Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: http://www.tandfonline.com/loi/iphb20

Synergistic anti-candidal activity and mode of

action of Mentha piperita essential oil and its
major components

Neha Samber, Amber Khan, Ajit Varma & Nikhat Manzoor

To cite this article: Neha Samber, Amber Khan, Ajit Varma & Nikhat Manzoor (2015) Synergistic
anti-candidal activity and mode of action of Mentha piperita essential oil and its major components,
Pharmaceutical Biology, 53:10, 1496-1504, DOI: 10.3109/13880209.2014.989623

To link to this article: http://dx.doi.org/10.3109/13880209.2014.989623

Published online: 08 Apr 2015.

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 http://informahealthcare.com/phb
ISSN 1388-0209 print/ISSN 1744-5116 online
Editor-in-Chief: John M. Pezzuto
Pharm Biol, 2015; 53(10): 1496–1504
! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.989623

ORIGINAL ARTICLE

Synergistic anti-candidal activity and mode of action of Mentha piperita

essential oil and its major components
Neha Samber1,2, Amber Khan1*, Ajit Varma2, and Nikhat Manzoor1
1
Medical Mycology Lab, Department of Biosciences, Jamia Millia Islamia, New Delhi, India and 2Amity Institute of Microbial Technology, Amity
University, Noida, India

Abstract Keywords
Downloaded by [Universitas Diponegoro] at 20:52 19 November 2017

Context: Mentha piperita L. (Lamiaceae) has been used in folk medicine since antiquity. Antifungal activity, Candida, carvone,
Its essential oil (mint EO) and major bioactive components have antimicrobial properties but menthol, menthone, plasma membrane
their mechanism of action is still not clear. H+-ATPase, synergy, virulence
Objective: The present work aims to elucidate M. piperita’s anti-Candida activity and mode of
action. History
Materials and methods: Chemical constituents of mint EO were identified by GC-MS by injecting
0.1 ml sample in a splitless mode. MIC was determined by the broth dilution method. Synergy Received 19 December 2013
with fluconazole (FLC) was evaluated by checkerboard assay and FICI. Mid log phase cells Revised 26 September 2014
harvested from YPD media were used for proton extrusion measurement and the rate of Accepted 15 November 2014
glucose-induced H+ efflux gives PM-ATPase activity. Cell membrane integrity was estimated by Published online 8 April 2015
total ergosterol content and scanning microscopy at respective MIC and sub-MIC values. In vitro
hemolytic activity was performed to rule out possible cytotoxicity of the test compounds.
Results: The MIC value of mint EO, carvone, menthol, and menthone was 225, 248, 500, and
4200 mg/ml, respectively. At their respective MICs, these compounds showed 47, 42, 35, and
29% decrease in PM-ATPase activity besides showing synergy with FLC. In case of FLC-resistant
strains, the decrease in H+ efflux was by 52, 48, 32, and 30%, a trend similar to the susceptible
cases. Exposed Candida cells showed a 100% decrease in the ergosterol content, cell membrane
breakage, and alterations in morphology.
Discussion and conclusion: Our studies suggest that mint EO and its lead compounds exert
antifungal activity by reducing ergosterol levels, inhibiting PM-ATPase leading to intracellular
acidification, and ultimately cell death. Our results suggest that mint EO and its constituents are
potential antifungal agents and need to be further investigated.

