Pterostilbene, a Natural Methoxylated Analog of
Resveratrol, Exhibits Antifungal Activity Induced
by Reactive Oxygen Species Production andPlasma Membrane Injury
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言語: English
出版者: MDPI
公開日: 2024-06-20
キーワード (Ja):
キーワード (En): antifungal, pterostilbene, reactive
oxygen species, plasma membrane injury,
Saccharomyces cerevisiae
作成者: Mizuhara, Naoko, Inoue, Moe, Kurotaki, Hideki,Matsumoto, Kazuyori, Ogita, Akira, Fujita, Ken-Ichi
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http://hdl.handle.net/10466/0002000963
This work is licensed under a Creative Commons
Attribution 4.0 International License.

Communication

Pterostilbene, a Natural Methoxylated Analog of Resveratrol,
Exhibits Antifungal Activity Induced by Reactive Oxygen
Species Production and Plasma Membrane Injury
Naoko Mizuhara 1 , Moe Inoue 2 , Hideki Kurotaki 2 , Kazuyori Matsumoto 2 , Akira Ogita 1,3 and Ken-Ichi Fujita 1, *
1

2

3

*

Graduate School of Science, Osaka Metropolitan University, Osaka 558-8585, Japan;
mizuhara@omu.ac.jp (N.M.); ogita@omu.ac.jp (A.O.)
Advanced Technology Research Center, Okuno Chemical Industries Co., Ltd., Osaka 538-0044, Japan;
m-manbo01@okuno.co.jp (M.I.); h-kurotaki01@okuno.co.jp (H.K.); k-matsumoto01@okuno.co.jp (K.M.)
Research Center for Urban Health and Sports, Osaka Metropolitan University, Osaka 558-8585, Japan
Correspondence: fujita@omu.ac.jp; Tel.: +81-6-6605-2580

Abstract: Pterostilbene has been shown to exhibit antifungal activities. However, the detailed
action mechanism is unknown. Here, we analyzed the antifungal mechanism of pterostilbene
against Saccharomyces cerevisiae. The minimum growth inhibitory and fungicidal concentrations ofpterostilbene were 120 and 240 µM in a YPD medium, respectively. Although pterostilbene produced
reactive oxygen species (ROS), resveratrol did not. This effect was maximized at 120 µM. Furthermore,
α-tocopherol acetate inhibited ROS production and reversed pterostilbene-induced growth inhibition.
At 240 µM, pterostilbene showed fungicidal effects accompanied by the leakage of intracellularpotassium ions, suggesting the involvement of membrane injury in addition to oxidative stress infungicidal action. Nevertheless, the antioxidants gradually reversed the decrease in cell viability
caused by pterostilbene, suggesting that ROS production mainly contributed to the lethal effect. As
Pterocarpus marsupium extract, mainly containing pterostilbene, is used as a traditional medicine,pterostilbene has great potential for development as a preservative with fewer adverse effects.
Citation: Mizuhara, N.; Inoue, M.;
Kurotaki, H.; Matsumoto, K.;

Keywords: antifungal; pterostilbene; reactive oxygen species; plasma membrane injury; Saccharomyces cerevisiae

Ogita, A.; Fujita, K.-I. Pterostilbene, aNatural Methoxylated Analog of
Resveratrol, Exhibits Antifungal
Activity Induced by Reactive Oxygen
Species Production and Plasma
Membrane Injury. Appl. Microbiol.
2023, 3, 666–674. https://doi.org/
10.3390/applmicrobiol3030045
Academic Editor: Ian Connerton
Received: 12 June 2023
Revised: 21 June 2023
Accepted: 26 June 2023
Published: 28 June 2023

Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Controlling fungal growth is essential to prevent frequent food spoilage, which leads tosignificant economic losses [1]. Despite advancements in food preservatives, food spoilage
caused by yeasts such as Saccharomyces cerevisiae and Wickerhamomyces anomalus cannot
be entirely prevented. These yeasts are major sources of spoilage in preserved foods andbeverages due to their high tolerance to weak acid preservatives and ethanol [2,3]. The
brewing yeast S. cerevisiae is the primary microorganism involved in alcoholic fermentation.
Among film-forming yeasts, W. anomalus frequently causes spoilage in beer [4,5] and bakeryrelated products [6,7]. Furthermore, non-Saccharomyces yeasts, including W. anomalus,
contribute to the complex aroma of wine [8]. Traditional preservatives such as sorbic acidand its potassium salt are added to wine with residual sugars to prevent re-fermentationby yeasts such as S. cerevisiae and W. anomalus, but their use is limited due to their slightodor and fatty acid-like taste. Therefore, safe and effective antifungal preservatives are
required to control these yeasts. Although several theories have been proposed to inhibit
yeast growth using this class of compounds, the weak antifungal activities of such organic
acid-based preservatives have not yet been overcome [9]. Thus, there is a pressing need todevelop effective fungus-targeting preservatives to reduce food losses.
Pterostilbene is a type of trans-stilbene, which is a natural methoxylated analog of resveratrol (Figure 1). It can be derived from several plants, including Pterocarpus marsupium (Indiankino tree), Pterocarpus santalinus (red sandalwood), Vitis vinifera (common grape vine), andAppl. Microbiol. 2023, 3, 666–674. https://doi.org/10.3390/applmicrobiol3030045

https://www.mdpi.com/journal/applmicrobiol

Appl. Microbiol. 2023, 3

Pterostilbene is a type of trans-stilbene, which is a natural methoxylated analog oresveratrol (Figure 1). It can be derived from several plants, including Pterocarpus marsu
pium (Indian kino tree), Pterocarpus santalinus (red sandalwood), Vitis vinifera667(common
grape vine), and Vaccinium ashei (rabbit-eye blueberry) [10–13]. Pterostilbene, in addition
to resveratrol, exhibits various biological activities that are beneficial to human health
Vaccinium
ashei(rabbit-eyeanti-inflammatory,
blueberry) [10–13]. Pterostilbene,
in addition
to resveratrol,
exhibits
includinganticancer,
antioxidant,
and analgesic
properties[14–17]. I
various biological activities that are beneficial to human health, including anticancer, antialso has anti-biofilm activity against the human pathogenic fungus Candida albicans [18]inflammatory, antioxidant, and analgesic properties [14–17]. It also has anti-biofilm activity
In addition
to itspathogenic
human healththis stilbene
exhibits to
antifungal
againstthe human
fungusbenefits,
Candida albicans[18]. In addition
its human effects
health agains
severalplant
pathogens,including
Phomopsis
viticola,
Phomopsis
obscurans,
andBotrytis ci
benefits, this stilbene exhibits antifungal effects against several plant pathogens, including
nerea [19–21].
However,
antifungal
mechanismof pterostilbene
fungi, includ
Phomopsis
viticola,
Phomopsis the
obscurans,
and Botrytis
cinerea [19–21].
However, against
the antifungal
ing food spoilage
yeasts, has
notfungi,
yet been
thoroughly
investigated.
Therefore,
we analyzed
mechanismof pterostilbene
againstincludingfood spoilage
yeasts, has
not yet beenthoroughly
analyzed
its action
mechanism using
the fungal
its action investigated.
mechanism Therefore,
using thewe
fungalmodel
microorganism
S. cerevisiae
atmodelthe molecula
microorganism
S. cerevisiae at the molecular level.
level.

