822acssdJournal of Agricultural and Food ChemistryJ Agric Food ChemPMC1195114611951146119511464005972210.1021/acs.jafc.4c11955Chemical Compositions
and Antibacterial Activities
of Litsea cubeba Essential Oil and Its Distillates
Prepared by Vacuum Fractional Distillation and Molecular DistillationChenQingqing†LinXiaocai†ZhangJia†LiuShichen†‡ZouZiqing†ChunJiong†‡*College
of Life Sciences, Gannan Normal University, Ganzhou 341000, P.R. ChinaJiangxi
Provincial Key Laboratory of Pest and Disease Control of Featured
Horticultural Plants, Gannan Normal University, Ganzhou 341000, P.R. China
Litsea cubeba essential
oil (LCEO)
has various bioactivities and wide applications. However, most reported
LCEOs are directly extracted from plants, and studies on further processing
of LCEO to enrich bioactive components using modern separation techniques
are scarce. In this study, LCEO was extracted by hydrodistillation
and further processed via vacuum fractional distillation (VFD) and
molecular distillation (MD). The chemical compositions of LCEO and
seven distillates were analyzed, and the activities of the EOs and
eight individual constituents against seven bacteria were tested.
Distillates VFD3 and MDH2 showed the best activity against Escherichia coli, Staphylococcus aureus, and Agrobacterium
rhizogenes. VFD3 exerted antibacterial action against A. rhizogenes by inhibiting biofilms, damaging the
cell membrane and cell wall, and perturbing metabolic pathways. VFD
and MD are effective processing methods for changing the chemical
composition and enhancing the bioactivity of LCEO, which might be
used to improve the quality and extend the applications of LCEO and
other EOs.
Received 2024 Dec 3; Accepted 2025 Feb 28; Revised 2025 Feb 28; Collection date 2025 Mar 26.
Introduction
Litsea
cubeba (Lour.) Pers. (L. cubeba) is an important natural woody plant that
is widely distributed in Southeast Asia, China, and Japan.1,2 The extract of L. cubeba is rich
in various natural components that are widely used in the food, medicine,
and cosmetics industries.3,4 The essential oil (EO)
of L. cubeba has received much attention
because of its wide range of bioactivities.5 The chemical compositions of EOs extracted from different parts
of L. cubeba are quite different, and
the highest yield of EO is from the fruit.6,7 The
origin, extraction, and processing methods of LCEO affect its components
and yield.7,8 China is a major producer of LCEO in the
world, with an annual output of 1500–2000 tonnes.8 Chinese customs have reported that from 2019
to 2023, the total export volume of LCEO reached 1682 tonnes.9 Therefore, conducting research on LCEO can help
researchers better utilize the advantages of high LCEO production
in China.
LCEO is mainly composed of oxygenated monoterpenes,
monoterpenes,
and sesquiterpenes. Citral, an oxygenated monoterpene, is a major
component of LCEO.10 Zhao et al. used the
hydrodistillation (HD) method to extract LCEO from L. cubeba fruit collected in Hangzhou City, China,
and reported that geranial (α-citral) and neral (β-citral)
accounted for 41.09 and 34.70%, respectively, of the total EO.10 LCEO has antioxidant, anticancer, antimicrobial,
and anthelmintic activities, which are affected by its chemical composition.4 Citral exhibits bacteriostatic activity against
methicillin-resistant Staphylococcus aureus (MRSA), and the activity is antagonistically modulated by the accompanying
components.11 Therefore, the key to improving
the application of LCEO and enhancing its biological activity is determining
how to better enrich its active components. LCEO samples used in most
reported studies were extracted using the HD method without further
processing.12 In this study, LCEO was extracted
from the fruits of L. cubeba by HD,
and then, vacuum fractional distillation (VFD) and molecular distillation
(MD) were performed to further fractionate the LCEO and enrich the
bioactive components. Distillation is the most important technique
for extracting and separating fluid mixtures in the process industry.
The main methods used to extract EO from the plant material include
HD, cold pressing, supercritical fluid extraction, etc.4 HD is the most common method used to extract
EO without organic solvents. The process involves immersing the plant
material in a water-filled container and heating it to a boil. The
resulting water–oil vapor is condensed, and the oil and water
phases are separated.13 The main distillation
methods used to process EO into different fractions include normal
atmospheric distillation, vacuum distillation (VD), fractional distillation
(FD), VFD, and MD. VFD is a physical separation process for liquid
mixtures based on differences in the boiling points of the components
in the mixture. This decreases the boiling temperatures of the compounds
under vacuum conditions. It is an efficient process that takes advantage
of the preservation of thermally sensitive compounds.14 MD is a specialized high-vacuum technique used to separate
thermally unstable and high-boiling compounds based on different mean
free path lengths of the molecules at different pressures and temperatures.15
The application of VFD to separate the
constituents of EO has rarely
been reported. Do et al. separated lemongrass EO by VFD to obtain
fractions containing high citral contents.16 Silvestre et al. used VFD to fractionate rosemary (Rosmarinus officinalis L.) EO and green mandarin
(Citrus deliciosa Tenore) EO; they
reported that VFD effectively separated compounds belonging to different
chemical classes.14,17 However, bioactivity tests of
these fractions have not been reported. In our study, LCEO was separated
by VFD into a low-boiling-point fraction as VFD1, a medium-boiling-point
fraction as VFD2, and a high-boiling-point fraction as VFD3. Next,
double MD was conducted to fractionate LCEO.18 By conducting the first MD, the light phase fraction and heavy phase
fraction were obtained and expressed as MDL1 and MDH1, respectively.
MDH1 was used as the feed during the second MD. After distillation,
the light phase fraction and heavy phase fraction were expressed as
MDL2 and MDH2, respectively. Some studies have used only LCEO samples
prepared via HD; we further processed LCEO by VFD and MD to obtain
different fractions and compared the chemical compositions and antibacterial
activities of the fractions.
