Fitoterapia 82 (2011) 1012–1020
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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Spice oil cinnamaldehyde exhibits potent anticandidal activity against fluconazole resistant clinical isolates Sheikh Shreaz a, Rimple Bhatia a, Neelofar Khan a, Sumathi Muralidhar b, Seemi F. Basir a, Nikhat Manzoor a, Luqman Ahmad Khan a,⁎ a b
Department of Biosciences, Jamia Millia Islamia, New Delhi, 110025, India Regional Sexually Transmitted Disease Centre, Safdarjung Hospital, New Delhi, 110029, India
a r t i c l e
i n f o
Article history: Received 17 December 2010 Accepted in revised form 7 June 2011 Available online 25 June 2011 Keywords: Cinnamaldehyde Candida Ergosterol Plasma membrane ATPase
a b s t r a c t Fluconazole resistance is becoming an important clinical concern. We studied the in vitro effects of cinnamaldehyde against 18 fluconazole-resistant Candida isolates. MIC90 of cinnamaldehyde against different Candida isolates ranged 100–500 μg/ml. Growth and sensitivity of the organisms were significantly affected by cinnamaldehyde at different concentrations. The rapid irreversible action of this compound on fungal cells suggested membrane-located targets for its action. Insight studies to mechanism suggested that cinnamaldehyde exerts its antifungal activity by targeting sterol biosynthesis and plasma membrane ATPase activity. Inhibition of H+-ATPase leads to intracellular acidification and cell death. Toxicity against H9c2 rat cardiac myoblasts was studied to exclude the possibility of further associated cytotoxicity. The observed selectively fungicidal characteristics against fluconazole-resistant Candida isolates signify a promising candidature of this essential oil as an antifungal agent in treatments for candidosis. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In recent years‚ drug resistance in human pathogenic fungi has been reported from various parts of the world [1,2]. Drug resistant fungal pathogens have complicated the treatment of infectious diseases in immunocompromised, AIDS and cancer patients [3,4]. Fluconazole resistance is becoming an important clinical concern [5–10]. Decreased fluconazole susceptibility has been noted especially in all human immunodeficiency virus (HIV)-infected patients with recurrent oropharyngeal candidiasis and in patients infected with yeasts other than Candida albicans[11–13]. Therefore, there exists a clear demand of more effective treatment of infections caused by these emerging fungal pathogens. Three general mechanisms of azole resistance have been described for species of Candida. The first is an alteration in the ⁎ Corresponding author at: Department of Biosciences, Jamia Millia Islamia, New Delhi, 110025, India. Tel.: +91 11 2698 1717x3410; fax: +91 11 2698 0229. E-mail address:
[email protected] (L.A. Khan). 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.06.004
target enzyme, 14α-demethylase. Inhibition of this enzyme by azoles causes an accumulation of C14 methylated sterols which likely disrupt membrane structure [14]. In some resistant organisms, there is overexpression of the 14 α-demethylase gene and/or the enzyme is less susceptible to azole inhibition [15–17]. The second mechanism is decreased drug accumulation, mediated by either diminished uptake or increased efflux of the drug [18,19]. The third mechanism of resistance is the presence of a deficiency in C5(6) desaturase. Organisms deficient in this enzyme produce 14-methylfecosterol and remain viable when 14 α-demethylase activity is inhibited [20,21]. Spices have traditionally been used to preserve foods, and to enhance flavour and odour. The antimicrobial activity of spice oils has been attributed to a number of substituted aromatic molecules, such as cinnamaldehyde, eugenol, and carvacrol [22–24]. Cinnamaldehyde is effective in inhibiting growth of bacteria, yeast and filamentous moulds. The chemical structure of this compound differs from existing antifungals, including azoles (Fig. 1). It is tentatively thought to act by inhibiting ATPases [25–27], cell wall biosynthesis [28], and by changing membrane structure and integrity [29–32]. Our previous study
