Fitoterapia 81 (2010) 1157–1162

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Proton translocating ATPase mediated fungicidal activity ofeugenol and thymol

Aijaz Ahmad 1, Amber Khan 1, Snowber Yousuf, Luqman A. Khan, Nikhat Manzoor⁎Department of Biosciences, Jamia Millia Islamia, New Delhi-110025, India.

a r t i c l e i n f o

⁎ Corresponding author. Department of BiosciencesNew Delhi, India.

1 These authors contributed equally to this work.

0367-326X/$ – see front matter © 2010 Elsevier B.V.doi:10.1016/j.fitote.2010.07.020

a b s t r a c t

Article history:Received 13 March 2010Accepted in revised form 20 July 2010Available online 24 July 2010

Eugenol (1) and thymol (2) exhibit excellent fungicidal activity against pathogenic yeasts,including isolates resistant to azoles. The rapid irreversible action of compound-1 andcompound 2 on fungal cells suggested a membrane-located target for their action. Weinvestigated their effect on H+-ATPase mediated H+-pumping by various Candida species. Bothcompounds inhibit H+-ATPase activity at their respective MIC values — 500 and 100 μg/ml.Glucose stimulated H+-extrusion was also inhibited significantly by compound 1 andcompound 2. Inhibition of H+-ATPase leads to intracellular acidification and cell death.Inhibition of cell growth and H+-efflux by test compounds suggests that their antifungalproperties are related to their inhibitory effects on H+-ATPase.

© 2010 Elsevier B.V. All rights reserved.

Keywords:EugenolThymolPlasma membrane H+-ATPaseIntracellular pHCandida

1. Introduction

Opportunistic infections caused by Candida species andother pathogenic fungi are widespread in immunocompro-mised patients [1–4]. These commensals can cause bothsuperficial and severe invasive systemic disease. Riskfactors that increase incidence of Candida infections includecompromised immunity, hormonal imbalances, prolongeduse of broad spectrum antibiotics and oral contraceptives,pregnancy, metabolic and nutritional disorders [5,6].Candida albicans is the predominant cause of virtually alltypes of Candidiasis [7]; but other Candida species,including C. glabrata, C. krusei, C. tropicalis, C. parapsilosisare equally important [8,9]. Existing classes of systemicantimycotic agents have low efficacy, high toxicity andfrequently lead to drug resistance. Treatment options forinvasive infections are limited and almost always involvethe use of nephrotoxic amphotericin B and azoles, basicallyfluconazole, which leads to resistance on prolong use [10].There is thus a critical need to develop more effective

, Jamia Millia Islamia,

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therapies to deal with such infections and natural productsoffer a safer alternative.

The antifungal effect of thyme and clove essential oils hasbeen described in several studies [11,12]. Eugenol (1) andThymol (2) (Fig. 1), the major phenolic components of cloveand thyme essential oils have potent antimicrobial andantioxidant properties [13,14]. Both the compounds wereshown to possess very low cytotoxicity against humanerythrocytes [13]. There are number of studies attributingthe antimicrobial effects of these and other essential oilcomponents to their interaction with cell membranes [15,16].Eugenol (1) profoundly affects cellular ATP concentrations,glucose uptake or glucose utilization against gram positiveand gram negative bacteria [17].

The fungal plasma membrane H+ ATPase is a promisingnew antifungal target [18,19]. It is a predominant membraneprotein that belongs to P-type ATPase family of ion translo-cating ATPases. This enzyme plays a crucial role in fungal cellphysiology as it maintains electrochemical proton gradientacross cell membrane necessary for nutrient uptake. Itregulates intracellular pH, cell growth and has been impli-cated in the pathogenicity of fungi through its effects ondimorphism, nutrient uptake and medium acidification[18,20]. The enzyme activity and H+ efflux is regulated bysome nutrients, most notably by glucose [21].

Fig. 1. Structures of compound 1 and compound 2.

Table 1Isolates used in the study.

