16.8%), α-terpineol (8.7%), isoborneol (8.1%), β-phellandrene (5.1%), and camphor (5.0%). The EO
had good activity against MRSA, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Salmonella galli-
narum and Escherichia coli. MRSA was selected as the model bacterium to illustrate antibacterial mechanism of
action of the EO, and the MIC and MBC values was 0.8 and 1.6 mg/mL, respectively. Apoptosis rate of MRSA
increased in a concentration-dependent manner after the addition of EO. The cell morphology was damaged by
the EO. There were 74 significantly different metabolites, including 29 upregulated and 45 downregulated
metabolites in the result of metabolomics evaluation. Seven pathways were enriched by shared differential
metabolites. The EO enhanced the activity of ICDH by 47.35%, while weaken MDH, SDH and α-KGDH by
72.63%, 31.52% and 63.29%, respectively.
Conclusions: The EO from C. camphora showed anti-MRSA activity via damaging cell membranes and disturbing
the amino metabolism.

1. Introduction bacterial (Yeh et al., 2009), anti-oxidative, anti-fungal (Pragadheesh

Cinnamomum camphora (Linn.) Presl, a subtropical evergreen tree
species, which is widely cultivated in the south of China as a common
Chinese medicine (Chen et al., 2018; Zhou et al., 2017). Pharmacolo-
gical studies showed that the EO from C. camphora had a wide spectrum
of bio-physiological functions (Singh and Jawaid, 2012) including anti-
et al., 2013), anti-inflammatory (Kang et al., 2019), insecticidal (Chen
et al., 2014) and repellent activities (Jiang et al., 2016). In traditional
Chinese medicine and the folk medicine, essential oil has been em-
ployed for the treatment of bacterial infection for a long history
(Marasini et al., 2015; Singh et al., 2008). Essential oils derived from
medicinal plants have come to be a promising alternative to traditional

∗
Corresponding author.
E-mail address: lssydp@mail.sysu.edu.cn (D. Yang).