The most common pathogenic bacteria
responsible for foodborne outbreaks
around the world are Escherichia coli (E. coli), Legionella spp., Listeria
monocytogenes, Bacillus cereus, Salmonella spp., Shigella spp.,
and S. aureus. They cause considerable
damage to the food industry and threaten human health.19,20 According to the World Health Organization, an estimated 600 million
people fall ill, and 0.42 million people (including 0.125 million
children) die due to the consumption of unsafe food each year.21 Foodborne pathogens such as E. coli and S. aureus greatly contribute to foodborne illness. This infection may cause
significant and even life-threatening health risks.20 Some bacteria not only affect humans but also threaten
plant growth. Agrobacterium is a soil-borne Gram-negative
plant pathogen that transfers tumor-inducing or root-inducing plasmids
into plant cells and causes crown gall and hairy root diseases; these
diseases are incurable, and infected plants remain for their entire
lives and continuously release opines.22,23Agrobacterium mainly affects dicotyledonous plants (apples and roses), grape vines,
shade trees, herbaceous perennials, and even hydroponically grown
tomatoes and cucumbers. It interferes with plant metabolism and decreases
nutrient accumulation and fruit production, resulting in large economic
losses.24−26 The disease was first reported in the United States
in the 1870s and was subsequently reported in South America, Asia,
and European countries.27 Therefore, effective
control of foodborne and plant pathogens is important for human health
and agricultural practices. Antibiotics are widely used in human medicine
and agriculture, and misusing these drugs can lead to the development
of multidrug resistance, which increases the risk of treating infections
caused by pathogenic bacteria and poses a hazard to human and plant
health.20,28 To reverse the threat posed by antibiotics,
the search for natural plant-derived agents to control foodborne and
plant diseases is inevitable. EOs are potential sources of novel natural
bacteriostatic agents.1,4 LCEO, which is generally regarded
as safe (GRAS) and capable of reducing risks to the environment and
human health, has various biological activities, especially antimicrobial
activities.29 Its use as a natural antimicrobial
agent has been applied in areas such as fruit, vegetable, and meat
preservation. Dai et al. added LCEO to different vegetable juices,
and the bacterial inhibition rate remained above 99.9% for 4 days.1 Su et al. reported that after cherry tomatoes
were treated with an LCEO nanoemulsion for 9 min, the Salmonella count decreased by 6.50 ± 0.20 log CFU/mL.30 Kunová et al. reported that the number of coliform
bacteria in deer meat decreased from 1.02 log CFU/mL to 0 under 1%
LCEO treatment.3 Thielmann et al. demonstrated
that LCEO has high antibacterial activity against S.
aureus and E. coli and has been used to preserve strawberries.31 Our study, in which processed LCEO was used
to examine its activity against foodborne pathogens, is an extension
of this field. We also studied the inhibitory activity of LCEO and
seven distillates against Agrobacterium, which has
not been previously reported. Some studies have shown that EOs can
exert bacteriostatic effects by inhibiting biofilm formation and causing
irreversible damage to cell membranes and cell morphology.1,32,33 We also assessed the mechanism
by which the most active fraction, VFD3, inhibits Agrobacterium
rhizogenes (A. rhizogenes) based on biofilm inhibition, ROS levels, AKP activity, and cell
morphology. Metabolomics is a powerful tool for evaluating secondary
metabolites and antibacterial mechanisms and has been widely used
in various fields.33 Hu et al. reported
that the inhibitory activity of LCEO on MRSA was related to the disruption
of cell membranes, the inhibition of the hexose monophosphate pathway,
and the activity of key enzymes.34 Chen
et al. showed that LCEO significantly affected Cutibacterium
acnes by altering 86 metabolites and perturbing 34
metabolic pathways.35 To elucidate its
bacteriostatic mechanism, we used UPLC-QTOF-MS-based untargeted metabolomics
and UPLC-QQQ-MS-based targeted metabolomics as the standard methods
to conduct qualitative and quantitative analysis of the effects of
VFD3 on metabolites of A. rhizogenes.36 Our study may help optimize the processing
of EOs, enrich their active compounds, improve their bioactivities,
and explore the application of EOs as natural antibacterial agents.
Materials and MethodsMaterials
Fruits of L. cubeba (Lour.) Pers. were collected in Anshun
City, Guizhou Province, China, in mid-June 2022 and were dried in
the shade at room temperature. Other materials including the bacterial
and authentic compounds used are shown in Supporting Information 1.1.
LCEO Extraction and ProcessingLCEO Extraction by HD
L. cubeba fruits (5.0 kg) were placed in a Clevenger-type
distillation device, and 15.0 L of H2O was added. HD was
performed for 12 h. The upper LCEO layer was collected and dried with
sodium sulfate (Na2SO4). After filtration, LCEO
was weighed and stored in a refrigerator at 4 °C before further
processing.
Vacuum Fractional Distillation of LCEO
LCEO (231.40 g) was added to a distillation flask, and a fractionating
column was attached to the flask. The temperature of the cooling water
was 3 °C. When the pressure stabilized at 0.4 kPa, the heating
was turned on and the VFD process started. The distilled fractions
were collected. Three distillates, VFD1, VFD2, and VFD3, were collected
at boiling temperatures ranging from 38 to 44, 44 to 76, and 76 to
97 °C, respectively.
Molecular Distillation of LCEO
The double MD process was conducted with a 2 inch. Pope Wiped-Film
Molecular Evaporator (Pope Scientific Inc., Saukville, WI, USA). In
the first stage, the distillation temperature, cooling temperature,
vacuum pressure, wiper speed, and feed rate were set at 55 °C,
3 °C, 0.6 kPa, 350 rpm, and 4 mL/min, respectively. The light
and heavy phase fractions were expressed as MDL1 and MDH1, respectively.
MDH1 was used as the feed at the second MD stage, and similar conditions
were set at 65 °C, 3 °C, 0.5 kPa, 400 rpm, and 4 mL/min.
After distillation, the light phase fraction and heavy phase fraction
were expressed as MDL2 and MDH2, respectively.
Gas Chromatography and Mass Spectrometry (GC-MS)
Characterization of EOs
GC-MS was performed to analyze and
identify the compositions of LCEO and seven distillates, according
to Peng et al., with minor modifications.37 The experimental details are given in Supporting Information 1.2. The constituents of the EOs were identified
by comparing their mass spectra with the National Institute of Standards
and Technology data library (2010 version) and the retention index
(RI). The RI was measured by injecting C8–C20 n-alkanes with n-hexane-diluted EO via GC-MS under
the same conditions described above. The major compounds were also
identified by comparing the mass spectra and RI values with those
of commercially available authentic compounds injected into the GC-MS
apparatus.
Antibacterial Activity of LCEO, Seven Distillates,
and Eight Individual ConstituentsPreparation of Bacterial Cultures
Four foodborne pathogens, S. aureus, E. coli, Pseudomonas aeruginosa (P. aeruginosa), and Salmonella typhimurium (S.
typhimurium), two phytopathogens, Agrobacterium tumefaciens (A. tumefaciens)
and A. rhizogenes, and Bacillus subtilis (B. subtilis, a foundational model organism
in microbiology research38) were used to
determine the antimicrobial activity of EOs. Foodborne bacteria and B. subtilis were activated
on nutrient agar (NA) overnight at 37 °C. Agrobacterium was activated on yeast extract peptone (YEP) broth medium at 28
°C. Bacteria were diluted and used to determine the inhibition
zone diameter (IZD) by using the filter paper diffusion method and
the minimum inhibitory concentration (MIC). To avoid the growth of
other bacteria, rifampicin (25 μg/mL) was added to the growth
medium of A. tumefaciens, and streptomycin sulfate (50 μg/mL) was added to the growth
medium of A. rhizogenes.