Classification of isolates Type of isolate Source

Susceptible (standard n=5)ATCC 44829 C. albicans JNUATCC 10261 C. albicans JNUATCC 90028 C. albicans SHATCC 750 C. tropicalis IIIMATCC 90030 C. glabrata IIIM

Susceptible (clinical n=35)HIV patients (n=9) C. krusei, C. albicans,

C. glabrataSH

Cutaneous (n=5) C. tropicalis, C. krusei SHENT, burn patients(n=10) C. glabrata, C. tropicalis SHCandidemia (n=11) C. krusei, C. tropicalis SH

Resistant (clinical n=19)Hospital acquired (postoperated)(n=8)

C. tropicalis, C.glabrata

Fluconazoleresistant

SH

HIV patients (n=6) C. krusei, C.glabrata

Fluconazoleresistant

SH

ENT, burn patients (n=5) C. albicans, C.parapsilosis

Fluconazoleresistant

SH

JNU: Jawahar Lal Nehru University, India; IIIM: Indian Institute of IntegrativeMedicine, India; SH: Safdarjung Hospital India.Organisms are classified as susceptible if the fluconazole MIC is ≤8 μg/mland as resistant if it is ≥64 μg/ml.

1158 A. Ahmad et al. / Fitoterapia 81 (2010) 1157–1162

The P-type ATPase family of ion pumps includes the Na+

K+-ATPase and the H+ K+-ATPase, which are moleculartargets for several clinically important therapeutics [22].Earlier studies have reported that the mechanism ofbactericidal action of plant oil aromatics involve inhibitionof ATP generation and membrane disruption [17,23]. Manynatural products have shown binding affinity to otherrelated ATPases [24–26]. There are only a few antifungaldrugs available largely due to the eukaryotic nature offungal cells and hence the difficulty in identifying uniqueantifungal targets not shared with human hosts. Plasmamembrane H+ ATPase is unique and crucial to fungal cellsand hence is a promising antifungal target. It will help inthe development of new mechanism based drugs andnatural compounds serve this purpose.

The objective of this study was to further elucidate theantimicrobial mechanism of action of compound 1 andcompound 2 by determining their effect on the activity ofH+ATPase located in the membranes of pathogenic Candidaspecies. We have evaluated H+ ATPase activity and corre-lated it with the growth and intracellular pH (pHi) of Candidain presence of these two natural phenols.

2. Materials and methods

2.1. Strains and media

The isolates used in this study are listed and classified inTable 1. The strainswere grown in YPDmedia containing (w/v)1% Yeast Extract, 2% Peptone and 2% Dextrose and main-tained on YPD plates with 2.5% (w/v) agar. Eugenol (1),thymol (2) and vanadate were purchased from ALDRICHchemicals (Germany), whereas all inorganic chemicals wereof analytical grade and procured from E. Merck (India).Compounds 1, 2 and fluconazole were dissolved in 1% DMSOand vanadate was dissolved in water to make up the desiredconcentrations.

2.2. Minimum inhibitory concentration

The Minimum Inhibitory Concentration (MIC) was de-fined as the lowest concentration of test molecule that causesinhibition of visible growth of Candida cells. MIC90 wasdetermined in vitro in liquid medium by micro broth dilutionmethod as described by NCCLS M27-A2 [27].

2.3. Growth studies

Before the tests were performed, the Candida specieswere sub-cultured at least twice and grown for 24 h at 37 °Con SDA plates. For growth studies, 106 cells (optical density

,

A595=0.1) of Candida strains were grown aerobically in50 ml media on automated shaker at 37 °C with agitation of200 rpm. Test compounds with final concentrations of0×MIC, 0.5×MIC, and 1×MIC for each Candida isolatewere also added to the cultures along with positive andnegative controls. At a pre-determined time (after 0, 2, 4, 6,8, 12, 24 and 48 h) aliquots were removed and growth wasfollowed turbidometrically at 595 nm using Lab MedSpectrophotometer (USA). Optical density was recordedfor each concentration against time in hours. The growthrate is equivalent to the slope of log (optical density) versustime during the exponential phase. Average growth rateversus two concentrations of each inhibitor is shown as bardiagram in Fig. 2.

2.4. Disc diffusion assay

Strains were inoculated into liquid YPD medium andgrown overnight at 37 °C. The cells were then pelleted andwashed three times with distilled water. Approximately105cells/ml were inoculated in molten agar media at 40 °Cand poured into 100-mm-diameter petri plates. The testcompounds initially dissolved in 10% DMSO were furtherdiluted in distilled water to concentration ranges of 10 fold oftheir respective MICs. 4-mm-diameter sterile filter discs wereimpregnated with the test compounds. 1% DMSO (solvent)and 100 μg/ml of fluconazole were also applied on the discs toserve as negative and positive controls, respectively. Thediameter of zone of inhibition was recorded in millimetersafter 48 h and was compared with that of control. The type ofthe halo produced depicts fungicidal/static characteristic oftest entity [23]. This experiment was performed on flucona-zole sensitive [standard (n=5), clinical (n=10)], andfluconazole resistant (n=5) isolates randomly selected out

