Antibacterial mode of action of the daucus carota essential oil active compounds against campylobacter jejuni and efflux-mediated drug resistance in gram-negative bacteria

Article abstract of DOI:10.3390/MOLECULES25225448

Today, an alarming rise of bacterial gastroenteritis in humans resulting from consuming Campylobacter-tainted foods is being observed. One of the solutions for mitigating this issue may be the antibacterial activity of essential oils. In the present research, we propose to study the antibacterial activity against Campylobacter and other Gram-negative bacteria of Daucus carota essential oil and its active molecules. In addition, a few chemically synthesized molecules such as (E)-methylisoeugenol, Elemicin, and eugenol were also studied. The results showed that the essential oil itself and its most active component, (E)-methylisoeugenol, exhibited bactericidal effects. Similar effects were detected using purified and chemically synthesized molecules. Also, it was observed that the Daucus carota essential oil and its active molecules affected intracellular potassium and intracellular ATP contents in Campylobacter cells. Inhibition of the membrane bound F_O F₁-ATPase was also observed. Eventually, for the first time, the efflux mechanism of active molecules of Daucus carota essential oil was also identified in gamma proteobacteria and its specific antibacterial activity against Campylobacter jejuni was associated with the lack of this efflux mechanism in this species.

Full text of DOI:10.3390/MOLECULES25225448

molecules
Article
Antibacterial Mode of Action of the Daucus carota
Essential Oil Active Compounds against
Campylobacter jejuni and Efflux-Mediated Drug
Resistance in Gram-Negative Bacteria
1
2
3
3
3
Dedieu Luc , Brunel Jean Michel
, Lorenzi Vanina , Muselli Alain , Berti Liliane and
2
,
Bolla Jean Michel *
1
Avignon Univ, INRAE, UMR SQPOV, SporAlim Team, Domaine Saint-Paul–Site Agroparc,
4914 Avignon CEDEX 9, France; luc.dedieu@univ-avignon.fr
Aix-Marseille Univ, INSERM, SSA, IRBA, MCT, UMR_MD1, U-1261, 13005 Marseille CEDEX 5, France;
bruneljm@yahoo.fr
8
2
3
Université de Corse, UMR CNRS 6134, SPE Sciences Pour l’Environnement, Campus Grimaldi, 20250 Corte,
France; lorenzi_v@univ-corse.fr (L.V.); muselli_a@univ-corse.fr (M.A.); berti_l@univ-corse.fr (B.L.)
*
Correspondence: jean-michel.bolla@univ-amu.fr; Tel.: +33-491-324-440
Academic Editor: Simone Carradori
Received: 28 October 2020; Accepted: 17 November 2020; Published: 20 November 2020
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Today, an alarming rise of bacterial gastroenteritis in humans resulting from consuming
Campylobacter-tainted foods is being observed. One of the solutions for mitigating this issue may be
the antibacterial activity of essential oils. In the present research, we propose to study the antibacterial
activity against Campylobacter and other Gram-negative bacteria of Daucus carota essential oil and its
active molecules. In addition, a few chemically synthesized molecules such as (E)-methylisoeugenol,
Elemicin, and eugenol were also studied. The results showed that the essential oil itself and its most
active component, (E)-methylisoeugenol, exhibited bactericidal effects. Similar effects were detected
using purified and chemically synthesized molecules. Also, it was observed that the Daucus carota
essential oil and its active molecules affected intracellular potassium and intracellular ATP contents in
Campylobacter cells. Inhibition of the membrane bound F F -ATPase was also observed. Eventually,
O
1
for the first time, the efflux mechanism of active molecules of Daucus carota essential oil was also
identified in gamma proteobacteria and its specific antibacterial activity against Campylobacter jejuni
was associated with the lack of this efflux mechanism in this species.
Keywords: natural compounds; chemical synthesis; efflux pump; ATP monitoring; Elemicin;
E-methylisoeugenol
1
. Introduction
Campylobacter is one of the leading causes of bacterial gastroenteritis worldwide [1]. The mode
of transmission is documented and is known to originate mainly from the ingestion of undercooked
poultry products. Campylobacter, mostly Campylobacter jejuni, and to a lesser degree, Campylobacter coli
are isolated in 100% of the layer and broiler flocks. These bacteria colonize the animal’s intestine
and are later transferred to the carcass during their processing [2]. Therefore, in order to control the
contagion in carcasses, it is important to reduce contamination among animals during their breeding
or slaughter. In addition, with ever-increasing multi-drug resistant bacteria, consumers have now
been opting for organic food, including antibiotic-free meat and poultry products. Thus, there is an
urgent need to develop new strategies to manage contamination. Recently, pertaining to this context,
essential oils (EOs) extracted naturally from plants have been highlighted as a promising solution [3].
Molecules 2020, 25, 5448; doi:10.3390/molecules25225448
www.mdpi.com/journal/molecules