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showed that cinnamaldehyde possessed a good in vitro antifungal activity against C. albicans and C. tropicalis[26]. In the present work we have evaluated antifungal activity of cinnamaldehyde against 18 fluconazole-resistant Candida isolates belonging to five species. The objective of this study was to further elucidate the antimicrobial mechanism of action of cinnamaldehyde by determining its effect on ergosterol biosynthesis and the activity of H+ATPase located in the membranes of pathogenic Candida species. This work is an additional effort to the development of new therapeutic treatment of infection by using plant derived principle which is fungicidal, non toxic and prevents drug resistance. 2. Materials and methods 2.1. Strains and growth media The strains used in this study are listed in Table 1. The clinical isolates were collected from Regional Sexually Transmitted Disease (STD) Centre, Safdarjung Hospital and Institute of Pathology, Safdarjung Hospital, New Delhi, India. All the strains were stored in 15% (vol/vol) glycerol stocked at −80 °C. Before each experiment, the cells were freshly revived on yeast extract, peptone and dextrose (YPD) plates from this stock. All the strains were routinely grown on YPD medium containing 2% (w/v) glucose, 2% peptone, and 1% yeast extract. YPD agar plates containing 2.5% agar in addition were used to maintain the culture. Medium chemicals and fluconazole were obtained from HiMedia (Mumbai, India). Cinnamaldehyde was purchased from ALDRICH chemicals (Germany), and all inorganic chemicals used in this study were of analytical grade and procured from E. Merck (India). 2.2. Determination of MIC90 2.2.1. Microtiter assay Cells were grown for 48 h at 30 °C to obtain single colonies, which were resuspended in a 0.9% normal saline solution to give an optical density at 600 nm (OD600) of 0.1. The cells were then diluted 100-fold in yeast nitrogen base (YNB) medium containing 2% glucose, and diluted cell suspensions were added to the wells of round-bottomed 96-well microtiter plates (100 μl/well) containing equal volumes of medium and different concentrations of test compound [33]. Fluconazole was included as positive control. In addition to this a drug-free control was also included. The plates were incubated at 30 °C for 24 h. The MIC test end point was evaluated both visually and by observing the OD620 in a microplate reader (BIO-RAD, iMark,
O
H
Fig. 1. Chemical structure of cinnamaldehyde.
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Table 1 Minimum Inhibitory concentration (MIC90) of cinnamaldehyde against fluconazole-resistant Candida isolates. Fungal species
STD no
Origin
MICs
C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.
1138 1131 977 41 53 126/09 1116 911 1004 985 608/09 2728 1121 64 77 1413 1342 1689
Invasive Invasive Invasive Cutaneous Respiratory Respiratory Cutaneous Cutaneous Cutaneous Invasive Respiratory Cutaneous Respiratory Invasive Respiratory Invasive Cutaneous Cutaneous
250 400 150 300 100 100 500 150 150 200 150 200 100 200 150 200 200 400
albicans albicans albicans albicans albicans albicans albicans albicans albicans tropicalis glabrata glabrata glabrata glabrata glabrata krusei krusei guilliermodii
US) and is defined as the lowest compound concentration that gave ≥90% inhibition of growth compared to the controls. 2.3. Disc diffusion halo assays Strains were inoculated into liquid YPD medium and grown overnight at 37 °C. The cells were then pelleted and washed three times with distilled water. Approximately 10 5 cells/ml were inoculated in molten agar media at 40 °C and poured into 100-mm diameter petriplates. Filter discs were kept on solid agar and test compound was spotted on the disc. Test compound (10 fold more than MIC) dissolved in 10% dimethyl sulphoxide (DMSO), or solvent control (10% DMSO) was pipetted onto 4-mm-diameter filter disc as described earlier [34]. 