Fig. 2. Representative growth rate plots for – (A) Candida standard ATCC isolates – (absolute growth rate log ΔO.D. /t for control was 1.05), (B) Candida Flu-sensitive clinical isolates (Absolute growth rate, log ΔO.D. /t for control was 1.03), (C) Candida Flu-resistant isolates (Absolute growth rate, log ΔO.D. /t for controlwas 0.78). Isolates were grown for 48 h in YEPD broth containing 0 (a), 250 (b) or 500 (d) μg/ml of compound 1 and 50 (c) or 100 (e) μg/ml of compound 2, or 64(f) μg/ml of fluconazole. The absence of error bars indicates that the error was too small to allow display of the error in those data.

1159A. Ahmad et al. / Fitoterapia 81 (2010) 1157–1162

of total 59 isolates. Results are reported as mean±standarderror of mean (SEM) of all three respective categories.

2.5. Proton efflux measurements

The proton pumping activity of Candida species wasdetermined by monitoring acidification of external mediumby measuring the pH as described previously [28]. Briefly,mid-log phase cells harvested from YEPD medium werewashed twice with distilled water and routinely 0.1 g cellswere suspended in 5 ml solution containing 0.1 M KCl,0.1 mM CaCl2 in distilled water. Suspension was kept in adouble-jacketed glass container with constant stirring. Thecontainer was connected to a water circulator at 25 °C. InitialpH was adjusted to 7.0 using 0.01 M HCl/NaOH. 100 μl ofcompound 1 or compound 2was added to achieve the desiredconcentration of 500 μg/ml and 100 μg/ml respectively in5 ml solution. For glucose stimulation experiments, 100 μl ofglucosewas added to achieve a final concentration of 5 mM intotal volume of suspension. H+ extrusion rate was calculatedfrom the volume of 0.01 N NaOH consumed. The experimentwas also performed with 5 mM vanadate – a potent inhibitorof the H+-ATPase and 5 μg/ml each of fluconazole andamphotericin B – two commonly used antifungal drugs.

2.6. Measurement of intracellular pH (pHi)

Intracellular pH was measured as done earlier [20] withmodifications. Mid-log phase cells grown in YEPD mediumwere harvested and washed twice with distilled water. Cells(0.1 g) were suspended in 5 ml solution containing 0.1 M KCland 0.1 mM CaCl2. Desired concentrations of test compounds(their respective MIC values) were added to the suspensionand pHwas adjusted to 7.0 in each case. Following incubationfor 30 min at 37 °C with constant shaking at 200 rpm, pH wasagain adjusted to 7.0. Nystatin (20 μM) dissolved in DMSOwas added to the unbuffered cell suspension and incubated at37 °C for 1 h. The change in pH of suspensionwas followed onpH meter with constant stirring. The value of external pH atwhich nystatin permeabilization induced no further shift wastaken as an estimate of pHi [29].

3. Results

3.1. Minimum inhibitory concentration

TheMinimum Inhibitory Concentrationwas defined as thelowest concentration of compound 1 or compound 2 thatcauses 90% decrease in absorbance (MIC90) compared withthat of the control (no test compound). The MIC90 values ofcompound 1 and compound 2 against both fluconazole-susceptible as well as fluconazole-resistant Candida isolateswere 450–500 μg/ml and 100–125 μg/ml, respectively.

3.2. Growth studies

Fig. 2(A), (B) and (C) show relative growth rates ofstandard, clinical and resistant Candida isolates respectively,in the presence of compound 1 and compound 2 at MIC andsub-MIC concentrations. Growth rates have been tabulated aslog ΔOD/t for the exponential growth phase. All the Candidaisolates were found to be susceptible to the test compounds.At sub-MIC values of test compounds, all the isolates showedmore than 50% growth with respect to control; whereas morethan 90% inhibition was observed at MIC values. On molarbasis compound 2 exerted greater inhibitions compared tocompound 1.