Figurestructuresof resveratrol(A) and(B).
Figure1.1.Chemical
Chemicalstructuresof resveratrol(A)pterostilbene
and pterostilbene(B).

2. Materials and Methods
2. Materials
2.1.
Chemicals and Methods

2.1. Pterostilbene
Chemicals and resveratrol were purchased from Tokyo Chemical Industry Co. Ltd.

(Tokyo,Japan). α-Tocopherol
acetate and20 ,70 -dichloro-fluoresceindiacetate(DCFH-DA)
Pterostilbene
and resveratrol
were
purchased from Tokyo
ChemicalIndustry Co
werepurchasedfrom
Sigma-Aldrich(St.
Louis,MO,
USA).
N,N-dimethylformamide(DMF) (DCFH
Ltd. (Tokyo, Japan). α-Tocopherol acetate and 2′,7′-dichloro-fluorescein diacetate
was obtained from Wako Pure Chemicals (Osaka, Japan). Pterostilbene and resveratrolDA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N,N-dimethylformamid
were dissolved in DMF before use. α-Tocopherol acetate and DCFH-DA were diluted in(DMF) was obtained from Wako Pure Chemicals (Osaka, Japan). Pterostilbene andethanol before use. The K+ ion assay kit was purchased from HACH (Loveland, CO, USA).
resveratrol
were dissolved
DMF beforeAllthe other chemicals
were ofin
analyticalgrade.use. α-Tocopherol acetate and DCFH-DA wer

diluted in ethanol before use. The K+ ion assay kit was purchased from HACH (Loveland

2.2.
and
CultureConditions
CO,Strain
USA).
All
the otherchemicals were of analytical grade.

The strain S. cerevisiae BY4741 was purchased from Thermo Fisher Scientific (Waltham,
MA,
cells
were grown in YPD broth, consisting of 1% Bacto yeast extract
2.2. USA).
Strain The
and yeast
CultureConditions(Difco Laboratories, Detroit, MI, USA), 2% Bactopeptone (Difco Laboratories), and 2%
The strain S. cerevisiae BY4741 was purchased from Thermo Fisher Scientific (WalD-glucose at 30 ◦ C for 16 h without shaking prior to experiments, unless otherwise stated.
tham, MA, USA). The yeast cells were grown in YPD broth, consisting of 1% Bacto yeas
All experiments, including an assay for cellular reactive oxygen species (ROS) levels, wereextract (Difco
Detroit, MI, USA), 2% Bactopeptone (Difco Laboratories), andperformed
in theLaboratories,dark.

2% D-glucose at 30 °C for 16 h without shaking prior to experiments, unless otherwis

2.3.
Measurementof Cell Growthand Viability
stated.
All experiments,
includingan assay for cellular reactive oxygen species (ROS) lev
obtainan initialin
cell
densityels, To
wereperformed
thedark. of approximately 106 cells/mL, the precultured cells

were inoculated into freshly prepared YPD medium in a test tube (diameter, 10 mm) andincubated
at 30 ◦ C ofwithout
shaking
in Viability
the presence or absence of the test compound at
2.3. Measurement
Cell Growthandvarious concentrations. After 24 h of culturing, we measured cell turbidity at 595 nm to
To theobtainan initialcell inhibitory
density ofconcentration
approximately
106 MIC
cells/mL,
the precultured
cell
estimate
minimum
growth(MIC).
was definedas thewere
inoculated
intofreshly
prepared
YPD
medium
in
a
test
tube
(diameter,
10
mm)
and
lowest concentration of the test compound that causes a decrease in cell turbidity of lessincubated
at 30°C without
in the presenceor absenceof the test
compoundathan
80% in theabsenceof drugs.shaking
After measuring
cell turbidity,
100 µL portionsof thecellvariousconcentrations.
After
24 h containingof culturing,measuredcell
at 595 nm t
culturewereplated on a YPD
medium
1.8%we(w/v)
agar. After48 turbidity
h of incubation
at30 ◦ C, colony
formationgrowth
was assessedto confirm
cell viability
and MIC
to determine
the as th
estimate
the minimum
inhibitory
concentration(MIC).
was definedminimumfungicidal concentration.
The minimum(MFC)
was of leslowest concentrationof the test compound
thatfungicidal
causes a concentration
decrease in cell
turbidity
determinedas thethe lowest
concentrationof the measuring
test compound
prevent colony
formation
than 80% in
absenceof drugs. After
celltoturbidity,
100 µL
portions of th
on agar plates.

cell culture were plated on a YPD medium containing 1.8% (w/v) agar. After 48 h of incu
bation at 30 °C, colony formation was assessed to confirm cell viability and to determin
the minimum fungicidal concentration. The minimum fungicidal concentration (MFC