100 μg/ml of fluconazole was also on the discs to serve as positive control. The diameter of zone of inhibition was recorded in millimetres, after 48 h and was compared with that of control. The results are presented as mean ± standard deviation of at least three replicate experiments performed on separate days. 2.4. Time-kill studies Candida cells were subcultured at least twice and grown for 24 h at 35 °C on Sabouraud dextrose agar (SDA) plates as described earlier [35]. The adjusted inoculum suspension of 5 × 106 cfu/ml was diluted 1:10 in media to give a final concentration of 5 × 105 cfu/ml. Each concentration of test compound was diluted 1:10 in media containing 5 × 10 5 cfu/ml. Final concentration of test compound was MIC, MIC/2 and MIC/3 for each Candida isolate. Candida cells (1 ml final volume) were incubated at 35 °C with agitation (200 rpm) in water bath. At pre-determined time points (0, 2, 4, 8, 12 and 24 h) 100 μl aliquots were transferred to Eppendorf tubes were then centrifuged (3900 g at 4 °C for 5 min), supernatant was discarded and pellets were washed twice with 0.9 ml of sterile distilled water. Pellets were suspended in 500 μl of sterile distilled water. An appropriate volume (20 μl) was spread onto
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SDA plates and incubated at 35 °C for 24 h to determine the numbers of cfu/ml. 2.5. Ergosterol extraction and estimation assay Total intracellular sterols were extracted as reported earlier with slight modifications [36]. Briefly, a single Candida colony from an overnight Sabouraud dextrose agar plate culture was used to inoculate 50 ml of Sabouraud dextrose broth (Hi Media) containing different concentrations of cinnamaldehyde. The cultures were incubated for 16 h with shaking at 35 °C. The stationary-phase cells were harvested by centrifugation at 2700 rpm (Sigma 3 K30) for 5 min and washed once with sterile distilled water. The net wet weight of the cell pellet was determined. Three millilitres of 25% alcoholic potassium hydroxide solution (25 g of KOH and 35 ml of sterile distilled water, brought to 100 ml with 100% ethanol), was added to each pellet and vortex mixed for 1 min. Cell suspensions were transferred to sterile borosilicate glass screw-cap tubes and were incubated in an 85 °C water bath for 1 h. Following incubation, tubes were allowed to cool to room temperature. Sterols were then extracted by addition of a mixture of 1 ml of sterile distilled water and 3 ml of n-heptane followed by vigorous vortex mixing for 3 min. The heptane layer was transferred to a clean borosilicate glass screw-cap tube and stored at −20 °C for 24 h. Prior to analysis, a 1-ml aliquot of sterol extract was diluted fivefold in 100% ethanol and scanned spectrophotometrically between 230 and 300 nm using LABOMED, INC Spectrophotometer (USA). The presence of ergosterol and the late sterol intermediate 24(28) DHE in the extracted sample resulted in a characteristic fourpeaked curve. The absence of detectable ergosterol in extracts was indicated by a flat line. Ergosterol content was calculated as a percentage of the wet weight of the cell by the following equations: % ergosterol + % 24ð28ÞDHE = ½ðA281:5 = 290Þ × F = pelletweight; % 24ð28ÞDHE = ½ðA230 = 518Þ290 × F = pelletweight; % ergosterol = ½% ergosterol + % 24ð28ÞDHE−% 24ð28Þ DHE;
where F is the factor for dilution in ethanol and 290 and 518 are the E values (in percentages per centimetre) determined for crystalline ergosterol and 24(28) DHE, respectively. Values were shown in terms of mean± standard error of mean (SEM) of all three respective categories. 2.6. Proton efflux measurements Mid-log phase cells harvested from YEPD medium were washed twice with distilled water, and 100 mg cells were suspended in 5 ml solution containing 0.1 M KCl and 0.1 mM CaCl2. Suspension was kept in a double-jacketed glass container with constant stirring. Test compound was added to achieve the desired concentration (MIC90) in this 5 ml solution. For glucose stimulation experiments, 100 μl of glucose was added to achieve a final concentration of 5 mM. The container was connected to a water circulator at 25 °C. H+ extrusion rate was calculated from the volume of 0.01 N NaOH consumed as described earlier [26, 34, 37].