3.3. Disc diffusion assay

The results summarized in Table 2 give the sensitivityassay, using standard discs of eugenol (1), thymol (2) andfluconazole. All the three types of Candida isolates showedhigh degree of sensitivity as is evident from the zone ofclearance in each case. Index of sensitivity defined as

Σ Zone diameter mmð Þ= concentration mg =mlð Þ = clearing mmð Þ=mg

is greater (3.27) for standard Candida isolates when treatedwith thymol and is least (1.41) for resistant isolates whentreated with compound 1. The discs impregnated with DMSO(negative control) showed no zone of inhibition and hence 1%DMSO had no effect on the strains tested in the present study.

Table 2In vitro sensitivity of compound 1 and compound 2 against Candida isolatesas determined by disc diffusion assay.

Candida isolates Sensitivity index (mm/mg)

Eugenol Thymol Fluconazole

Standard (n=5) 1.61±0.54 3.27±0.88 2.77±0.21Clinical (n=10) 1.54±0.43 2.28±0.14 2.48±0.29Resistant (n=5) 1.41±0.40 2.22±0.22 0.16±0.13

Sensitivity index is expressed as mean±SD and was calculated as diameterof zone of inhibition (mm)/concentration of drug (mg/ml). n is number oisolates. Each isolate was tested in duplicate.

Table 3Effect of compound 1 and compound 2 on the rate of H+-efflux by variousCandida isolates at pH 7.0: Cells were suspended in 0.1 mM CaCl2 and 0.1 MKCl at 25 °C.

Incubationwith

Range of relative H+-efflux rate(×10−11 mol min−1 mg cells1)

Standard Clinical Resistant

Control 1 1* 1**Eugenol(500 μg/ml)

1.95±0.27 (64) 2.6±0.42 (50) 4.69±0.59 (46)

Thymol(100 μg/ml)

1.53±0.44(72) 1.93±0.52 (63) 3.93±0.7 (54)

Glucose(5 mM)

27.37±1.1 27.8±1.154 31.5±1.3

Glucose+eugenol

15.35±1(44) 15.07±1.57 (46) 19.2±2.94 (39)

Glucose+thymol

11.02±1.3 (60) 12.75±1.4 (54) 16.98±2.7 (46)

Vanadate(5 mM)

0±0 (100) 0.43±0.45 (91) 0.3±0.1 (94)

Fluconazole(5 μg/ml)

4.15±0.08 (24) 4.2±0.14 (19) 6.47±0.93 (25)

Amphotericin B(5 μg/ml)

4.6±0.14 (16) 4.35±0.07 (16) 7.17±0.89 (17)

Control had an average (of 4 independent recordings) H+ efflux rate o5.44±1.18 nmol/min/mg cells in standard isolates (1); 5.18±0.41 nmolmin/mg yeast cells in clinical isolates (1*) and 8.567±1.04 nmol/min/mgyeast cells in resistant isolates (1**). Values in parentheses give the %-ageinhibition of H+-efflux w.r.t. control.

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3.4. Proton efflux measurements

Candida cells susceptible to the test compounds wereexamined for the ability to pump intracellular protons to theexternal medium (as measured by the alteration of pH of theexternal medium) in the presence of compound 1 andcompound 2. Table 3 gives relative rates of H+-efflux byCandida sp. in presence of eugenol (1) (500 μg/ml), thymol(2) (100 μg/ml), vanadate (5 mM), fluconazole (5 μg/ml) andAmphotericin B (5 μg/ml). Retention of H+-efflux in standardisolates was 36% and 28% when treated with compound 1 andcompound 2, respectively. H+-efflux rate was also decreasedto 50% and 37% respectively for compound 1 and compound 2in case of clinical isolates. In case of resistant isolates per-centage efflux decreased to 54% and 36% when cells weretreated with compound 1 and compound 2 respectively.Glucose (5 mM) stimulated H+-efflux in all the strains by 4–5folds. Glucose stimulated H+-efflux was also inhibited bycompound 1 and compound 2. Glucose-stimulated H+-effluxrates in standard, clinical and resistant isolateswith respect to

Fig. 3. Intracellular pH in presence of test compounds in Candida sp. Midlogarithmic cells were incubated with MIC of compound 1 and compound 2Remarkable decline in pH as shown in figure is indicative of induced acidity

f/

control, were 56%, 54% and 61% in the presence of compound1 and 40%, 46% and 54% in presence of compound 2.Experiments were also performed at 1/2×MIC's (not shownbecause the results were almost similar to those of control).In addition, the effect of vanadate, fluconazole and ampho-tericin B were also analyzed on the proton pumping activity.H+-extrusion in every case was inhibited by 91–100%following addition of 5 mM vanadate-a well known inhibitorof H+-ATPase, whereas neither fluconazole nor AmphotericinB had any significant effect on the acidification of the externalmedium.

