ORIGINAL RESEARCH

published: 24 May 2021

doi: 10.3389/fchem.2021.679116

Chemical Composition, Antimicrobial

and Insecticidal Activities of Essential
Oils of Discarded Perfume Lemon and
Leaves (Citrus Limon (L.) Burm. F.) as
Possible Sources of Functional
Botanical Agents
Panpan Wu 1,2, Xiaowen Tang 1,2, Rongchao Jian 1,2, Jiahao Li 1,2, Maoyu Lin 1,2, Huachao Dai 1,2,
Kangpeng Wang 1,2, Zhaojun Sheng 1,2, Baizhong Chen 3, Xuetao Xu 1,2, Chen Li 1,2,
Zhongze Lin 1,2, Qingmin Zhang 1,2, Xi Zheng 1,2, Kun Zhang 1,2, Dongli Li 1,2* and
Weiqian David Hong 1,2,4*
1
School of Biotechnology and Health Sciences, Wuyi University, Jiangmen, China, 2International Healthcare Innovation Institute
Edited by:
(Jiangmen), Jiangmen, China, 3Guangdong Xinbaotang Biotechnology Co. Ltd., Jiangmen, China, 4Department of Chemistry,
Guigen Li,
University of Liverpool, Liverpool, United Kingdom
Texas Tech University, United States
Reviewed by:
Zhu Liang, Two essential oils were isolated from discarded perfume lemon and leaves (Citrus limon (L.)
South China University of Technology,
Burm. F.) by hydro-distillation with good yield (0.044% for perfume lemon and 0.338% for
China
Sarita Kumar, leaves). Their biological activities were evaluated against five selected bacterial strains and
University of Delhi, India Aedes albopictus (Ae. albopictus, Diptera: Culicidae). Chemical composition indicated that
*Correspondence: both essential oils were rich in essential phytochemicals including hydrocarbons,
Dongli Li
[email protected]
monoterpenes and sesquiterpene. These constituents revealed some variability among
Weiqian David Hong the oils displaying interesting chemotypes (R)-(+)-limonene (12.29–49.63%), citronellal
[email protected]
(5.37–78.70%) and citronellol (2.98–7.18%). The biological assessments proved that the
two essential oils had similar effect against bacterial (inhibition zones diameter ranging from
Specialty section:
This article was submitted to 7.27 ± 0.06 to 10.37 ± 0.15 mm; MICs and MBCs ranging from 1.6 to 6.4 mg/mL); against
Organic Chemistry, Ae. albopictus larvae (LC50 ranging from 384.81 to 395.09 ppm) and adult mosquito (LD50
a section of the journal
Frontiers in Chemistry
ranging from 133.059 to 218.962 μg/cm2); the activity of the two chemotypes ((R)-
Received: 12 March 2021
(+)-limonene and citronellal): larvae (LC50 ranging from 267.08 to 295.28 ppm), which
Accepted: 06 May 2021 were all presented in dose-dependent manners. Through this work, we have showcased
Published: 24 May 2021
that recycling and reusing of agriculture by-products, such as discarded perfume lemon
Citation:
and leaves can produce eco-friendly alternatives in bacterial disinfectants and mosquito
Wu P, Tang X, Jian R, Li J, Lin M, Dai H,
Wang K, Sheng Z, Chen B, Xu X, Li C, control product.
Lin Z, Zhang Q, Zheng X, Zhang K, Li D
and Hong WD (2021) Chemical Keywords: chemical composition, antimicrobial, insecticidal, essential oil, perfume lemon, leaves
Composition, Antimicrobial and
Insecticidal Activities of Essential Oils of
Discarded Perfume Lemon and Leaves
INTRODUCTION
(Citrus Limon (L.) Burm. F.) as Possible
Sources of Functional
Many mosquito-borne tropical and subtropical diseases, such as malaria, yellow fever, filariasis,
Botanical Agents. dengue, and viral encephalitis (Rouis et al., 2013; Duguma, et al., 2020; Wilson, et al., 2020)
Front. Chem. 9:679116. contribute to a larger proportion of public health problems and a major economic burden within
doi: 10.3389/fchem.2021.679116 disease-endemic countries (Bhatt et al., 2013; Pavela, 2015). Left unchecked, insecticide resistance

Frontiers in Chemistry | www.frontiersin.org 1 May 2021 | Volume 9 | Article 679116

Wu et al. Eco-Friendly Sources of Functional Botanicals

FIGURE 1 | Recycling and reusing of the discarded perfume lemon and leaves.

could lead to a substantial increase in mosquito-borne diseases MATERIALS AND METHODS

incidence and mortality (Weill et al., 2003; Liu, 2015; Chang et al.,
2017). Herein, urgent action is required to prevent the further Plant Material and Chemicals
development of resistance (Ranson and Lissenden, 2016) and to Discarded perfume lemon and leaves (Citrus limon (L.) Burm. F.
maintain the effectiveness of existing vector control interventions Appraised by Professor Junxia Zheng of Guangdong University of
(Shaw and Catteruccia, 2019; Wilson et al., 2020). In addition to Technology) were obtained from the local farm (Xinghong farm,
that, it would provide significant added value if the newly Enping, Jiangmen, Gaungdong, China). Mueller-Hinton agar
developed products can be used to control bacterial infection (MHA) and Mueller-Hinton broth (MHB) were purchased
e.g. caused by scratching of the irritated skin or other from Guangdong Huankai Microbial Technology Co., Ltd.
inflammation reactions after mosquito bites (Ivory et al., 2015; (Guangdong, China), custom alkanes solution of C8-C40
Derua et al., 2019). n-alkanes standard (Reagent Brand: o2si) was bought from
More and more studies have been carried out and suggested that ANPEL Laboratory Technologies (Shanghai, China).
plant extractions are effective against mosquitoes at various stages of Deltamethrin was purchased as analytical reagent from J&K
development (Bakkali et al., 2008; Gross et al., 2017). Citrus fruits Scientific (Beijing, China). (R)-(+)-Limonene and citronellal
(family Rutaceae) are among the most widely produced fruits all over were purchased from Aladdin chemical company (Shanghai,
the world (Zhang et al., 2017). The perfume lemon is a major genus of China). Other reagents were obtained as analytical reagents
the family Citrus limon, which has been widely cultured in southern from Tansoole (Shanghai, China).
China. As shown in Figure 1, the discarded perfume lemon used in
this study primarily resulted from commercial demand of a fixed
range of fruit size, especially in the process of fruit tea preparation.
Extraction and Chemical Analysis of
Perfume lemon leaves came mainly from the pruning of perfume Discarded Fresh Perfume Lemon and
lemon trees, which were often discarded as a waste product, with some Leaves Essential Oils
adverse effects on the local environment and ecology. Thus, reusing of The discarded fresh perfume lemon and leaves were suspended in
discarded perfume lemon and leaves is not only beneficial to the distilled water with a solid-liquid ratio of 1: 4 (W/V), and the
comprehensive utilization of natural resources, but also can turn waste aliquot was placed in a 2.5 L round-bottomed sample flask which
into treasure and increase the added value of the agricultural process. was then hydro-distilled for 1.5 h after the water and sample
The two essential oils of discarded perfume lemon and leaves mixture started to boil. The essential oils were collected by oil-
were obtained by hydro-distillation; their chemical compositions water separator, which then dried over anhydrous MgSO4 and
were determined by gas chromatography-mass spectrometry filtered through a microporous membrane.
(GC-MS) analyses (Peng et al., 2014). The aim of this study Chemical components of the two essential oils were analyzed
was to investigate the antibacterial and larvicidal potentials of the on a Thermo Scientific TRACE 1300 Gas Chromatograph
perfume lemon and leaf essential oils against the selected bacteria, coupled to an ISQ Qd Mass Spectrometer and equipped with
the larval and adult Aedes albopictus (Ae. albopictus, Diptera: a TG-5 MS capillary column (30 m × 0.25 mm i.d., 0.25 μm film
Culicidae). Comparison of active components and their active thickness, Thermo Scientific). Helium was used as the carrier gas
ingredients were also discussed. The reduced usage of synthetic with a flow rate of 1 ml/min. The oven temperature was
insecticides by replacement of plant origin product is being programmed for 2 min at 60°C, then raised to 160°C at 5°C/
considered as safe alternatives for environment and human min and held for 2 min, then increased to 260°C at 20°C/min and
health (Pavela, 2015; Pavela and Benelli, 2016). maintained for 20 min. The temperature of injector and MS