Introduction antifungal compounds are attracting much interest as natural

alternatives owing to their versatile applications (Ahmad
Infections caused by opportunistic fungal pathogens have
et al., 2010b; Iwu et al., 1999).
become increasingly common in immunocompromised
Essential oils and their major components have been
patients. Candida albicans is the predominant organism
explored for antimicrobial properties (Matasyoh et al., 2009).
associated with candidiasis; but other Candida species,
Mentha piperita L. (Lamiaceae) essential oil (mint EO) has
including Candida glabrata, Candida parapsilosis, and
been used in traditional medicine and its biological activity
Candida krusei, are now emerging as serious hospital-
may be due to its major components, carvone, menthol,
acquired infections (Maschmeyer, 2006; Pfaller & Diekema,
and menthone (Figure 1). Recent studies have shown that it
2004). Available antifungal drugs can treat superficial fungal
inhibits the formation of Candida and other microbial
infections, but only a few treatments are available for invasive
biofilms (Carretto et al., 2010; Derwich et al., 2011;
diseases. Polyenes cause serious host toxicity (Cohen, 1998)
Saharkhiz et al., 2012; Sandasi et al., 2011), decreasing
whereas azoles are fungistatic and their prolonged use leads
the production of virulence-associated exoproteins by
to drug resistance (Sanglard et al., 2003). Due to a dramatic
Staphylococcus aureus at sub-inhibitory concentrations in a
rise in fungal infections and the current trend toward
dose-dependent manner (Li et al., 2011).
increasing awareness in traditional medicine, plant-derived
The fungal plasma membrane H+ ATPase is a predominant
membrane protein that belongs to P-type ATPase family of
*Present address: Department of Pharmacy and Pharmacology, Faculty ion translocating ATPases (Perlin et al., 2006; Set-Young
of Health Sciences, Medical School, University of Witwatersrand,
Johannesburg, South Africa
et al., 1997). These include Na+ K+-ATPase and H+ K+-
ATPase, which are molecular targets for several clinically
Correspondence: Nikhat Manzoor, Department of Biosciences, Jamia
Millia Islamia, New Delhi 110025, India. Tel: +91 11 2698 1717x3410. important therapeutics (Yatime et al., 2009). H+ ATPase plays
Fax: +91 11 2698 0229. E-mail: nikhatmanzoor@yahoo.co.in a crucial role in fungal cell physiology as it maintains
 DOI: 10.3109/13880209.2014.989623
Figure 1. Chemical structures of the lead molecules present in mint
essential oil.
electrochemical proton gradient across cell membranes
necessary for nutrient uptake. It regulates intracellular pH,
cell growth, and has been implicated in the pathogenicity of
fungi through its effects on dimorphism, nutrient uptake, and
medium acidification (Manzoor et al., 2002; Set-Young et al.,
1997). The enzyme activity and H+ efflux are regulated by
some nutrients, most notably by glucose (Serrano, 1983).
Plasma membrane H+ ATPase is unique and crucial to fungal
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cells and hence is a promising antifungal target. It will help in
the development of new mechanism-based drugs.
The present work investigates the anti-Candida activity of
mint EO and its lead molecules carvone, menthol, and
menthone against five Candida strains; C. albicans (ATCC
10261, ATCC 44829, and ATCC 90028), C. tropicalis (ATCC
750), and C. glabrata (ATCC 90030). The fungicidal efficacy
of these compounds was evaluated by studying the effect of
growth and virulence of Candida. The antifungal mode of
action of mint EO and its lead molecules was further
elucidated by determining their effect on PM-ATPase-
mediated H+ efflux, ergosterol biosynthesis, and the ultra-
structure of Candida cells using scanning electron micros-
copy (SEM).
Materials and methods
Strains and media
Three Candida species were used in the present study:
C. albicans (ATCC 10261, ATCC 44829, and ATCC 90028),
C. tropicalis (ATCC 750), and C. glabrata (ATCC 90030).
All the strains were grown on the YPD medium containing
1% yeast extract, 2% peptone, and 2% dextrose (w/v)
(Hi Media, Mumbai, India). YPD agar plates containing
2.5% agar in addition were used to maintain the culture. Extra
pure grade of mint EO (ISO 9001:2008) extracted from
Mentha piperita was purchased from Mohan Perfumery Co.
(Delhi, India), and stored at 4  C. Carvone, menthol, and
menthone were purchased from SAFC (St. Louis, MO),
Aldrich (Munich, Germany) and MP Biomedicals (Santa Ana,
CA), respectively, whereas all inorganic chemicals were of
analytical grade and procured from E. Merck (Mumbai,
India). Fluconazole (FLC) was purchased from HiMedia
(Mumbai, India).
Minimum inhibitory and fungicidal concentrations
The MIC values was defined as the lowest concentration of
the oil/test molecules that caused inhibition of visible growth
of Candida cells and was determined in vitro in the
liquid medium by the broth dilution method as described
by Clinical and Laboratory Standards Institute (CLSI, 2008).
Synergy and mode of action of Mentha piperita 1497
To determine the MFC values after reading the corresponding
MIC values, 20 ml samples from all optically clear tubes
(complete growth inhibition) plus the last tube showing
growth were sub-cultured on YEPD agar plates. The plates
were incubated at 35  C for a minimum of 3 d, until growth
was clearly visible in the control samples, and MFC values
were determined as the lowest concentration of the mint
EO and its three major components for which there was no
visible growth.
Checkerboard assay (FIC index, FICI)
Checkerboard synergy testing was performed as reported
earlier (Ahmad et al., 2010a). Fractional inhibitory concen-
trations (FIC) were calculated as follows: FIC of drug
A ¼ MIC of drug A in combination/MIC of drug A alone;
FIC of drug B ¼ MIC of drug B in combination/MIC of drug
B alone. FICI was defined as the sum of FIC of drug A and
FIC of drug B and was interpreted as follows: synergy,
FICI  0.5; antagonism, FICI44.0; and indifferent,
0.55FICI54.0.
GC-MS analysis
Mint EO was analyzed by GC-MS as described previously
(Khan et al., 2010a). The chemical components of oil were
identified by comparing the retention times of the chroma-
tographic peaks with those of authentic compounds using the
WILEY8.LIB and NIST05s.LIB.
Proton extrusion measurement
The proton pumping activity of Candida cells was determined
as described previously (Ahmad et al., 2010c; Manzoor et al.,
2002). Briefly, mid-log phase cells harvested from the YPD
medium were washed twice with distilled water and routinely
100 mg cells were suspended in 5 ml solution containing
0.1 M KCl, 0.1 mM CaCl2 in distilled water. Suspension was
kept in a double-jacketed glass container with constant
stirring. The container was connected to a water circulator
at 25  C. Initial pH was adjusted to 7.0 using 0.01 M HCl/
NaOH. Test compounds were added to achieve the desired
concentrations (MIC90) in 5 ml solution. For glucose stimu-
lation experiments, 100 ml of glucose was added to achieve a
final concentration of 5 mM in total volume. H+ extrusion rate
was calculated from the volume of 0.01 N NaOH consumed.
The experiment was also performed with 5 mM vanadate
(a potent inhibitor of the H+ ATPase) and 5 mg/ml FLC used
as the standard antifungal drug.
Sterol quantification method
The total intracellular sterols were extracted as reported
earlier with slight modifications (Ahmad et al, 2011;
Arthington-Skaggs et al., 1999). Briefly, a single Candida
colony from an overnight sabouraud dextrose agar plate
culture was used to inoculate 50 ml of sabouraud dextrose
broth (HiMedia, Mumbai, India) containing 0.056, 0.112, and
0.225 mg/ml of mint EO; 0.062, 0.124, and 0.248 mg/ml of
carvone; 0.125, 0.25, and 0.5 mg/ml of menthol and 2.1, 4.2,
and 8.4 mg/ml of menthone along with a positive control
(FLC). The cultures were incubated for 16 h with shaking at
 1498 N. Samber et al. Pharm Biol, 2015; 53(10): 1496–1504