Molecules 2020, 25, 5448
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Many vegetable extracts from herbs and plants from all over the world have been used ancestrally
in traditional medicines for different properties, including their antimicrobial activity. Several EOs
have antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts, and filamentous
fungi [
4,
5]. The interest for the antibacterial activity of EOs increased due to the threat toward the
food industry, with zoonotic pathogens such as Salmonella, Shigella, Listeria, and Campylobacter [
6].
However, once a new plant extract has been established which carries an effective anti-pathogenic
activity, it is still necessary to identify the active molecule and/or the combination of active molecules
and to further determine their mechanism of action. To date, many antimicrobial molecules including
phenolic compounds such as vanillin, thymol, and carvacrol have been found in several EOs [
They have been studied as natural antimicrobial agents since the use of antibiotics in animal food
farms is restricted [ 11 13]. There are well-documented works of literature that have reported that
7–10].
7
,8
,
–
compounds like eugenol, cinnamaldehyde, and carvacrol alter the ATP level in the cell and cause cell
membrane disruption [11–13].
In a previous study, a panel of 28 EOs from Corsica was screened for antibacterial activity against
five bacterial strains from the species Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa,
Staphylococcus aureus, and Campylobacter jejuni. Results showed that the Daucus carota essential oil
(DCEO) was one of the more active oils against C. jejuni [14]. Moreover, DCEO is specifically active on
C. jejuni and does not affect the other bacteria screened in this study [14]. The chemical composition
of DCEO has been investigated at different stages of development [15] and in different geographical
locations, such as Corsica and Portugal [15,16]. In a second study conducted by Rossi et al., DCEO
was fractionated by liquid chromatography and the resulting fractions were analyzed by GC and
GC/MS. The oxygenated fraction containing the antibacterial activity was further submitted to column
chromatography. Among several fractions, two separate fractions containing 97% (E)-methylisoeugenol
(
EMIE) and 98% Elemicin were obtained, respectively. Minimum inhibitory concentration (MIC)
of these fractions against C. jejuni was 0.125 and 0.250 g/mL, respectively. Structural analogues
have been used to classify the molecular characteristics that are essential for antibacterial activity [ ].
µ
9
To date, however, no information on the type of impact (bactericidal or bacteriostatic), the target or the
mechanism of action is available.
In the present study, we examined the mode of action of EMIE and Elemicin and compared it to
the mode of action of the entire DCEO. Using E. coli efflux pump mutants, we have demonstrated that
similar effects are observed with said strains and C. jejuni wild type strains suggesting that the target is
shared by both species. Decisively, we can state that due to lack of E. coli-like efflux mechanism DCEO
and its most active component, EMIE, exhibited activity specific to C. jejuni.
2
. Results
2
.1. Synthesis of Elemicin (4) and EMIE (6)
As shown in Scheme 1, 2,6-dimethoxyphenol (1) was easily protected with allyl bromide to give
2
3
-(allyloxy)-1,3-dimethoxybenzene (
-(4-hydroxy-3,5-dimethoxyphenyl)-1-propene (
, Elemicin) in 92% yield. In a similar way, isoeugenol (5)
2
, yield, 94%), which underwent a Claisen rearrangement to form
3). A subsequent methylation of the hydroxy group
gave 3-(3,4,5-tri-methoxyphenyl)-1-propene (
4
was easily methylated in 82% yield to form of the expected compound
NMR spectra are shown in Supplementary Figure S3.
6 (EMIE). The corresponding

Molecules 2020, 25, 5448
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Scheme 1. Synthetic pathway for the preparation of Elemicin (4) and EMIE (6).
2
.2. MIC and MBC Determination
To characterize the antibacterial effect of the DCEO against the C. jejuni F38011 strain, the EO
was prepared in our laboratory as previously described [14] and the DCEO was fractioned by two
column chromatography steps on silica gel as previously described [ ]. The MIC of the entire oil,
9
the oxygenated fraction (Oxy-Fr), and the EMIE and Elemicin fractions were determined using the
standard broth-dilution method and the agar-dilution method. The MIC in broth and agar indicated as
MIC( and MIC , respectively are given in Table 1. Only the MIC value obtained for the DCEO
(
L)
(S)
(L)
(
S)
500 µg/mL) differs from the MIC (250 µg/mL). In addition, only one dilution difference was observed
between the two methods suggesting a good correlation within them.
Table 1. Susceptibility and type of antibacterial effect of the DCEO, the DCEO fractions, and synthesized
molecules against the C. jejuni F38011 strain. Data resulted from three independent experiments.
MIC(S)
*
MIC(L)
*
MBC *
MBC/MIC
Effect
DMSO
DCEO
Oxy-Fr
>1000
250
250
>1000
125
250
ND
ND
0,125
0,5
ND
250
>1000
500
250
>1000
125
250
125
250
0,125
0,5
31.25
250
>1000
1000
500
>1000
125
250
125
250
1
ND
2
2
ND
1
1
1
1
8
2
ND
Bactericidal
Bactericidal
ND
HC-Fr
EMIE-Fr
Elemicin-Fr
EMIE
Elemicin
Erythromycin
Gentamicin
Carvacrol
Eugenol
Bactericidal
Bactericidal
Bactericidal
Bactericidal
Bacteriostatic
Bactericidal
Bactericidal
Bactericidal
Bactericidal
1
62.5
250
250
2
1
1
Isoeugenol
125
250
MIC and MBC values are expressed in µ
g/mL. MIC(S) values were obtained with the solid method and MIC
(L)
*
(L)
values were obtained with the liquid method. MBC/MIC ratios were calculated with the MIC values and was
used to determine the type of antibacterial effect (see materials and methods section). Oxy-Fr, oxygenated fraction;
HC-Fr, hydrocarbon (apolar) fraction; EMIE-Fr, EMIE purified fraction; Elemicin-Fr, Elemicin purified fraction; ND,
Not determined.
DMSO alone and the hydrocarbon fraction of EO were found to be inactive, irrespective of the
method used. The MBC was also determined using the liquid method. The MBC/MIC ratios were
calculated for the active compounds and compared to erythromycin and gentamicin, classically termed
as bacteriostatic and bactericidal antibiotics, respectively [17,18]. Carvacrol was used as a non-antibiotic
positive control to achieve bactericidal effect [ 12 13 19]. We obtained a ratio of 2 for the DCEO and
8, , ,