Frontiers in Chemistry | www.frontiersin.org 2 May 2021 | Volume 9 | Article 679116

Wu et al. Eco-Friendly Sources of Functional Botanicals

transfer line was set to 280°C; temperature of ion source was set to diluted essential oils were prepared with DMSO as the solvent
320°C. Mass spectra were recorded in the electron impact by two-fold serial dilution starting from a stock solution of
ionization (EI) at 70 eV. The scan range was from 35 to 512 mg/mL. The final concentrations of the test essential oils
450 m/z. Identification of the components of the two essential were obtained in a range between 1 mg/mL and 512 mg/mL.
oils was based on the search results of NIST mass spectral library, Each well received 5 μL of a specific concentration of the
together with comparisons of commercially available standards essential oils and 195 μL of MHB inoculated with the test
and Kovats indices (KI). The KI was determined relative to the microorganism (1.5 × 10 5 CFU/mL); the final concentrations
retention times of a C8-C40 n-alkanes standard, and calculated as of the examined essential oils were reached. Gatifloxacin was
Eq. 1. Then the calculated KI was compared with the KI of NIST used as positive control and DMSO was used as negative
Chemistry WebBook. control (Wu et al., 2021). The microplates were incubated in a
bacteriological oven for 24 h at 37 °C, and the susceptibility
100(tx − tn )
KI  100n + . (1) results of tested samples were monitored by measuring the
tn+1 − tn absorbance at 600 nm using a Multimodel Plate Reader
In Eq. 1 tx means the retention time of analyzed component (Infinite 200). The lowest concentration without visible
(min); tn means the retention time of n-alkane in which carbon growth was defined as the MIC (Wu et al., 2021).
number is n; tn+1 means the retention time of n-alkane in which The minimum bactericidal concentrations (MBCs) were
carbon number is n+1; and tn＜tx＜tn+1. determined based on the MIC results (Jabrane et al., 2010;
Chouaib et al., 2015; Wu et al., 2018; Wu et al., 2021): serial
Microorganisms and Culture Media sub-cultivation of a 5 μL aliquots near the MIC with 195 μL fresh
The bacterial strains of two Staphylococcus aureus (ATCC 6538 MHB were uniformly inoculated onto MHA solidified in 90 mm
and ATCC 29213), Staphylococcus epidermidis (ATCC 12228), Petri dishes; incubation for 24 h at 37°C. The lowest
Salmonella typhimurium (CMCC 50115) and Escherichia coli concentration of antimicrobial agent that killed at least 99.9%
(CMCC 44102) were obtained from Guangdong Culture of the starting inoculum was defined as the MBC endpoint, which
Collection Center (Guangdong, People’s Republic of China). was determined as the lowest concentration with no visible
All the five strains were cultured in Mueller-Hinton agar growth of bacterial colony on the Petri dishes (Wu et al.,
(MHA) and Mueller-Hinton broth (MHB). 2021). All experiments were conducted in triplicate. The final
concentration of DMSO in the 96-well plate had no effect on
bacterial growth.
Agar Disk Diffusion Assay of the Two
Essential Oils
The antimicrobial of essential oils were determined according to
Killing Kinetic Studies of the Two Essential
the standard agar disk diffusion method with a slight Oils
modification (Luangtongkum et al., 2007; Gaudreau et al., The killing kinetic studies of the two essential oils on tested
2008; Benamrouche et al., 2014; Wu et al., 2018; Wu et al., bacteria strains (Theophel et al., 2014; Phee et al., 2015; Meng
2021). A 0.5 McFarland (1 × 107 to 1 × 108 CFU/mL) et al., 2016; Wu et al., 2018), including two strains of
concentration of the bacterial suspension was uniformly Staphylococcus aureus (ATCC 6538 and ATCC 29213),
inoculated onto MHA solidified in 120 mm Petri dishes. Once Staphylococcus epidermidis (ATCC 12228), Salmonella
the dishes were prepared, 6 mm-diameter discs of filter paper typhimurium (CMCC 50115) and Escherichia coli (CMCC
containing 5 μL of the examined essential oil solutions, which had 44102), were performed in 96-well plates, respectively. Four
been diluted with dimethyl sulfoxide (essential oil: DMSO 1:9 v/ different concentrations (negative, 0.5 ×MIC, 1 ×MIC,
v), were pressed gently against the surface of the agar. Discs 2 ×MIC) of each essential oil were studied. The microplates
containing gatifloxacin (1 nmol) was used as positive control, were incubated for 24 h at 37°C, and the growth of bacteria was
while DMSO was used as the negative control. The dishes were monitored by measuring the absorbance at 600 nm using a
incubated in a constant temperature incubator at 37°C for 24 h. Multimodel Plate Reader (Infinite 200) every 1 h (Wu et al.,
The inhibition zone diameter was measured by a vernier caliper. 2021).
All the experiments were performed in triplicate (Wu et al., 2021).
Mosquitoes
A local strain of Ae. albopictus larvae were collected at Huangpu
Broth Microdilution Assay of the Two District, Guangzhou, Guangdong Province, China, and had been
Essential Oils maintained consecutively since 2013 in the laboratory. The
The minimum inhibitory concentration (MIC) and the species were reared at 14:10 light/dark photoperiod, 70 ± 5%
minimum bactericidal concentration (MBC) were relative humidity at 26 ± 2°C, in the laboratory of International
determined by a microdilution method in 96-well plates Healthcare Innovation Institute (Jiangmen), Jiangmen, China.
according to Clinical and Laboratory Standards Institute Larvae were fed daily with fish food, and adults were fed with 5%
(CLSI), with a slight modification (Sader et al., 2006; Meng glucose solution. The fourth instar larvae and two to five-days-old
et al., 2016; Wu et al., 2018; Wu et al., 2021). A series of female mosquitoes were used in this bioassay.