2.7. MTT cell viability assay H9c2 rat cardiac myoblasts were cultured and maintained as monolayer in Dulbecco's modified Eagle's medium (DMEM), high glucose, supplemented with 10% foetal bovine serum (heat inactivated), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin B, at 37 °C in humidified incubator with 5% CO2[38]. For treatments, compound stock solutions were prepared in DMSO and added to wells to give the indicated final concentrations. Final DMSO concentration was 0.2% in all wells including the untreated cells (control) and fluconazole controls. Cells were incubated for 48 h at 37 °C in 5% CO2 humidified incubator together with untreated control sample. After incubation cells were washed in PBS and incubated with MTT solution for 45 min at 37 °C. After discarding the supernatant MTT crystals were dissolved with acid isopropanol and the absorbance was measured at 570 nm. All assays were performed in triplicate. Percent viability was defined as the relative absorbance of treated versus untreated control cells. Plates were analysed in an ELISA plate reader (Labsystems Multiskan RC, Helsinki, Finland) at 570 nm with a reference wavelength of 655 nm. 3. Results 3.1. Minimal inhibitory concentration (MIC90) Table 1 summarises the in vitro susceptibilities of 18 fluconazole-resistant Candida species against cinnamaldehyde. The data are reported as MIC's required to inhibit 90% growth of the Candida cell population. All the isolates exhibited fluconazole MIC ≥ 64 μg/ml, and were considered as resistant. MIC varied with isolates and was strain specific. No trend was observed with respect to location or species of isolates. 3.2. Disc diffusion C. glabrata, C. krusei, C. albicans and C. parapsilosis are inherently resistant or rapidly acquire resistance to azoles [39]. All the fluconazole-resistant Candida isolates showed high degree of sensitivity as is evident from large inhibition zone in Table 2. Our results show that the growth was inhibited by cinnamaldehyde at different concentrations, and the halo was completely clear, indicating potential fungicidal activity, whereas in contrast fluconazole showed a turbid halo and in most of the cases inhibition zone was absent, an indication of its fungistatic nature. It was noticed that fungicidal activity of cinnamaldehyde on solid media increased when treated against C. guilliermondii. Results obtained demonstrated that the ability to kill Candida species is dependent on the concentration of the test compound. The discs impregnated with 10% DMSO (negative control) showed no zone of inhibition and hence 10% DMSO had no effect on the strains tested in the present study. 3.3. Time kill curves Fig. 2(A–E) shows the killing activity of cinnamaldehyde against five fluconazole resistant Candida species: C. albicans
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Table 2 Disc diffusion assay of cinnamaldehyde (mg/ml) against fluconazole-resistant Candida isolates. Zone of inhibition (mm) Fungal species
STD no
Origin
5 mg/ml
3 mg/ml
1 mg/ml
C. albicans C. albicans C. albicans C. albicans C. albicans C. tropicalis C. tropicalis C. tropicalis C. tropicalis C. tropicalis C. glabrata C. glabrata C. glabrata C. glabrata C. glabrata C. krusei C. krusei C. guilliermondii
1138 1131 977 41 53 126/09 1116 911 1004 985 608/09 2728 1121 64 77 1413 1342 1689
Invasive Invasive Invasive Cutaneous Respiratory Respiratory Cutaneous Cutaneous Cutaneous Invasive Respiratory Cutaneous Respiratory Invasive Respiratory Invasive Cutaneous Cutaneous
09 09 15 09 17 16 04 14 15 12 15 12 17 11 15 13 13 19
04 04 09 05 12 10 02 09 09 07 10 07 11 05 10 08 08 13
02 02 06 02 08 06 01 05 05 04 06 04 07 07 09 04 04 09
(± 0.5) (± 0.1) (± 0.1) (± 0.5) (± 0.4) (± 0.1) (± 0.0) (± 0.7) (± 1.0) (± 0.9) (± 0.2) (± 0.2) (± 0.5) (± 0.1) (± 1.0) (± 0.2) (± 0.2) (± 0.2)
(± 0.2) (± 0.0) (± 0.5) (± 0.8) (± 0.9) (± 0.3) (± 0.1) (± 0.0) (± 0.4) (± 0.5) (± 0.5) (± 0.3) (± 0.2) (± 0.0) (± 0.4) (± 0.1) (± 0.2) (± 0.0)
(± 0.1) (± 0.1) (± 0.9) (± 0.2) (± 0.0) (± 0.1) (± 0.0) (± 0.4) (± 0.1) (± 0.2) (± 0.2) (± 0.1) (± 0.5) (± 0.5) (± 0.3) (± 0.1) (± 0.6) (± 0.3)
The data represents (Mean ± S.D) of three sets of experiments.