Appl. Microbiol. 2023, 3668

2.4. Leakage of K+ Ions
Yeast cells cultured overnight in YPD medium were harvested by centrifugation, washedtwice, and resuspended in 50 mM succinate buffer (pH 6.0) at a cell density of 5 × 107 cells/mL.
The cell suspensions were supplemented with or without each compound and incubatedwith vigorous shaking at 30 ◦ C for 3 h. The supernatant obtained after removing the cells by
centrifugation was used for the quantification of K+ ions released from the cells. The K+ ionsfrom the cells react with sodium tetraphenylborate to form potassium tetraphenylborate, aninsoluble white solid. The amount of turbidity produced is proportional to the K+ concentration.
The measurement wavelength of the spectrophotometer was 650 nm. Quantification was
performed using a K+-ion assay kit based on the tetraphenylborate method [22].
2.5. Determination of Cellular Reactive Oxygen Species (ROS) Levels
Cellular ROS production was measured using the DCFH-DA method, which is dependent on intracellular deacetylation and oxidation of 20 ,70 -dichlorodihydrofluorescein(DCFH) to the fluorescent compound 20 ,70 -dichlorofluorescein (DCF) [23,24]. This probe
is highly reactive with hydrogen peroxide and has been used to evaluate ROS generationin yeast. Following preincubation (1 × 107 CFU/mL) in YPD medium with 40 µM of
DCFH-DA at 30 ◦ C for 60 min, 3 mL of cell suspension was withdrawn and further treatedwith the test compounds against the yeast cells for 60 min. After treatment, the cells were
washed and resuspended in 330 µL of phosphate-buffered saline buffer. The fluorescenceintensity of the cell suspension was measured using a GENios (TECAN, Grödig, AT, USA)
(excitation at 485 nm and emission at 535 nm). Arbitrary units were based on fluorescenceintensity per 107 cells.
2.6. Statistical Analysis
Statistical evaluation was performed using Student’s t-test; p < 0.05 was considered torepresent statistical significance.
3. Results and Discussion
Pterostilbene constitutes approximately 90% of the extract from the dried heartwoodof P. marsupium [11]. The Pterocarpus extract (Sabinsa Japan Corp., Tokyo, Japan) was preliminarily found to restrict the colony formation of W. anomalus NBRC (Biological Resource
Center, NITE, Tokyo, Japan) 10213 and S. cerevisiae NBRC 100929 on potato dextrose agar
plates after 48 h incubation at concentrations more than 100 µg/mL. Therefore, pterostilbene is thought to be the active constituent of the extract and is expected to be a candidate
preservative with antifungal properties. To analyze the antifungal mechanism of pterostilbene, we evaluated the antifungal activities of resveratrol and pterostilbene after 24 h ofincubation with the drugs based on cell turbidity in liquid YPD media. Although resveratrol
did not show growth-inhibitory effects on S. cerevisiae at concentrations up to 480 µM (datanot shown), the MIC of pterostilbene was 120 µM, as shown in Figure 2. After determining
the MIC, the MFC was determined using cell suspensions. The MFC of pterostilbene was
240 µM. These results indicate that the two methoxy residues contribute to the antifungal
activity of pterostilbene compared with the chemical structure of resveratrol. The two
methoxy residues of pterostilbene are demethylated in resveratrol (Figure 1). In resveratrol,
the hydroxyl residues are distributed at the ends of the molecule. Additionally, due to thepresence of two methoxy residues at one end of the molecule and one hydroxy residue at theother end, pterostilbene possibly exhibits nonionic surfactant-like characteristics, with bothhydrophobic and hydrophilic properties within the molecule. As an example of nonionicsurfactants, hydroxyl groups in n-alkanols would interact with hydrophilic groups thruston the membrane to form hydrogen bonds [25]. Nonpolar carbon chains in the alkanols
might be folded into membrane bilayers [26–28]. Therefore, nonionic surfactants such asn-alkanols and alkyl gallates physically disrupt the orderly structure of the lipid bilayer inthe plasma membrane, thereby affecting membrane permeability [26–28]. Therefore, we
examined the effect of pterostilbene on membrane permeability in S. cerevisiae cells.

Appl. Microbiol. 2023, 3

with
with hydrophilichydrophilic groups
groups thrustthrust on
on thethe membrane
membrane toto form
form hydrogenhydrogen bonds
bonds [25].
[25]. Nonpolar
Nonpolarcarboncarbon chainschains inin thethe alkanols
alkanols might
might bebe folded
folded into
into membrane
membrane bilayers
bilayers [26–28].
[26–28]. Therefore,
Therefore,
nonionic
nonionic surfactants
surfactants such
such asas n-alkanols
n-alkanols andand alkyl
alkyl gallates
gallates physicallyphysically disrupt
disrupt thethe orderly
orderly
structure
of
the
lipid
bilayerintheplasmamembrane,
therebyaffectingmembranestructure of the lipid bilayer in the plasma membrane, thereby affecting membrane perper669
meability
meability [26–28].
[26–28]. Therefore,
Therefore, we
we examinedexamined thethe effecteffect ofof pterostilbene
pterostilbene onon membrane
membrane perpermeability
meability inin S.
S. cerevisiae
cerevisiae cells.
cells.

Figure2.
Effectofpterostilbene
onthegrowthofS.
cerevisiae.
The
exponentiallygrowing
yeast
cells
FigureFigure2.
2.Effect
Effectof
ofpterostilbene
pterostilbeneon
onthe
thegrowth
growthof
ofS.
S.cerevisiae.
cerevisiae.The
Theexponentiallyexponentiallygrowing
growingyeast
yeastcells
cells
◦
were
incubated
in
YPD
broth
with
0–240
µM
of
pterostilbene
at
30
°C
for
24
h.
After
incubation,
the
were
C for
wereincubatedincubatedininYPD
YPDbrothbrothwith
with0–240
0–240µMµMofofpterostilbene
pterostilbeneatat30
30 °C
for24
24h.h.After
Afterincubation,
incubation,thethecellturbiditywasmeasured
at
595
nm
to
evaluate
growthinhibitory
effectofpterostilbene.
Each
cell
at at
595595nmnm
to evaluate
growthinhibitory
effecteffectof pterostilbene.
Each data
cellturbidity
turbiditywas
wasmeasured
measuredto evaluate
growthinhibitoryof pterostilbene.
Each
data
represents
the± SD ofassays.
data point
point
represents
the meanmeanof triplicate
triplicate
assays.
point
represents
the mean± SD ±ofSD
triplicate
assays.