Frontiers in Chemistry | www.frontiersin.org 3 May 2021 | Volume 9 | Article 679116

Wu et al. Eco-Friendly Sources of Functional Botanicals

Larvicidal Assay GC-MS was employed to identify the constituents and their
The larvicidal activity was tested by immersion method in a 24- relative content in the essential oils by the method of Kovats
well plate according to the standard procedures recommended by indices (Retention indices), and the results were as presented in
the World Health Organization, with slight modifications (WHO, Table 1 and Figure 2. The chemical structures of major
2005; Seo et al., 2015; dos Santos Moreira et al., 2018). Firstly, the components in the two essential oils were as presented in
two essential oils, (R)-(+)-limonene and citronellal are diluted in Figure 3.
acetone to prepare an acetone solution with a series of As indicated in Table 1 and Figure 2, 26 components were
concentration of 0–800 ppm. Then, five larvae were transferred identified from the two essential oils, representing almost 100% of
into the series testing solution each well for testing with no the total amount. The predominant component of the fresh
feeding during the screening. Mortality was determined after 24 h perfume lemon essential oil was (R)-(+)-limonene,
of exposure. Three replicates were carried out for each sample. representing 49.63%, only 12.24% of this component was
The mortalities of each essential oil sample ranging from 0 to detected in the fresh leaves. In comparison, it was found that
100% were obtained. At least 10 concentrations were selected to the component of citronellal in the essential oil extracted from
determine the LC20, LC50, and LC80. Three replicates were carried leaves was the highest, reaching 78.35%. Other types of five
out for every sample at every concentration. Deltamethrin was alcohols and two aldehydes were detected in fresh perfume
used as positive control while blank reference was used as lemon essential oil, whilst 7.18% of citronellol was also found
negative control. in leaves. An ester, citronellyl acetate was identified in the
Adjusted mortality is calculated by Eq. 2: essential oil extracted from the leaf, but not from the fruit.
mortality of testing group − mortality of blank group
Apart from the predominant component, the lemon essential
Adjusted mortality (%)  ×100. oil is composed of d-limonene, γ-terpinene and β-pinene,
1 − mortality of blank group
(2) followed by four other monoterpene hydrocarbons (myrcene,
sabinene, α-pinene and p-cimene) and other compounds in five
different classes (sesquiterpene hydrocarbons, carbonyl
Adulticidal Bioassays compounds, alcohols, esters and oxides). In comparison to
Adulticidal bioassays were assayed using Tarsal contact assay other previously reported analysis of essential oils from similar
according to the reported method with slight modification sources, most of the identified components are the same, but the
(Chansang et al., 2018; Lees et al., 2019). Firstly, a graduated series composition of these components is different. For example, the
of concentrations (0∼200 μg/m2) of deltamethrin, essential oils and percentages of citronellol and citronellal determined from the
the two chemotypes ((R)-(+)-limonene and citronellal) sample essential oils in this study are higher than in previous report and
solution in acetone were prepared. Then, the sample solution was some of the less volatile components are also slightly different
covered on the surface of 60 mm glass Petri dishes evenly. Bioassays (Ciriminna et al., 2017; Yazgan et al., 2019).
were conducted immediately after the 4 h drying period, at 26 ± 2°C
and ambient humidity. Ten female adults (2–5 days old) were exposed
in each Petri dish. A 25 mL plastic deli pot with a hole melted through Antibacterial Activity of the Two Essential
the base was fixed onto each Petri dish with Parafilm. Mosquitoes Oils
were introduced through the hole and, after exposing them to the The antibacterial activity of the two essential oils was assayed
treated surface for 1 h, they were aspirated out and transferred to against five microorganisms, including two Gram-negative
holding cups. Then mosquitoes were put in an incubator with bacteria strains and three Gram-positive bacteria strains. All
constant temperature (26 ± 2°C) and humidity (70 ± 5%), and fed the bacterial strains were cultured in Muller Hinton agar at
with sugar solution. Mortality was determined after 24 h of 37°C overnight (Wu et al., 2021).
incubation. Three duplicate trials were carried for every
concentration. Blank reference was treated as negative control Agar Disk Diffusion Assay of the Two Essential Oils
while deltamethrin was used as positive control. The adjusted The antimicrobial activity of the two essential oils against the five
mortality was calculated by the formula mentioned in Larvicidal different microorganisms are summarized in Table 2. 5 μL of
Assay (Eq. 2). The concentration is expressed as μg of deltamethrin pure essential oil or 512 mg/mL acetone solution of essential oil
per m2 of surface of culture dishes. Regression analysis of adjusted on the filter paper was tested in this agar disk diffusion assay. The
mortalities vs. the concentrations of each examined sample to give sizes of the inhibition zone diameters assay suggest that the
toxicity regression equations. antibacterial activity of the two essential oils against Gram-
positive strains is higher than that against Gram-negative
strains. The results also indicate that the perfume lemon
RESULTS AND DISCUSSION essential oil was more potent than the leaves essential oil, in
which the inhibition zones diameter were in the range from 7.27 ±
Chemical Composition of the Two Essential 0.06 to 10.37 ± 0.15 mm of three tested Gram-positive
Oils Staphylococcus strains. However, both essential oils displayed
The essential oils extracted from discarded perfume lemon and no inhibitory activity against the two selected Gram-negative
leaves were prepared by traditional hydro-distillation with yield bacteria strains. Interesting, while testing the one of main
of 0.044% (w/w) for perfume lemon and 0.338% (w/w) for leaves. components of the essential oils, (R)-(+)-limonene, showed

Frontiers in Chemistry | www.frontiersin.org 4 May 2021 | Volume 9 | Article 679116

Wu et al. Eco-Friendly Sources of Functional Botanicals

TABLE 1 | Chemical compositions of fresh perfume lemon and leaves essential oils.