https://doi.org/10.1016/j.jep.2020.112652
Received 18 October 2019; Received in revised form 11 January 2020; Accepted 3 February 2020
Available online 06 February 2020
0378-8741/ © 2020 Elsevier B.V. All rights reserved.
J. Chen, et al.
Abbreviations
ATP adenosine triphosphate
CFU colony forming units
CLSI Clinical and Laboratory Standards Institute
CS citrate synthetase
DCFH-DA 2,7-dichlorodihydrofluorescein diacetate
EO essential oil
FESEM field emission scanning electron microscopy
GC-MS Gas chromatography-mass spectrometry
ICDH isocitrate dehydrogenase
α-KGDH α-ketoglutarate dehydrogenase
antimicrobials in recent years for their excellent antibacterial properties
(He et al., 2019; Winska et al., 2019). The chemical constituents of the
EO from C. camphora have been studied in detail; however, there are
few reports on the mechanism of action of the EO from C. camphora.
Methicillin-resistant Staphylococcus aureus (MRSA), an important
pathogen, could lead to a variety of diseases such as skin, soft tissue and
life-threatening systemic infections (Dantes et al., 2013). More and
more public attention was paid to MRSA because of its complex epi-
demiology and its ability to acquire novel antibiotic resistance me-
chanisms. Furthermore, studies have shown that existing antibiotics are
becoming more and more limited, which results in the need of
searching for more additional new agents (Gould et al., 2012).
It has been reported that C. camphora showed strong antibacterial
activity (Marasini et al., 2015). However, its antibacterial mechanism is
still unclear. Recently, metabolomics has become effective tool for
understanding the antibacterial effects of different agents on bacteria
deeply and systematically (Campos and Zampieri, 2019; Zampieri,
2018). Metabolomics provides a multiparametric description of drug
action and detects dynamic changes in the abundance of metabolite in
response to drug treatment (Pang et al., 2019). In addition, identifica-
tion of key biomarkers through pathway analysis is used to detect
changes in metabolites during antimicrobial activity (Beyoglu and Idle,
2013; Ibanez et al., 2017; Wishart, 2016). Studies on the metabolomics
of MRSA showed that the tricarboxylic acid (TCA) cycle of MRSA was
often affected by external conditions (Rutowski et al., 2019; Schelli
et al., 2017). Liebeke et al. (2011) discovered a significant increase in
concentrations of endocellular amino acids and alterations in the levels
of enzymes of TCA cycle. Thus, metabolomics was also applied to ex-
plore the antibacterial mechanism of the EO in this study.
In order to discover new antimicrobials and explain the anti-
bacterial mechanism of action of the EO, this study was designed to
investigate the antibacterial effect of the EO from C. camphora. The
effects of the EO on MRSA were evaluated by growth curve, apoptosis,
field emission scanning electron microscopy (FESEM) observation,
transmission electron microscope (TEM) observation and the integrity
of cell membrane assays. To further investigate the possible mechanism,
we used GC-MS-based metabolomics and measured the enzyme activity
of MRSA in TCA cycle. Through these assays, we desire to provide
better insight into the antibacterial mechanism of action of the EO.
2. Materials and methods
2.1. Materials
Bacterial peptone and yeast extract were obtained by Guangdong
Huankai Microbial Sci. & Tech. Co., Ltd. (Guangzhou, China). Glucose,
sodium chloride and sodium hydroxide were from Tianjin zhiyuan
chemical reagent Co., Ltd (Tianjin, China). Methoxyamine hydro-
chloride, pyridine and N-methyl-N-(trimethylsilyl) trifluoroacetamide
(MSTFA) were obtained from Sigma-Aldrich (Sy.Louis, USA).
Anhydrous sodium sulfate, sodium phosphate dibasic dihydrate,
2
Journal of Ethnopharmacology 253 (2020) 112652
LB Luria-Bertani
MBC minimal bactericidal concentration
MIC minimum inhibitory concentration
MRSA methicillin resistant Staphylococcus aureus
NIST National Institute of Standards and Technology
SDH succinate dehydrogenase
TCA tricarboxylic acid
MDH malate dehydrogenase
NADH nicotinamide adenine dinucleotide;
NADP+ nicotinamide adenine dinucleoside phosphorus
NADPH nicotinamide adenine dinucleotide phosphate
TEM transmission electron microscope
magnesium sulfate and calcium chloride were purchased from
Guangzhou chemical reagent factory (Guangzhou, China). Ethanol,
tertiary butyl ethanol, acetate and glutaraldeyde were from Tianjin
damao chemical reagent factory (Tianjin, China). HPLC methanol was
from Oceanpak Alexative Chemical., Ltd (Goteborg, Sweden).
Succinate dehydrogenase (SDH), malic dehydrogenase (MDH), iso-
citrate dehydrogenase (ICDH) and α-ketoglutarate dehydrogenase (α-
KGDH) reagent kits were purchased from Solarbio Science &
Technology Co., Ltd. (Beijing, China). Enhanced BCA Protein Assay Kit
was from Beyotime Institute of Biotechnology (Shanghai, China).
2.2. Plant material and distillation of the EO
Leaves of C. camphora. were collected in March 2019 from adult
trees located in the campus of the Sun Yat-Sen University and identified
by Professor Depo Yang, School of Pharmaceutical Sciences, Sun Yat-
Sen University. A voucher specimen for C. camphora. (NO. A20190301)
has been deposited at School of Pharmaceutical Sciences, Sun Yat-Sen
University. The EO was extracted by the hydrodistillation in a
Clevenger-type apparatus for 2 h, and then dehydrated by filtration
with anhydrous sodium sulfate. The recovered EO samples were stored
in the dark at 4 °C until analysis.
2.3. Bacterial strains and growth conditions
The antibacterial activity of the EO was tested against 7 bacteria,
including 4 gram-positive (Staphylococcus aureus (S. aureus) ATCC
25923, methicillin-resistant Staphylococcus aureus (MRSA) ATCC
43300, Enterococcus faecalis (E. faecalis) ATCC29212 and Bacillus subtilis
(B. subtilis) ATCC 21332) and 3 gram-negative bacteria (Pseudomonas
aeruginosa (P. aeruginosa) ATCC 27853, Salmonella gallinarum (S. galli-
narum) CVCC 79207 and Escherichia coli (E. coli) ATCC 25922). These
bacteria were provided by microbial culture collection center of
Guangdong Institute of Microbiology and stored in glycerin at −80 °C.
All strains were cultured at 37 °C on Luria-Bertani (LB) medium.
2.4. Chemical compositions of the EO
The EO was diluted 1:99 (v/v) in ethyl acetate. The compositions of
the EO were analyzed by an Agilent 7890A gas chromatographic (GC)
instrument coupled to an Agilent 5975 mass spectrometer (MS) (Agilent
Technologies, Inc., Santa Clara, CA, USA), equipped with an HP-5MS
capillary column (30.00 m × 250.00 μm × 0.25 μm film thickness).
The carrier gas, helium, was injected at a constant flow rate of 1.0 mL/
min. The temperature of injector was 280 °C. The sample (1 μL) was
injected in the split mode (1:5). The initial oven temperature was 40 °C
and was maintained at 40 °C for 5 min, then the oven temperature
would be gradually raised to 280 °C at 5 °C/min and was finally
maintained for 5 min. The mass spectrometer was operated at an io-
nization voltage of 70 eV and a mass range of 40–600 m/z. The re-
tention indices were calculated for all constituents using a homologous
J. Chen, et al.
series of n-alkanes C7–C40. The essential oil constituents were identi-
fied by comparing their GC retention indices and the National Institute
of Standards and Technology 17 (NIST17) library in combination with