Determination of the Inhibition Zone Diameter
The filter paper diffusion method was used to evaluate the inhibitory
activity of LCEO and the distillates on four foodborne pathogens and Bacillus subtilis (B. subtilis), as described
by Hao et al.39 In total, 150 μL
of each pathogen suspension was spread on a solid medium in a Petri
dish with a diameter of 90 mm. The IZD of Agrobacterium was determined using the method described by Long et al.40 The YEP medium was melted to a certain temperature,
after which Agrobacterium was added, vortexed, mixed,
and poured into a 90 mm diameter Petri dish. Next, 5 μL of EO
was added to the center of the filter paper (6 mm in diameter) and
then incubated at 37 °C for 24 h (four foodborne pathogens and B. subtilis) or at 28 °C
for 36 h (Agrobacterium). Ampicillin (100 μg/mL)
was used as a positive control. The IZD was measured with a caliper.
Each bacterium was measured three times in parallel.
Determination of the Minimum Inhibitory
Concentrations
The MICs of all EOs were determined by the
broth microdilution method.17 EOs were
dissolved in Tween 80 at 1% v/v and diluted in a 2-fold gradient in
nutrient broth (NB) or YEP. Each well consisted of 100 μL of
each sample at each concentration, 80 μL of NB or YEP, and 20
μL of bacterial suspension. Ampicillin was used as the positive
control, and bacteria enriched with only NB or liquid YEP or with
Tween 80 (1% v/v) without EO were used as the negative control. After
incubation at 37 °C for 24 h or at 28 °C for 36 h, 10 μL
of 20 mg/mL 2,3,5-triphenyltetrazolium chloride (TTC) was added to
each well and incubated at 37 or 28 °C for 30 min. The MIC was
the minimum EO concentration of the wells that did not turn red. Each
set of experiments was repeated three times.
Antibacterial Mechanism of VFD3 against <italic>A. rhizogenes</italic>
The antibacterial mechanism of VFD3
on A. rhizogenes was investigated by
measuring the biofilm inhibition rate,33 accumulation of ROS,41 AKP activity,41 and SEM images of the cell morphology.29 The experimental details are shown in Supporting Information 1.3.
Analysis of Metabolites of <italic>A. rhizogenes</italic>Sample Preparation
Suspensions
of A. rhizogenes from the VFD3 (at
a concentration of MIC)-treated group and the control group (CK) were
collected by centrifugation at 6000 r/min for 5 min at 4 °C.32 The samples were washed twice with 2 mL of phosphate-buffered
saline (0.01 M, pH 7.0), which was rapidly placed in liquid nitrogen
for 5 min and then on dry ice for 5 min. The samples were thawed on
ice and vortexed for 2 min. The thawed samples were submerged and
completely suspended in 80% methanol. The supernatant was obtained
by vortexing three times at intervals of 5 min and centrifuged twice
at 12,000 rpm and 4 °C. An aliquot of the supernatant from each
sample was pipetted and mixed into a linear tube of the corresponding
injection vial for LC-MS analysis. Three replicates were maintained
for the VFD3 treatment group and the control group.
Untargeted Analysis of the VFD3 Treatment
Group by UPLC-QTOF-MS
The metabolites of A.
rhizogenes after VFD3 treatment were determined by
a nontargeted method based on UPLC-QTOF-MS. The experimental details
are shown in Supporting Information 1.4.1.
Target Analysis of the VFD3 Group by UPLC-QQQ-MS
The UPLC-QQQ-MS technique was used to quantitatively analyze the
metabolites. The experimental details are shown in Supporting Information 1.4.2.
Screening of Differential Metabolites
The metabolites of the VFD3 and CK groups were screened by a combination
of variable importance projection (VIP) derived from orthogonal partial
least-squares-discriminant analysis (OPLS-DA) and the p-values from Student’s t tests. The screening
criteria for differential metabolites (DMs) were VIP > 1 and p < 0.05. The VIP value indicates the strength of the
effect of intergroup differences in the corresponding metabolite on
the classification of each group of samples. The Kyoto Encyclopedia
of Genes and Genomes (KEGG) database was used to analyze the metabolic
pathways.
Statistical Analysis
All experiments
were repeated three times. Except for the MIC values, the results
were expressed as the mean ± the standard deviation (SD). One-way
analysis of variance (ANOVA) and Tukey’s post hoc tests were
performed using IBM SPSS Statistics 27 software, and p < 0.05 was considered to indicate a statistically significant
difference.
Results and DiscussionYields and Chemical Compositions of LCEOs
The extraction yield of LCEO by HD may be affected by factors such
as origin and extraction conditions, and the oil yield from fresh
fruit is 3–6%.6 In this study, LCEO
was extracted by HD with a yield of 3.5%. After the VFD of LCEO, fractions
VFD1, VFD2, and VFD3 were obtained in yields of 24.36, 37.45, and
26.88%, respectively. After the MD of LCEO, fractions MDL1 and MDH1
were obtained in yields of 20.47 and 19.32%, respectively. After the
second MD using MDH1 as the feed, MDL2 and MDH2 were collected in
9.22 and 48.44% yields, respectively.
The chemical compositions
and relative contents of LCEO and seven fractions are shown in Table 1. In total, 36 compounds
were detected in all EOs by GC-MS. The main components of the LCEO
were β-citral (14.69%), citronellal (14.00%), limonene (11.40%),
linalool (9.25%), α-citral (7.56%), and 1,3,4-trimethyl-3-cyclohexene-1-carboxaldehyde
(7.18%). The diversity of the chemical composition and content of
the LCEO is related to climate conditions, soil types, and other factors.42 Gogoi et al. extracted the LCEO by HD in Assam,
India, and reported that the relatively high-content compounds were
sulcatone (30.9%), limonene (23.14%), β-citral (14.00%), α-citronellol
(6.52%), linalool (2.26%), and caryophyllene (1.73%). α-Citral
was not detected.43 Sattayakhom et al.
identified 25 compounds of the LCEO produced in Nonthaburi, Thailand,
and reported that α-citral (33.36%) and β-citral (26.78%)
were the predominant components.44 The
total ion chromatograms are shown in Figure S1, with the top 10 high-content compounds in each EO labeled with
numbers, with each number corresponding to the serial number of the
compound in Table 1.
Chemical Compositions of LCEO and
Seven Distillates Identified by GC-MS<xref rid="t1fn1" ref-type="table-fn"><sup>a</sup></xref>
composition
(%)
no.
RIexp.