No Componenta RTb (min) KIc KI litd Perfume lemon Perfume lemon

(%) leaves (%)
Name CAS no

1 β-myrcene 123–35–3 7.546 990 990 1.49 0.56

2 octanal 124–13–0 7.871 1,002 1,002 0.49 ND
3 (R)- (+)-limonene 5,989–27–5 8.626 1,030 1,028 46.04 11.38
4 ocimene mixture of isomers 3,338–55–4 9.102 1,047 1,047 0.19 0.47
5 3,8-p-menthadiene 586–67–4 9.746 1,070 1,071 0.34 ND
6 alpha, p-dimethylstyrene 1,195–32–0 10.288 1,090 1,090 0.20 ND
7 linalool 78–70–6 10.562 1,099 1,098 2.29 0.75
8 nonanal 124–19–6 10.678 1,104 1,104 0.44 0.28
9 (−)-isopulegol 89–79–2 11.92 1,148 1,150 3.87 ND
10 citronellal 106–23–0 12.067 1,153 1,153 4.71 75.34
11 (1R,2R,5S)-5-methyl-2-(prop-1- — 12.229 1,159 — 2.39 ND
en-2-yl) cyclohexanol
12 2-(4-methyl-2,4-cyclohexadienyl)- 1,686–20–0 12.523 1,169 1,168 1.40 ND
2-propanol
13 terpinen-4-ol 562–74–3 12.898 1,182 1,181 0.71 ND
14 2-(4-methylphenyl) propan-2-ol 1,197–01–9 13.03 1,187 1,187 0.43 ND
15 α-terpineol 98–55–5 13.197 1,193 1,189 4.04 ND
16 decanal 112–31–2 13.541 1,205 1,205 0.37 0.79
17 (Z)-carveol,2-methyl-5-(1-methy 1,197–06–4 13.658 1,209 1,210 0.19 ND
lethenyl)-2-cyclohexen-1-ol,
cis-mentha-1,8-dien-6-ol
18 citronellol 106–22–9 14.18 1,229 1,228 2.70 6.68
19 neral 106–26–3 14.56 1,242 1,241 10.57 0.51
20 nerol 106–25–2 14.92 1,256 1,259 0.59 ND
21 geranial 141–27–5 15.371 1,272 1,271 15.79 0.50
22 citronellyl acetate 150–84–5 17.545 1,353 1,353 0.07 1.70
23 neryl acetate 141–12–8 18.336 1,383 1,381 0.13 0.51
24 β-caryophyllene 87–44–5 19.415 1,426 1,425 ND 0.52
25 2,6-dimethyl-6-(4-methyl-3-pent 17,699–05–7 19.75 1,439 1,440 0.22 ND
enyl)bicyclo[3.1.1]hept-2-ene
26 β-bisabolene 495–61–4 21.538 1,510 1,510 0.32 ND
a
Components are listed in the order of KI values. Only major components (content >0.1%) are listed in the table.
b
Kovats indices (min).
c
Linear Kovats indices homologous series of C8-C30 alkanes performed on a TG-5MS column.
d
Linear Kovats indices were taken from https://webbook.nist.gov/chemistry/.

FIGURE 2 | Chemical compositions of the two essential oils.

similar activity against Gram-positive bacteria, this compound Table 3. The similar MIC and MBC values suggested that
displayed higher antibacterial activity against Gram-negative the two essential oils had inhibitory activity through a
bacteria than the essential oils. bactericidal effect against three Gram-positive strains of
Staphylococcus, with both MICs and MBCs ranging from
Broth Microdilution Assay of the Two Essential Oils 1.6 to 6.4 mg/mL. However, in contrast to the results from
Using the micro-dilution method, the essential oils extracted the agar disk diffusion assay, the results from the broth assays
from discarded perfume lemon and leaves were tested in the indicated that the essential oil extracted from the fruit was
MICs and MBCs assays and the results are presented in also active against the two tested strains of Gram-negative

Frontiers in Chemistry | www.frontiersin.org 5 May 2021 | Volume 9 | Article 679116

Wu et al. Eco-Friendly Sources of Functional Botanicals

FIGURE 3 | Chemical structures of major components in the two essential oils.

TABLE 2 | Biological evaluation of the two essential oils expressed in the inhibition zone (mm).

Sample code Bacterium and inhibition zone (mm)a

Gram-positive bacteria strains Gram-negative bacteria strains
Staphylococcus aureus Staphylococcus aureus Staphylococcus Salmonella Escherichia
(ATCC (ATCC epidermidis (ATCC typhimurium (CMCC coli (CMCC
6538) 29213) 12228) 50115) 44102)

Perfume lemon essential oil 9.87 ± 1.15 8.57 ± 0.61 9.10 ± 0.78 ＜6b ＜6
(2.56 mg)
Perfume lemon essential oil 10.37 ± 0.67 10.20 ± 0.52 10.37 ± 0.15 ＜6 ＜6
(100%)c
Perfume lemon leaves 8.97 ± 1.10 7.27 ± 0.06 7.53 ± 0.61 ＜6 ＜6
essential oil (2.56 mg)
Perfume lemon leaves 8.20 ± 0.26 7.37 ± 0.12 8.23 ± 0.31 ＜6 ＜6
essential oil (100%)c
(R)-(+)-limonene (2.56 mg) ND＃ ND ND ND ND
(R)-(+)-limonene (100%)c 6.77 ± 0.23 6.70 ± 0.50 7.13 ± 0.25 11.67 ± 0.32 7.93 ± 0.35
Citronellal (2.56 mg) ND ND 7.70 ± 0.10 ND ND
Citronellal (100%)c ND ND 8.60 ± 0.26 ND ND
Gatifloxacind 19.12 ± 0.73 17.13 ± 0.64 18.67 ± 0.25 20.92 ± 0.72 20.78 ± 1.06
a
Results are expressed as the diameter of inhibition zone (mm), values represent the means of three independent replicates ±SD.
b
＜6, no obvious inhibition zone was detected.
c
Pure perfume lemon and its leaf essential oil with no dilution.
#ND, not detected.
d
Gatifloxacin was used as a positive control, the dosage of gatifloxacin used in the inhibition zone assay is 1 nmol.