Leakagepotassium
ions
from
cells
was
confirmedtoeffectofLeakage
ions
from
cells
was
confirmed
to evaluate
the theeffectof pterostilbene
Leakageofof
ofpotassium
potassium
ions
from
cells
was
confirmed
to evaluate
evaluatetheeffectof pterostilpterostilbene
onmembranepermeability.
A
traditional
polyeneantifungal,
amphotericin(AmB),
onmembranepermeability.
A
traditional
polyeneantifungal,
amphotericin
B(AmB),
bene on membrane permeability. A traditional polyene antifungal, amphotericin B
Binteracts(AmB),
interacts
withfungalergosterols,
therebyforming
pores
to
leak
potassiumions
selectively
withfungalergosterols,
thereby forming
pores
to leakpotassium
selectively
It wasinteracts
withfungal ergosterols,
therebyforming
pores
to leakions
potassiumions[29].
selectively
[29].
It
was
used
as
a
positive
controlinthisstudy,
causinganincreaseinmembraneperusedasapositive
controlinthisstudy,
causinganincreaseinmembranepermeability.
[29]. It was used as a positive control in this study, causing an increase in membraneAmB
permeability.
AmB
significantly
of
with
at
MFC significantly
inducedleakage induced
after 120leakage
min of after
incubationwiththe cells (Figure3).
meability.
AmB atat MFCMFC
significantly
inducedleakageafter 120
120 min
minof incubation
incubation
with thethecells(Figure3).
Similarly,
pterostilbene
at
MFC
also
significantly
inducedleakage,but
theSimilarly,
pterostilbene
at
MFC
also
significantly
inducedleakage,but
theleakagewas
gradualcells (Figure 3). Similarly, pterostilbene at MFC also significantly induced leakage, but theleakage
was
and
at
that
AmB
Therefore,
andat lowerlevels thanin thelevels
AmBthantreatment.
Therefore,
the fungicidal
effect theofleakage
was gradual
gradualandthat
at lowerlower
levelsthanthat inin thethe
AmB treatment.
treatment.
Therefore,thefungicidal
effectofpterostilbene
cannotbeexplainedsolelybyitseffectonmembranepterostilbene
cannotbeexplainedsolelybyitseffectonmembranepermeability,
and
other
fungicidal effect of pterostilbene cannot be explained solely by its effect on membrane
permeability,andfactors
factors
are possibly
involved.
permeability,
and otherotherfactors areare possibly
possibly involved.
involved.

+ ion leakage from cells. Exponentially growing yeast cells
FigureEffectsofpterostilbene
onFigure3.3.
3.Effects
Effectsof
ofpterostilbene
pterostilbeneon
onKK
K++ ionionleakage
leakagefrom
fromcells.
cells.Exponentially
Exponentially growing
growingyeast
yeastcells
cells
Figurewere
incubated
at
30
°C
in
S-bufferalone(closedcircle)andthebuffercontaining
10
µM
of
AmB
wereincubatedincubatedat
at30
30◦°C
inS-buffer
S-bufferalone
alone(closed(closedcircle)
circle)andandthethebufferbuffercontaining
containing10
10µM
µMof
ofAmB
AmB
were
C
in
(open
(open square)square) oror 240240 µMµM ofof pterostilbene
pterostilbene (open(open circle).
circle). Each
Each data
data point
point represents
represents thethe meanmean ±± SD
SD ofof(open square) or 240 µM of pterostilbene (open circle). Each data point represents the mean ± SD of
triplicate assays. Asterisks indicate significant differences compared to the cells incubated in S-buffer
without drugs (p < 0.05).

Dodecanol is a medium-chain normal aliphatic alcohol that also shows nonionicsurfactant-like characteristics, attributed to a hydrophobic tail alkyl chain and a hydrophilic
head hydroxyl moiety. This alcohol induces oxidative stress by inducing the productionof cellular reactive oxygen species (ROS) against S. cerevisiae [30]. In addition, AmB

triplicate assays. Asterisks indicate significant differences compared to the cells incubated in Sbuffer without drugs (p < 0.05).

Appl. Microbiol. 2023, 3

Dodecanol is a medium-chain normal aliphatic alcohol that also shows nonionic sur670
factant-like characteristics, attributed to a hydrophobic tail alkyl chain and a hydrophilic
head hydroxyl moiety. This alcohol induces oxidative stress by inducing the productionof cellular reactive oxygen species (ROS) against S. cerevisiae [30]. In addition, AmB also
induces
cellular
ROSROS
production
againsthuman
pathogenic
yeastyeast
species,
including
Canalsoinduces
cellularproduction
againsthuman
pathogenic
species,
includingCandidaalbicansThus,
we evaluatedthe effects
of resveratrol
and pterostilbene
ondida albicans[31].[31].
Thus,
we evaluatedthe effects
of resveratrol
and pterostilbene
on cellular
cellular
ROS production
using
a ROS fluorescence
detection fluorescenceprobe, DCFH-DA.
Prior
ROS production
using a ROS
detectionprobe, DCFH-DA.
Prior to determin◦ C for 1 h in thetodetermining
yeast
cells wereincubated
atin30theing
cellular ROScellularlevels, ROS
yeast levels,
cells wereincubated
at 30°C for 1 h
YPD broth with
YPD
broth withor without
the drugs.
not induce
ROS production
or without
the drugs.
Resveratrol
did notResveratrol
induce ROSdid
production
at concentrations
upat
toconcentrations
up
to
240
µM
(Figure4).
Incontrast,
pterostilbene
significantly
induced240 µM (Figure 4). In contrast, pterostilbene significantly induced ROS production at 120
ROS
at 120 and240µM,
indicatingthat the drugcausedoxidative
stress in yeast
and production
240 µM, indicating
that
the
drug
caused oxidative
stressin yeast
cells. Maximum
ROS
cells.
Maximum
production
was observed
at aµM.
concentration
ofis120µM. However,production
was ROSobserved
at a concentrationof 120However, it
unclearwhy ROSitproductionis unclear was
whyreduced
ROS productionin thetreatment.
240 µM pterostilbene
in the 240wasµM reduced
pterostilbene
The nonionictreatment.
surfactant
Thenonionic
surfactant may
natureof pterostilbene
may haveleakedthereby
DCFH-DAmolecules,
natureof pterostilbene
haveleaked DCFH-DA
molecules,
reducing
the inthereby
reducing
theintracellularconcentrationofthefluorescentprobe.
As
oneoftracellular concentration of the fluorescent probe. As one of the other possibilities, thethe
reotherpossibilities,
the reaction
rate point
may reacha plateaupoint in thewhere
ROS production,
where
actionrate may reacha plateau
in theROS production,
all of the oxygen
allof thewas
oxygensourcebywasconsumed by pterostilbene.
sourceconsumedpterostilbene.