STD No 41, C. tropicalis STD No 1004, C. glabrata STD No 2728, C. krusei STD No 1342 and C. guilliermondii STD No 1689. At their respective MIC90 values the fungicidal activity of cinnamalde-
B
C 2500
2500
2000
2000
1500
1500
1000 500 0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (hrs)
1000 500
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (hrs)
Control
100µg/ml
Control
50µg/ml
150µg/ml
300µg/ml
75µg/ml
150µg/ml
D
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (hrs) Control 100µg/ml
66.6µg/ml 200µg/ml
E 2500
2500
2000
2000
1500
1500
Cells
Cells
Cells
1800 1600 1400 1200 1000 800 600 400 200 0
Cells
Cells
A
hyde was fast against all the Candida isolates tested. Significant and pronounced effect was observed for other species (data not shown). Results obtained demonstrated that the ability to kill
1000 500 0
1000 500
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (hrs) Control 100µg/ml
66.6µg/ml 200µg/ml
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (hrs) Control 200µg/ml
133.3µg/ml 400µg/m
Fig. 2. Representative time-kill plots for fluconazole-resistant Candida isolates following exposure to cinnamaldehyde (A) C. albicans STD no 41 (B) C. tropicalis STD no 1004 (C) C. glabrata STD no 2728 (D) C. krusei STD no 1342 (E) C. guilliermondii STD no 1689. At MIC90 values (300 μg/ml, 150 μg/ml, 200 μg/ml, 200 μg/ml and 400 μg/ml) almost complete cessation of growth was observed for all the yeast species.
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Candida species is dependent on the concentration of the test compound. The fungicidal endpoints for fluconazole resistant isolates were reached after 24 h of incubation at MIC90 of cinnamaldehyde. No systematic difference was observed between isolates from various locations.
3.4. Ergosterol extraction and estimation assay Total ergosterol content was determined for each isolate grown in varying concentrations of cinnamaldehyde. A dosedependent decrease in ergosterol production was observed when isolates were grown in the presence of cinnamaldehyde. Fig. 3(A–E) gives typical scans of ergosterol assay, for C. albicans STD No 41, C. tropicalis STD No 1004, C. glabrata STD No 2728, C. krusei STD No 1342 and C. guilliermondii STD No 1689. Percent decrease in total cellular ergosterol content for resistant isolates ranged 10.16–83.24% after exposure to 30– 500 μg cinnamaldehyde/ml respectively (Table 3). Average decrease in total cellular ergosterol content when treated with fluconazole (100 μg/ml) was measured to be 17.34% only. From the results, it is clear that with increase in cinnamaldehyde concentrations, % ergosterol inhibition increases and finally at MIC value, almost flat line was observed indicating absence of ergosterol in the sample. High level of cinnamaldehyde drastically reduced ergosterol content of cell membrane. MIC90 values of cinnamaldehyde co-relates very well with % age inhibition of sterol biosynthesis.
3.6. Toxicity profile To examine the toxicity level of the compounds H9c2 rat cardiac myoblast cells were treated with increasing concentrations of the test compound and the number of viable cells was
B
C 1.6
1.4
1.4
1.4
1.2
1.2
1.2
0.8 0.6
1 0.8 0.6
0.4
0.4
0.2
0.2
D
0.6
0.2
Wavelength (nm) Control 100µg/ml
100µg/ml 300µg/ml
0.8
0.4
0 230 235 240 245 250 255 260 265 270 275 280 285 290 295 300
Wavelength (nm) Control 200µg/ml
1
Absorbance
1
0 230 235 240 245 250 255 260 265 270 275 280 285 290 295 300
50µg/ml 150µg/ml
0 230 235 240 245 250 255 260 265 270 275 280 285 290 295 300
Wavelength (nm) Control 100µg/ml
50µg/ml 200µg/ml
E 1.4
0.8
1.2
0.7
1
0.6
Absorbance
Absorbance
H+ efflux is an immediate event associated with H+ ATPase activity. Proton-pumping ability of fungi mediated by the H+ ATPase at the expense of energy is crucial for the regulation of internal pH and growth regulation of fungal cell. Fungal cells depleted of carbon-source when exposed to glucose, rapidly acidify medium to generate proton motive force for nutrient uptake. Untreated Candida cells (control) in the presence of 0.1 mM CaCl2 and 100 mM KCl showed acidification starting from pH 7.0 (Table 4.1). Acidification decreased in the presence of cinnamaldehyde, at respective MIC90 values. Reduction in H+ efflux in the presence of the cinnamaldehyde alone and cinnamaldehyde with glucose ranged 48–89% and 26–61%. Respective inhibition by fluconazole alone (64 μg/ml) ranged 9–24% respectively (Table 4.2). Presence of glucose (5 mM) stimulated H+-efflux in the range of 3–4 fold, with respect to control in all the tested species. Glucose-stimulated H +-efflux was inhibited by the cinnamaldehyde but the inhibition was significantly less (Table 4.2). Detailed studies of test compound on the same can give us more insight into the possible mechanisms of action.