bacterial (MIC and MBC  3.2 mg/mL), which mighty cause Meng et al., 2016; Wu et al., 2018). Figure 4 displays the
by some of the more volatile components, such as alcohols time-kill curves of the two essential oils against five bacteria
and aldehydes in the essential oil behaving differently in strains, including three Gram-positive Staphylococcus
these two types of assays. Since both essential oils were strains (ATCC 6538, ATCC 29213, ATCC 12228), and two
derived from plant waste products, these findings Gram-negative strains of Salmonella typhimurium (CMCC
indicated that recycling and reusing of these by-products 50115) and Escherichia coli (CMCC 44102). As presented in
can produce useful material with antibacterial activity. Figure 4, all the bacterial strains could be effectively
Moreover, it suggests that the physiochemical properties of inhibited at the MIC of each essential oil but with a slight
the essential oils and their components may have further growth after 10 h’ examination, which may be because of
implication when apply this type of essential oils in future their volatility during the assay. In addition, the bacterial
research and development. growth could be effectively inhibited or killed at higher
concentrations of 2 ×MICs and 4 ×MICs, even till the end
Killing Kinetic Studies of the Two Essential Oils of 24 h assay, which is also in generally accordance with the
The time killing kinetic studies were assayed over a period of MBCs results. While the bacteria strains were incubated with
24 h at 37 °C according to previous study with a slightly the 0.5 ×MICs of both essential oils, the bacterial
modification (Theophel et al., 2014; Phee et al., 2015; concentration was initially inhibited to a certain level for

Frontiers in Chemistry | www.frontiersin.org 6 May 2021 | Volume 9 | Article 679116

Wu et al. Eco-Friendly Sources of Functional Botanicals

TABLE 3 | Biological Evaluation of the two essential oils expressed in MICa and MBCb (mg/mL).

Sample code MICs and MBCs of selected bacterium (mg/mL or ng/mL)

Gram-positive bacteria strains Gram-negative bacteria strains
Staphylococcus Staphylococcus Staphylococcus Salmonella Escherichia coli
aureus (ATCC aureus (ATCC epidermidis typhimurium (CMCC 44102)
6538) 29213) (ATCC 12228) (CMCC 50115)
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

Perfume lemon essential oil (mg/mL) 3.2 3.2 1.6 3.2 1.6 1.6 3.2 3.2 3.2 3.2
Perfume lemon leaves essential oil (mg/mL) 6.4 6.4 3.2 3.2 6.4 6.4 >12.8 >12.8 >12.8 >12.8
(R)-(+)-limonene (mg/mL) 4 NT# 512 NT 256 NT 2 NT 256 NT
Citronellal (mg/mL) 1 NT 16 NT 4 NT 128 NT 128 NT
Gatifloxacinc (ng/mL) 75.08 75.08 75.08 75.08 75.08 75.08 75.08 75.08 75.08 75.08
a
MICs (mg/mL), minimum inhibitory concentrations, i.e., the lowest concentration of the compound that completely inhibits the growth of bacteria.
b
MBCs (mg/mL), minimum bacterial concentrations, i.e., the lowest concentration of the compound that completely kills the bacteria.
#NT, not tested.
c
Gatifloxacin (ng/mL) was chosen as a positive control.

FIGURE 4 | The time killing kinetic studies of the oil extracted from discarded perfume lemon and leaves against five bacteria strains. Including Staphylococcus
aureus (ATCC 6538) (A,F), Staphylococcus epidermidis (ATCC 12228) (B,G), Staphylococcus aureus subsp. Aureus (ATCC 29213) (C,H), Salmonella typhimurium
(CMCC 50115) (D), Escherichia coli (CMCC 44102) (E) and exposed to four different concentrations of the oil extracted from discarded perfume lemon (Figures 3A–E)
and the oil extracted from discarded perfume leaves (Figures 3F–H) according to their respective MICs (n  4), gatifloxacin was also conducted as positive control
along with the two tested essential oils.

several hours then gradually increased, and no inhibitory toxicity regression equations (produced by the software of
effect was observed at the end of assay. Similar growth Origin 8.5), LC 20 , LC 50 and LC 80 values were calculated from
inhibition patterns were generally observed for the five the curve fitting equations. The larvicidal activity of the oil
selected bacteria strains in dose-dependent manner extracted from discarded perfume lemon was LC 20 
during the time killing kinetic studies of the two 248.60 ppm, LC 50  384.81 ppm and LC 80  595.64 ppm.
essential oils. The larvicidal activity of perfume lemon leaves essential oil
was LC 20  195.73 ppm, LC 50  395.09 ppm and LC 80 
Larvicidal Activity of the Two Essential Oils 797.52 ppm. The larvicidal activity of the (R)-
The larvicidal activities of the essential oils extracted from (+)-limonene was LC 20  237.88 ppm, LC 50  295.28 ppm
discarded perfume lemon and leaves and the two main and LC 80  364.26 ppm and the activity of citronellal was
components, (R)-(+)-limonene and citronellal in serial LC 20  198.53 ppm, LC 50  267.08 ppm and LC 80 
concentrations ranged from 0 to 650 ppm were tested 382.92 ppm. The activity of these two main chemical
against fourth instar larvae of Ae. albopictus. As shown in components is comparable but slightly better than the
Table 4 and Figure 5, they all showed dose-dependent corresponding essential oils, which may suggest that (R)-
manner for larvicidal potency and according to the (+)-limonene and citronellal play a key role in the effect of
nonlinear fitting curves of the acute toxicity study of essential oils on larvae.

Frontiers in Chemistry | www.frontiersin.org 7 May 2021 | Volume 9 | Article 679116

Wu et al. Eco-Friendly Sources of Functional Botanicals

TABLE 4 | Acute toxicity of the two essential oils and two chemotypes against fourth instar larvae of Ae. albopictus (ppm).

Sample code Toxicity regression equations 4th instar larvae of Ae. albopictus
a
LC20 (ppm) LC50 (ppm)a (95% CI) LC80 (ppm)a

Perfume lemon essential oil y  118.942 − 120.457/(1 + (x/384.808)3.173 ) (R2  0.985) 248.60 384.81 (278.464–437.534) 595.64
Perfume lemon leaves essential oil y  138.471 − 141.968/(1 + (x/395.094)1.974 ) (R2  0.970) 195.73 395.09 (204.929–383.510) 797.52
(R)-(+)-limonene y  105.266 − 108.298/(1 + (x/297.369)5.864 ) (R2  0.975) 237.88 295.28 (255.816–328.304) 364.26
Citronellal y  103.437 − 128.585/(1 + (x/240.245)3.219 ) (R2  0.995) 198.53 267.08 (207.284–345.576) 382.92
Deltamethrinb y  118.440 − 153.112/(1 + (x/0.531)1.247 ) (R2  0.992) 0.175 0.531 (0.055–0.148) 1.615
a
14 serial concentrations were selected to determine LC20, LC50 and LC80 values. Mortality of each examined concentration was performed three replicates, five larvae each well was
applied in this high throughput assay.
b
Deltamethrin was treated as positive control.

FIGURE 5 | Toxicity curve of the two chemotypes against Ae. albopictus.

TABLE 5 | Acute toxicity of the two essential oils against Ae. albopictus (μg/cm2).