the published data, while the relative content of each component was
calculated according to the area of the chromatographic peaks based on
the FID.
2.5. Antibacterial activity
2.5.1. Determination of minimum inhibitory concentration (MIC) and
minimal bactericidal concentration (MBC)
MIC and MBC tests were performed following the Clinical and
Laboratory Standards Institute (2017) (CLSI 2017) guidelines using the
broth microdilution method. The EO was dissolved in 1%Tween 80
(Mazzarrino et al., 2015) and prepared in sterile LB medium ranging
from 0.0125 to 12.8 mg/mL. Bacterial suspensions containing 1 × 107
colony forming units (CFU) /mL(10 μL) were added in a 96-well plate,
and 1%Tween 80 was used as a control. (Mazzarrino et al., 2015; Silva
et al., 2019). Bacteria were incubated at 37 °C for 18 h. MBC was the
minimum concentration that has no visible bacterial growth after in-
cubating 50 μL subcultures from each well on LB agar plates at 37 °C for
24 h. All tests were performed in triplicate.
2.5.2. Effect of the EO on the growth curve
The growth curve protocol was used to investigate the bactericidal
effects of the EO. Bacteria were treated with the EO at 1/4 MIC, 1/2
MIC and 1 MIC, respectively. The control group only contained 1%
tween 80. Then the bacteria were cultivated at 37 °C with shaking
180 rpm for 0, 2, 4, 6, 8, 10, 12, 24 and 30 h, respectively. At selected
time intervals, the OD600 of supernatants was determined by UV–Vis
spectrophotometer (Huang et al., 2018). The experiment was done in
triplicate.
2.6. Antimicrobial mechanism
2.6.1. Apoptosis analysis
Logarithmic growth phase cells of MRSA were treated with different
concentrations of the EO at 37 °C for 6 h and then washed with PBS.
Cells were stained with Annexin V-FITC and PI (BD Pharmingen, San
Diego, CA, USA) following the manufacturer's instructions. Bacterial
samples were analyzed by a BD Biosciences FACSCalibur. Apoptosis
analysis was based on the method described by previous scholar (Zhang
et al., 2017). Tests were performed in triplicate.
2.6.2. Field emission scanning electron microscope and transmission
electron microscope
FESEM and TEM were used to observe the morphological changes of
bacteria induced by the EO (Liu et al., 2019). Logarithmic growth phase
cells of MRSA were incubated at 37 °C for 6 h with the EO at different
concentrations, and then centrifuged and washed three times with
Milloning's phosphate buffer (0.1M, pH7.2).
For FESEM observation, firstly, these samples were fixed with glu-
taraldeyde (2.5% v/v) in Milloning's phosphate buffer (0.2M, pH7.2) at
4 °C for 12 h. Secondly, samples were dehydrated by 30%, 50%, 70%,
90% and 100% alcohol for 15 min each. Thirdly, the ethanol was then
replaced by 100% tertiary butyl alcohol. Finally, the samples were
dried and sputtered with gold then visualized under field emission
scanning electron microscope (FE - SEM, JEOL JSM-6330F, JAPAN).
For TEM observation, treated cells were fixed with 2.5% glutar-
aldehyde and 1% osmic acid for 12 h and 2 h, respectively. After
washing with PBS (0.1M,pH7.0), the samples were dehydrated in a
series of graded ethanol solutions (30%,50%,70%, 80%, 90%, 95% and
100%), then treated with pure acetone for 20min, finally infiltrated in
Spurr812 epoxide resin, embedded, and left to polymerize overnight at
70 °C in an oven. Each sample was cut into thin sections, ~70-90 nm,
using an ultramicrotome, and double stained with 50% uranyl acetate,
3
Journal of Ethnopharmacology 253 (2020) 112652
and lead citrate. Morphology and ultrastructure of bacteria were ob-
served and photographed by transmission electron microscope (FEI
Tecnai Spirit TEM, USA) at 100 kv.
2.6.3. Integrity of cell membrane
The integrity of the cell membrane could be evaluated by releasing
cytoplasmic constituents from the supernatant. In brief, logarithmic
growth phase cells of MRSA were washed three times with sterile saline,
collected by centrifugation at 5000 rpm for 10 min and resuspended in
M9 minimal media (1 × 109 CFU/mL) (Rugbjerg et al., 2018). Samples
were treated with the EO at 1/4 MIC, 1/2 MIC and 1 MIC value except
the control group. Samples were incubated at 37 °C for 2, 6, 18 and 24 h
respectively, and then immediately filtered through 0.22 μm Millipore
filter (Meng et al., 2016). To determine the amounts of the DNA and
RNA released from the cytoplasm, the supernatant was used to measure
the optical density at 260 nm with Thermo Scientific NanoDrop 2000.
The protein content was determined by the Enhanced BCA Protein
Assay Kit. Each experiment was performed in triplicate.
2.6.4. Metabolomics
Logarithmic growth phase bacteria were collected by centrifugation
at 5000 rpm for 10 min, washed three times with PBS, and resuspended
in LB (1 × 109 CFU/mL). Samples were treated with the EO at 37 °C for
6 h followed by washing with PBS. The bacteria were collected by
centrifugation and ground with liquid nitrogen. Ground cells were first
suspended in methanol, and then were centrifuged at 12000 rpm at 4 °C
for 10 min. Supernatant of each sample was collected, and 10 μL of
0.1 mg/mL ribitol was included as an analytical internal standard. In
addition, samples only with the reagents were used as a control. The
supernatants were concentrated in a rotary vacuum centrifuge device
(LABCONCO). The dried extracts were used for GC-MS analysis.
GC–MS analysis was performed with a little bit adjustment based on
the technique from previous study (Du et al., 2017; Su et al., 2018; Zeng
et al., 2017). Samples were derivatized. Methoximation was carried out
to protect carbonyl moieties through a 90 min 37 °C reaction with 80 μL
of 20 mg/mL methoxyamine hydrochloride in pyridine. Then deriva-
tization of acidic protons was performed through a 30 min reaction at
37 °C with the addition of 80 μL of N-methyl-N-(trimethylsilyl) tri-
fluoroacetamide (MSTFA). The derivatized sample (1 μL) was injected
into a DB-5MS capillary column (30 m × 250 μm i.d. × 0.25 μm) by
using splitless model, and analysis was fulfilled on an Agilent 7890A/
5975MSD system. The temperature of the GC oven was initially kept at
70 °C for 5 min, and then increased to 270 °C at a rate of 2 °C/min, and
finally held for 5 min. Electron impact ionization was of 70 eV energy.
Helium was used as the carrier gas with flow rate of 1 mL/min. The
mass spectrometer was operated at 60–600 m/z. Each sample had six
biological and two technical replicates.
2.6.5. Measurement of enzyme activity
Bacteria were collected by centrifugation at 5000 rpm for 10 min,
washed three times with PBS, and resuspended in LB (1 × 109 CFU/
mL). Then samples were incubated at 37 °C with the EO for 6 h and the
control group only contained 1% tween 80. Cells were collected by
centrifugation at 8000 rpm for 5 min at 4 °C. Cells were washed by PBS
three times. Then, the isocitrate dehydrogenase (ICDH), α-ketoglutarate
dehydrogenase (α-KGDH), succinic dehydrogenase (SDH) and malic
dehydrogenase (MDH) activities in MRSA cells with different con-
centrations of the EO were analyzed spectrophotometrically by using
commercially available kits according to manufacturer's instructions.
Inhibition rate % = [(sample - control) /control] *100%. The experi-
ment was done in triplicate.
2.7. Data analysis
2.7.1. Statistical analysis
All data were expressed as mean ± SD with the statistical method
J. Chen, et al. Journal of Ethnopharmacology 253 (2020) 112652