1.6
Absorbance
Absorbance
A
3.5. H +-extrusion studies
0.8 0.6 0.4
0.5 0.4 0.3 0.2
0.2
0.1
0 230 235 240 245 250 255 260 265 270 275 280 285 290 295 300
0 230 235 240 245 250 255 260 265 270 275 280 285 290 295 300
Wavelength (nm)
Wavelength (nm)
Control 100µg/ml
Control 200µg/ml
50µg/ml 200µg/ml
100µg/ml 400µg/ml
Fig. 3. Typical UV spectrophotometric sterol profile in the presence of cinnamaldehyde. (A) C. albicans STD no 41 in concentration range of 100–300 μg/ml. (B) AgainstC. tropicalis STD no 1004 in concentration range of 50–150 μg/ml. (C) AgainstC. glabrata STD no 2728 in concentration range of 50–200 μg/ml. (D) AgainstC. krusei STD no 1342 in concentration range of 50–200 μg/ml. (E). AgainstC. guilliermondii STD no 1689 in concentration range of 100–400 μg/ml.
S. Shreaz et al. / Fitoterapia 82 (2011) 1012–1020
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Table 3 Percentage decrease in ergosterol content of control cells and treated samples expressed in (μg/ml). Cinnamaldehyde STD no 1413
STD no 126/09
STD no 1116
STD no 1138
Sample control
% decrease 0
Sample control
% decrease 0
Sample control
% decrease 0
Sample control
% decrease 0
50 100 200
15.21 ± 1.427 43.00 ± 0.613 63.54 ± 0.746
30 60 100
22.23 ± 0.734 56.11 ± 0.278 82.02 ± 1.311
100 300 500
10.16 ± 0.570 25.43 ± 0.5680 47.00 ± 0.341
100 200 250
14.21 ± 0.456 39.62 ± 0.209 57.11 ± 0.328
STD no 1131
STD no 608/09
STD no 2728
STD no 1121
Control
0
Control
0
Control
0
Control
0
100 200 400
13.01 ± 1.324 35.49 ± 1.269 51.11 ± 1.200
50 100 150
20.01 ± 1.112 48.19 ± 0.478 75.31 ± 1.456
50 100 200
16.03 ± 1.451 46.19 ± 0.877 62.55 ± 0.651
30 60 100
21.13 ± 0.435 49.09 ± 0.129 76.32 ± 1.302
STD no 1689
STD no 1342
STD no 977
STD no 911
Control
0
Control
0
Control
0
Control
0
100 200 400
12.43 ± 1.167 35.61 ± 0.548 53.22 ± 0.344
50 100 200
14.00 ± 0.234 45.16 ± 0.455 60.54 ± 0.287
50 100 150
19.32 ± 0.987 45.00 ± 0.210 72.17 ± 0.277
50 100 150
18.48 ± 1.402 42.53 ± 0.289 69.03 ± 0.879
STD no 1004
STD no 985
STD no 41
STD no 53
Control
0
Control
0
Control
0
Control
0
50 100 150
18.04 ± 0.378 46.00 ± 0.423 70.24 ± 0.926
50 100 200
15.01 ± 1.213 43.55 ± 0.611 60.11 ± 0.234
100 200 300
13.01 ± 0.255 36.34 ± 0.302 55.00 ± 0.211
30 60 100
25.14 ± 0.139 53.08 ± 0.120 83.24 ± 0.235
STD no 64
STD no 77
Control
0
Control
0
50 100 200
13.01 ± 1.342 40.24 ± 0.342 59.00 ± 1.175
50 100 150
16.01 ± 1.342 44.63 ± 0.966 65.42 ± 0.145
The data represents (Mean ± S.D) of three sets of experiments.