RIlit.
compounds
LCEO
VFD1
VFD2
VFD3
MDL1
MDH1
MDL2
MDH2
1
927
927
α-thujene
0.16 ± 0.02 b
0.56 ± 0.03 a
—
—
0.44 ± 0.12 a
—
0.21 ± 0.01 b
—
2
933
929
α-pinene
4.51 ±
0.20 d
13.02 ± 0.69 b
1.01 ± 0.14 ef
0.17 ± 0.08 g
16.07 ± 0.21 a
1.37 ± 0.01 e
6.36 ± 0.23 c
0.31 ± 0.01 fg
3
947
951
camphene
0.99 ± 0.03 d
3.21 ± 0.06 b
0.30 ± 0.04 e
—
3.39 ± 0.03 a
0.35 ± 0.00 e
1.60 ± 0.05 c
—
4
974
970
sabinene
5.82 ± 0.16 d
14.99 ± 0.12 b
2.67 ± 0.23 e
—
16. 44 ± 0.36 a
2.25 ± 0.09 e
10.16 ± 0.30 c
0.70 ± 0.05 f
5
977
972
β-pinene
2.23 ± 0.19 c
8.19 ± 0.73 a
1.75 ± 0.38 c
—
8.86 ± 0.60 a
1.79 ± 0.08 c
6.82 ± 0.17 b
0.59 ± 0.01 d
6
991
988
sulcatone
3.88 ± 0.05 c
8.26 ± 0.33 a
5.19 ± 0.25 b
—
8.29 ± 0.98 a
2.16 ± 0.40 d
9.19 ± 0.29 a
0.97 ± 0.45 de
7
994
992
β-myrcene
3.00 ± 0.16 c
7.95 ± 0.28 a
2.97 ± 0.80 c
—
6.48 ± 0.82 b
2.53 ± 0.20 cd
6.58 ± 0.46 b
1.32 ± 0.12 d
8
1006
1006
α-phellandrene
0.26 ± 0.01 b
0.51 ± 0.04 a
—
—
0.17 ± 0.01 c
—
—
—
9
1027
1030
limonene
11.40 ± 0.33 d
27.45 ± 0.49 a
13.18 ± 1.44 c
0.19 ± 0.03 g
23.22 ± 0.31 b
7.10 ± 0.17 e
24.53 ± 0.03 b
3.44 ± 0.26 f
10
1029
1023
eucalyptol
1.45 ± 0.05 d
—
—
—
4.03 ± 0.02 b
2.23 ± 0.14 c
5.25 ± 0.22 a
1.38 ± 0.06 d
11
1040
1039
trans-β-ocimene
0.17 ± 0.01 c
0.44 ± 0.04 a
0.27 ± 0.02 bc
—
0.28 ± 0.03 b
—
0.36 ± 0.10 ab
—
12
1050
1050
α-ocimene
0.18 ± 0.01 d
0.44 ± 0.05 a
0.26 ± 0.01 c
—
0.27 ± 0.01 c
—
0.36 ± 0.00 b
—
13
1060
1060
γ-terpinene
0.19 ± 0.01 d
0.45 ± 0.05 a
0.27 ± 0.01 c
—
0.31 ± 0.02 bc
0.14 ± 0.03 d
0.35 ± 0.02 b
—
14
1071
1074
cis-sabinene
hydrate
0.10 ± 0.00 d
—
0.24 ± 0.01 a
—
0.08 ± 0.02 d
0.13 ± 0.01 c
0.18 ± 0.01 b
—
15
1089
1089
terpinolene
0.38 ± 0.01 c
0.72 ± 0.08 a
0.61 ± 0.10 ab
—
0.41 ± 0.03 c
—
0.57 ± 0.04 b
—
16
1108
1102
linalool
9.25 ± 0.04 c
4.99 ± 0.33 e
20.05
± 0.62 a
6.18 ± 0.46 d
4.21 ± 0.06 e
12.54
± 0.26 b
10.06 ± 0.17 c
13.02 ± 0.70 b
17
1112
1116
α-cyclocitral
0.31 ± 0.04 b
0.63 ± 0.00 a
—
—
0.16 ± 0.04 bc
0.51 ± 0.10 a
0.30 ± 0.10 b
0.53 ± 0.07 a
18
1125
1123
trans-p-mentha-2,8-dienol
0.19 ± 0.02 b
—
0.30 ± 0.05 a
—
—
0.16 ± 0.02 b
—
—
19
1140
1139
trans-limonene oxide
0.29 ± 0.07 b
0.18 ± 0.02 bc
0.57 ± 0.02 a
—
0.09 ± 0.05 cd
—
0.25 ± 0.10 b
—
20
1144
1143
camphor
0.15 ± 0.00 b
—
0.37 ± 0.01 a
—
0.06 ± 0.01 c
0.21 ± 0.02 b
0.16 ±
0.07 b
0.18 ± 0.04 b
21
1146
1152
isopulegol
0.54 ± 0.08 b
—
—
—
0.07 ± 0.02 c
0.71 ± 0.03 ab
0.18 ± 0.07 c
0.91 ± 0.20 a
22
1151
1146
β-pinene oxide
0.36 ± 0.07 bc
—
0.45 ± 0.11 b
1.32 ± 0.04 a
—
0.24 ± 0.04 cd
0.15 ± 0.03 d
—
23
1159
1155
citronellal
14.00 ± 1.00 d
3.26 ± 0.32 f
14.04 ± 0.51 d
16.23 ± 1.23 c
2.24 ± 0.09 f
18.40 ± 0.42 b
5.35 ± 0.15 e
21.32 ± 1.11 a
24
1169
1170
cis-verbenol
4.30 ± 0.40 bc
0.20 ± 0.04 e
1.65 ±
0.10 d
8.14 ± 0.45 a
0.12 ± 0.02 e
3.99 ± 0.10 c
0.29 ± 0.16 e
4.91 ± 0.26 b
25
1181
1178
terpinen-4-ol
1.46 ± 0.03 cd
0.51 ± 0.08 e
2.68 ± 0.50 a
1.76 ± 0.30 bc
0.36 ± 0.00 e
1.57 ± 0.03 bc
0.88 ± 0.03 de
2.15 ± 0.27 ab
26
1188
1171
1,3,4-trimethyl-3-cyclo-hexene-1-carboxaldehyde
7.18 ± 0.58 bc
0.52 ± 0.11 e
3.71 ± 0.11 d
10.48 ± 0.83 a
0.20 ± 0.11 e
6.38 ± 0.35 c
0.67 ± 0.03 e
7.66 ± 0.31 b
27
1196
1191
α-terpineol
1.44 ± 0.03 b
-
2.15 ± 0.75 ab
2.47 ± 0.11 a
0.11 ±
0.02 c
1.41 ± 0.08 b
0.35 ± 0.13 c
1.88 ± 0.05 ab
28
1249
1249
β-citral
14.69 ± 1.12 e
2.39 ± 0.30 g
17.01 ± 1.15 d
30.03 ± 1.38 a
2.07 ± 0.18 g
19.80 ± 0.14 c
5.86 ± 0.31 f
22.64 ± 0.66 b
29
1278
1273
α-citral
7.56 ± 0.64 c
0.53 ± 0.04 d
6.06 ± 0.86 c
17.04 ± 1.07 a
0.35 ± 0.12 d
9.83 ± 0.27 b
1.33 ± 0.39 d
10.82 ± 0.53 b
30
1373
1375
copaene
0.53 ± 0.03 c
—
0.68 ± 0.13 bc
1.00 ± 0.09 a
0.14 ±
0.19 d
0.71 ± 0.04 bc
0.20 ± 0.02 d
0.83 ± 0.05 ab
31
1427
1428
β-caryophyllene
0.32 ±
0.03 bc
—
0.24 ±
0.05 c
0.71 ± 0.08 a
—
0.42 ± 0.02 b
—
0.84 ± 0.10 a
32
1465
1465
α-caryophyllene
0.17 ± 0.00 d
—
—
0.39
± 0.02 a
—
0.24
± 0.02 c
—
0.29
± 0.03 b
33
1471
1473
β-farnesene
0.15 ± 0.01 c
—
—
0.37 ± 0.06 a
—
0.24 ± 0.02 b
—
0.28 ± 0.03 b
34
1498
1482
γ-elemene
0.23 ± 0.16 bc
—
—
0.61 ± 0.22 a
—
0.44 ± 0.02 ab
—
0.52 ± 0.05 a
35
1514
1511
β-bisabolene
0.23 ± 0.07 b
—
—
0.36
± 0.06 ab
—
0.28
± 0.03 ab
—
0.41
± 0.11 a
36
1591
1583
caryophyllene oxide
0.73 ± 0.07 c
—
—
0.76 ± 0.09 c
—
0.93 ± 0.04 b
—
1.11 ± 0.12 a
total
98.82
99.40
98.67
98.19
98.92
99.07
98.54
99.01
monoterpene
hydrocarbons
29.30
77.92
23.28
0.35
76.34
15.53
57.89
6.36
oxygenated monoterpenes
56.10
12.69
65.56
83.17
13.95
71.72
30.58
79.75
sesquiterpene hydrocarbons
1.63
0.00
0.92
3.43
0.14
2.33
0.20
3.17
oxygenated sesquiterpenes
0.73
0.00
0.00
0.76
0.00
0.93
0.00
1.11
others
11.07
8.78
8.90
10.48
8.50
8.55
9.86
8.63
RIexp., experimental
retention index on an HP-5MS column. RIlit., literature
retention index. “—”: not detected. The results
are expressed as the mean area percentage (%) ± SD of three independent
injections. The mean values in the same row followed by different
superscript letters are significantly different (p < 0.05), according to ANOVA followed by Tukey’s post hoc
test.
The effects of the VFD and MD on the individual compounds
in the
eight EOs were illustrated by OPLS-DA (Figure 1). The contribution of compounds with VIP
> 1 to the differences between EOs was significant. We identified