of one-way analysis of variance (ANOVA) and Student's t-test using the 1 MIC separately. While the rate of necrotic bacterial cells increased
SPSS 19.0 software. A P value less than 0.05 was considered as a sta- from 1.00% to 31.50%. These results suggested that the EO could in-
tistically significant difference. duce apoptosis in MRSA cells in a concentration-dependent manner,
respectively.
2.7.2. Metabolomics data analysis
The mass fragmentation spectrum was analyzed by Agilent
Technologies MSD Productivity ChemStation software to identify the 3.3.2. Field emission scanning electron microscopy and transmission
compounds by matching the data with the NIST 17 library. Metabolite electron microscope
data were normalized by ribitol as internal standard and scaled by the In order to understand the direct influence of the EO on the mor-
quartile range in the sample for subsequent analysis. Scaling each phological and physical changes of MRSA cells, FESEM and TEM was
metabolite in terms of a reference distribution and calculating on the used in our study. The FESEM and TEM photograph demonstrate that
basis of the mean and standard deviation of reference sets control there was morphological damage when cells were treated with the EO
through Z-score analysis. Hierarchical clustering was carried out on the (Fig. 2B&C). As concentrations of the EO increased, cells membranes of
quartile normalize date, completed in the R platform with the package MRSA also exhibited relatively increasing damage. On the contrary, the
gplots (http://www.r-project.org/) by using the distance matrix. SPSS control group exhibited a spherical shape with a complete and smooth
19.0 and GraphPad Prism 5.0 were used to draw the histogram of the surface.
scatter plot. Multivariate statistical analysis included principal com-
ponent analysis (PCA) and orthogonal-PLS-DA (OPLS-DA) (SIMCA-
P+12.0.1), which was used to discriminate sample patterns. The Kyoto
Table 1
Encyclopedia of Genes and Genomes (KEGG) analysis of the metabo- Chemical composition of the EO.
lomics results were performed using Metaboanalyst (https://www.
No Compounds RI Percentage (%)
metaboanalyst.ca/MetaboAnalyst/faces/docs/Resources.xhtml).
1 Linalool 1099 26.6
3. Results 2 Eucalyptol 1032 16.8
3 α-Terpineol 1190 8.7
3.1. Chemical compositions of the EO 4 Isoborneol 1157 8.1
5 β-Phellandrene 1005 5.1
6 Camphor 1144 5.0
The chemical compositions of the EO were analyzed by GC–MS and 7 Caryophyllene 1419 3.3
the results are showed in Table 1. There were 48 constituents identified, 8 Naphthalene 1179 3.1
which was 98.4% of the EO. Linalool was the major compound (26.6%), 9 α-Pinene 937 2.2
10 Humulene 1579 1.9
followed by eucalyptol (16.8%), α-terpineol (8.7%), isoborneol (8.1%),
11 γ-Elemene 1434 1.3
β-phellandrene (5.1%), and camphor (5.0%). The components were 12 β-Myrcene 991 1.3
similar to the previous reports (Chen et al., 2014; Guo, et al., 2016b; 13 Terpinen-4-ol 1182 1.1
Jiang et al., 2016; Yu et al., 2019). 14 5-Isopropyl-2-methylbicyclo[3.1.0]hexan-2-ol 1075 1.1
15 (−)-Spathulenol 1576 0.9
16 Germacrene D 1432 0.8
3.2. Antibacterial activity 17 Propanoic acid, ethyl ester < 700 0.8
18 β-Pinene 937 0.7
3.2.1. MIC and MBC of the EO 19 Caryophyllene oxide 1581 0.6
The antibacterial activities of the EO are shown in Table 2. The 20 α-Phellandrene 1005 0.6
21 Cyclohexane 1391 0.6
results showed that the EO displayed remarkable antibacterial activity
22 Nerolidol 1564 0.5
against both tested Gram-positive and Gram-negative bacteria. The 23 Camphene 952 0.5
MICs of the EO against MRSA, S.aureus, B. subtilis, E. faecalis, E. coli and 24 2-Furanmethanol < 700 0.5
S. gallinarum were 0.8, 1.6, 1.6, 1.6, 3.2 and 1.6 mg/mL, respectively. 25 Methyl Isobutyl Ketone < 700 0.5
While the EO has no antibacterial effect on P.aeruginosa. 26 γ-Terpinene 1060 0.5
27 β-Ocimene 1038 0.5
28 γ-Muurolene 1477 0.4
3.2.2. Effect of the EO on the growth curve of MRSA 29 2,4,6-Octatriene, 2,6-dimethyl- 1131 0.4
As shown in Fig. 1A, the control group exhibites a typical growth 30 2-Carene 1001 0.4
curve including lag, exponential and stabilization period; however, 31 Cis-Calamenene 1524 0.3
32 Neointermedeol 1660 0.3
there was no distinct decline period because the OD600 value contained
33 Geraniol 1255 0.3
both live and dead bacteria (Guo et al., 2019). Groups treated with the 34 Acetic acid < 700 0.3
EO exhibited strong inhibition on the growth of cells in varying degrees. 35 Longipinane 1398 0.3
The control group reproduced from the beginning, but 1/4 MIC and 1/2 36 (+)-4-Carene 1009 0.3
MIC groups did not reproduce in the initial treatment period until 6 h. 37 Trans-Carveol 1090 0.3
38 Cis-Linaloloxide 1086 0.2
Unlike the changing trend of the number of viable cells at 1/4 MIC and 39 Isospathulenol 1549 0.2
1/2 MIC groups, which of the groups with treatment of 1 MIC group did 40 β-Eudesmol 1649 0.2
not increased throughout the experiment. 41 Copaene 1376 0.2
42 1H-Imidazol-2-amine < 700 0.2
43 Aromandendrene 1440 0.2
3.3. Antibacterial mechanism of action of the EO
44 Alloaromadendrene 1461 0.2
45 Isopropyl acetate < 700 0.1
3.3.1. Apoptosis analysis 46 3-Methyl-3-buten-1-ol < 700 0.1
Cell apoptosis assay was performed to determine the apoptosis-sti- 47 Phytol 2114 0.1
mulating effects of the EO against MRSA. As shown in Fig. 2A, the rates 48 Valerenol 1695 0.1
49 Others 1.6
of living cells of MRSA (Q4) declined from 97.0% to 17.8% with in- Total 100.0
creasing EO concentration from 0 to 0.8 mg/mL (1MIC). The apoptotic
rate of the control was 2.03% and those of EO treatment groups were RI: the retention index was calculated for all volatile constituents using a
3.95%, 28.07% and 50.73% at concentrations of 1/4 MIC, 1/2 MIC, and homologous series of n-alkanes C7–C40 on HP-5MS column.