Figure4.4.Effects
Effectsofofpterostilbene
pterostilbeneon
oncellular
cellularROS
ROSproduction.
production.(A)(A)Effects
Effectsofofresveratrol
resveratrol(closed)(closed)andand
Figurepterostilbene (open) concentrations on cellular ROS production. The DCFH-DA pre-stained cells
pterostilbene (open) concentrations on cellular ROS production. The DCFH-DA pre-stained cells
were incubated in YPD broth with each drug at 30 °C for 1 h prior to the measurement of DCF
were incubated in YPD broth with each drug at 30 ◦ C for 1 h prior to the measurement of DCF
fluorescence. Each data point represents the mean ± SD of triplicate assays; (B) effect of tocopherol
fluorescence.
Each data point represents
the mean ±The
SD DCFH-DAof triplicatepre-stained
assays; (B) cells
effectwereof tocopherol
acetate on pterostilbene-induced
ROS production.
incubatedacetateonpterostilbene-induced
ROS
production.
The
DCFH-DApre-stained
cells
were
in
in YPD broth with 120 µM of pterostilbene and 120 µM of tocopherol acetate at 30 °Cincubatedfor 1 h prior◦ C for 1 h prior to the
YPD
broth
with
120
µM
of
pterostilbene
and
120
µM
of
tocopherol
acetateat30
to the measurement of DCF fluorescence. Each data point represents the mean ± SD of triplicate
measurementof DCF fluorescence. Each data point represents the mean ± SD of triplicate assays.
assays.

Cellularoriginates
mainlyfromthe themitochondrial
respiratory
chainchain[32],
CellularROS
ROSproduction
production
originates
mainlyfrommitochondrial
respiratory
in[32],
additiontolipidperoxidationoftheplasmamembrane[33].
Moreover,inprevious
studies,in addition to lipid peroxidation of the plasma membrane [33]. Moreover, in previous
cellularproduction
was effectively
inhibited byinhibitedthe additionof antioxidants
to the yeast
studies,ROS
cellular
ROS production
was effectively
by theaddition of antioxidants
cultureTherefore,
we investigatedtheinvestigatedeffect of hydrophilicandantioxidants
to the [34,35].
yeast culture[34,35].
Therefore, we
the effectoflipophilichydrophilic
and lipoon
pterostilbene-induced
ROS
production.
TomeasureROS
levels,pterostilbene
atphilic antioxidants on pterostilbene-induced ROS production. To measure was
ROSusedlevels,a concentration of 120 µM, because the maximum level of ROS production was observed at
this concentration. Yeast cells were co-treated with each antioxidant and the indicated drugs
at 30 ◦ C for 1 h. The hydrophilic antioxidant ascorbic acid (1.0 mM) reduced ROS production
by 23% (data not shown). In contrast, the lipophilic antioxidant, 120 µM of α-tocopherol
acetate, reduced ROS production by 63% (Figure 4B). Next, we examined the effects ofthe two antioxidants on the decrease in cell turbidity induced by 120 µM of pterostilbene.
Pterostilbene at 120 µM reduced cell turbidity by 90% and 97%, respectively, compared to drug-Appl. Microbiol. 2023, 3

pterostilbene was used at a concentration of 120 µM, because the maximum level of ROS
production was observed at this concentration. Yeast cells were co-treated with each antioxidant and the indicated drugs at 30 °C for 1 h. The hydrophilic antioxidant ascorbic acid(1.0 mM) reduced ROS production by 23% (data not shown). In contrast, the lipophilic
antioxidant, 120 µM of α-tocopherol acetate, reduced ROS production by 63% (Figure 4B).
671
Next, we examined the effects of the two antioxidants on the decrease in cell turbidity
induced by 120 µM of pterostilbene. Pterostilbene at 120 µM reduced cell turbidity by 90%
and 97%, respectively, compared to drug-free treatment after 24 and 48 h of incubation,
respectively.
ascorbic(1.0 mM)
did not restore
the decrease
cell (1.0
turbidityfreetreatmentAlthough
after 24 and48 h ofacid
incubation,
respectively.
Althoughascorbicinacid
mM)
(data
not
shown),
120µM
ofα-tocopherol
acetaterestoredcellturbidity
by
up
to
and
did not restore the decrease in cell turbidity (data not shown), 120 µM of α-tocopherol30%
acetate90% after
24turbidity
and 48 hby
of up
incubation,
respectively5).hThese
results indicatedthat
restoredcell
to 30% and
90% after (Figure
24 and 48
of incubation,
respectively
the hydrophilic
antioxidant
ascorbicacid
did not affectpterostilbene-induced
ROSaffectpro(Figure5). These results
indicatedthat thehydrophilic
antioxidant
ascorbic acid did notduction or growth inhibition,
whereasα-tocopherol
acetatereducedROS propterostilbene-induced
ROS production
or lipophilic
growth inhibition,
whereaslipophilic
α-tocopherol
ductionreduced
and restored
growth inhibition.
As ascorbic
acid probably
behavesacid
as aprobably
dissociacetate
ROS production
and restored
growth inhibition.
As ascorbic
ated
ion
in
media
such
as
a
YPD
broth,
itisdifficultfor
dissociationtopenetrate
the cellbehaves as a dissociated ion in media such as a YPD broth, it is difficult for dissociation
membranebe incorporated
intocytoplasm.into
Therefore,
higher Therefore,
concentrationsoftopenetrate andthe cellmembrane andbe theincorporated
the cytoplasm.
higherconcentrationsof ascorbic
acid may
be required
to induce its
antioxidant
effects.α-tocopherol
In contrast,
ascorbic acid may
be required
to induce
its antioxidant
effects.
In contrast,
α-tocopherol
acetatereadily thepenetrates
the cell membrane,gets deacetylated,
and integrates
acetate readily
penetrates
cell membrane,gets deacetylated,
and integrates
into theinto
the membranes.
Therefore,
α-tocopherol
probably
inhibits
ROS production
originating
membranes.
Therefore,
α-tocopherol
probably
inhibits
ROS production
originating
from
from
membranesof cellorganelles,
suchthemitochondrial
mitochondrialinner
innermembrane,
membrane, where ROS
membranesof cellorganelles,
such
as as
the
ROS
productioniscausedbytheblockadeofelectrontransferincomplexes
I
and
III
of
the
electronproduction is caused by the blockade of electron transfer in complexes I and III of thetransport
chain [36,37].
the otherregardto treatment
with a withfungicidal
electron transport
chain On[36,37].
On thehand,
otherwith
hand,
with regard
to treatment
a funconcentrationof pterostilbene,
α-tocopherol
acetate atacetate
240 µM
growthgicidal concentrationof pterostilbene,
α-tocopherol
atdid
240 notµMrestore
did notyeast
restoreyeast
after
24 hafterof incubation,
but afterbut
72 afterh incubation,
the growth
was restored
to 85% toof 85%
thegrowth24 h of incubation,
72 h incubation,
the growth
was restoredcontrolcondition(data not(datashown),
that it takesthat
a longtime torecovergrowthafterof the control
conditionnotindicating
shown), indicating
it takes
a longtime
to recovertreatment
withtreatment
the lethal withconcentration.
growth after
the lethal concentration.