13 differential compounds (marked in red in Figure 1), including β-citral, linalool, 1,3,4-trimethyl-3-cyclohexene-1-carboxaldehyde,
etc. OPLS-DA can efficiently separate EOs into different areas of
the coordinate chart. β-Citral, 1,3,4-trimethyl-3-cyclohexene-1-carboxaldehyde,
and α-citral were enriched in the VFD3 area in quadrant 1. Linalool
and citronellal were the main components of VFD2 and MDH2 in the fourth
quadrant, respectively. α-Pinene, sabinene, and β-pinene
were mainly concentrated in the same quadrant as MDL1 and VFD1 at
close distances, indicating that these compounds were similar. The
seven distillates were a certain distance from the LCEO (Figure 1), indicating that
VFD and MD could effectively separate the compounds in the LCEO. The
relative contents of β-citral and α-citral increased from
14.69 and 7.56%, respectively, in LCEO to 30.03 and 17.04%, respectively,
in VFD3, and to 22.64 and 10.82%, respectively, in MDH2.
OPLS-DA score
scatter plot of eight EOs and individual constituents.
The 36 compounds were categorized into five groups,
and the compounds
with the most significant effects on the relative contents of VFD
and MD included oxygenated compounds, sesquiterpenes, and monoterpenes.
Regarding the VFD fractions, the relative content of monoterpene hydrocarbons
decreased considerably from 77.92% in VFD1 to 23.28% in VFD2, and
only 0.35% was found in VFD3. However, the relative content of oxygenated
monoterpenes increased considerably: 12.69% in VFD1, 65.56% in VFD2,
and 83.17% in VFD3. In the MD process, compared to that in the first
distillation, the monoterpene content of the light phase fractions
decreased in the second distillation, from 76.34% in MDL1 to 57.89%
in MDL2. However, the percentage of oxygenated monoterpenes increased
from 71.72% in MDH1 to 79.75% in MDH2. Compared to those in LCEO,
oxygenated compounds in VFD3 and MDH2 increased by 27.07 and 23.65%,
respectively, and monoterpenes decreased by 28.95 and 22.94%, respectively.
The contents of sesquiterpenes in VFD3 and MDH2 were about twice as
high as those in LCEO. Overall, the VFD and MD processes greatly increased
the relative contents of oxygenated monoterpenes and decreased the
relative contents of monoterpene hydrocarbons in VFD3 and MDH2. These
results indicated that the VFD and MD changed the composition and
relative content of the LCEO, which could be used to enrich the active
compounds for other EOs.
Analysis of the Bacteriostatic Activity of
LCEO, Seven Distillates, and Eight Individual Constituents
The activity of the EOs and individual constituents against seven
bacteria was determined by the filter paper diffusion method and the
broth microdilution method. Based on the criteria defined in another
study,45 bacterial sensitivity to EOs can
be classified as follows: IZD ≤ 8.0 mm, insensitive; IZD between
8.0–14.0 mm, moderately sensitive; IZD between 14.0–20.0
mm, sensitive; and IZD ≥ 20.0 mm, extremely sensitive.
As shown in Tables 2 and 3, eight EOs displayed different antibacterial
activities. The activities of LCEO and the three VFD fractions were
ranked as follows: VFD3 > VFD2 > LCEO > VFD1. The order of
antibacterial
activity of the MD distillates was MDH2 > MDH1 > MDL2 > MDL1. B. subtilis and S. aureus were extremely sensitive to VFD2, VFD3, and MDH2, with IZD values
greater than 20.00 mm and MIC values ranging from 0.20 to 1.56 μL/mL.
Most studies on the bacteriostatic activity of LCEO have focused on
foodborne pathogens.1 Nguyen et al. studied
the bacteriostatic activity of LCEO extracted by HD in Vietnam and
reported MIC values ranging from 2800 to 5500 μg/mL against B. subtilis, S. aureus, and E. coli.46 The activity of the LCEO in that study was close to that
of the LCEO found in this study; however, it was weaker than VFD3
and MDH2. Ambrosio et al. assessed the activity of 28 types of EOs
against S. aureus and B. subtilis and reported that orange EO had the best
activity, with IZDs of 28.40 and 27.90 mm, respectively, whereas the
IZDs of VFD3 were greater than 90.00 mm for both bacteria.45 Miladinović et al. reported the inhibitory
activities of six EOs against S. aureus and E. coli, with MICs ranging from
1326 to 5375 μg/mL, which were comparable to the MICs of VFD3
and MDH2 (0.78–1.56 μL/mL).47 In this study, the bacteriostatic effects of eight EOs against A. tumefaciens and A. rhizogenes were determined. Hsouna et al. reported that peppermint (Mentha piperita L.) EO had good antibacterial activities
against the phytopathogenic bacteria A. tumefaciens, with an MIC of 12.50 mg/mL,48 which
was weaker than those of LCEO, VFD2, VFD3, and MDH2, with MICs of
1.56–3.13 μL/mL. Like foodborne pathogens, VFD3 also
showed the best bacteriostatic activity against both plant pathogens,
with the same MIC of 1.56 μL/mL. Lee et al. investigated 50
EOs and reported that cinnamon bark (Cinnamomum verum) EO had the best activity against A. tumefaciens, with an MIC of 250 μg/mL, which was better than those of
VFD3 and MDH2.28 VFD2 and MDH2 also exhibited good bacteriostatic
activity against the two plant pathogens, with MICs of 3.13 μL/mL
for A. rhizogenes and 1.56 μL/mL
for A. tumefaciens.