measured after 48 h by MTT cell viability assay. The concentration range of the test compound was 20–620 μg/ml. Fig. 4 depicts that the cinnamaldehyde showed a viability of 100% at the concentration range of 20 μg/ml whilst as the reference compound fluconazole showed 91% viability. At concentrations of 40, 80, 160, 320 and 640 μg/ml fluconazole showed 82%, 68%, 53%, 42% and 28% viability whilst cinnamaldehyde offered a remarkable viability of 100%, 96%, 89%, 84% and 79% respectively. In comparison to fluconazole, cinnamaldehyde showed almost negligible toxicity even at value approaching MIC90. These properties reveal the potential therapeutic application of this compound.
4. Discussion Resistance usually arises after long periods in the presence of the drug, conditions that may support the gradual, stepwise development of resistance. It is more likely that resistance will gradually evolve over time as a result of several alterations due to continuous selective pressure from the drug. Current drug treatments are largely effective, but resistant strains and intrinsically resistant species are emerging fast. Moreover, the treatment cost, associated host cytoxicity and the fact that most available antifungal drugs have only fungi-static activity justify the search for new strategies.
This is a significant finding as cinnamaldehyde is a normal component of food and has a high human consumption in India. As Candida is a normal resident of oral cavity and genitourinary tract, it receives direct exposure of cinnamaldehyde. Our findings suggest options for expanding the utility of essential oil components as antifungal agents. The antimicrobial and antifungal effect of many aromatic plant essential oils [40,41] including cinnamaldehyde [26–30] has been described in several studies. Although the activity of cinnamaldehyde against yeasts has been demonstrated previously [28,29], its mode of action is not clearly understood. We demonstrated that cinnamaldehyde exhibits fungicidal and not fungistatic activity, by halo assay and time kill studies against recently obtained clinical isolates. MIC90 results obtained in this study show that cinnamaldehyde exhibited varying degrees of antifungal activity against fluconazole-resistant Candida isolates. Anticandidal activity order of cinnamaldehyde on solid medium leads to a similar conclusion. Generally, all the fluconazole-resistant Candida isolates investigated were found to be sensitive to cinnamaldehyde. The use of total mean MICs obtained gave a good indication of the overall antimicrobial effectiveness of cinnamaldehyde. This may indicate that the yeast physiology may not be better equipped to counteract the antifungal properties of cinnamaldehyde. On solid media growth was inhibited by cinnamaldehyde, and the halo produced was completely clear, an indication of potential fungicidal activity,
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Table 4.1 Effect of cinnamaldehyde on rate of H+-efflux in different fluconazole-resistant Candida cells at pH 7.0. Relative H+-efflux rate (nmoles/mg yeast cells/min) Fungal species
STD no
Origin
Control
Cinnamaldehyde alone
Glucose alone
Cinnamaldehyde Glucose
C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.
1138 1131 977 41 53 126/09 1116 911 1004 985 608/09 2728 1121 64 77 1413 1342 1689
Invasive Invasive Invasive Cutaneous Respiratory Respiratory Cutaneous Cutaneous Cutaneous Invasive Respiratory Cutaneous Respiratory Invasive Respiratory Invasive Cutaneous Cutaneous
5.9 6.1 6.0 5.8 5.1 5.3 5.0 6.3 6.1 5.3 5.9 5.6 5.6 6.2 5.7 5.0 5.7 5.0
0.43 0.50 0.27 0.45 0.12 0.15 0.52 0.28 0.20 0.41 0.30 0.35 0.10 0.40 0.59 0.41 0.36 0.47
3.23 3.93 3.85 3.79 3.93 3.73 4.00 3.80 3.77 4.33 3.40 3.78 3.78 3.72 3.86 4.00 3.47 3.72
2.13 2.63 1.92 2.42 1.67 1.60 2.48 1.82 1.62 2.82 1.66 2.00 1.47 2.19 1.85 2.48 1.94 2.26
albicans albicans albicans albicans albicans tropicalis tropicalis tropicalis tropicalis tropicalis glabrata glabrata glabrata glabrata glabrata krusei krusei guilliermondii
Control stands for cells without any compound, incubated in 0.1 mM CaCl2 and 0.1 M KCl at 25 °C.