Molecules 2020, 25, 5448
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the Oxy-Fr and a ratio of 1 for the purified EMIE and Elemicin molecules indicating, a bactericidal
effect for the entire EO and all the active fractions purified along the purification process (Table 1).
(L)
The EMIE and Elemicin molecules were chemically synthesized and their MIC and MBC were
tested against the C. jejuni F38011 strain. Similar results (125 g/mL for EMIE and 250 g/mL for
µ
µ
Elemicin) were obtained using the purified and the synthesized molecules (Table 1). Both the methods
were used to study commercially available solutions of eugenol and isoeugenol as they are very similar
to the active molecules. As observed, the MIC( and MIC of Isoeugenol was found to be 250
L)
(S)
µg/mL
and 125
µg/mL, respectively with only is different by just one dilution. Taken together, these results
indicate that all the active compounds had a bactericidal effect and that the purified EMIE molecule and
the synthesized EMIE molecule are the most active compounds compared to Elemicin and the Oxy-Fr.
2
.3. MIC and MBC Determination
To evaluate the effect of Campylobacter inoculum size on the MIC of DCEO, Oxy-Fr, and purified
(L)
4
7
molecules, variable inoculum sizes ranging from 1
were carried out according to the previously described protocol. As observed, no significant changes
were observed in MIC of any test samples (Table 2). MIC of DCEO, the Oxy-Fr fraction and
Elemicin with inoculum size of 1
×
10 to 1
×
10 cells/mL were assessed. Experiments
(
L)
(L)
4
×
10 CFU/mL shifted slightly by one dilution however, MIC findings
were the same for EMIE, for all the inoculum size measured.
Table 2. Effect of the inoculum size on MIC.
4
5
6
7
Inoculum Size
1 × 10
1 × 10
1 × 10
1 × 10
DCEO-MIC
Oxy-Fr-MIC
EMIE-MIC
250
125
125
125
500
250
125
250
500
250
125
250
500
250
125
250
Elemicin-MIC
Effect of the inoculum size on the MIC(L) values for the DCEO, the oxygenated fraction (Oxy-Fr), and the synthesized
4
molecules against the C. jejuni F38011 strain. MIC values are expressed in
to 1 × 10 cells per mL. Data resulted from three independent experiments.
µ
g/mL. Inoculum size ranged from 1
×
10
7
2
.4. Characterization of the Rapid Bactericidal Effect
As we observed a bactericidal effect, time-kill assays were performed on mid-log phased
Campylobacter cells to determine the kinetics of the cell death. DCEO concentrations of 500, 1000,
and 2000 g/mL corresponding to MIC, 2 MIC, and 4 MIC respectively, were used to treat the cells
for 30 min.
µ
×
×
Survival of the bacteria was determined by colony forming units (CFU) counting. The results
of test samples were compared to that of negative control i.e., dimethyl sulfoxide (DMSO) treated
cells. We observed a dose-dependent response and a DCEO concentration of 2
induced a strong decrease in CFU count within 30 min (<1% of the bacterial CFU count). Similar
µ
g/mL (4
×
MIC)
results were observed using EMIE (Figure 1A). Same experiments were performed with Oxy-Fr,
Elemicin, and carvacrol. Correspondingly, a strong decrease was reached in 30 min with 4
×
MIC for
all the treatments (Figure 1B). Also, similar results were obtained when experiments were performed
on stationary growth phased Campylobacter cells (Figure S1). These results suggest that the active
molecules acted not specifically on the synthesis of macromolecules necessary during cell growth and
are in agreement with the bactericidal effect observed.

Molecules 2020, 25, 5448
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(
A)
(B)
Figure 1. Rapid bactericidal effect of the DCEO and its active molecules against mid-log grown cells of
C. jejuni F38011 using time kill assays for 30 min with concentrations corresponding to MIC, 2 × MIC,
and 4
×
MIC. Control cells were treated with DMSO and carvacrol was used as a positive standard.
(
A
) effect of the entire DCEO and EMIE. (
B
) effect of Oxy-Fr and Elemicin. Data are the means
±
SEM
from three independent experiments.
2
.5. Cell Lysis Determination by O.D. Measurements and SDS-PAGE
The decrease in CFU observed could result from bacterial lysis. In order to assess bacterial
lysis, cell density was adjusted to 0.5 OD600 unit, and cells were treated with DCEO, EMIE, Elemicin,
and carvacrol for 30 min. OD measurements were performed after 3, 10, and 30 min and compared with
OD measurements of DMSO treated cells (negative standard). SDS and TCA were used to treat samples
and were consider as positive standard molecules for cell lysis. We observed that OD600 obtained after
3
0 min for cells treated with DCEO, EMIE, and Elemicin were not significantly different (all p-values
were >0.2) from OD600 obtained with untreated cells (Table S1). On the contrary, OD600 obtained
after 30 min for cells treated with carvacrol were 20% lower than OD600 obtained with untreated cells.
Control cells treated with SDS and TCA showed OD600 values 90% lower than OD600 obtained with
untreated cells (Table S1).
To further examine the cell integrity, suspensions containing treated and untreated cells were
centrifuged and protein content of the pellets and the supernatants were analyzed by SDS-PAGE.
As observed in Figure 2, untreated cells had negligible protein content in the supernatant and most
of the protein content was detected in the cell pellet. Same result was obtained with DCEO, EMIE,
Elemicin, and carvacrol treated cells. Cell suspensions treated with SDS (positive controls) showed a
substantial protein content in the supernatant and almost no protein in the pellet suggesting severe cell
lysis. Immunodetection using Omp50 outer membrane protein-specific antibodies [20] was performed
on pellets and supernatants of treated and untreated cell suspensions (Figure S2). Although this
method is more sensitive, no specific signals were obtained in the supernatant for cell suspensions
treated with DCEO, EMIE, and Elemicin indicating that no membrane disruption occurred during the
treatment. Collectively, these data showed that there was no cell lysis in these conditions.

Molecules 2020, 25, 5448
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1
2
3
4
5
6
7
Figure 2. Cell lysis analyzed by SDS-PAGE on cell pellets and supernatants after incubation with
different DCEO concentrations. Lane 1, molecular weight; lanes 2, 4, 6, and 8, cell pellets after treatment
with DMSO (negative standard), DCEO at 2
Lanes 3, 5, 7, and 9, supernatants after treatment with DMSO (negative standard), DCEO at 2
DCEO at 4 × MIC, and SDS 0.02% (w/v) respectively.
×
MIC, DCEO at 4
×
MIC, and SDS 0.02% (w/v) respectively.
×
MIC,
2
.6. Measurement of the Potassium Release
Although no cell lysis was observed after treatment with DCEO and its active molecules, a partial
alteration of membrane functions could explain the cell death. In intact cells, a high intracellular
potassium level is maintained compared to the extracellular level. To investigate the cellular potassium
gradient integrity, we performed potassium level measurements outside of the cells after treatment
(
Figure 3). DMSO-treated cells were used as negative standard and heated cells were used as positive
standard of cell lysis. We detected a drastic increase of the potassium concentration outside of the cells
after treatment with carvacrol. An increase of the potassium concentration was also observed after
treatment with DCEO, EMIE and Elemicin. After erythromycin and gentamicin treatment, no increase
of the potassium concentration outside of the cells was detected (data not shown). This indicates that
in Campylobacter the potassium gradient was strongly altered by DCEO and its active molecules.
Figure 3. Extracellular potassium level measured by flame emission spectroscopy after treatment with
DCEO, oxygenated fraction and purified active molecules. Data are the means
independent experiments.
±
SEM from three
2
.7. Cellular ATP Content and ATP Release Measurements
In addition to potassium release, we investigated the ATP concentration inside and outside of
cells after treatments. After DCEO, Oxy-Fr, EMIE, Elemicin, and carvacrol treatments, a negligible ATP
level was detected outside of cells. The same result was observed for untreated cells (data not shown).
On the contrary, ATP concentration inside the cells substantially decreases post-treatment as compared
to the DMSO, erythromycin, and gentamicin treated cells (Figure 4).