Inhibition Zone Diameter (IZD, mm)
of EOs and Individual Constituents
bacterial
B. subtilis
S. aureus
E. coli
P.
aeruginosa
S. typhimurium
A. rhizogenes
A. tumefaciens
EOs
LCEO
>90.00 a
16.08 ± 0.03 g
8.11 ± 0.09 e
7.68 ± 0.18 a
7.94 ± 0.27 e
8.35 ± 0.08 i
14.68 ± 0.06 d
VFD1
12.99 ± 0.30 g
12.98 ± 0.16 i
6.00 ± 0.00 i
6.00 ± 0.00 e
7.25 ± 0.11 f
11.25 ± 0.04 e
7.50 ± 0.08 j
VFD2
>90.00 a
34.85
± 0.08 b
11.74 ± 0.20 b
6.57 ± 0.29 c
10.24
± 0.09 b
17.69 ± 0.13 b
13.67 ± 0.13 e
VFD3
>90.00 a
>90.00 a
13.41 ± 0.09 a
6.83 ± 0.02 b
11.50 ± 0.23 a
21.53 ± 0.11 a
21.48 ± 0.11 a
MDL1
13.64 ± 0.05 f
13.90 ± 0.07 h
6.54 ± 0.04 g
6.00 ± 0.00 e
6.30 ± 0.04 h
7.18 ± 0.01 l
7.50 ± 0.04 j
MDH1
90.00 ± 0.00 a
17.46 ± 0.36 f
8.01 ± 0.01 e
6.14 ± 0.01 de
6.99 ± 0.03 fg
9.49 ± 0.11 g
12.52 ± 0.22 f
MDL2
21.42 ± 0.08 c
17.33 ± 0.09 f
7.67 ± 0.02 f
6.09 ± 0.01 de
6.83 ± 0.03 g
10.85 ± 0.07 f
7.76 ± 0.01 j
MDH2
>90.00 a
20.46 ± 0.08 e
9.43 ± 0.09 d
6.28 ± 0.03 d
7.18 ± 0.01 f
16.14 ± 0.10 d
18.65 ± 0.05 c
citral
>90.00 a
32.24 ± 0.08 c
6.00 ± 0.00 i
6.22 ± 0.04 de
8.59 ± 0.03 c
16.70 ± 0.09 c
19.57 ± 0.13 b
citronellal
>90.00 a
10.67 ± 0.16 j
6.00 ± 0.00 i
6.00 ± 0.00 e
6.00 ± 0.00 i
7.79 ± 0.05 j
8.75 ± 0.20 i
comp. 26a
>90.00 a
7.23 ± 0.06 m
6.00 ± 0.00 i
6.00 ± 0.00 e
6.00 ± 0.00 i
6.56 ± 0.08 m
6.59 ± 0.15 k
linalool
9.22 ± 0.09 h
9.35 ± 0.07 k
11.20 ± 0.02 c
6.00 ± 0.00 e
8.28 ± 0.07 d
9.38 ± 0.07 g
12.48 ± 0.01 f
α-terpineol
15.20 ± 0.04 e
10.43 ± 0.09 j
11.81
± 0.07 b
6.00 ± 0.00 e
8.39 ± 0.01 cd
7.43
± 0.14 k
12.21 ± 0.07 g
terpinen-4-ol
16.29 ±
0.11 d
8.40 ± 0.07 l
13.28 ± 0.09 a
6.00 ± 0.00 e
11.36 ± 0.06 a
8.61 ± 0.17 h
10.15 ± 0.06 h
β-pinene oxide
7.50 ± 0.02 i
7.12 ± 0.03 m
6.28 ± 0.05 d
6.00 ± 0.00 e
6.00 ± 0.00 i
6.27 ± 0.02 n
6.27 ± 0.07 l
caryophyllene
oxide
7.30 ± 0.02 i
6.53
± 0.07n
6.00 ± 0.00 i
6.00 ± 0.00 e
6.00 ±
0.00 i
6.00 ± 0.00°
6.00 ± 0.00 m
ampicillin
22.15 ± 0.28 b
28.06 ± 0.18 d
6.00 ± 0.00 h
6.00 ± 0.00 e
8.37 ± 0.23 cd
11.46 ± 0.10 e
12.06 ± 0.10 g
Comp. 26 indicates 1,3,4-trimethyl-3-cyclohexene-1-carboxaldehyde
and is shown in Table 1. The IZD values are presented as the mean ± SD for at least
three experiments in mm, including a 6.0 mm disk diameter. The mean
values in the same column followed by different superscript letters
were significantly different (p < 0.05), according
to ANOVA followed by Tukey’s post hoc test.
Minimum Inhibitory Concentration (MIC,
μL/mL) of EOs and Individual Constituents against Seven Bacterial
Strains
bacteria
B. subtilis
S. aureus
E. coli
P. aeruginosa
S. typhimurium
A. rhizogenes
A. tumefaciens
EOs
LCEO
0.78
6.25
6.25
200.00
12.5
6.25
3.13
VFD1
3.13
12.50
100.00
200.00
100.00
25.00
12.50
VFD2
0.20
1.56
6.25
200.00
3.13
3.13
1.56
VFD3
0.39
0.78
1.56
100.00
0.78
1.56
1.56
MDL1
3.13
1.56
12.50
100.00
100.00
25.00
12.50
MDH1
0.39
1.56
1.56
50.00
3.13
6.25
6.25
MDL2
1.56
3.13
3.13
50.00
6.25
25.00
12.50
MDH2
0.39
0.78
1.56
50.00
1.56
3.13
1.56
citral
0.39
0.39
0.78
400.00
6.25
1.56
1.56
citronellal
0.39
0.78
3.13
400.00
6.25
12.50
12.50
comp. 26a
0.39
6.25
6.25
200.00
12.50
3.13
3.13
linalool
3.13
3.13
3.13
400.00
6.25
3.13
3.13
α-terpineol
3.13
0.78
0.78
400.00
0.78
3.13
3.13
terpinen-4-ol
0.78
1.56
1.56
400.00
0.78
3.13
3.13
β-pinene oxide
12.5
6.25
3.13
400.00
12.50
3.13
3.13
caryophyllene oxide
12.5
6.25
3.13
400.00
12.50
12.50
12.50
ampicillin (μg/mL)
25.00
0.10
3.13
800.00
1.56
250.00
625.00
Comp. 26 indicates 1,3,4-trimethyl-3-cyclohexene-1-carboxaldehyde
and is shown in Table 1.