35  C. The stationary-phase cells were harvested by centrifu- for crystalline ergosterol and 24 (28) DHE, respectively.
gation at 2700 rpm (Sigma 3K30, Sigma, St. Louis, MO) for This experiment was performed on FLC-sensitive and FLC-
5 min and washed once with sterile distilled water. The net resistant strains. The values are shown in terms of the
wet weight of the cell pellet was determined. Three milliliters mean ± STD of all three respective categories.
of 25% alcoholic potassium hydroxide solution (25 g of KOH
and 35 ml of sterile distilled water, brought to 100 ml with Scanning electron microscopy
100% ethanol) was added to each pellet and vortex mixed for
Candida cell suspensions from overnight cultures were
1 min. Cell suspensions were transferred to 16  100 mm
prepared in YPD medium. Test compound, at the MIC
sterile borosilicate glass screw-cap tubes and were incubated
value, was added to the cell culture (1  106) and incubated
in an 85  C water bath for 1 h. Following incubation, tubes
for 14 h at 30  C and prepared for electron microscopy. All
were allowed to cool to room temperature. Sterols were then
Candida cells were fixed with 2% glutaraldehyde in 0.1%
extracted by the addition of a mixture of 1 ml of sterile
phosphate buffer for 1 h at 20  C (Kaneshima et al., 1977;
distilled water and 3 ml of n-heptane followed by vigorous
Mares, 1989). Cells were washed with 0.1 M phosphate buffer
vortex mixing for 3 min. The heptane layer was transferred to
(pH 7.2) and post fixed with 1% OsO4 in 0.1 M phosphate
a clean borosilicate glass screw-cap tube and stored at 20  C
buffer for 1 h at 4  C. Samples were dehydrated in acetone and
for as long as 24 h. Prior to analysis, a 20 ml aliquot of sterol
dropped on round glass cover slip with HMDS and dried at
extract was diluted five-fold in 100% ethanol and scanned
room temperature followed by sputter coating with gold and
between 240 nm and 300 nm using Systronics UV–Vis 117
observed under the electron microscope (Zeiss EV040, Carl
Downloaded by [Universitas Diponegoro] at 20:52 19 November 2017

spectrophotometer (Systronics 117, Mumbai, India). The

presence of ergosterol and the late sterol intermediate 24
(28) DHE in the extracted sample resulted in a characteristic
four-peaked curve (Figure 2). A dose-dependent decrease in
the height of the absorbance peaks was evident and corres-
ponded to decreased ergosterol concentration. The absence of
detectable ergosterol in extracts was indicated by a flat line.
The ergosterol content was calculated as percentage of wet
weight of the cell by the following equations:
½ðA281:5 =290Þ  F 
% ergosterol þ %24 ð28Þ DHE ¼
pellet weight
½ðA230 =518Þ  F 
% 24 ð28Þ DHE ¼ , and
pellet weight
% ergosterol ¼ ½%ergosterol þ % 24 ð28Þ DHE
 % 24 ð28Þ DHE,
where F is the factor for dilution in ethanol and 290 and 518
are the E values (in percentages per centimeter) determined
Zeiss Meditec Inc., Dublin, CA).
Hemolytic activity
Hemolytic activity of mint EO and its lead compounds on
human red blood cells was performed in vitro as described
earlier (Ahmad et al., 2011). Human erythrocytes from
healthy individuals were collected in tubes containing EDTA
as an anti-coagulant. The erythrocytes were harvested by
centrifugation for 10 min at 2000 rpm at 20  C, and washed
three times in PBS. The pellet was suspended in PBS to yield
10% (v/v) erythrocyte/PBS suspension. The 10% suspension
was then diluted 1:10 in PBS. From each suspension, 100 ml
was added in triplicate to 100 ml of a different dilution series
of test compounds (and FLC) in the same buffer in eppendorf
tubes. Total hemolysis was achieved with 1% Triton X-100.
The tubes were incubated for 1 h at 37  C and then centrifuged
for 10 min at 2000 rpm at 20  C. From the supernatant fluid,
150 ml was transferred to a flat-bottomed microtiter plate