Molecules 2020, 25, 5448
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Figure 4. Intracellular ATP level in Campylobacter cells after treatment with the entire DCEO,
the oxygenated fraction and purified active molecules. Data are the means SEM from three
independent experiments.
±
A crucial decrease of the ATP level inside of cells without increase of the extracellular ATP
level indicated that ATP molecules were not released from the cells to the medium. Taken together,
the measurements of the potassium and ATP levels indicate either a consumption of ATP or an
inhibition of the ATP synthesis. To address this question, we investigated the in vitro inhibition of
the membrane bound F F -ATPase activity after incubation of purified membrane fractions with the
O
1
DCEO and its active molecules. In vitro, the F F -ATPase can hydrolyze exogenous ATP added in
O
1
the samples in ADP and inorganic phosphate leading to a decrease in ATP level. Inhibition of this
enzyme would result in a slow decrease in ATP concentration [13]. We purified membrane fractions of
C. jejuni and E. coli strains as previously described [21,22]. These preparations were incubated 5, 10
or 20 min with DCEO and ATP was added to the preparation just before ATP levels measurements.
Without molecule addition (incubation with DMSO), a rapid and significant decrease in ATP level was
observed with membrane fractions of C. jejuni and E. coli demonstrating the quality of the preparations
both containing active F F -ATPase able to maintain its activity (hydrolysis of ATP) for 20 min to
O
1
perform the tests (Figure 5). Incubation with DCEO and EMIE inhibited the ATPase activity of E. coli
and Campylobacter preparations (Figure 5). After 20 min, the inhibition rate of 4 MIC carvacrol on
E. coli ATPase activity was 46%. Similar effect was observed on the Campylobacter ATPase with 44%
inhibition. After 20 min, the inhibition rate of 4 MIC EMIE on E. coli ATPase activity was 42% and
×
×
a comparable effect was observed on the Campylobacter ATPase with 36% inhibition suggesting an
analogous type of effect on the two bacteria.
(
A)
Figure 5. Residual ATPase activity after incubation with DCEO, EMIE, and carvacrol. (
on E. coli membrane fractions. ( ) Measured on Campylobacter membrane fractions. Data are the
means ± SEM from three independent experiments.
(B)
A
) Measured
B

Molecules 2020, 25, 5448
8 of 16
2
.8. Specificity of EMIE and Related Molecules Against C. jejuni
Our data showed that the active molecules of DCEO inhibit the action of F F -ATPase in cells of
O
1
C. jejuni and E. coli. As previously stated, the specificity of DCEO against Campylobacter [14] could not
be explained by such a mode of action since F F -ATPase is ubiquitous. Without a definite target in
O
1
C. jejuni, the specificity of DCEO could be explained by a particular resistance mechanism that is absent
in C. jejuni. To address this query, we determined the MIC( of the DCEO and the active molecules
against a collection of C. jejuni and E. coli strains with different efflux levels (Table 3).
L)
Table 3. MIC of DCEO and active molecules against Gram negative bacteria.
Strain
PABN
DCEO
32,000
500
EMIE
32,000
125
Elemicin Erythromycin *
−
+
−
+
32,000
500
ND
ND
ND
ND
Wild type E. coli
(
AG100)
-
500
1000
125
1000
250
AcrAB E. coli
(
AG100A)
Wild type C. jejuni
F38011)
500
−
500
125
250
0.125
(
−
+
−
+
500
250
250
125
250
125
125
62.5
500
62.5
250
0.25
Efflux activated C. jejuni
(81176)
0.0015
0.125
-
CmeB C. jejuni
(
81176B)
Helicobacter pylori
HpJ99)
Enterobacter aerogenes
ATCC13048)
Pseudomonas aeruginosa (CIP A22)
62.5
0.0015
−
500
250
ND
ND
(
−
−
32,000
32,000
32,000
32,000
ND
ND
ND
ND
(
*
Erythromycin was used as a positive standard molecule for efflux by a PABN sensitive efflux system and as a
negative standard molecule for CmeABC activity. Data resulted from three independent experiments.
According to previous work using the entire DCEO [14], neither DCEO nor the active molecules
tested in present study demonstrated any antibacterial activity against Pseudomonas aeruginosa,
Enterobacter aerogenes and E. coli wild type strain since the MIC value was higher than 32,000 µg/mL
which is over the solubility limit of the compounds (Table 3). When E. coli wild type strain was
treated with the PABN efflux system inhibitor [23], MIC values of DCEO and Elemicin was found to be
5
00 µg/mL and 125 µg/mL, respectively.
MIC highly influenced by treatment with PABN indicated that DCEO and its active molecules
were transported by a PABN-sensitive efflux mechanism in E. coli. The E. coli strain AG100A is devoid
of AcrAB efflux system. The MIC of DCEO, EMIE and Elemicin in this strain were highly affected by the
mutation suggesting that the EO and the molecules have been partly transported by the AcrAB-TolC
efflux system. We also used a set of Campylobacter strains for MIC determination with or without
PABN. MICs for EMIE and Elemicin on Campylobacter strain 81,176 which is a well characterized
efflux-activated strain [24] were not significantly different from those obtained with the F38011 strain
(
Table 4). The MIC of DCEO and EMIE was not significantly changed by the use of PABN on this strain
or by the use of an isogenic mutant strain deleted from the CmeB efflux component [25]. Compared to
untreated cells, only the MIC of Elemicin was significantly changed (by three dilutions) in PABN treated
cells. This three dilutions variation is a minor effect compared to the variation obtained with or without
PABN for Elemicin MIC in E. coli (i.e., at least six dilutions) and compared to the variations obtained
with or without PABN for erythromycin MIC (seven dilutions) which is in agreement with the lower
activity of Elemicin compared to EMIE. As previously described, erythromycin was used as a positive