Figure5.5. Effect
Effectof
oftocopherol
tocopherolacetate
acetateon
onpterostilbene-induced
pterostilbene-inducedgrowth
growthinhibition.
inhibition. The
The cells
cellswere
were
Figureincubated in YPD broth with or without 120 µM of pterostilbene supplemented with or without 120
incubated in YPD broth with or without 120 µM of pterostilbene supplemented with or withoutµM of tocopherol acetate at 30 °C. After incubation, the cell turbidity was measured at 595 nm. Each
12 µM of tocopherol acetate at 30 ◦ C. After incubation, the cell turbidity was measured at 595 nm.
data point represents the mean ± SD of triplicate assays.
Each data point represents the mean ± SD of triplicate assays.

Recently,thetheinvolvement
involvementofofoxidativeoxidative
stresslethaleffects
beenreportedin
Recently,
stressin inlethaleffectshashasbeenreportedin anantimicrobial
drugs[38,39].
The
drugs
have
been
believedtodisrupt
cellularmetabolism
timicrobial drugs [38,39]. The drugs have been believed to disrupt cellular metabolism andandmembrane
the membraneas primarytargets.
In contrast,
bacteriostatic
not induce
oxitheas primarytargets.
In contrast,
bacteriostatic
drugsdrugs
do notdo
induceoxidative
dativesuch
stress,as the productionof hydroxyl
radicals[39]. Although
antifungal
AmB
stress,as suchthe productionof hydroxyl
radicals [39].
Althoughantifungal
AmB induces
plasma membrane injury, such as the formation of pores and leakage of potassium ionsagainst fungi, including human pathogenic Candida albicans, the fungicidal action of AmB
is related to membrane depolarization, decreasing metabolic activity, and increasing ROS
production in addition to membrane injury [40]. A sesquiterpene dialdehyde, polygodial,also acts primarily by damaging the permeability barrier of S. cerevisiae cells [41]. In addition, this dialdehyde also exerts fungicidal action via ROS production caused by thedepletion of intracellular glutathione [42]. In this case, α-tocopherol can lower the cellularlevels of ROS produced, thereby partly reducing the fungicidal effect. Following treatment
with fungistatic concentrations of pterostilbene, cellular ROS were produced at maximum

Appl. Microbiol. 2023, 3

672

levels, but membrane injury was not observed (data not shown). Pterostilbene induced
fungicidal action via membrane injury and ROS production, the levels of which were lower
than those induced by the fungistatic concentration. The involvement of cellular damage
induced by two or more pathways in fungicidal action was consistent with previously
reported AmB and polygodial fungicidal mechanisms, although slightly different. Farnesol
showed growth-inhibitory effects against S. cerevisiae, and the mechanism involved ROS
production without membrane injury [43]. This is consistent with our results showing thatpterostilbene-induced fungistatic effects are dependent on ROS production.
Although pterostilbene has been reported to show antioxidative activities [14–16]
also, including the blockade of ROS’s surge in mammalian cell lines [44], its antioxidative
effects seem to be inconsistent with its ability to induce ROS production in fungi. This
discrepancy was also reported for other compounds. When phenolic compounds withpotent antioxidant activity react with ROS and scavenge radicals, they produce highly
active phenoxy and hydroxyl radicals, which in turn, produce new ROS [45–47]. Whether
pterostilbene exhibits an antioxidant effect or exerts oxidative stress may depend on thespecies of drug applied. Further investigations are needed to clarify the possibility ofpterostilbene-induced fungus-specific oxidative stress.
4. Conclusions
We showed that pterostilbene exerts fungistatic effects via cellular ROS production
and fungicidal effects via plasma membrane injury in addition to ROS production. ROS
contributes to both fungistatic and fungicidal activities. Phenolic compounds derived
from plants, such as flavonoids and dihydroxycinnamic acids, also induce ROS production
depending on the concentrations used [47]. Therefore, pterostilbene and Pterocarpus extract
may serve as potential preservatives when used to restrict fungal growth at concentrations
that do not cause side effects in humans. The replacement of certain preservatives withpterostilbene might contribute to a reduction in food losses.
Author Contributions: Conceptualization, K.M. and K.-I.F.; methodology, N.M., M.I., H.K., K.M.,
A.O. and K.-I.F.; validation, K.M., A.O. and K.-I.F.; formal Analysis, N.M. and A.O.; investigation,
N.M., M.I., H.K., K.M., A.O. and K.-I.F.; data curation, A.O.; writing—original draft preparation, A.O.
and K.-I.F.; writing—Review and editing, N.M., M.I., H.K., K.M., A.O. and K.-I.F.; supervision, K.M.
and K.-I.F.; project administration, K.M. and K.-I.F.; funding acquisition, K.M., A.O. and K.-I.F. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was partially funded by the Japan Society for the Promotion of Science,
Grants-in-Aid for Scientific Research (C) K19K05799 and 21K07029. This study was partly funded by
Okuno Chemical Industries Co., Ltd.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data supporting the findings of this study are available from thecorresponding author upon reasonable request.
Acknowledgments: We thank Editage “https://www.editage.jp/ (accessed on 10 June 2023)” fortheir linguistic assistance during the preparation of this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

References
1.
2.
3.

Loureiro, V.; Querol, A. The prevalence and control of spoilage yeasts in foods and beverages. Trends Food Sci. Technol. 1999, 10, 356–365.
[CrossRef]
Stratford, M.; Vallières, C.; Geoghegan, I.A.; Archer, D.B.; Avery, S.V. The preservative sorbic acid targets respiration, explaining
the resistance of fermentative spoilage yeast species. mSphere 2020, 5, e00273-20. [CrossRef] [PubMed]
Sundh, I.; Melin, P. Safety and regulation of yeasts used for biocontrol or biopreservation in the food or feed chain. Antonie Van
Leeuwenhoek 2011, 99, 113–119. [CrossRef] [PubMed]

Appl. Microbiol. 2023, 3

4.
5.
6.
7.
8.
9.
10.
11.

12.
13.

14.

15.

16.

17.
18.
19.
20.
21.

22.
23.

24.

25.
26.
27.
28.
29.