Figure 2. UV spectrophotometric sterol pro-

files of representative fluconazole-suscep-
tible (A) and fluconazole-resistant (B)
Candida strains. Strains were grown for 16 h
in Sabouraud dextrose broth containing
MIC/4, MIC/2, and MIC values of mint EO
(curves c, d, and e); carvone (curves f, g, and
h); menthol (curves i, j, and k), and menthone
(curves l, m, and n), respectively. Curve ‘a’ is
control (untreated cells) while curve ‘b’
shows positive control (FLC). Sterols were
extracted from the cells and spectral profiles
between 240 and 300 nm were determined.
 DOI: 10.3109/13880209.2014.989623 Synergy and mode of action of Mentha piperita 1499

(BIO-RAD, iMark, Hercules, CA), and the absorbance was Table 1. GC-MS analysis of mint essential oil.
measured spectrophotometrically at 450 nm. Hemolysis per-
Retention time Area% Compound
centage was calculated by following equation:
7.107 0.15 alpha-Pinene
ðA450 of test compound treated sample 8.760 0.14 beta-Pinene
A450 of buffer treated sampleÞ 9.001 0.08 Sabinene
% hemolysis ¼ 9.905 0.10 beta-Myrcene
ðA450 of 1% Triton X  100  treated samples
11.096 3.32 Limonene
A450 of buffer treated sampleÞ 11.407 5.16 Eucalyptol
13.314 0.06 p-Cymene
 100
17.398 0.13 3-Octanol
20.697 9.10 Menthone
20.800 0.05 1-Octanol acetate
Results 21.316 0.32 Cyclohexanol
21.750 5.32 Isomenthone
Antifungal activity of mint EO 23.452 0.05 Linalool
The test compounds were found to be active against all the 23.843 0.05 1-Octanol
24.331 1.64 Menthyl acetate
strains tested. The MIC value was defined as the concentra- 24.641 0.86 Isopulegol
tion required to reduce 90% cell growth. MIC and MFC 24.852 0.06 cis-Myrtanol
values of mint EO against all the Candida strains used in the 25.254 0.16 Terpineol 5delta-4
present study were 225 mg/ml. The MIC of carvone was 25.578 5.44 Neoisomenthol
Downloaded by [Universitas Diponegoro] at 20:52 19 November 2017