Molecules 2020, 25, 5448
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standard molecule for efflux by a PABN sensitive efflux system and as a negative standard molecule
for CmeABC [24]. Interestingly, the MIC of DCEO and EMIE observed against Helicobacter pylori
was found to be 500 and 250, respectively. Taken together, these results indicate that, in contrast to
E. coli and other gamma proteobacteria, Campylobacter did not resist to DCEO treatment because EMIE,
the more active compound, is not extruded by any efflux pump.
3
. Discussion
Since Campylobacter species are detected in 100% of chickens on farms and in more than 75% of
commercialized chicken carcasses [ ], it is of significant importance to reduce the bacterial load to
improve food safety in zoonotic conditions. In this context, it may be useful to study the antimicrobial
activity of natural products such as plant-extracted molecules [ ]. Here, the MIC of the Daucus carota
essential oil, its fractions, and chemically synthesized molecules were determined using a solid and a
2
3
liquid method against C. jejuni. The comparison between MIC( and MIC yielded very comparable
values, suggesting a strong correlation between the two techniques. Similar MIC values were also
obtained with purified molecules and chemically synthesized, demonstrating the consistency of the
preparations for either chemically synthesized molecules or natural purified molecules. Therefore,
we could be assured that no impurities play a major role in the antibacterial activity. In the previous
research, MIC of DCEO against Campylobacter was studied and EMIE and Elemicin were identified as
L)
(S)
active molecules [9] but no minimum bactericidal concentration (MBC) value and no mode of action
information was obtained because the agar dilution method used to assess the MIC was not suitable
for MBC determination. In the present study, it was observed that MBC/MIC ratio of DCEO, EMIE,
Elemicin fractions, and chemically synthesized molecules against the C. jejuni F38011 strain exhibited
bactericidal effect. Moreover, a ratio of 1 (same values for MIC and MBC) was obtained for synthesized
and purified EMIE and Elemicin. On the contrary, we obtained ratios of 2 for the entire DCEO and the
Oxy-Fr. This resulted probably due to impurities in the entire EO and in the Oxy-Fr. A bactericidal
effect has been already observed for other phenolic compounds such as carvacrol and cinnamaldehyde
against Listeria innocua, Bacillus cereus and E. coli O157:H7 [19,26,27].
Using time-kill assays, we observed the same kinetics of death for the DCEO and the EMIE
and Elemicin fractions. This supported our hypothesis that these molecules are active compounds.
In addition, time-kill experiments performed with DCEO, Oxy-Fr, EMIE, Elemicin, and carvacrol
on stationary and mid-log phased Campylobacter cells gave similar results signifying that the active
molecules did not specifically acted on the synthesis of macromolecules which occur during cell
growth viz. peptidoglycan, lipids or protein. This was found to be in agreement with the bactericidal
activity observed since differential effects would be observed for bacteriostatic molecules. Since we
did not observe cell lysis, studies of essential bacterial functions, such as potassium gradient and ATP
production, were carried out to decode the mechanism of action. We found that the potassium gradient
and the ATP production processes were both altered. These two phenomena occur with analogous
kinetics (linear for 10 min and maximum at 20 min) suggesting that they were not triggered by each
other. On the contrary, the kinetics obtained with carvacrol was not similar. Also, the rapid decrease
observed in the potassium level could explain ATP depletion observed later. These different kinetics
(
effect of carvacrol on the potassium release before ATP depletion and effect of DCEO molecules on
ATPase and a potassium release at the same time) could indicate different mode of actions. One could
hypothesize that potassium release caused by carvacrol induces ATP depletion to restore the potassium
gradient which is necessary for bacteria to survive but this hypothesis could not be explained using
the results obtained with DCEO molecules.
Depletion of ATP have been already observed with carvacrol on E. coli and L. monocytogenes [13].
In that study, Gill and Holley showed that incubations of 10 min with 5 mM and 10 mM carvacrol
inhibited the ATPase activity of E. coli and L. monocytogenes by 35% and 40%, respectively, and that
a 10 min incubation with 1mM carvacrol had no effect on the ATPase activity. Here, we used a
concentration of 4
×
MIC corresponding to 1.5 mM carvacrol and observed inhibition of the ATPase of

Molecules 2020, 25, 5448
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E. coli and Campylobacter of 39% and 32%, respectively. Using a concentration of 4
to 2.8 mM EMIE, we observed inhibition of the ATPase of E. coli and Campylobacter of 37% and 28%,
×
MIC corresponding
respectively. However, the rates of inhibition after 20 min of treatment with EMIE were higher, reaching
4
2% on E. coli and 36% on C. jejuni.
The species specificity of the DCEO and EMIE antibacterial activity was assessed using gamma
proteobacteria including Enterobacter aerogenes, Pseudomonas aeruginosa and E. coli. The results showed
that neither the EO nor the purified EMIE molecule tested here showed any antibacterial activity.
These results are in agreement with our previous study using the agar dilution method [14]. Moreover,
we showed that treatment with the PABN inhibitor on the wild type E. coli (strain AG100) and that
deletion for the AcrAB efflux system in the E. coli (strain AG100A) strongly affected the MIC of
DCEO, EMIE, and Elemicin. This suggested that DCEO active molecules are partly transported by the
AcrAB-TolC efflux system. Furthermore, for the E. coli AG100A strain treated with the PABN efflux
system inhibitor, the MIC was similar to that obtained for the PABN treated E. coli wild type strain.
These results suggested that in E. coli the molecules could be transported by at least two efflux systems
that are PABN-sensitive. Surprisingly, the increase in susceptibility observed after PABN treatment
and after deletion differed for DCEO, EMIE and Elemicin. This could be explained by molecules in
the mixture that could interact with the efflux system or compete with EMIE and Elemicin. The lack
of antibacterial activity against E. coli was also observed in a study conducted by Degirmenci and
collaborators using Daucus carota juice on the pathogenic O157:H7 E. coli strain [28].
On the contrary, in the Campylobacter strains, the mutation of the CmeB efflux component and
the use of PABN did not significantly modify the MIC values for DCEO and EMIE. Only the MIC of
Elemicin was slightly modified (three dilutions) after PABN treatment. This three dilutions variation
is negligible compared to the variation obtained with or without PABN for Elemicin MIC in E. coli
(
at least six dilutions) and compared to the variations obtained with or without PABN for erythromycin
MIC (seven dilutions) which is not a substrate for CmeABC but is transported by a PABN sensitive
efflux system [24]. Cumulatively, these results indicated that, contrary to E. coli, Campylobacter did not
resist DCEO treatment because EMIE, its major and most active compound, is not transported by the
efflux pumps present in Campylobacter. The MIC observed against Helicobacter pylori (500 for the DCEO
and 250 for the EMIE) suggested that bacteria phylogenetically close to Campylobacter jejuni could be
sensitive to DCEO and its active molecules. This was in agreement with the data of a previous study on
antibacterial activity of mastic gum essential oil against drug-resistant strains of Helicobacter pylori [29].
In addition, as reported by Costa and Iraola [30], new methods of isolation and identification allowed
the description of emerging Campylobacter species known to be underestimated, that could become
other target for the compounds described here.
In this study, a strong decrease of CFU from Campylobacter was observed using DCEO, EMIE,
and Elemicin at 4
×
MIC for 30 min which is a short time. In addition, no effect of inoculum size was
observed. These two data could be of importance because they can minimize resistance acquisition
during treatment for improving the safety of poultry meat. The use of plant-derived GRAS (generally
recognized as safe) antimicrobial molecules as feed supplements for reducing fecal pollutants in
chickens have been already tested on Salmonella enteritidis and C. jejuni [31] and could be considered
with DCEO or DCEO molecules. In this context, studies about cytotoxicity, showed that DCEO was
not cytotoxic against dendritic cells [16]. Other studies have indicated that Elemicin is not directly
toxic but could be metabolized in vivo and produce alkaloid metabolites that could be responsible for
hallucinogenic effects [32]. In addition, the effect could be different on mixed populations compared
to pure culture as already observed for many molecules including EMIE [33]. The DCEO and EMIE
should probably not be considered efficient enough to be used alone. Studies using these molecules in
combination with other antibacterial molecules as already described using carvacrol and endolysin
LysSA97 against Staphylococcus aureus should be considered [34]. Hence, synergistic properties of these
compounds with well-established antibiotics should be tested in near future.