Sample code Toxicity regression equations Ae. Albopictus (2–5 days)

2 a
LD20 (μg/cm ) LD50 LD80 (μg/cm2)a
(μg/cm2)a (95% CI)

Perfume lemon essential oil y  107.424 − 107.878/(1 + (x/133.059)4.138 ) (R2  0.996) 95.180 133.059 (106.631–142.248) 186.014
Perfume lemon leaves essential oil y  140.278 − 136.307/(1 + (x/218.962)3.367 ) (R2  0.985) 145.071 218.962 (140.992–202.215) 330.489
Deltamethrinb y  127.489 − 128.998/(1 + (x/0.0659)1.292 ) (R2  0.996) 0.0225 0.0659 (0.027–0.059) 0.193
a
At least nine serial concentrations were selected to determine LD20, LD50 and LD80 values. Mortality of each examined concentration was performed three replicates, 10 adult female
mosquitoes each concentration was applied in this assay.
b
Deltamethrin was treated as positive control.

Tarsal Contact Test lemon was LD20  95.180 μg/cm2, LD50  133.059 μg/cm2, LD80
A direct tarsal contact assay was performed for quantification of  186.014 μg/cm2. The adulticidal activity of perfume lemon
the anti-mosquito activity. The adulticidal bioassay of the leaves was LD20  145.071 μg/cm2, LD50  218.962 μg/cm2 and
essential oils extracted from discarded perfume lemon and LD80  330.489 μg/cm2, which were less active than the essential
leaves and the two main components (R)-(+)-limonene and oil extracted from the fruit. Surprisingly, the two components
citronellal in serial concentrations ranged from 0 to 650 μg/cm2 with good activity against larvae when tested alone failed to
against 2–5 days old female adults of Ae. albopictus were produce promising activity against any of the mosquito larvae
performed. As shown in Table 5 and Figure 6, both essential (mortality >50% was observed only at the higher test dosage). In
oils revealed dose-dependent adulticidal potency and according the present study, GC-MS data (Table 1) reveals that besides
to the nonlinear fitting curves of the acute toxicity study of (R)-(+)-limonene and citronellal there are many other
toxicity regression equations (produced by the software of terpenoids and related compounds present in the oil. Thus,
Origin 8.5), LD20, LD50 and LD80 values were calculated the activity of the two essential oils against the adult mosquito
according to the fitting curve equations. The adulticidal (LD50  133.059–218.962 μg/cm2) may be attributed to the
activity of the essential oils extracted from discarded perfume additive or synergistic or blend effect of many/some of the

Frontiers in Chemistry | www.frontiersin.org 8 May 2021 | Volume 9 | Article 679116

Wu et al. Eco-Friendly Sources of Functional Botanicals

FIGURE 6 | Toxicity curve of the two essential oils against Ae. albopictus.

constituents. This effect has previously been observed in quality control are other challenge in the development
some other essential oils, whose activity is due to the process. The study of molecular mechanisms of the active
combination of the main components, but no significant components may potentially guide the development of more
activity has been found with single components alone efficient new vector control or antibacterial agents. Relevant
(Papachristos et al., 2004). Overall, these two essential research is undergoing and the results will be reported in due
oils have certain insecticidal activity against adult course.
mosquitoes.
Statistical Analysis
The results are expressed as the mean ± standard deviation (SD).
CONCLUSION
In summary, discarded perfume lemon and leaves, which DATA AVAILABILITY STATEMENT
were often disposed as waste by-produces, were recycled and
reused during this study. The two essential oils were The original contributions presented in the study are included in
produced by traditional hydro-distillation with good yields. the article/Supplementary Material, further inquiries can be
Kovats indices method was applied to identify the directed to the corresponding authors.
constituents and their relative content in the essential oils
by GC-MS. 26 chemical components were identified,
representing almost 100% of the total amount. The AUTHOR CONTRIBUTIONS
bioassay results implied that both essential oils present
antibacterial activity, especially for the Gram-positive PW: Conceptualization, Methodology, Software, XT:
Staphylococcus bacterial strains. Moreover, the anti- Writing- Reviewing and Editing, Software, RJ: Writing-
mosquito potential of the two essential oils was also clearly Original draft preparation, JL: Data curation, ML: Data
demonstrated in their larvicidal and adulticidal activities. curation, HD: Validation, KW: Writing- Original draft
Therefore, the development of the essential oils extracted preparation, ZS: Original draft preparation; BC:
from discarded perfume lemon and leaves is not only friendly Conceptualization, XX: Software, Validation, CL: Data
to the environment and ecology, but also can provide added curation, ZL: Conceptualization, QZ: Data curation, XZ:
value to the agricultural process. Writing- Reviewing and Editing, KZ: Writing- Reviewing
As far as the comparison between (R)-(+)-Limonene and and Editing, DL: Supervision, Writing- Reviewing and
Citronellal is concerned, the insecticidal activity of the Editing, WH: Supervision, Writing- Reviewing and Editing.
oxygenated monoterpenoids is higher than that of
containing hydrocarbons. This conclusion is consistent with
the previous findings (Regnault-Roger et al., 1993; FUNDING
Papachristos et al., 2004). However, these two chemicals
showed significantly lower adulticidal activity than that of This study was supported by National Natural Science
essential oils in this study. This finding suggests the Foundation of China (No. 81803390), Guangdong Basic and
insecticidal activity of the researched essential oil is not Applied Basic Research Foundation (No. 2021A1515010221),
directly related to the content of its main components. In Special Fund Project of Science and Technology Innovation
addition, the mode of action of these two essential oils from Strategy of Guangdong Province 2018 and 2020 (No. Jiangke
this study and from other similar studies remains to be (2018)352 and Jiangke (2020)182), the project of Jiangmen city
elucidated (Sheng et al., 2020). This is one of the social welfare innovation platform construction (No.
significant barriers for the further development of plant 2016350100170008351, 2018090103460022105). The authors
origin essential oils whilst chemical standardization and are also grateful to the foundation of Department of

Frontiers in Chemistry | www.frontiersin.org 9 May 2021 | Volume 9 | Article 679116

Wu et al. Eco-Friendly Sources of Functional Botanicals

Education of Guangdong Province (No. 2020KZDZX1202, ACKNOWLEDGMENTS

2018KTSCX236, 2017KSYS010 and 2016KCXTD005) and
Jiangmen Program for Innovative Research Team (No. Sincere and heartfelt thanks must go to Mrs. Sulian Liang who has
2018630100180019806) and the Youth Foundation of Wuyi given generous suggestions on the English language of the
University (No. 2017td01). manuscript.