673

Dufour, J.; Verstrepen, K.; Derdelinckx, G. Brewing yeasts. In Yeasts in Food–Beneficial and Detrimental Aspects; Boekhout, T., Robert,
V., Eds.; Behr’s Verlag: Hamburg, Germany, 2003; pp. 347–388.
Timke, M.; Wang-Lieu, N.Q.; Altendorf, K.; Lipski, A. Identity, beer spoiling and biofilm forming potential of yeasts from beerbottling plant associated biofilms. Antonie Van Leeuwenhoek 2008, 93, 151–161. [CrossRef]
Bonjean, B.; Guillaume, L.D. Yeasts in bread and baking products. In Yeasts in Food–Beneficial and Detrimental Aspects; Boekhout, T.,
Robert, V., Eds.; Behr’s Verlag: Hamburg, Germany, 2003; pp. 289–307.
Lanciotti, R.; Sinigaglia, M.; Gardini, F.; Guerzoni, M.E. Hansenula anomala as spoilage agent of cream-filled cakes. Microbiol. Res.
1998, 153, 145–148. [CrossRef] [PubMed]
Padilla, B.; Gil, J.V.; Manzanares, P. Past and future of non-Saccharomyces yeasts: From spoilage microorganisms to biotechnologicaltools for improving wine aroma complexity. Front. Microbiol. 2016, 7, 411. [CrossRef]
Ullah, A.; Orij, R.; Brul, S.; Smits, G.J. Quantitative analysis of the modes of growth inhibition by weak organic acids in
Saccharomyces cerevisiae. Appl. Environ. Microbiol. 2012, 78, 8377–8387. [CrossRef]
Bulle, S.; Reddyvari, H.; Nallanchakravarthula, V.; Vaddi, D.R. Therapeutic potential of Pterocarpus santalinus L.: An update.
Pharmacogn. Rev. 2016, 10, 43–49. [PubMed]
Majeed, M.; Majeed, S.; Jain, R.; Mundkur, L.; Rajalakshmi, H.R.; Lad, P.S.; Neupane, P. An open-sabel single-arm, monocentric
study assessing the efficacy and safety of natural pterostilbene (Pterocarpus marsupium) for skin brightening and antiaging effects.
Clin. Cosmet. Investig. Dermatol. 2020, 13, 105–116. [CrossRef]
Nagarajan, S.; Mohandas, S.; Ganesan, K.; Xu, B.; Ramkumar, K.M. New Insights into dietary pterostilbene: Sources, metabolism,and health promotion effects. Molecules 2022, 27, 6316. [CrossRef]
Vaňková, E.; Paldrychová, M.; Kašparová, P.; Lokočová, K.; Kodeš, Z.; Mat’átková, O.; Kolouchová, I.; Masák, J. Natural
antioxidant pterostilbene as an effective antibiofilm agent, particularly for gram-positive cocci. World J. Microbiol. Biotechnol. 2020,
36, 101. [CrossRef] [PubMed]
Abd-Elmawla, M.A.; Abdelalim, E.; Ahmed, K.A.; Rizk, S.M. The neuroprotective effect of pterostilbene on oxaliplatin-induced
peripheral neuropathy via its anti-inflammatory, anti-oxidative and anti-apoptotic effects: Comparative study with celecoxib. Life
Sci. 2023, 315, 121364. [CrossRef] [PubMed]
Amin, A.; Akhtar, M.F.; Saleem, A.; Sharif, A.; Shah, S.; Khan, M.I.; Anwar, F.; Abbas, G.; Zubair, H.M.; Sohail, M.F. Pterostilbene
improves CFA-induced arthritis and peripheral neuropathy through modulation of oxidative stress, inflammatory cytokines andneurotransmitters in Wistar rats. Inflammopharmacology 2022, 30, 2285–2300. [CrossRef] [PubMed]
Rimando, A.M.; Pezzuto, J.M.; Farnsworth, N.R.; Santisuk, T.; Reutrakul, V. Revision of the NMR assignments of pterostilbene
and of dihydrodehydrodiconieferyl alcohol: Cytotoxic constituents from Anogeissus acuminata. Nat. Prod.Lett. 1994, 4, 267–272.
[CrossRef]
Sun, C.; Li, Y.; Zhang, Y.; Huang, H.; Chen, H.; Chen, J.; Han, L.; Chen, X.; Chen, X.; Zhang, Y. Subacute oral toxicology andtoxicokinetics of pterostilbene, a novel Top1/Tdp1 inhibiting anti-tumor reagent. Drug Chem. Toxicol. 2023, 46, 392–399. [CrossRef]
Li, D.D.; Zhao, L.X.; Mylonakis, E.; Hu, G.H.; Zou, Y.; Huang, T.K.; Yan, L.; Wang, Y.; Jiang, Y.Y. In vitro and in vivo activities ofpterostilbene against Candida albicans biofilms. Antimicrob. Agents Chemother. 2014, 58, 2344–2355. [CrossRef]
Adrian, M.; Jeandet, P.; Veneau, J.; Weston, L.A.; Bessis, R. Biological activity of resveratrol, a stilbenic compound from grapevines,
against Botrytis cinerea, the causal agent for gray mold. J. Chem. Ecol. 1997, 23, 1689–1702. [CrossRef]
Pont, V.; Pezet, R. Relation between the chemical structure and the biological activity of hydroxystilbenes against Botrytis cinerea.
J. Phytopathol. 1990, 130, 1–8. [CrossRef]
Sobolev, V.S.; Khan, S.I.; Tabanca, N.; Wedge, D.E.; Manly, S.P.; Cutler, S.J.; Coy, M.R.; Becnel, J.J.; Neff, S.A.; Gloer, J.B. Biologicalactivity of peanut (Arachis hypogaea) phytoalexins and selected natural and synthetic stilbenoids. J. Agric. Food Chem. 2011, 59, 1673–1682.
[CrossRef]
Yamada, N.; Murata, W.; Yamaguchi, Y.; Fujita, K.I.; Ogita, A.; Tanaka, T. Enhancing the fungicidal activity of amphotericin B via
vacuole disruption by benzyl isothiocyanate, a cruciferous plant constituent. Lett. Appl. Microbiol. 2021, 72, 390–398. [CrossRef]
Fujita, K.; Tatsumi, M.; Ogita, A.; Kubo, I.; Tanaka, T. Anethole induces apoptotic cell death accompanied by reactive oxygen
species production and DNA fragmentation in Aspergillus fumigatus and Saccharomyces cerevisiae. FEBS J. 2014, 281, 1304–1313.
[CrossRef]
Tsukuda, Y.; Mizuhara, N.; Usuki, Y.; Yamaguchi, Y.; Ogita, A.; Tanaka, T.; Fujita, K. Structure-activity relationships of antifungal
phenylpropanoid derivatives and their synergy with n-dodecanol and fluconazole. Lett. Appl. Microbiol. 2022, 74, 377–384.
[CrossRef] [PubMed]
Chiou, J.S.; Ma, S.M.; Kamaya, H.; Ueda, I. Anesthesia cutoff phenomenon: Interfacial hydrogen bonding. Science 1990, 248, 583–585.
[CrossRef]
Fujita, K.; Kubo, I. Antifungal activity of octyl gallate. Int. J. Food Microbiol. 2002, 79, 193–201. [CrossRef] [PubMed]
Fujita, K.; Kubo, I. Plasma membrane injury induced by nonyl gallate in Saccharomyces cerevisiae. J. Appl. Microbiol. 2002, 92, 1035–1042.
[CrossRef] [PubMed]
Kubo, I.; Fujita, T.; Kubo, A.; Fujita, K.I. Modes of antifungal action of alkanols against Saccharomyces cerevisiae. Bioorg. Med. Chem.
2003, 11, 1117–1122. [CrossRef]
Carolus, H.; Pierson, S.; Lagrou, K.; Van Dijck, P. Amphotericin B and other polyenes: Discovery, clinical use, mode of action anddrug resistance. J. Fungi 2020, 6, 321. [CrossRef]