Molecules 2020, 25, 5448
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In conclusion, the data obtained here highlight the potential of essential oils (EOs) extracted
naturally from plants in the context of food safety. In addition, the use of chemically synthesized
molecules allowed us to be assured that these molecules are the active compounds and that no impurities
play a major role in the antibacterial activity. The bactericidal effect was observed specifically against
Campylobacter and Helicobacter strains, but no effect was observed against gamma-proteobacteria.
This provide three interesting outcomes (i) these molecules were not transported efficiently by
Campylobacter efflux pumps, (ii) the use of antibacterial molecules with specific antibacterial effect
should be useful because they exert no effect on human gut microbiota, (iii) the use these molecules
could be of interest to target emerging Campylobacter species.
4
. Materials and Methods
4
.1. General Information
All solvents were purified according to reported procedures, and reagents were used as
◦
commercially available. Methanol, ethyl acetate, dichloromethane, and petroleum ether (35–60 C)
were purchased from Merck (Saint Quentin Fallavier, France) and used without further purification.
Column chromatography was performed on silica gel (70–230 mesh, Macherey-Nagel, Bethlehem, PA,
USA). Commercial solutions of erythromycin, gentamicin, eugenol, isoeugenol, and carvacrol were
purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). The EMIE and Elemicin purified
fractions and the EMIE and Elemicin chemically synthesized molecules were analyzed by NMR.
1
13
H-NMR and C-NMR spectra were recorded as previously described [9] in CDCl on an AC 400
3
spectrometer (Bruker, Billerica, MA, USA) working at 400 MHz and 100 MHz, respectively (the usual
abbreviations used in text are: s; singlet, d; doublet, t; triplet, q; quadruplet, m; multiplet). All chemical
shifts are given in ppm.
4
.2. Synthesis of 2-(Allyloxy)-1,3-dimethoxybenzene (2)
To a solution of 2,6-dimethoxyphenol (syringol, 1, 3 g, 0.016 mol) in acetone (50 mL) was added
potassium carbonate (2.5 g, 0.018 mol) and allyl bromide (2.17 g, 0.018 mol). After stirring the mixture
overnight, the solvent was removed and 3N HCl (15 mL) was added to it. The extraction was performed
with ethyl acetate, the organic fraction so obtained was washed with brine solution and then dried
over anhydrous Na SO . The solvents were removed at reduced pressure. Purification of the residue
2
4
was performed by flash chromatography (eluent: petroleum ether/ethyl acetate, 15:1, v/v) to yield
1
compound
2
(4.74 g, 94%); colorless oil: H-NMR (400 MHz, CDCl ):
δ
(ppm) = 3.84 (s, 6H), 4.47
3
(
(
1
d, 2H, J = 6.0 Hz), 5.13 (dd, 1H, J = 10.4, 1.0 Hz), 5.25 (dd, 1H, J = 17.2, 1.6 Hz), 6.03 (m, 1H), 6.49
d, 2H, J = 12.0 Hz), 6.91 (t, 1H, J = 12.0 Hz). 13C-NMR (100 MHz, CDCl ):
δ
(ppm) = 53.16, 74.65,
3
+
)
05.43, 116.59, 123.36, 135.14, 136.50, 153.81. MS (EI), m/z 194 (M .
4
.3. Synthesis of 3-(4-Hydroxy-3,5-dimethoxyphenyl)-1-propene (3)
Compound
◦
2
(1.25 g, 6.5 mmol) was heated in the absence of solvents to 200 C for 3 h and the
residue was then later purified using flash chromatography (eluent: petroleum ether/ethyl acetate,
1
8:1, v/v) to yield compound
3
(1.2 g, 91%); a colorless oil. H-NMR (400 MHz, CDCl ):
δ
(ppm) = 2.02
3
(
(
s, 2H), 5.08 (dd, 1H, J = 10.2, 1.0 Hz), 5.10 (dd, 1H, J = 17.6, 1.6 Hz), 5.57 (s, 1H), 5.91 (m, 1H), 6.40
s, 2H). MS (EI), m/z 194 (M ).
+
4
.4. Synthesis of 3-(3,4,5-Trimethoxyphenyl)-1-propene (Elemicin) (4)
To a solution of 3-(4-hydroxy-3,5-dimethoxyphenyl)-1-propene (3, 1 g, 5.1 mmol) in acetone
(50 mL), potassium carbonate (0.9 g, 6.5 mmol) was added. The suspension was stirred for 10 min
and iodomethane (1.1 g, 7.8 mmol) was subsequently added and stirred overnight. The solvent was
then evaporated, 3 N HCl (10 mL) was added, and the mixture was later extracted with ethyl acetate.
The organic fraction was then washed with water, dried over Na SO and the solvent was removed.
2
4