4
J. Chen, et al. Journal of Ethnopharmacology 253 (2020) 112652

Table 2 metabolites detected), lipids (24metabolites detected) and others. As

MICs and MBCs of the EO against tested bacteria. compared to the control group, the abundance of 74 metabolites in EO
Microorganisms MICs MBCs group was different (Fig. 4A). The Z-value based on the control group
was calculated for comparative study, which displayed variations of
EO Antibiotic EO (mg/mL) Antibiotic these metabolites. The Z-Score plot showed that it spanned from
(mg/ (μg/mL) (μg/mL) −23.29 to 327.14 in the EO group (Fig. 4B). Specifically, there were 45
mL)
metabolites decreased and 29 metabolites increased in the EO group.
Gram-positive MRSA 0.8 8.0a 1.6 32 In order to investigate the metabolic differences between the EO-
S. aureus 1.6 0.5a 3.2 1.0 treated cells and control cells, orthogonal partial least-squares dis-
B. subtilis 1.6 0.8b 1.6 0.8 criminant analysis (OPLS-DA) was applied for the recognition of the
E. faecalis 1.6 3.2b 1.6 3.2
sample patterns, followed by ranking the altered metabolites in loading.
Gram-negative E. coli 3.2 0.8b 3.2 0.8
S. gallinarum 1.6 3.2b 1.6 3.2 The EO group was separated from control group clearly (Fig. 5 A).
P. aeruginosa ND 3.2b ND 3.2 Discriminating variables were present with S-plot (Fig. 5 B) when cut-
off values were set as greater or equal to the 0.05 and 0.5 for the ab-
ND: Not detected. solute value of covariance p and correlation p (corr), respectively. As-
a
Oxacillin Sodium.
b
partic acid, glutamic acid, proline and urea had the largest correlations
Gentamycin.
and covariances in predictive component between the control and the
EO group (Fig. 5 C).
3.3.3. Integrity of cell membrane
Seven pathways were enriched by shared differential metabolites,
As shown in Fig. 1B, the DNA of supernatant from MRSA cells
including alanine, aspartate and glutamate metabolism, sulfur meta-
treated with the EO (1/4 MIC) was 11.3, 2.7, 2.4 and 1.5 times as high
bolism, arginine and proline metabolism, cysteine and methionine
as that of the control for 2, 6, 18 and 24 h, respectively. They increased
metabolism, aminoacyl-tRNA biosynthesis, cyanoamino acid metabo-
by 23.9, 4.8, 4.0 and 2.5 times as high as that of the control when
lism and streptomycin biosynthesis between the two groups (Fig. 6A).
treatment with the EO (1/2 MIC) for 2, 6, 18 and 24 h, respectively,
The first five pathways were the most impacted pathways, where amino
while they increased by 34.5, 7.1, 6.2 and 3.5 times, respectively when
acids metabolism was the most significantly adjusted pathway. Ac-
treated 1 MIC. The results of RNA and protein leakage (Fig. 1C&D) in
cording to metabolic difference, four of those were obviously accounted
culture medium were similar.
to cell metabolism, which included alanine, aspartate and glutamate
metabolism, arginine and proline metabolism, cysteine and methionine
3.3.4. Metabolomics analysis metabolism, and aminoacyl-tRNA biosynthesis (Fig. 6B). Most of me-
In this study, six biological and two technical replicates yielded 24 tabolites enriched in the four pathways decreased. As shown in Fig. 6A,
data. After data processing, there were 80 metabolites for subsequent alanine aspartate and glutamate metabolism pathway was the biggest
analysis. KEGG was used to retrieve the biological categories of iden- factor impacting altered metabolic activity under the EO stress with
tified metabolites. These detected metabolites can be categorized into considerable enrichment (p < 0.05). These results indicate that the EO
five major classes (Fig. 3), including carbohydrates (13 metabolites showed antibacterial activity via disturbing the cell metabolism.
detected), amino acids (26 metabolites detected), nucleotides (3

Fig. 1. (A) The growth curve of MRSA treated with the EO at 0, 1/4 MIC, 1/2 MIC and 1 MIC. Leakage of (B) DNA, (C) RNA and (D) protein from MRSA treated with
the EO at 0, 1/4 MIC, 1/2 MIC and 1 MIC for 2, 6, 18, 24 h. Data were expressed as mean ± SEM, n = 3. **p ≤ 0.01; ***p ≤ 0.001 vs control group. All tests were
performed in triplicate.

5
J. Chen, et al.
Fig. 2. (A) The apoptosis of MRSA treated with the EO at 0, 1/4 MIC, 1/2 MIC and 1 MIC for 6 h. (B) FESEM of MRSA (magnification × 20,000) treated with the EO
at 0, 1/4 MIC, 1/2 MIC and 1 MIC for 6 h. (C) TEM of MRSA (magnification × 68,000) treated with the EO at 0, 1/4 MIC, 1/2 MIC and 1 MIC for 6 h. All tests were
performed in triplicate.
Fig. 3. Categories of metabolites detected.
3.3.5. Enzyme activity analysis
To further demonstrate the effect of the EO on MRSA metabolism,
we measured the activities of ICDH, α-KGDH, MDH and SDH, key en-
zymes associated with the TCA cycle. As shown in Fig. 7B, compared to
the untreated one, the activities for MDH, SDH and α-KGDH decreased
by 72.63, 31.52 and 63.29%, respectively, after treatment with EO at 1/
2 MIC for 6 h, whereas, ICDH increased by about 47.35%.
6
Journal of Ethnopharmacology 253 (2020) 112652
4. Discussion
Cell membrane integrity plays an important role in cell growth. Our
results showed that the EO of C. camphora had serious effects on cell
walls and plasma membrane(Fig. 2B&C). EO significantly increased the
release of cytoplasmic components in a concentration and time de-
pendent manner (Fig. 1B–D) in our study, and there was no significant
difference between 18 and 24 h owing to the bacteria growth got into
the stationary period. We suggested that the leakage of intracellular
substances might be caused by the damage of the cell membranes, and
it was consistent with the previous study (Fang et al., 2019). FESEM and
TEM (Fig. 2B&C) analysis demonstrated that MRSA cells treated with
the EO exhibited collapsed surfaces and anomalous shapes, and there
were cell fragments around, which validated membrane was damaged
combining the leakage of intracellular substances of MRSA cells.
Studies have shown that not only damage of cell membranes but
also metabolic disturbance can lead to bacteria death (He et al., 2019;
Xu et al., 2017). The level of bacterial metabolites can be used as an
important indicator to reflect the inhibition or activation of related
metabolic pathways (An et al., 2019). As shown in the Z-Score plot,
there were 74 significantly different metabolites, including 29 upre-
gulated and 45 downregulated metabolites in the treatment group
compared with the control group (Fig. 4B). Furthermore, PCA and S-
plot score plot (Fig. 5) showed that the sample points of the two groups
were separated and clustered well in a certain range, which means
normal physiological metabolism of bacteria was disturbed after
treatment of the EO. Analysis of the metabolic categories of the selected
differential metabolites showed various percentages (Fig. 3) with a
ranking of amino acids > lipids > carbohydrates > nucleotides in
J. Chen, et al. Journal of Ethnopharmacology 253 (2020) 112652