25.837 1.05 Caryophyllene

248 mg/ml for all the strains except for C. glabrata ATCC 26.094 0.39 neoiso (ISO)-Pulegol
90030 (497 mg/ml) while the MFC value was 2 MIC. Menthol 26.757 0.68 (+)-Isomenthol
gave an MIC value of 500 mg/ml and an MFC value of 27.422 34.82 Menthol
27.706 0.47 Pulegone
1000 mg/ml (2 MIC). Menthone was the weaker antifungal
28.226 0.68 beta-Terpineol
giving an MIC of 4.2–8.4 mg/ml and an MFC double that of 28.350 0.05 Isocarveol 5trans-4
its MIC. Mint EO and carvone were the most effective with 28.521 0.11 Geraniol
low MIC and MFC values. No systematic differences were 29.361 0.16 Terpineol 5alpha-4
30.738 0.19 Piperitone
observed between FLC sensitive and resistant strains. 31.131 19.54 Carvone
34.365 9.54 Anethole 5(E)-
GC-MS analysis 39.747 0.05 Caryophyllene oxide
41.206 0.07 Aubepine
Mint EO was characterized by a detailed GC-MS analysis and
the results are shown in Table 1. Thirty-three compounds
Table 2. Effect of mint essential oil and its lead compounds on the rate
were identified representing 100% oil. The oil was character- of H+-efflux by Candida cells at pH 7.0: Cells were suspended in
ized by high amounts of menthol (34.82%), carvone (19.54%), 0.1 mM CaCl2 and 0.1 M KCl at 25  C.
and menthone (9.10%) as the main components.
Range of relative H+-efflux rate
(1011 mol/min/mg cells)
Proton extrusion measurements
Incubation with Standard Resistant
The plasma membrane H+ ATPase regulates intracellular pH
Control 1a 1b
(Stewart et al., 1988), maintains ion balance, and generates Mint EO 0.53 ± 0.012 (47) 0.47 ± 0.015 (52)
the electrochemical proton gradient crucial for nutrient uptake Carvone 0.58 ± 0.017 (42) 0.52 ± 0.005 (48)
and morphogenesis in fungal cells. The inhibition of its Menthol 0.64 ± 0.021 (35) 0.64 ± 0.03 (36)
activity correlates with the cessation of cell growth. Hence Menthone 0.70 ± 0.035 (29) 0.69 ± 0.015 (30)
Glucose (5 mm) 22.48 ± 2.34 12.04 ± 0.85
PM-ATPase-mediated H+ efflux in C. albicans was deter- Glucose + mint EO 2.31 ± 0.106 (44) 0.98 ± 0.85 (38)
mined in the presence of mint EO and its lead molecules. Glucose + carvone 2.67 ± 0.118 (35) 1.03 ± 0.08 (35.5)
Table 2 gives relative rates of H+ efflux by Candida cells in Glucose + menthol 2.77 ± 0.106 (34) 1.08 ± 0.05 (32)
the presence of MIC of mint EO, carvone, menthol, and Glucose + menthone 3.12 ± 0.296 (24) 1.22 ± 0.01 (23)
Vanadate (5 mM) 0 ± 0 (100) 0 ± 0 (100)
menthone. The effect of FLC (5 mg/ml) on H+ efflux was also FLC (5 mg/ml) 0.76 ± 0.07 (24) 0.755 ± 0.94 (25)
studied. The inhibitory effect of mint EO on the rate of H+
efflux was determined in nmoles/min/mg of Candida cells. Values in parentheses are percentage inhibition with respect to corres-
ponding control (without and with 5 mM glucose).
The control (cells only) showed H+ efflux rate of 5.1– a
5.39 ± 0.25 nmoles/min/mg of standard Candida cells.
5.5 nmoles/min/mg yeast cells. Mint EO, carvone, menthol, b
7.51 ± 0.13 nmoles/min/mg of resistant Candida cells.
and menthone showed about 47, 42, 35, and 29% inhibition,
respectively. In the case of FLC-resistant strains, the decrease
in efflux was by 52, 48, 36, and 30%, respectively, when resistant Candida strains was 44 and 38%, respectively, with
treated with the same compounds, a trend similar in the respect to control at pH 7.0. The values were 35 and 35.5% in
susceptible cases. In the presence of 5 mM glucose, H+ efflux the presence of carvone; 34 and 32% in the presence of
was enhanced by 4.2-fold in susceptible cells and two-fold in menthol, and 24 and 23% in the presence of menthone,
resistant cells. This is in conformity with previous observa- respectively. The above inhibition studies were done at
tions (Manzoor et al., 2002; Serrano, 1983). Inhibition of respective MIC values of mint EO and its main constituents.
glucose induced H+ efflux by mint EO in standard and Experiments were also performed at ½ MIC values but data
 1500 N. Samber et al. Pharm Biol, 2015; 53(10): 1496–1504

are not shown as the results were similar to that of the control.
Also, H+ extrusion in every case was inhibited by 100%
following the addition of 5 mM orthovandate (positive
control), a specific inhibitor of H+-ATPase, suggesting it to
be the major agency responsible for H+ extrusion whereas
FLC did not have a significant effect on the acidification of
the extracellular medium.
Sterol quantification method
Ergosterol is specific to fungi and is the major sterol
component of the fungal cell membranes, being responsible
for maintaining cell function and integrity. The primary
mechanism of action by which azole antifungal drugs inhibit
fungal cell growth is the disruption of normal sterol
biosynthetic pathways, leading to a reduction in ergosterol
biosynthesis. Figure 2(A) and (B) shows UV spectrophoto-
metric sterol profiles of standard and FLC-resistant Candida
strains when grown in SD broth for 16 h in the presence of
Downloaded by [Universitas Diponegoro] at 20:52 19 November 2017
the decrease in the ergosterol content varied. At half MIC
values, mint EO showed the maximum decrease by 59% in
sensitive and 42% in resistant strains, carvone was next with
46% in sensitive, and 32% in resistant strains. Menthol and
menthone showed a decline in the ergosterol content by only
15–25%. At even lower concentration (MIC/4), mint EO
showed a decline in ergosterol content by 46% in sensitive
and 38% in resistant while carvone showed a decline by 30%
in sensitive and 22% in resistant Candida strains. The
decrease was not significant in case of menthol and menthone
at MIC/4, the values being less than 17%.
Combination studies of FLC with mint EO
Azoles have excellent efficacy–toxicity profiles and the most
widely used azole in both the treatment and the prevention
of candidiasis is FLC (Vanden Bossche, 1985). However,
treatment failures are increasing, especially in AIDS patients,