Antibacterial Activities of the Root Oil of Ridolfia Segetum (L.) Moris from
REFERENCES Tunisia. Nat. Product. Res. 24, 491–499. doi:10.1080/14786410802228520
Lees, R., Praulins, G., Davies, R., Brown, F., Parsons, G., White, A., et al. (2019). A
Bakkali, F., Averbeck, S., Averbeck, D., and Idaomar, M. (2008). Biological Effects Testing cascade to Identify Repurposed Insecticides for Next-Generation
of Essential Oils - A Review. Food Chem. Toxicol. 46, 446–475. doi:10.1016/j.fct. Vector Control Tools: Screening a Panel of Chemistries with Novel Modes
2007.09.106 of Action against a Malaria Vector. Gates Open Res. 3, 1464. doi:10.12688/
Benamrouche, N., Lazri, M., Tali-Maamar, H., and Rahal, K. (2014). Comparaison gatesopenres.12957.2
de la Sensibilité aux Antibiotiques de Corynebacterium Diphtheriae par les Liu, N. (2015). Insecticide Resistance in Mosquitoes: Impact, Mechanisms, and
Mméthodes de Dilution en Bouillon et de Diffusion (E-test et Disques). Méd. Research Directions. Annu. Rev. Entomol. 60, 537–559. doi:10.1146/annurev-
Mal. Infect. 44, 392–393. doi:10.1016/j.medmal.2014.07.007 ento-010814-020828
Bhatt, S., Gething, P. W., Brady, O. J., Messina, J. P., Farlow, A. W., Moyes, C. L., Luangtongkum, T., Morishita, T. Y., El-Tayeb, A. B., Ison, A. J., and Zhang, Q.
et al. (2013). The Global Distribution and burden of Dengue. Nature 496, (2007). Comparison of Antimicrobial Susceptibility Testing of Campylobacter
504–507. doi:10.1038/nature12060 Spp. By the agar Dilution and the agar Disk Diffusion Methods. J. Clin.
Chang, K.-S., Kim, H.-C., Klein, T. A., and Ju, Y. R. (2017). Insecticide Resistance Microbiol. 45, 590–594. doi:10.1128/jcm.00986-06
and Cytochrome-P450 Activation in Unfed and Blood-Fed Laboratory and Meng, Y., Hou, X., Lei, J., Chen, M., Cong, S., Zhang, Y., et al. (2016). Multi-
Field Populations of Culex pipiens Pallens. J. Pest Sci. 90, 759–771. doi:10.1007/ functional Liposomes Enhancing Target and Antibacterial Immunity for
s10340-016-0820-1 Antimicrobial and Anti-biofilm against Methicillin-Resistant Staphylococcus
Chansang, A., Champakaew, D., Junkum, A., Jitpakdi, A., Amornlerdpison, aureus. Pharm. Res. 33, 763–775. doi:10.1007/s11095-015-1825-9
D., Aldred, A. K., et al. (2018). Synergy in the Adulticidal Efficacy of Papachristos, D. P., Karamanoli, K. I., Stamopoulos, D. C., and Menkissoglu-
Essential Oils for the Improvement of Permethrin Toxicity against Aedes Spiroudi, U. (2004). The Relationship between the Chemical Composition of
aegypti L. (Diptera: Culicidae). Parasites & Vectors 11, 417. doi:10.1186/ Three Essential Oils and Their Insecticidal Activity againstAcanthoscelides
s13071-018-3001-7 obtectus(Say). Pest Manag. Sci. 60, 514–520. doi:10.1002/ps.798
Chouaïb, K., Hichri, F., Nguir, A., Daami-Remadi, M., Elie, N., Touboul, D., et al. Pavela, R., and Benelli, G. (2016). Essential Oils as Ecofriendly Biopesticides?
(2015). Semi-synthesis of New Antimicrobial Esters from the Natural Oleanolic Challenges and Constraints. Trends Plant Sci. 21, 1000–1007. doi:10.1016/j.
and Maslinic Acids. Food Chem. 183, 8–17. doi:10.1016/j.foodchem.2015. tplants.2016.10.005
03.018 Pavela, R. (2015). Essential Oils for the Development of Eco-Friendly Mosquito
Ciriminna, R., Fidalgo, A. M., Carnaroglio, D., Tamburino, A., Cravotto, G., Larvicides: A Review. Ind. Crops Prod. 76, 174–187. doi:10.1016/j.indcrop.2015.
Ilharco, L. M., et al. (2017). Lemon Essential Oil of Variable Composition 06.050
by Changing the Conditions of the Extraction from Lemon Peel via Microwave Peng, C., Zhao, S.-Q., Zhang, J., Huang, G.-Y., Chen, L.-Y., and Zhao, F.-Y. (2014).
Hydrodiffusion and Gravity. ChemistrySelect 2, 7123–7127. doi:10.1002/slct. Chemical Composition, Antimicrobial Property and Microencapsulation of
201701471 Mustard (Sinapis alba) Seed Essential Oil by Complex Coacervation. Food
Derua, Y. A., Kweka, E. J., Kisinza, W. N., Githeko, A. K., and Mosha, F. W. (2019). Chem. 165, 560–568. doi:10.1016/j.foodchem.2014.05.126
Bacterial Larvicides Used for Malaria Vector Control in Sub-saharan Africa: Phee, L. M., Betts, J. W., Bharathan, B., and Wareham, D. W. (2015). Colistin and
Review of Their Effectiveness and Operational Feasibility. Parasites Vectors 12, Fusidic Acid, a Novel Potent Synergistic Combination for Treatment of
426. doi:10.1186/s13071-019-3683-5 Multidrug-Resistant Acinetobacter Baumannii Infections. Antimicrob. Agents
dos Santos Moreira, A. M., Bittencourt, V. C. E., Costa, F. L. S., Elena de Lima, M., Chemother. 59, 4544–4550. doi:10.1128/aac.00753-15
Lopes, M. T. P., Borges, W. S., et al. (2018). Hydrophobic Nanoprecipitates of Ranson, H., and Lissenden, N. (2016). Insecticide Resistance in African
β-Cyclodextrin/Avermectins Inclusion Compounds Reveal Insecticide Activity Anopheles Mosquitoes: A Worsening Situation that Needs Urgent Action
against Aedes aegypti Larvae and Low Toxicity against Fibroblasts. J. Agric. Food to Maintain Malaria Control. Trends Parasitol. 32, 187–196. doi:10.1016/j.pt.
Chem. 66, 7275–7285. doi:10.1021/acs.jafc.8b01300 2015.11.010
Duguma, D., Rueda, L. M., and Debboun, M. (2020). “Chapter 11 - Mosquito- Regnault-Roger, C., Hamraoui, A., Holeman, M., Theron, E., and Pinel, R. (1993).
Borne Diseases,” in Mosquitoes, Communities, and Public Health in Texas, Insecticidal Effect of Essential Oils from Mediterranean Plants
Editors M. Debboun, M. R. Nava, and L. M. Rueda (Cambridge, MA: Academic uponAcanthoscelides Obtectus Say (Coleoptera, Bruchidae), a Pest of
Press), 319–337. doi:10.1016/b978-0-12-814545-6.00011-0 Kidney Bean (Phaseolus vulgaris L.). J. Chem. Ecol. 19, 1233–1244. doi:10.
Gaudreau, C., Girouard, Y., Gilbert, H., Gagnon, J., and Bekal, S. (2008). Comparison 1007/bf00987383
of Disk Diffusion and Agar Dilution Methods for Erythromycin, Ciprofloxacin, Rouis, Z., Laamari, A., Abid, N., Elaissi, A., Cioni, P. L., Flamini, G., et al. (2013).
and Tetracycline Susceptibility Testing of Campylobacter Coli and for Chemical Composition and Larvicidal Activity of Several Essential Oils from
Tetracycline Susceptibility Testing of Campylobacter Jejuni Subsp. Jejuni. Aac Hypericum Species from Tunisia. Parasitol. Res. 112, 699–705. doi:10.1007/
52, 4475–4477. doi:10.1128/aac.00767-08 s00436-012-3189-y
Gross, A. D., Norris, E. J., Kimber, M. J., Bartholomay, L. C., and Coats, J. R. (2017). Sader, H. S., Fritsche, T. R., and Jones, R. N. (2006). Daptomycin Bactericidal
Essential Oils Enhance the Toxicity of Permethrin againstAedes Activity and Correlation between Disk and Broth Microdilution Method
aegyptiandAnopheles Gambiae. Med. Vet. Entomol. 31, 55–62. doi:10.1111/ Results in Testing of Staphylococcus aureus Strains with Decreased
mve.12197 Susceptibility to Vancomycin. Aac 50, 2330–2336. doi:10.1128/aac.01491-05
Ivory, M. O., Birchall, J. C., and Piguet, V. (2015). Early Dengue Virus Infection in Seo, S.-M., Jung, C.-S., Kang, J., Lee, H.-R., Kim, S.-W., Hyun, J., et al. (2015).
Human Skin: A Cycle of Inflammation and Infectivity. J. Invest. Dermatol. 135, Larvicidal and Acetylcholinesterase Inhibitory Activities of Apiaceae Plant
1711–1712. doi:10.1038/jid.2014.544 Essential Oils and Their Constituents againstAedes Albopictusand
Jabrane, A., Ben Jannet, H., Mastouri, M., Mighri, Z., and Casanova, J. (2010). Formulation Development. J. Agric. Food Chem. 63, 9977–9986. doi:10.
Chemical Composition and In Vitro Evaluation of Antioxidant and 1021/acs.jafc.5b03586