Among the single constituents of VFD3 and MDH2, which
had the best
inhibitory activity against most bacteria, citral (47.07, 33.46%),
citronellal (16.23, 21.32%), 1,3,4-trimethyl-3-cyclohexene-1-carboxaldehyde
(10.48, 7.66%), linalool (6.18, 13.02%), α-terpineol (2.47,
1.88%), terpinen-4-ol (1.76, 2.15%), and caryophyllene oxide (0.76,
1.11%) were the common constituents in both distillates with the highest
contents. To explore the contributions of individual constituents
to EOs, the bacteriostatic activities of the above seven constituents
and β-pinene oxide, which had relatively high contents in VFD3,
were tested. Like the eight EOs, the above eight compounds inhibited
all bacteria except P. aeruginosa with MICs ranging from 0.39 to 12.50 μL/mL, as shown
in Table 3. Oxygenated
monoterpenes, such as citral, linalool, α-terpineol, terpinen-4-ol,
and β-pinene oxide, displayed strong activity against A. rhizogenes and A. tumefaciens, with MICs ranging from 1.56 to 3.13 μL/mL. However,
caryophyllene oxide, as the only representative oxygenated sesquiterpene
in the EOs tested, showed the weakest activity among the eight constituents
tested, with an MIC of 12.5 μL/mL against the above two plant
pathogens. Rasoul et al. reported that linalool displayed bacteriostatic
activity against A. tumefaciens, with an MIC of 2500 mg/L.49 Kotan et al. reported that citronellal, linalool, and α-terpineol
had antibacterial effects on A. tumefaciens, with IZD values ranging from 7 to 8 mm.50 In our study, linalool (20.05% in VFD2) had an MIC of 3.13
μL/mL against A. tumefaciens, with less inhibitory activity than VFD2 (MIC of 1.56 μL/mL),
suggesting that the inhibitory effect may be a result of the combined
action of several compounds rather than a single compound. The results
of the bacteriostatic activity of eight individual compounds further
proved that the VFD and MD were efficient methods for enriching bioactive
constituents.
Spearman correlation analysis was performed to
reveal the relationships
between the individual compounds and the bacteriostatic activity of
the eight EOs to identify highly active compounds. The clustered heat
map (Figure 2) revealed
a positive or negative correlation between the bacteriostatic activities
(MICs) of the different compounds. β-Citral, citronellal, linalool,
1,3,4-trimethyl-3-cyclohexene-1-carboxaldehyde, α-citral, α-terpineol,
and terpinen-4-ol were negatively correlated with the MICs against
different bacteria, and these compounds had relatively high contents
in VFD3 and MDH2, which presented the best bacteriostatic activity.
Citral may play an important role in the bacteriostatic effects of
VFD3 (47.07%) and MDH2 (33.46%) against B.
subtilis, S. aureus, and E. coli, with
MICs of 0.39–0.78 μL/mL. Guo et al. reported that citral,
terpineol, and linalool had good antibacterial activity against B. subtilis, S. aureus, and E. coli, with MICs ranging from 0.48 to 15.84 μL/mL.51 Similar findings were reported in this study,
with MICs ranging from 0.39 to 3.13 μL/mL for the three constituents
against the three bacteria. Among the five classes of compounds, oxygenated
monoterpenes presented significant negative correlations with all
bacteria except P. aeruginosa. Monoterpenes were significantly positively correlated
with seven species of bacteria. Other studies have shown that some
oxygenated compounds in EOs have better antibacterial activity than
terpenes.52 Sidorova et al. reported that
the two monoterpenes limonene and α-pinene did not inhibit A. tumefaciens even at relatively
high concentrations (400 μmol and 600 μmol); however,
2-octanone and isoamyl alcohol strongly inhibited A. tumefaciens.53 However, the contents of monoterpene hydrocarbons in VFD1 (77.92%)
and MDL1 (76.34%) were significantly greater than those in VFD3 (0.35%)
and MDH2 (6.36%). The contents of oxygenated monoterpenes in VFD3
and MDH2 were 10 times and 6 times greater than those in VFD1 and
MDL1, respectively (Table 1), suggesting that oxygenated monoterpenes may be the dominant
factor facilitating the inhibitory effects of VFD3 and MDH2 on the
foodborne pathogens and plant pathogens tested. As the antibacterial
activities of VFD3, VFD2, and MDH2 were better than those of LCEO,
we concluded that VFD and MD are extremely efficient methods to fractionate
LCEO and may be used for other EOs and practical applications. This
study revealed that some LCEO distillates, such as VFD3 and MDH2,
and individual constituents not only effectively inhibited foodborne
pathogens but also had good antibacterial effects on plant pathogens.
Considering the few studies on LCEO and individual compounds targeting A. rhizogenes and A. tumefaciens, this study provided a theoretical basis for the use of
LCEO distillates and active individual compounds as plant-derived
bacteriostatic agents to control plant diseases in agriculture. Qiu
et al. reported that LCEO is a new promising natural cosmetic ingredient.54 Chen et al. reported that citral-rich LCEO may
help treat inflammatory diseases.55 Therefore,
the results of this study may deepen our understanding of the bioactivity
of LCEO and its distillates and broaden its application prospects
in the medical and cosmetic industries.
Spearman correlation
analysis. The correlation values showed positive
(above zero) and negative (below zero) associations between chemical
compositions, eight EOs and their antibacterial activity. “*”,
“**”, and “***” indicate a significant
difference at the p < 0.05, p < 0.01, and p < 0.001 level, respectively.
Analysis of Antibacterial Mechanism of VFD3
against <italic>A. rhizogenes</italic>
The growth and reproduction
of pathogens are tightly dependent on biofilm formation. The hydrophobic
components of EOs might permeate the cell membrane to affect biofilm
formation.41 The effects of different concentrations
of VFD3 on biofilm formation were investigated by a crystal violet
staining assay. The effect of inhibiting biofilms was more prominent
with increasing concentrations of VFD3 (Figure 3A), and the inhibition rates at 1 MIC and
2 MIC reached 58.58 and 82.46%, respectively.
Effects of different
concentrations of VFD3 on biofilm activity,
AKP activity, and ROS levels in A. rhizogenes. (A) Biofilm inhibition rate at different concentrations of VFD3;
MIC = 1.56 μL/mL; CK was the blank control. (B) Effects of different
concentrations of VFD3 on ROS levels. (C) Effects of different concentrations
of VFD3 on AKP activity. Different lowercase letters above the columns
within (A–C) indicate significant statistical differences at p < 0.05.