MIC and sub-MIC values of mint EO, carvone, menthol, and
menthone. Their effect on ergosterol biosynthesis in both
FLC-sensitive and resistant Candida strains is summarized in
Table 3. A dose-dependent decrease in ergosterol synthesis
was observed when the yeast cells were grown in the presence
of test compounds. The total ergosterol content in Candida
cells grown with mint EO and its bioactive lead molecules
at their respective MIC values showed a 100% decrease in all
the strains used in the present study. Cells grown in
FLC (8 mg/ml) also showed 100% decrease in susceptible
cells which inhibit ergosterol synthesis by interacting with
lanosterol 14a-demethylase. As expected in the case of FLC-
resistant strains, the inhibition observed was only 16% when
cells were exposed to 64 mg/ml FLC (MIC of resistant
strains). It was encouraging to see that mint EO and its lead
molecules inhibited the ergosterol content in resistant strains
to the same extent. At sub-MIC values of the test compounds,
as prolonged use of this drug has led to resistance in Candida
spp. probably due to its fungistatic rather than fungicidal
nature of antifungal action (Sanglard et al., 2003; Uppuluri
et al., 2008). New therapeutic strategies are required to reduce
the toxicity of these drugs.
The perspective on antifungal drug resistance and other
problems suggest several possible ways of overcoming, or
preventing, drug resistance. The efficacy of FLC and other
antifungal agents can be improved by using combination
therapy (Ghannoum et al., 1995; Mukherjee et al., 2005; Pinto
et al., 2009; Tariq et al., 1995). Combinational therapy can be
used to attack more than one target simultaneously with a low
probability so that resistance to both drugs will arise, or to
attack a drug target and its resistance mechanism. Synergistic
antifungal activity of carvone and essential oils rich in
carvone, with azoles, is well described against Candida
(Dudhatra et al., 2012).

Table 3. Reduction in ergosterol content by mint essential oil, carvone, menthol and menthone in Candida cells.

Standard strains Resistant strains

Control
0 0.0128 ± 0.0004 0.0101 ± 0.0005
Mean ergosterol contenta of cells grown with mint EO
MIC/4 0.007 ± 0.0005 (46)b 0.0062 ± 0.0008 (38)b
MIC/2 0.0053 ± 0.0004 (59)b 0.0058 ± 0.0009 (42)b
MIC 0 ± 0 (100)b 0 ± 0 (100)b
Mean ergosterol contenta of cells grown with carvone
MIC/4 0.009 ± 0.0034 (30)b 0.0078 ± 0.0007 (22)b
MIC/2 0.007 ± 0.0008 (46)b 0.0069 ± 0.0017 (32)b
MIC 0 ± 0 (100)b 0 ± 0 (100)b
Mean ergosterol contenta of cells grown with menthol
MIC/4 0.0108 ± 0.0003 (17)b 0.0091 ± 0.0015 (10)b
MIC/2 0.0095 ± 0.0008 (26)b 0.0081 ± 0.0011 (19)b
MIC 0 ± 0 (100)b 0 ± 0 (100)b
Mean ergosterol contenta of cells grown with menthone
MIC/4 0.0112 ± 0.0007 (13)b 0.0092 ± 0.0013 (08)b
MIC/2 0.0100 ± 0.001 (22)b 0.0086 ± 0.001 (14)b
MIC 0 ± 0 (100)b 0 ± 0 (100)b
Mean ergosterol contenta of cells grown with FLCc 0 ± 0 (100)b 0.0084 ± 0.0009 (16)b
a
Expressed as percentage wet weight of cell ± standard deviation of the mean (followed in parentheses by the
percent reduction in mean cellular ergosterol content compared with that of control cells grown without test
entity).
b
Significant reduction compared with controls (p50.05) after Student’s t test.
c
8 mg/ml in susceptible cells and 64 mg/ml in resistant cells.
 DOI: 10.3109/13880209.2014.989623 Synergy and mode of action of Mentha piperita 1501

SYN
SYN
SYN
SYN
SYN

SYN
SYN
SYN
SYN
SYN
INT
To assess the interaction of drug combinations (mint EO
plus FLC, carvone plus FLC, menthol plus FLC, and
menthone plus FLC), the MIC data were further analyzed

Menthone + FLC
using the fractional inhibitory concentration index (FICI)
(Berenbaum, 1989). The FICI was calculated by the formula,

0.47

0.41
0.42

0.47
0.49
0.47
0.49
0.47
0.5
0.5
MICA in combination MICB in combination
FICI ¼ þ
MICA tested alone MICB tested alone

SYN
SYN
SYN
SYN
SYN

SYN
SYN
SYN
SYN
SYN
INT
where MICA and MICB are the MICs of drugs A and B,

Table 4. Synergistic antifungal activity (MIC and FICI) of mint EO and its major compounds against fluconazole-sensitive standard strains and fluconazole-resistant strains.
respectively. The interaction was defined as synergistic if the
FICI was 0.5, indifferent if the FICI was 40.5 and 4, and

Menthol + FLC
antagostic if the FICI was 44.0 (Cantón et al., 2005).
MIC value of FLC against the standard susceptible and

0.38
0.37
0.36
0.41

0.29
0.49
0.26
0.36
0.42
0.4
resistant strains ranged from 7.5 to 8.0 and 64 to 80 mg/ml,
respectively. FICI of mint EO in combination with FLC