Frontiers in Chemistry | www.frontiersin.org 10 May 2021 | Volume 9 | Article 679116

Wu et al. Eco-Friendly Sources of Functional Botanicals

Shaw, W. R., and Catteruccia, F. (2019). Vector Biology Meets Disease Control: Wu, P., Tu, B., Liang, J., Guo, S., Cao, N., Chen, S., et al. (2021). Synthesis and Biological
Using Basic Research to Fight Vector-Borne Diseases. Nat. Microbiol. 4, 20–34. Evaluation of Pentacyclic Triterpenoid Derivatives as Potential Novel Antibacterial
doi:10.1038/s41564-018-0214-7 Agents. Bioorg. Chem. 109, 104692. doi:10.1016/j.bioorg.2021.104692
Sheng, Z., Jian, R., Xie, F., Chen, B., Zhang, K., Li, D., et al. (2020). Screening of Yazgan, H., Ozogul, Y., and Kuley, E. (2019). Antimicrobial Influence of
Larvicidal Activity of 53 Essential Oils and Their Synergistic Effect for the Nanoemulsified Lemon Essential Oil and Pure Lemon Essential Oil on
Improvement of Deltamethrin Efficacy against Aedes albopictus. Ind. Crops Food-Borne Pathogens and Fish Spoilage Bacteria. Int. J. Food Microbiol.
Prod. 145, 112131. doi:10.1016/j.indcrop.2020.112131 306, 108266. doi:10.1016/j.ijfoodmicro.2019.108266
Theophel, K., Schacht, V. J., Schluter, M., Schnell, S., Stingu, C. S., Schaumann, R., Zhang, H., Xie, Y., Liu, C., Chen, S., Hu, S., Xie, Z., et al. (2017). Comprehensive
et al. (2014). The Importance of Growth Kinetic Analysis in Determining Comparative Analysis of Volatile Compounds in Citrus Fruits of Different
Bacterial Susceptibility against Antibiotics and Silver Nanoparticles. Front. Species. Food Chem. 230, 316–326. doi:10.1016/j.foodchem.2017.03.040
Microbiol. 5, 544. doi:10.3389/fmicb.2014.00544
Weill, M., Lutfalla, G., Mogensen, K., Chandre, F., Berthomieu, A., Berticat, C., Conflict of Interest: BC was employed by the company Guangdong Xinbaotang
et al. (2003). Insecticide Resistance in Mosquito Vectors. Nature 423, 136–137. Biotechnology Co. Ltd.
doi:10.1038/423136b
WHO (2005). Guidelines for Laboratory and Field Testing of Mosquito Larvicides. The remaining authors declare that the research was conducted in the absence of
Geneva, Switzerland: World Health Organization. WHO/CDS/WHOPES/ any commercial or financial relationships that could be construed as a potential
GCDPP/2005.13 conflict of interest.
Wilson, A. L., Courtenay, O., Kelly-Hope, L. A., Scott, T. W., Takken, W., Torr, S. J.,
et al. (2020). The Importance of Vector Control for the Control and Elimination Copyright © 2021 Wu, Tang, Jian, Li, Lin, Dai, Wang, Sheng, Chen, Xu, Li, Lin,
of Vector-Borne Diseases. Plos Negl. Trop. Dis. 14, e0007831. doi:10.1371/ Zhang, Zheng, Zhang, Li and Hong. This is an open-access article distributed under
journal.pntd.0007831 the terms of the Creative Commons Attribution License (CC BY). The use,
Wu, P.-P., He, H., Hong, W. D., Wu, T.-R., Huang, G.-Y., Zhong, Y.-Y., et al. distribution or reproduction in other forums is permitted, provided the original
(2018). The Biological Evaluation of Fusidic Acid and its Hydrogenation author(s) and the copyright owner(s) are credited and that the original publication
Derivative as Antimicrobial and Anti-inflammatory Agents. Infect. Drug in this journal is cited, in accordance with accepted academic practice. No use,
Resist. 11, 1945–1957. doi:10.2147/idr.s176390 distribution or reproduction is permitted which does not comply with these terms.

Frontiers in Chemistry | www.frontiersin.org 11 May 2021 | Volume 9 | Article 679116