Fig. 4. Metabolic profiles of MRSA in response to the EO. (A) Heat maps of differential metabolites (row). Yellow and blue indicate an increase and decrease of the
metabolites relative to the mean and standard deviation of the row metabolite level, respectively (see color scale). (B) Z-score scatter diagrams of significant
differentially expressed metabolites (p < 0.05) in the presence or absence of the EO. Metabolites are showed on the y-axis. (For interpretation of the references to
color in this figure legend, the reader is referred to the Web version of this article).

the EO groups. These results indicate that amino acid metabolism plays
a crucial role in the antibacterial activity.
The alanine, aspartate and glutamate metabolism had the greatest
impact on the seven metabolic pathways (Fig. 6A). TCA cycle, the
metabolic process by which ATP is generated, is an important part of
the alanine, aspartate and glutamate metabolism (Fig. 7A). Our results
showed that two upstream metabolites (citrate and succinate) detected
decreased, whereas two downstream metabolites (fumarate and ma-
late) increased in TCA cycle, indicating that the TCA pathway might be
disrupted. TCA cycle contains several key enzymes (ICDH, α-KGDH,
SDH, MDH) and produces critical intermediates such as isocitrate,
succinate, malate, and α-ketoglutarate. The activities of ICDH regulate
the influx of isocitrate into the TCA cycle; α-KGDH reduces production
of NADH through the TCA cycle and the succeeding ability of the cell to
synthesize ATP (Guo,. et al., 2016a); SDH catalyzes the oxidation of
succinic acid to fumaric acid and transfers electrons from succinic acid
to ubiquinol, playing an obbligato role in the energy metabolism of
bacteria, and its activity reflects the energy metabolism (Xu et al., 2017;
Zheng et al., 2015); MDH, catalyzes the interconversion of malate to
oxaloacetate. NADP+ is a coenzyme of dehydrogenases, which forms
nicotinamide adenine dinucleotide phosphate (NADPH) by accepting
hydrogen from metabolites during biosynthesis; To further demonstrate
the effect of the EO on TCA cycle, we measured the activity of the key
enzymes (ICDH, α-KGDH, SDH, MDH). The increased activity of ICDH
promoted the conversion of citrate to α-ketoglutarate and led to the
reduction of citrate. The decreased activity of α-KGDH might lead to
reduced production of succinyl-CoA. Succinate was transformed from
succinyl-CoA, therefore, the decrease in succinate was indirectly due to
the decrease of α-KGDH. Similarly, the decrease of SDH activity was
expected to cause the decrease of fumarate; however, the result was
opposite, because fumarate not only could be converted from succinate
in TCA cycle but also could be converted from aspartate in the alanine,
aspartate and glutamate metabolism, which increased the abundance of
fumarate significantly. Nicotinamide adenine dinucleotide (NADH) is a

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J. Chen, et al. Journal of Ethnopharmacology 253 (2020) 112652

Fig. 5. PCA and S-plot score plot of features obtained from GC-MS of MRSA with or without the EO treatment. (A) PCA analysis of two groups. Each dot represents the
technological replicate analysis of samples. (B) S-plot generated from OPLS-DA. Predictive component p [1] and correlation p (corr) [1] differentiate the treatment
from the control. Dot represents metabolites, which are p [1] < −0.05 or > 0.05 and p (corr) [1] < −0.5 or > 0.5 and marked in red dot. (C) Scatter diagram of 4
biomarkers, each dot shows a technical replicate. Data were expressed as mean ± SEM, n = 6. ***p < 0.01 vs control group. (For interpretation of the references to
color in this figure legend, the reader is referred to the Web version of this article).

metabolic coenzyme that plays an important role in cell metabolism. 5. Conclusion

The activities of MDH decreased by 72.63% in our study. Refer to
previously study, we speculated MDH reduced the production of NADH
through the TCA cycle and the subsequent ability of the cell to syn-
thesize ATP, which caused the disturbance of TCA cycle (Xu et al.,
2017). When α-KGDH and MDH activity was inhibited significantly,
NADH generated in TCA cycle would decrease, which indicated energy
supply was blocked and TCA cycle was disturbed. Zheng showed that
Penicillium digitatum cells treated with citral suffered a decrease in the
activities of TCA-related enzymes and disrupted the TCA pathway
(Zheng et al., 2015). Moreover, Xu reported that the effect of tea tree oil
on Botrytis cinerea was achieved mainly by disrupting the TCA cycle by
decreasing the activities of ICDH, α-KGDH, SDH, MDH (Xu et al., 2017).
Similarly, our results verified that the EO interfered TCA cycle by af-
fecting the activities of key enzymes.
In summary, a metabolomics approach was adopted to investigate
the antibacterial mechanisms of action of the EO. Our studies showed
that the EO can increase the apoptosis rate and disrupt cell structures
including cell wall and membrane, leading to leakage of essential mo-
lecules such as DNA, RNA and protein. The EO can inhibit the re-
production of MRSA, via damaging the membrane, decreasing activities
of TCA-related enzymes and disrupting of TCA cycle. Therefore, the EO
from the leaves of C. camphora are potential antimicrobial agents to
solve the problem of bacterial resistance. Furthermore, these results
could contribute to a detailed understanding of the mechanisms that the
EO acts as an antibacterial agent against MRSA via damaging cell
membranes and disturbing the amino metabolism.

Fig. 6. (A) Enriched pathways in the presence of the EO significantly enriched pathways were selected to plot. (B) Heat map of the average levels of differential
metabolites in the four enriched pathways shared by the two group. Red and green, respectively, indicate increase and decrease in the metabolites scaled to mean and
standard deviation of row metabolite level (see the color scale). (For interpretation of the references to color in this figure legend, the reader is referred to the Web
version of this article).