Molecules 2020, 25, 5448
12 of 16
The residue was purified using flash chromatography with petroleum ether/ethyl acetate (10:1, v/v)
1
as eluent yielding compound
4
; a colorless oil (1.0 g, 92%): H-NMR (400 MHz, CDCl ): 3.30 (d, 2H,
3
J = 6.6 Hz), 3.95 (s, 9H), 5.03 (dd, 1H, J = 9.6, 1.2 Hz), 5.10 (dd, 1H, J = 17.2, 1.6 Hz), 5.91 (m, 1H), 6.34
(
1
s, 2H). 13C-NMR (100 MHz, CDCl ):
37.13, 153.09. MS (EI), m/z 208 (M ).
δ
(ppm) = 40.45, 55.92, 61.30, 104.59, 115.90, 123.58, 135.71, 136.19,
3
+
4
.5. Synthesis of (E)-methylisoeugenol (EMIE) (6)
In a two necked round flask, an amount of 3 g of (E)-isoeugenol (5, 1.8 mmol) was dissolved in
1
5 mL of acetone. To this solution, 5 g of K CO (3.6 mmol) and 5 mL of methyl iodide (3.6 mmol) were
2 3
subsequently added. The resulting mixture was stirred at room temperature for 48 h. The solvents were
then removed in-vacuo, and the crude residue was dissolved in ethyl acetate (50 mL). Later, the residue
was washed twice with water (50 mL) and brine (50 mL). The organic layer was dried over MgSO , and
4
the solvent was removed to afford a colorless oil which was purified by flash column chromatography
1
(
40 g SiO , eluent petroleum ether: ethyl acetate, 3:1). The product yield was 82%. NMR H (400 MHz,
2
1
CDCl3):
6
1
δ
H-NMR (400 MHz, CDCl ): 1.86 (d, 3H, J = 8.0 Hz), 3.84 (s, 3H), 3.87 (s, 3H), 6.03 (m, 1H),
3
1
3
.29 (dd, 1H, J = 20.0, 1.0 Hz), 6.75-6.89 (m, 3H). C-NMR (100 MHz, CDCl3):
δ
+
(ppm) = 55.64, 55.77,
08.45, 110.87, 118.58, 123.58, 130.58, 131.07, 148.10, 148.92. MS (EI), m/z 179 (M ).
4
.6. Bacterial Strains and Culture Conditions
The bacterial strains used in this study are listed in Table 4. Campylobacter strains were routinely
grown under microaerophilic conditions on Columbia agar or Mueller-Hinton (MH) agar supplemented
with or without 5% sheep blood (Bio-M
strain was routinely grown at 37 C under microaerophilic conditions on MH agar complemented
é
rieux, Marcy l’
é
toile-France). The Helicobacter pylori Hp99
◦
by 10% sheep blood. Microaerophilic conditions (5% O , 10% CO and 85% N ) were obtained using
2
2
2
GENBag microaer (BioMérieux). For experimental requirements (see below) and to improve bacterial
growth and survival during all the experiments, liquid cultures of Cj and Hp were performed in a
mixture (50:50) of MH Broth (Oxoid, Dardilly, France) and Minimal Eagle Medium (D-MEM broth,
◦
Sigma-Aldrich) [35
,
36]. E. coli, Pseudomonas, and Enterobacter strains were routinely grown at 37 C
on Luria Bertani agar (LB agar). E. coli AG100A strain is an efflux pump mutant (acrAB-) and a
kanamycin-resistant derivative of the AG100 strain [37].
Table 4. Strains used in this study.
Genera and Species
Strain Name
Relevant Phenotype
Reference
Campylobacter jejuni
Campylobacter jejuni
Campylobacter jejuni
Campylobacter jejuni
Campylobacter jejuni
Escherichia coli
F38011
81176
96C208
Wild type
[38]
[39]
[40]
[24]
[24]
[37]
[37]
[29]
[41]
[41]
Wild type, active efflux
Wild type, active efflux
CmeB mutant strain
CmeB mutant strain
Wild type
81176 CmeB-
96C208 CmeB-
AG100
AG100A
HpJ99
ATCC13048
CIP A22
R
Escherichia coli
Km , AcrAB-
Helicobacter pylori
Enterobacter aerogenes
Pseudomonas aeruginosa
Wild type
Wild type
Wild type
4
.7. Fractionation of Essential Oil
DCEO was fractioned by two steps of column chromatography on silica gels as previously
described [ ]. Briefly, the bulk oil was fractioned by column chromatography on silica gel
ICN 200–500 m) successively with pentane (n-C H ) and diethyl ether (Et O) to obtain the apolar
9
(
µ
5
12
2
hydrocarbon fraction and the oxygenated fraction, respectively. The hydrocarbon fraction was
inactive and thus retained to be used as negative controls in further experiments. The oxygenated