Appl. Microbiol. 2023, 3

30.
31.

32.
33.
34.

35.
36.

37.
38.
39.
40.
41.
42.
43.

44.
45.
46.
47.

674

Fujita, K.I.; Fujita, T.; Kubo, I. Anethole, a potential antimicrobial synergist, converts a fungistatic dodecanol to a fungicidal agent.
Phytother. Res. 2007, 21, 47–51. [CrossRef]
Mesa-Arango, A.C.; Trevijano-Contador, N.; Román, E.; Sánchez-Fresneda, R.; Casas, C.; Herrero, E.; Argüelles, J.C.; Pla, J.;
Cuenca-Estrella, M.; Zaragoza, O. The production of reactive oxygen species is a universal action mechanism of amphotericin B
against pathogenic yeasts and contributes to the fungicidal effect of this drug. Antimicrob. Agents Chemother. 2014, 58, 6627–6638.
[CrossRef]
Innokentev, A.; Kanki, T. Mitophagy in yeast: Molecular mechanism and regulation. Cells 2021, 10, 3569. [CrossRef]
Singh, S.; Brocker, C.; Koppaka, V.; Chen, Y.; Jackson, B.C.; Matsumoto, A.; Thompson, D.C.; Vasiliou, V. Aldehyde dehydrogenases
in cellular responses to oxidative/electrophilicstress. Free Radic. Biol. Med. 2013, 56, 89–101. [CrossRef]
Okai, Y.; Higashi-Okai, K.; Machida, K.; Nakamura, H.; Nakayama, K.; Fujita, K.; Tanaka, T.; Otani, S.; Taniguchi, M. Protective
effect of antioxidants against para-nonylphenol-induced inhibition of cell growth in Saccharomyces cerevisiae. FEMS Microbiol. Lett.
2000, 185, 65–70. [CrossRef] [PubMed]
Perrone, G.G.; Tan, S.X.; Dawes, I.W. Reactive oxygen species and yeast apoptosis. Biochim. Biophys. Acta. 2008, 1783, 1354–1368.
[CrossRef] [PubMed]
Fujita, K.; Tani, K.; Usuki, Y.; Tanaka, T.; Taniguchi, M. Growth inhibition dependent on reactive oxygen species generated by
C9-UK-2A, a derivative of the antifungal antibiotic UK-2A, in Saccharomyces cerevisiae. J. Antibiot. 2004, 57, 511–517. [CrossRef][PubMed]Shen, Z.F.; Li, L.; Zhu, X.M.; Liu, X.H.; Klionsky, D.J.; Lin, F.C. Current opinions on mitophagy in fungi. Autophagy. 2022, 19, 747–757.
[CrossRef]
Belenky, P.; Camacho, D.; Collins, J.J. Fungicidal drugs induce a common oxidative-damage cellular death pathway. Cell Rep.
2013, 3, 350–358. [CrossRef]
Kohanski, M.A.; Dwyer, D.J.; Hayete, B.; Lawrence, C.A.; Collins, J.J. A common mechanism of cellular death induced bybactericidal antibiotics. Cell 2007, 130, 797–810. [CrossRef]
Teixeira-Santos, R.; Ricardo, E.; Guerreiro, S.G.; Costa-de-Oliveira, S.; Rodrigues, A.G.; Pina-Vaz, C. New insights regarding yeast
survival following exposure to liposomal amphotericin B. Antimicrob. Agents Chemother. 2015, 59, 6181–6187. [CrossRef]
Taniguchi, M.; Yano, Y.; Tada, E.; Ikenishi, K.; Oi, S.; Haraguchi, H.; Hashimoto, K.; Kubo, I. Mode of action of polygodial, an
antifungal sesquiterpene dialdehyde. Appl. Biol. Chem. 1988, 52, 1409–1414.
Machida, K.; Tanaka, T.; Taniguchi, M. Depletion of glutathione as a cause of the promotive effects of polygodial, a sesquiterpene
on the production of reactive oxygen species in Saccharomyces cerevisiae. J. Biosci. Bioeng. 1999, 88, 526–530. [CrossRef]
Machida, K.; Tanaka, T.; Fujita, K.I.; Taniguchi, M. Farnesol-induced generation of reactive oxygen species via indirect inhibitionof the mitochondrial electron transport chain in the yeast Saccharomyces cerevisiae. J. Bacteriol. 1998, 180, 4460–4465. [CrossRef][PubMed]Kalamkar, S.D.; Bose, G.S.; Ghaskadbi, S.; Mittal, S. Andrographolide and pterostilbene inhibit adipocyte differentiation by
downregulating PPARγ through different regulators. Nat. Prod. Res. 2022, 14, 1–7. [CrossRef] [PubMed]
Bakkali, F.; Averbeck, S.; Averbeck, D.; Idaomar, M. Biological effects of essential oils–a review. Food Chem. Toxicol. 2008, 46, 446–475.
[CrossRef] [PubMed]
Inouye, S. Anti-microbial activity of essential oils involving reactive oxygen species. Aroma Res. 2010, 11, 307–311.
Sakihama, Y.; Cohen, M.F.; Grace, S.C.; Yamasaki, H. Plant phenolic antioxidant and prooxidant activities: Phenolics-induced
oxidative damage mediated by metals in plants. Toxicology 2002, 177, 67–80. [CrossRef]

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