8
J. Chen, et al.
Fig. 7. (A) Superimposed on metabolic pathways related to alanine, aspartate and glutamate metabolism. Red and black, respectively, indicate increase and decrease
Red, increased abundance of metabolites; Grey, indicate metabolites not detected; Bold, indicate key enzymes (ICDH, α-KGDH, SDH, MDH) in TCA cycle. (B)
Measurement of the EO on SDH, MDH, ICDH and α-KGDH activities in MRSA cells. Data were expressed as mean ± SEM, n = 3. **p ≤ 0.01; ***p ≤ 0.001 vs control
group. All tests were performed in triplicate. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this
article).
Author contributions
Jiali Chen designed the research, performed the experiment and
wrote the manuscript. Cailin Tang designed the research and reviewed
the manuscript. Rongfei Zhang and Shaoxia Ye revised drafts of the
manuscript. Depo Yang, Zhimin Zhao, Xinjun Xu and Yuquan Huang
supervised the work and reviewed the manuscript. All authors read and
approved the final manuscript.
Acknowledgments
This work was financially supported by the National Key R&D
Program of China (NO. 2017YFC1701100) and Guangzhou Science and
Technology Project (NO. 201904010203).
References
An, P., Yang, X., Yu, J., Qi, J., Ren, X., Kong, Q., 2019. α-terpineol and terpene-4-ol, the
critical components of tea tree oil, exert antifungal activities in vitro and in vivo
against Aspergillus Niger in grapes by inducing morphous damage and metabolic
changes of fungus. Food Contr. 98, 42–53.
Beyoglu, D., Idle, J.R., 2013. Metabolomics and its potential in drug development.
Biochem. Pharmacol. 85 (1), 12–20.
Campos, A.I., Zampieri, M., 2019. Metabolomics-driven exploration of the chemical drug
space to predict combination antimicrobial therapies. Mol. Cell. 74 (6), 1291–1303.
Chen, H.P., Yang, K., You, C.X., Lei, N., Sun, R.Q., Geng, Z.F., Ma, P., Cai, Q., Du, S.S.,
Deng, Z.W., 2014. Chemical constituents and insecticidal activities of the essential oil
of Cinnamomum camphora leaves against Lasioderma serricorne. J. Chem. 1–5 2014.
Chen, S., Zheng, T., Ye, C., Huannixi, W., Yakefu, Z., Meng, Y., Peng, X., Tian, Z., Wang,
J., Ma, Y., Yang, Y., Ma, Z., Zuo, Z., 2018. Algicidal properties of extracts from
Cinnamomum camphora fresh leaves and their main compounds. Ecotoxicol. Environ.
Saf. 163, 594–603.
Clinical and Laboratory Standards Institute, 2017. Performance Standard for
9
Journal of Ethnopharmacology 253 (2020) 112652
Antimicrobial Susceptibility Testing, vol. 52. pp. 177 Twenty-seventh Informational
Supplement CLSI document M100-S27.
Dantes, R., Mu, Y., Belflower, R., Aragon, D., Dumyati, G., Harrison, L.H., Lessa, F.C.,
Lynfield, R., Nadle, J., Petit, S., Ray, S.M., Schaffner, W., Townes, J., Fridkin, S.,
Emerging infections program-active bacterial core surveillance, M.S.I., 2013.
National burden of invasive methicillin-resistant Staphylococcus aureus infections,
United States, 2011. JAMA Intern. Med. 173 (21), 1970-1978.
Du, C.C., Yang, M.J., Li, M.Y., Yang, J., Peng, B., Li, H., Peng, X.X., 2017. Metabolic
mechanism for l-leucine-induced metabolome to eliminate Streptococcus iniae. J.
Proteome Res. 16 (5), 1880–1889.
Fang, Z., Xu, L., Lin, Y., Cai, X., Wang, S., 2019. The preservative potential of Octopus
scraps peptides−Zinc chelate against Staphylococcus aureus: its fabrication, anti-
bacterial activity and action mode. Food Contr. 98, 24–33.
Gould, I.M., David, M.Z., Esposito, S., Garau, J., Lina, G., Mazzei, T., Peters, G., 2012.
New insights into meticillin-resistant Staphylococcus aureus (MRSA) pathogenesis,
treatment and resistance. Int. J. Antimicrob. Agents 39 (2), 96–104.
Guo, H., Madzak, C., Du, G., Zhou, J., 2016a. Mutagenesis of conserved active site re-
sidues of dihydrolipoamide succinyltransferase enhances the accumulation of alpha-
ketoglutarate in Yarrowia lipolytica. Appl. Microbiol. Biotechnol. 100 (2), 649–659.
Guo, S., Geng, Z., Zhang, W., Liang, J., Wang, C., Deng, Z., Du, S., 2016b. The chemical
composition of essential oils from Cinnamomum camphora and their insecticidal ac-
tivity against the stored product pests. Int. J. Mol. Sci. 17 (11), E1836.
Guo, J., Gao, Z., Li, G., Fu, F., Liang, Z., Zhu, H., Shan, Y., 2019. Antimicrobial and
antibiofilm efficacy and mechanism of essential oil from Citrus Changshan-huyou Y.
B. chang against Listeria monocytogenes. Food Contr. 105, 256–264.
He, F., Li, K., Zhang, X., Yang, Y., Fang, Y., Xiang, F., 2019. Components and antibacterial
activity of a novel essential oil from the nutrient broth of Eremothecium ashbyii
H4565. LWT (Lebensm.-Wiss. & Technol.) 101, 389–394.
Huang, J., Qian, C., Xu, H., Huang, Y., 2018. Antibacterial activity of Artemisia asiatica
essential oil against some common respiratory infection causing bacterial strains and
its mechanism of action in Haemophilus influenzae. Microb. Pathogenesis 114,
470–475.
Ibanez, C., Simo, C., Palazoglu, M., Cifuentes, A., 2017. GC-MS based metabolomics of
colon cancer cells using different extraction solvents. Anal. Chim. Acta 986, 48–56.
Jiang, H., Wang, J., Song, L., Cao, X., Yao, X., Tang, F., Yue, Y., 2016. GCxGC-TOFMS
analysis of essential oils composition from leaves, twigs and seeds of Cinnamomum
camphora L. Presl and their insecticidal and repellent activities. Mol 21 (4), 423.
Kang, N.J., Han, S.C., Yoon, S.H., Sim, J.Y., Maeng, Y.H., Kang, H.K., Yoo, E.S., 2019.
J. Chen, et al.