Oxidative stress leads to the accumulation of high
levels of ROS,
disrupting bacterial cell membranes and inhibiting their growth.41 EOs kill bacteria through the accumulation of
intracellular ROS. A DCFH-DA fluorescent probe was used to detect
intracellular ROS production. The level of ROS accumulation was positively
correlated to the concentration of VFD3 (Figure 3B). After treatment of A.
rhizogenes with VFD3 at 1–4 MICs, the ROS level
was 5–7 times greater than that in the CK group.
Alkaline
phosphatase (AKP) is considered to be a cell wall damage
marker, and a leakage assay revealed that the permeability of the
cell wall increased after treatment with VFD3. AKP activity increased
in a concentration-dependent manner and was about 4-fold greater than
that of the control group after treatment with 4 MIC VDF3 (Figure 3C), suggesting that
VFD3 damaged the cell wall, which allowed the leakage of AKP into
the cell culture medium.
SEM analysis was performed to assess
the effect of VFD3 on the
morphology of A. rhizogenes cells (Figure S2). Untreated cells maintained a normal
and intact appearance with smooth surfaces (Figure S2A), whereas VFD3-treated cells became irregular, with collapsed
or shrunken cell surfaces (Figure S2B,C). These findings suggested that VFD3 can alter the morphology of A. rhizogenes cells, which might affect the integrity
of the cell membrane and the cell wall.
To summarize, VFD3 might
exert its antibacterial effect by inhibiting
cell biofilm formation, inducing the accumulation of ROS, increasing
extracellular AKP activity, and altering the cell morphology, leading
to disruption of the cell membrane and cell wall integrity.
Analysis of Metabolomics DataEffect of VFD3 on the Metabolism of <italic>A. rhizogenes</italic>
To assess the effects of VFD3 on A. rhizogenes, a combination of untargeted and targeted
UPLC-MS/MS techniques was used to quantitatively and qualitatively
analyze the metabolites. OPLS-DA was used to analyze differences in
metabolites between the VFD3 and CK groups (Figure 4A). The DMs of A. rhizogenes in the VFD3 group were visualized by using a volcano plot (Figure 4B). In total, 326
DMs were screened, of which 133 DMs were significantly upregulated
and 193 DMs were downregulated. The scatter plot (Figure S3) and clustered heat map (Figure S4) revealed large differences in the DMs between the VFD3
and CK groups. Among them, the DMs with significant changes were found
to be mainly amino acids and their metabolites, which were significantly
lower than those in the CK group. Our results agreed with those of
Chen et al., who reported that almost all amino acids were significantly
reduced in the cells of Cutibacterium acnes after LCEO treatment.56
Metabolomic analysis
of A. rhizogene with and without VFD3
treatment. (A) OPLS-DA score plot. (B) Volcano
plot. The red and green dots represent increases and decreases in
metabolite abundance, respectively, compared with those in the CK
group.
Metabolic Pathway Analysis of <italic>A.
rhizogene</italic> with VFD3 Treatment
Pathway analysis of
the 326 DMs screened via the KEGG database yielded 64 metabolic pathways
(Figure S5). According to the hypergeometric
test p-value, the top 20 ranked pathways are presented
from the smallest to largest, and the results are shown in the enrichment
analysis bubble (Figure 5). After VFD3 treatment, metabolites in the bacterial secretion system
pathway, which was related to membrane transport, were the most highly
enriched and upregulated.57 Guillín
et al. reported that the main DMs were involved in the pathways of
aminoacyl-tRNA biosynthesis, glutathione metabolism, and amino acid
metabolism.33 In this study, DMs were significantly
enriched in the pathways of glutathione metabolism, glycerophospholipid
metabolism, and peptidoglycan biosynthesis. The first two pathways
were downregulated in expression. In the glutathione metabolic pathway,
oxidized glutathione, cysteinylglycine, and the intermediate 5-oxoproline
were significantly downregulated, resulting in blocked transport.
A similar mode of action was reported by Zhao et al., who reported
that glycerophospholipid metabolism was significantly altered by menthone
treatment of MRSA. The structure of the cell membrane was disrupted
by interference with lipid synthesis, thus exerting an antibacterial
effect.58
Differential metabolite pathway enrichment
maps of A. rhizogene in response to
VFD3 treatment.
We found that in the VFD3-treated group, uridine-5′-diphospho-N-acetylgalactosamine-disodium-salt and UDP-N-acetylmuramic acid, which are related to peptidoglycan (PG), were
significantly downregulated. PG plays a crucial role in maintaining
the morphological, structural, and functional integrity of bacterial
cells, and impaired PG synthesis leads to severe growth defects, ultimately
leading to cell death.59,60 Therefore, the significant downregulation
of the PG biosynthetic pathway may also be a reason for the bacteriostatic
effect of VFD3.
These results suggested that VFD3 treatment
may affect A. rhizogenes by interfering
with the pathways of
glutathione metabolism, glycerophospholipid metabolism, and PG biosynthesis,
resulting in bacteriostatic effects.
To summarize, two separation
technologies, VFD and MD, were used
to process LCEO, and the chemical composition and antibacterial activity
of the seven distillates were significantly varied. Some active components,
such as citral, were strongly enriched in the VFD3 and MDH2 distillates,
resulting in better bacteriostatic effects on some foodborne pathogens,
such as E. coli and S. aureus. VFD3 and MDH2 had strong antibacterial
activity against two strains of plant pathogens, A.
rhizogenes, and A. tumefaciens, which have not been previously reported. VFD and MD techniques
played an important role in enhancing the content of the active ingredients
and the antibacterial activity of LCEO while retaining heat-sensitive
components under high-vacuum and low-temperature conditions. The antibacterial
mechanism of VFD3 on A. rhizogenes was
investigated by measuring the biofilm inhibition rate, accumulation
of ROS, AKP activity, and SEM images of cell morphology. Additionally,
the UPLC-MS/MS technique was used to qualitatively and quantitatively
analyze the metabolites. The effect of VFD3 might involve damage to
the cell membrane and cell wall integrity and interference with metabolic
pathways. The results of this study may provide a reference for EO
processing and separation research to increase the bioactivity and
improve the application potential of LCEO and other EOs.
Acknowledgments
This work was supported by 2023 Gan Poyang Talents
Support Program-Jiangxi Province High-level Overseas Talents Project
(20232BCJ25001), Jiangxi Provincial Education Department Project (GJJ2201256),
and Research Fund of Jiangxi Provincial Key Laboratory of Pest and
Disease Control of Featured Horticultural Plants (2024SSY04181).
Supporting Information Available
The Supporting Information
is
available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.4c11955.
Materials, experimental details of GC-MS characterization
of EOs, antibacterial mechanism of VFD3 against A.
rhizogenes, metabolite analysis, abbreviations used,
and Figures S1–S5 (PDF)
The authors
declare no competing financial interest.
Supplementary Material
jf4c11955_si_001.pdf
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