Molecules 2020, 25, 5448
13 of 16
fraction (Oxy-Fr) was further fractionated using silica gel (ICN 63–200 mm) with an elution gradient
n-C H /Et O, 95/5 to 0/100. The GC and GC/MS analysis of the 26 sub-fractions obtained by
5
12
2
column chromatography permitted the identification of two separate fractions containing EMIE and
Elemicin, respectively. As mentioned above [ ], The presence of active components was tested by
9
complementary NMR analysis, and GC/MS data verified that these fractions contained more than 95%
of antibacterial molecules.
4
.8. Component Identification
GC-FID and GC-MS analysis of essential oil and its fractions obtained by column chromatography
were performed under the experimental conditions described previously [9]. The identification was
carried out by comparison of their mass spectra with those compiled in our laboratory-built spectral
library, as well as by comparison of their retention indices with those of authentic samples or literature
data [42,43]. The GC retention indices (RI) on the polar and non-polar columns were determined
relative to the retention time of a series of n-alkanes with linear interpolation. Identification of the
major components were ensured by 13C-NMR, following a procedure developed and computerized
in our laboratories and based on the comparison of 13C-NMR chemical shifts of components in the
mixture with those of pure compounds compiled in a laboratory-built library [15].
4
.9. Determination of MIC and MBC
Susceptibility to Daucus carota essential oil (DCEO), its purified components, and chemically
synthesized molecules is expressed as a minimal inhibitory concentration (MIC) and as minimal
bactericidal concentration (MBC). The MIC( was performed on agar plates as previously described [14].
For E. coli, Enterobacter, and Pseudomonas, MIC experiments were conducted in 1 mL of liquid media
S)
(
L)
6
(
MIC ). The liquid media used in said experiment was MH Broth inoculated with 10 cells and
◦
(L)
incubated at 37 C for 18 h without agitation. For C. jejuni, and H. pylori, MIC experiments were
conducted in 10 mL volumes of a mixture (50:50) of MH Broth (Oxoid) and Minimal Eagle Medium
7
◦
(
D-MEM broth, Sigma-Aldrich) inoculated with 10 cells and incubated at 37 C for 18 h under agitation
4
7
in microaerophilic condition. For Campylobacter, the size of inocula from 10 to 10 cells were checked.
MIC ( g/mL) was defined as the lowest concentration required to completely inhibit the growth of
µ
bacteria. Inhibition was observed visually. For all experiments, the DCEO, the DCEO fractions, and
the chemically synthesized molecules were used two-fold diluted in dimethyl sulfoxide (DMSO) in
order to solubilize in the media. When specified, PABN was used at 10
µg/mL for Campylobacter and
Helicobacter, and at 15 g/mL for the other bacteria. MBC was defined as the lowest concentration
µ
required to kill more than 99% of the bacterial inoculum. The MBC/MIC ratios were calculated for the
active compounds including erythromycin and gentamicin, classically termed as bacteriostatic and
bactericidal antibiotics, respectively [20
bactericidal and if the ratio MBC/MIC was >4, the effect was described as bacteriostatic [20].
,
21]. If the ratio MBC/MIC was 4, the effect was considered
≤
4
.10. Time-Kill Assays
Campylobacter cells were grown in MH Broth and D-MEM (50:50). Cells were harvested at mid-log
phase, centrifuged, and resuspended in phosphate buffer saline (PBS), pH 7.4. For time-kill assays,
6
1
mL containing 5
compounds at 4
compared with those obtained with the negative standard.
×
10 cells were incubated with DMSO (negative standard) or with the active
×
MIC two-fold diluted in DMSO. CFUs were counted after 10, 30, and 60 min and
4
.11. Effect on Bacterial Lysis
Bacterial lysis was investigated on Campylobacter cells grown in MH Broth and D-MEM (50:50).
The cells were harvested at mid-log phase and resuspended in PBS, pH 7.4 to achieve a cell density
equivalent to 0.5 OD600. The cell pellets were then incubated with DMSO (negative standard) or with
the DMSO two-fold diluted active compounds. Cells were also treated with Sodium-dodecylsulfate

Molecules 2020, 25, 5448
14 of 16
(SDS) and trichloroacetic acid (TCA) as positive standard molecules for cell lysis. Bacterial lysis was
assessed by measuring OD600 after 10, 30 and 60 min. Values were compared with those obtained with
the negative standard. Bacterial lysis was also investigated by SDS-PAGE protein content analysis.
8
Briefly, cells (5
×
10 CFU/mL) were incubated with DMSO or active compounds for 10, 30 and 60 min
and then harvested by centrifugation at 10,000
20 C prior to analysis. The supernatants were precipitated using trichloroacetic acid (TCA) and later
centrifuged to recover the proteins. The extracted proteins were also frozen at
×
g for 10 min. The harvested pellets were frozen at
◦
−
◦
−
20 C prior to analysis.
4
.12. Determination of the Intra- and Extra-Cellular Potassium Content
Mid-log phased Campylobacter cells were grown in MH Broth and D-MEM (50:50). Cells were
centrifuged and resuspended to obtain a cell density of 1 OD600nm. Cells were energized by the
addition of 0.2% (w/v) glucose and incubated with DMSO (negative standard) or two-fold diluted
active compounds. After variable time intervals, cells were centrifuged, and respective supernatants
◦
were immediately cooled at
−
80 C for further analysis. The cell pellets so obtained were treated
under agitation with buffer containing 10% (w/v) TCA and 2 mM EDTA for 2 min. Post-treatment,
the disrupted cells were centrifuged, and their resulting supernatants were immediately cooled at
◦
−
80 C for further analysis. Intra- and extra-cellular potassium levels were determined by Atomic
Absorption Spectrometry using a flame photometer (VARIAN (Agilent), Les Ulis, France) set to read
potassium concentrations. Values were extrapolated against a standard calibration curve of KCl.
4
.13. Determination of the Intra- and Extra-Cellular ATP Content
ATP concentrations were determined in mid-log phased Campylobacter cells grown in MH Broth
and D-MEM broth (50:50) and membrane extracts of C. jejuni and E. coli prepared as mentioned
previously [21 22]. In both cases, ATP concentrations were determined using the rLuciferin-Luciferase
,
ATP assay kit (Promega, Lyon, France). The reaction was assayed over 10 ms period using a
chemiluminometer (Yelen S.A., Marseille, France). For two sets of experiments, three independent
assays were performed, and suitable controls were included to ensure that none of the elements
interfered with the assays. The intracellular ATP contents were presented as relative luminescence units
(
RLUs) and compared with the RLU of DMSO treated cells. The Residual activity of the F F -ATPase
O 1
was deduced from the ratio of the RLU obtained with treatment with tested molecules to the RLU of
the negative standard (DMSO).
Supplementary Materials: The following are available online. Figure S1: Kinetics of bactericidal effect, Figure S2:
Outer membrane protein detection, Figure S3: NMR spectra of synthesized compounds, Table S1: Effect of
compounds on cell integrity.
Author Contributions: B.J.M. (Brunel Jean Michel), B.J.M. (Bolla Jean Michel) and D.L. conceived and designed
the study. D.L. and B.J.M. (Brunel Jean Michel) carried out the experiments. B.J.M. (Brunel Jean Michel), B.J.M.
(
Bolla Jean Michel), M.A., D.L., L.V. and B.L. performed the data analysis. The manuscript was written by D.L.,
B.J.M. (Brunel Jean Michel) and B.J.M. (Bolla Jean Michel). All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by Aix-Marseille Université and Collectivité territorial de Corse (CTC).
Acknowledgments: We thank Jean-Marie Desjobert (UCPP) for GC-MS assistance, Nicolas Vidal (Yelen S.A.)
for bioluminescence material supports and technical assistance in ATP measurements, and Joseph Casanova for
RMN analyses. We also thank Paul-Georges Rossi and Jean Panighi for technical assistance and Mrunal Patil for
carefully improving the English writing of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2020, 25, 5448
15 of 16
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