Cinnamomum camphora leaves alleviate allergic skin inflammatory responses in vitro
and in vivo. Toxicol. Res. 35 (3), 279–285.
Liebeke, M., Dorries, K., Zuhlke, D., Bernhardt, J., Fuchs, S., Pane-Farre, J., Engelmann,
S., Volker, U., Bode, R., Dandekar, T., Lindequist, U., Hecker, M., Lalk, M., 2011. A
metabolomics and proteomics study of the adaptation of Staphylococcus aureus to
glucose starvation. Mol. Biosyst. 7 (4), 1241–1253.
Liu, M., Yang, K., Wang, J., Zhang, J., Qi, Y., Wei, X., Fan, M., 2019. Young astringent
persimmon tannin inhibits methicillin-resistant Staphylococcus aureus isolated from
pork. LWT (Lebensm.-Wiss. & Technol.) 100, 48–55.
Marasini, B.P., Baral, P., Aryal, P., Ghimire, K.R., Neupane, S., Dahal, N., Singh, A.,
Ghimire, L., Shrestha, K., 2015. Evaluation of antibacterial activity of some tradi-
tionally used medicinal plants against human pathogenic bacteria. 2015. BioMed
Res. Int 265425.
Mazzarrino, G., Paparella, A., Chaves-López, C., Faberi, A., Sergi, M., Sigismondi, C.,
Compagnone, D., Serio, A., 2015. Salmonella enterica and Listeria monocytogenes in-
activation dynamics after treatment with selected essential oils. Food Contr. 50,
794–803.
Meng, X., Li, D., Zhou, D., Wang, D., Liu, Q., Fan, S., 2016. Chemical composition, an-
tibacterial activity and related mechanism of the essential oil from the leaves of
Juniperus rigida Sieb. et Zucc against Klebsiella pneumoniae. J. Ethnopharmacol. 194,
698–705.
Pang, H., Jia, W., Hu, Z., 2019. Emerging applications of metabolomics in clinical
pharmacology. Clin. Pharmacol. Ther. 106 (3), 544–556.
Pragadheesh, V.S., Saroj, A., Yadav, A., Chanotiya, C.S., Alam, M., Samad, A., 2013.
Chemical characterization and antifungal activity of Cinnamomum camphora essential
oil. Ind. Crop. Prod. 49, 628–633.
Rugbjerg, P., Feist, A.M., Sommer, M.O.A., 2018. Enhanced metabolite productivity of
Escherichia coli adapted to glucose M9 minimal medium. Front. Bioeng. Biotechnol. 6,
166.
Rutowski, J., Zhong, F., Xu, M., Zhu, J., 2019. Metabolic shift of Staphylococcus aureus
under sublethal dose of methicillin in the presence of glucose. J. Pharmaceut.
Biomed. Anal. 167, 140–148.
Schelli, K., Rutowski, J., Roubidoux, J., Zhu, J., 2017. Staphylococcus aureus methicillin
resistance detected by HPLC-MS/MS targeted metabolic profiling. J. Chromatogr. B
Anal. Technol. Biomed. Life Sci. 1047, 124–130.
Silva, C.S., Figueiredo, H.M., Stamford, T.L.M., Silva, L., 2019. Inhibition of Listeria
monocytogenes by Melaleuca alternifolia (tea tree) essential oil in ground beef. Int. J.
Food Microbiol. 293, 79–86.
10
Journal of Ethnopharmacology 253 (2020) 112652
Singh, R., Jawaid, T., 2012. Cinnamomum camphora (Kapur): review. Phcog. J. 4, 1–5.
Singh, P., Srivastava, B., Kumar, A., Dubey, N.K., 2008. Fungal contamination of raw
materials of some herbal drugs and recommendation of Cinnamomum camphora oil as
herbal fungitoxicant. Microb. Ecol. 56 (3), 555–560.
Su, Y.B., Peng, B., Li, H., Cheng, Z.X., Zhang, T.T., Zhu, J.X., Li, D., Li, M.Y., Ye, J.Z., Du,
C.C., Zhang, S., Zhao, X.L., Yang, M.J., Peng, X.X., 2018. Pyruvate cycle increases
aminoglycoside efficacy and provides respiratory energy in bacteria. Proc. Natl.
Acad. Sci. U.S.A. 115 (7), E1578–E1587.
Winska, K., Maczka, W., Lyczko, J., Grabarczyk, M., Czubaszek, A., Szumny, A., 2019.
Essential oils as antimicrobial agents-myth or real alternative? Mol 24 (11), E2130.
Wishart, D.S., 2016. Emerging applications of metabolomics in drug discovery and pre-
cision medicine. Nat. Rev. Drug Discov. 15 (7), 473–484.
Xu, J., Shao, X., Li, Y., Wei, Y., Xu, F., Wang, H., 2017. Metabolomic analysis and mode of
action of metabolites of tea tree oil involved in the suppression of Botrytis cinerea.
Front. Microbiol. 8, 1017.
Yeh, R.Y., Shiu, Y.L., Shei, S.C., Cheng, S.C., Huang, S.Y., Lin, J.C., Liu, C.H., 2009.
Evaluation of the antibacterial activity of leaf and twig extracts of stout camphor tree,
Cinnamomum kanehirae, and the effects on immunity and disease resistance of white
shrimp, Litopenaeus vannamei. Fish Shellfish Immunol. 27 (1), 26–32.
Yu, H., Ren, X., Liu, Y., Xie, Y., Guo, Y., Cheng, Y., Qian, H., Yao, W., 2019. Extraction of
Cinnamomum camphora chvar. Borneol essential oil using neutral cellulase assisted-
steam distillation: optimization of extraction, and analysis of chemical constituents.
Ind. Crop. Prod. 141 111794.
Zampieri, M., 2018. From the metabolic profiling of drug response to drug mode of ac-
tion. Curr. Opin. Struct. Biol. 10, 26–33.
Zeng, Z.H., Du, C.C., Liu, S.R., Li, H., Peng, X.X., Peng, B., 2017. Glucose enhances tilapia
against Edwardsiella tarda infection through metabolome reprogramming. Fish
Shellfish Immunol. 61, 34–43.
Zhang, L.L., Zhang, L.F., Hu, Q.P., Hao, D.L., Xu, J.G., 2017. Chemical composition, an-
tibacterial activity of Cyperus rotundus rhizomes essential oil against Staphylococcus
aureus via membrane disruption and apoptosis pathway. Food Contr. 80, 290–296.
Zheng, S., Jing, G., Wang, X., Ouyang, Q., Jia, L., Tao, N., 2015. Citral exerts its antifungal
activity against Penicillium digitatum by affecting the mitochondrial morphology and
function. Food Chem. 178, 76–81.
Zhou, H., Ren, J., Li, Z., 2017. Antibacterial activity and mechanism of pinoresinol from
Cinnamomum Camphora leaves against food-related bacteria. Food Contr. 79,
192–199.