FICI
against all FLC sensitive and FLC-resistant Candida strains
studied, calculated from the checkerboard microtiter assay

SYN
SYN
SYN
SYN
SYN

SYN
SYN
SYN
SYN
SYN
INT
ranged from 0.28 to 0.41 and 0.26 to 0.35, respectively,
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indicating significant synergism (Table 4). Carvone in

Carvone + FLC
combination with FLC gave an FICI of 0.28–0.42 in
susceptible and 0.38–0.48 in resistant Candida strains. FICI

0.33
0.42
0.28
0.36
0.38

0.38
0.39
0.48
0.44
0.38
of menthol with FLC was 0.36–0.41 and 0.26–0.49, respect-
ively, in susceptible and resistant cells. Menthone did not
show significant synergism with FLC. Almost all FICI values
were showing indifferent interaction.

SYN
SYN
SYN
SYN
SYN

SYN
SYN
SYN
SYN
SYN
INT
Scanning electron microscopy
Alterations in the morphology of Candida cells in the Mint EO + FLC
presence of test compounds were studied using scanning

INT, interpretation; IND, indifference; SYN, synergism; FICI, fractional inhibitory concentration Index.
0.38
0.28
0.41

0.36

0.30
0.34
0.26
0.34
0.35
0.3
electron microscopy (SEM). Figure 3 shows SEM images of
C. albicans ATCC 90028 cells when exposed to test
compounds for 14 h at their respective MIC values.
Figure 3(A) shows the SEM micrographs of untreated cells
Menthone

having well-defined shape and normal smooth surfaces. Cells

8400
4200
4200
8400
4200

8400
4200
4200
8400
4200
treated with mint EO and its lead compound carvone at their
respective MIC values for 14 h showed extensive cell damage

MIC and FICI values are shown as a mean of three independent experiments.
and leakage of the intracellular content in large quantities
Menthol

(Figure 3B and C). Images show changes in the cell shape due
500
500
500
500
500

500
500
500
500
to the formation of deep wrinkles in both the cases. 500
MIC (mg/ml)

Hemolytic activity
Carvone

248
248
248
248
497

250
250
250
250
500

The effects of mint EO and its lead molecules were studied

on human red blood cells and are shown in Figure 4. At
2000 mg/ml, mint EO, carvone, menthol, and menthone
Mint EO

showed 6, 19, 27, and 42% hemolysis, respectively, while

225
225
225
225
225

225
225
225
230
225

FLC, a conventional antifungal therapeutic agent,

showed 85% hemolysis at the same concentration. Hence
hemolytic activity was in the following order:
FLC

7.5
8
8
8

64
80
64
70
64

FLC4menthone4menthol4carvone4mint EO. All the

compounds tested here showed less hemolysis in comparison
C. albicans ATCC 90028
C. albicans ATCC 10261
C. albicans ATCC 44829

C. glabrata ATCC 90030

C. albicans ATCC 90028

C. albicans ATCC 10261
C. albicans ATCC 44829

C. glabrata ATCC 90030

with a conventional drug even at very high concentrations.

C. tropicalis ATCC 750

C. tropicalis ATCC 750

Discussion
Although there has been a surge of improved antifungal drugs
FLC sensitive

FLC resistant

for the treatment of fungal infections, not many drugs are

effective against invasive fungal infections, and hence remain
Strains

a significant clinical problem. Infections caused by eukaryotic

organisms like yeasts are generally more problematic than
 1502 N. Samber et al. Pharm Biol, 2015; 53(10): 1496–1504
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Figure 3. Scanning electron micrographs of C. albicans ATCC 90028 untreated control cells (A) treated with mint EO (B) and treated with carvone
(C). Black arrow indicates damage and wrinkling of cell surface, white arrow indicates leakage of the cellular content.

100 Fluconazole diverse biological activities. Plants and plant products

Mint EO significantly contribute to antimicrobial drug discovery
80
Carvone (Khan et al., 2010a,b). Our results suggest that plant active
principles can be developed into potential antifungal agents.
Cell lysis(%)