3.5. Measurement of intracellular pH

The internal pH of yeast cells is maintained between 6.0and 7.5 by plasmamembrane H+-ATPase activity. We tried toinvestigate whether cells with normal H+-ATPase activitymaintains the constant internal pH as compared to cells withlowH+-ATPase activity. Fig. 3 shows changes in the pattern ofpHi with control and treated cells. Only yeast control cellswith normal H+ -ATPase activity maintain the pHi (6.5),while the treated cells show increase in internal acidification.The decrease in pHi wasmore in cells exposed to compound 2than compound 1.

4. Discussion

The antimicrobial and antifungal effect of aromatic plantessential oils has been described in several studies [12,30].The major phenolic components of some essential oils havebeen shown to have potent antimicrobial and antifungalactivity [31–33]. Although the activity of both eugenol (1)and thymol (2) against various pathogenic and non-pathogenic yeasts has been demonstrated previously, theirmode of action is not clearly understood. We have dem-onstrated fungicidal activity of compound 1 and compound2 by disc diffusion assay and their effect on growth ratesagainst clinical Candida isolates. In vitro studies have shownthat compound 1 and compound 2 significantly inhibitsboth fluconazole susceptible as well as fluconazole resistantCandida isolates.

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Low MIC values and low cytotoxicity obtained againstCandida with eugenol (1) and thymol (2) encouraged us tostudy their mode of action. The effect of both thecompounds was rapid and lethal. The rapid irreversibleaction of these compounds suggests that there may be acellular target(s) accessible to the compound externally.We, therefore, explored the effect of compound 1 andcompound 2 on H+ extrusion by plasma membrane H+-ATPase of various clinical Candida isolates. Candida isolatesshowing susceptibility to the test compounds also showedinhibition of H+-ATPase-mediated proton pumping sug-gesting that the two events are linked. It is to be noted thatthe inhibition of H+-ATPase function was achieved at theMIC concentrations of the compounds. The decrease in H+-extrusion being less when cells were exposed to the testcompounds in presence of glucose. Glucose inducedacidification of the external medium by yeast cells is aconvenient measure of H+-ATPase-mediated proton pumping[34]. The enzymemay be existing in a different conformationalstate in the two situations. Furthermore, the commonly usedantifungal agents like fluconazole and amphotericin B whichare known to interact with the sterol components of themembrane had no significant effect on H+-ATPase. Moreover,in presence of vanadate the complete inhibition of H+-efflux took place. These data suggest that interaction of anycompound with the plasma membrane is insufficient toaffect the function of the enzyme. It is thus possible that thetest compounds may be directly interacting with theenzyme, which serves as the primary reason for theirantifungal activity. Phytochemicals have been extensivelyshown to target different types of ATPases including P and Ftype. In general ATPases play a critical role in regulating cellfunction [26,35,36]. Thus, it would be useful to furtherinvestigate the interaction of the test compounds with thepurified PM-ATPase enzyme and study its activity in bothsteady state and pre-steady state.

Regulation of pHi, appears to be a fundamentalprerequisite for growth of Candida and activation of plasmamembrane ATPase as it is involved in maintenance of pHi[20]. We therefore studied the role of plasma membraneATPase activation in the regulation of pHi, in control as wellas treated cells. The pHi was near neutrality in absence oftest compounds and then declined to 6.31 and 6.23 atconcentrations inhibitory to H+-efflux and growth ofCandida. The H+ translocating ATPase mediated fungicidaleffects of naturally occurring plant active principles,eugenol (1) and thymol (2), suggest that the plasmamembrane H+-ATPase could be developed as a cell surfaceantifungal target. Rapidity of action, low MIC values, lethaleffect and negligible cytotoxicity demands more insightstudies into all other possible mechanisms of thesecompounds.

Acknowledgement

This work was partially supported by ICMR, India (IRIS ID2008-04780, Grant No. 59/24/2008/BMS/TRM) to Dr. N.Manzoor & Dr. L.A. Khan. Authors wish to thank Dr. MaliniR. Capoor, Safdarjung Hospital and Dr I. Xess, AIIMS, NewDelhi, India for providing clinical Candida isolates.

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