Menthol
60
Menthone
40
20
0 essential oils and their components have been studied
5 10 50 100 250 500 1000
Concentrations of test agents (µg ml−1)
Figure 4. Hemolysis caused by mint EO, carvone, menthol, menthone,
and fluconazole was determined by recording an absorbance at 450 nm
and compared with hemolysis achieved with 1% Triton X-100 (reference
for 100% hemolysis). The data shown are the mean of triplicate
experiments.
bacterial infections. There are only a few antifungal agents
that can identify targets unique to the fungus and not shared
with human host. The fact that most available antifungal
drugs are fungistatic leads to the emergence of intrinsically
resistant fungal species. Moreover, high treatment costs
and associated host cytotoxicity justify the search for new
strategies to combat fungal infections. Natural products
provide an unparalleled source of chemical scaffolds with
As reported earlier (Burt, 2004; Palmeira-de-Oliveira
et al., 2009), majority of the essential oils act by disrupting
microbial cytoplasmic membranes. In the present study, we
also show membrane damage as a mode of action of mint EO
and its major constituents. The antimicrobial activity of
2000 extensively (Cristani et al., 2007; Lucchini et al., 1990;
Xu et al., 2008), but not much is known about their
mechanism of action. This work is an attempt to assess the
antifungal role of mint EO, carvone, menthol, and menthone
by studying their effect on structural and functional aspects of
membrane integrity in terms of ergosterol content and PM-H+
ATPase activity.
PM-H+ ATPase plays a crucial role in fungal cell
physiology and maintains electrochemical proton gradient
across the cell membrane necessary for nutrient uptake.
It regulates pHi, changes which are widely regarded as of
crucial importance to cell physiology and morphogenesis
(Kaur et al., 1988; Manzoor et al., 2002). We explored
the effect of mint EO and its major constituents on
glucose-induced H+ efflux which is an estimate of PM-H+
ATPase activity. Inhibition of enzyme activity correlates well
 DOI: 10.3109/13880209.2014.989623
with the cessation of cell growth and this observation is in
accordance with previous studies as well. Azoles did not
affect medium acidification by yeast cells even at concentra-
tions higher than its MIC value (Ben Josef et al., 2000;
Manavathu et al., 1999) while mint EO, carvone, menthol,
and menthone could do so at MIC values. It may be
conjectured that the test compounds are possibly interactive
with the enzyme directly, serving as the primary reason for
their antifungal activity. Our studies also suggest the presence
of cellular target that is accessible to the compounds
externally. Thus, it would be useful to further investigate
the interaction of the test compounds with purified
PM-ATPase enzyme. Existing classes of antifungals have
low efficacy, high toxicity, and frequently lead to drug
resistance. There is thus a critical need to develop more
effective therapies to deal with such infections and natural
essential oils like mint EO and its active components offer a
safer alternative. Our results suggest that PM H+-ATPase can
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be explored as a potential surface active antifungal target for
these and other potential drugs.
Although natural antifungal compounds act by disrupting
cytoplasmic membranes of fungi (Ahmad et al., 2011),
specific mechanisms involved in their mode of action remain
poorly characterized. It was interesting to observe that cell
leakage induced by mint EO and its main constituents was far
greater than that induced by FLC which is an established
membrane-targeting antifungal drug. Ergosterol is a unique
membrane sterol of fungi. The selective cytotoxic behavior
and affinity of test compounds for this particular target hints
towards their interference with ergosterol biosynthesis path-
way. Analysis of ergosterols obtained from FLC-sensitive and
resistant Candida strains showed no major difference in the
sterol content or pattern in comparison with the untreated
control. Growth of Candida in the presence of sub-inhibitory
concentrations of the test compounds altered the sterol
patterns of the FLC-sensitive and resistant strains in a similar
manner. These plant active principles completely blocked
ergosterol biosynthesis at MIC values. Studying the effect of
these compounds at various sub-inhibitory concentrations on
the sterols of the FLC-resistant Candida strains showed that
they act in a dose-dependent fashion in decreasing the
ergosterol content (Figure 2 and Table 3). In the present study,
very significant inhibition of ergosterol biosynthesis was
observed even at the sub-MIC concentration of mint EO and
its lead constituents. The effect on membrane integrity, thus,
appears to originate from ergosterol biosynthesis inhibition in
a manner similar to that of FLC. The antifungal effects were
attributed to their ability to inhibit ergosterol biosynthesis and
to make the cytoplasmic membrane porous in both sensitive
and resistant strains. SEM analysis performed in the present
study on a representative Candida strain ATCC 90028 cells
treated with compounds for 14 h clearly demonstrates
damaging effect of mint EO and its active compound carvone
on membrane and cell wall structure.
Conclusion
This work suggests that naturally occurring plant active
principles – mint EO, carvone, menthol, and menthone, could
be developed into promising antifungal drugs and also
Synergy and mode of action of Mentha piperita 1503
advocates the determination of their optimal concentrations
for clinical application, as an alternative to FLC therapy for
the treatment and prevention of candidiasis. Mint EO and its
major constituents are fungicidal bioactive compounds that
have a profound effect on PM-ATPase of Candida species
making this membrane protein a very good potential
antifungal target. These compounds penetrate the cell mem-
brane and target ergosterol biosynthesis pathway, thus,
impairing its biosynthesis. Simultaneously they react with
the membrane itself with their reactive hydroxyl moiety, and
the extensive lesion on membrane is a combined effect of the
two events. Although the hypothesis requires identification/
screening of the target enzyme(s) of the pathway, involvement
of hydroxyl group in the formation of hydrogen bonds and the
acidity of these phenolic compounds may provide possible
explanations. The excellence of these compounds demand
more insight studies into all possible mechanisms of these
compounds.
Declaration of interest
The authors report that they have no conflicts of interest. This
work was supported by Indian Council of Medical Research,
India (59/24/2008/BMS/TRM [2008-04780]).
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