whereas in contrast fluconazole showed a turbid halo an indication of fungistatic nature of the same [42]. Results obtained in time kill assays demonstrated that increase in concentration of cinnamaldehyde leads to significant killing activity. The fungicidal endpoints were reached after 48 h of incubation at MIC90 of cinnamaldehyde and no systematic difference was observed between isolates from various locations. Subconfluent populations of H9c2 rat cardiac myoblast cells provide a handy tool for toxicity studies of the compounds. The in vitro MTT cell viability assay is a possible screening assay for gauging in vivo toxicity to host cells. Of note is that at the concentration of 320 μg/ml of cinnamaldehyde which had profound effect on ergosterol biosynthesis and plasma membrane ATPase activity of almost all Candida isolates, the viability observed was 84%. Respective viability shown by fluconazole
on the same concentration was only 42%. It is already reported that in 72 h ID 50 value, human KB cells had 3.6 times higher value than Saccharomyces cervisiae to cinnamaldehyde [43]. Thus the toxicity rankings of cinnamaldehyde determined in our study at MIC90 values show a good parallel. Minimal toxicity shown by cinnamaldehyde against H9c2 rat cardiac myoblast cells encouraged us to study its mode of action. This work constitutes the first attempt to assess the antifungal role of cinnamaldehyde by studying its effect on sterol biosynthesis and plasma membrane ATPase activity of fungi. Plasma membrane ATPase, an important fungal pump, generates membrane gradient which is employed for nutrient transport. The pump is activated in the presence of glucose to extrude more H+. The rapid irreversible action of cinnamaldehyde suggests that there may be a cellular target(s) accessible to
Table 4.2 Percentage inhibition of H+-efflux with respect to control in different fluconazole-resistant Candida isolates. % age inhibition of H+-efflux Fungal species
STD no
Origin
Cinnamaldehyde alone
Cinnamaldehyde with glucose
Fluconazole alone
C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.
1138 1131 977 41 53 126/09 1116 911 1004 985 608/09 2728 1121 64 77 1413 1342 1689
Invasive Invasive Invasive Cutaneous Respiratory Respiratory Cutaneous Cutaneous Cutaneous Invasive Respiratory Cutaneous Respiratory Invasive Respiratory Invasive Cutaneous Cutaneous
56.11 ± 1.12 50.00 ± 1.54 73.00 ± 1.81 55.00 ± 1.67 87.06 ± 3.11 84.16 ± 2.20 48.00 ± 1.87 71.12 ± 2.88 79.02 ± 1.43 58.12 ± 0.99 70.0 ± 1.149 65.00 ± 1.54 89.11 ± 3.13 60.00 ± 2.27 74.04 ± 2.29 59.00 ± 2.43 63.16 ± 0.79 3.00 ± 3.20
34.04 ± 1.35 33.00 ± 0.97 50.00 ± 1.09 36.00 ± 3.00 59.01 ± 1.33 57.03 ± 3.00 26.00 ± 1.00 52.00 ± 1.19 57.02 ± 2.20 35.00 ± 0.89 51.05 ± 1.88 47.03 ± 3.19 61.03 ± 1.67 41.01 ± 2.00 52.03 ± 3.54 38.00 ± 1.22 44.05 ± 1.46 39.04 ± 2.44
13.00 ± 0.00 09.00 ± 0.00 17.00 ± 0.00 11.00 ± 0.00 22.00 ± 0.00 24.00 ± 0.00 10.00 ± 0.00 20.00 ± 0.00 21.00 ± 0.00 14.00 ± 0.00 18.00 ± 0.00 15.00 ± 0.00 21.00 ± 0.00 13.00 ± 0.00 16.00 ± 0.00 17.00 ± 0.00 18.00 ± 0.00 10.00 ± 0.00
albicans albicans albicans albicans albicans tropicalis tropicalis tropicalis tropicalis tropicalis glabrata glabrata glabrata glabrata glabrata krusei krusei guilliermondii
The data represents (Mean ± S.D) of three sets of experiments.
S. Shreaz et al. / Fitoterapia 82 (2011) 1012–1020
more insight studies into all other possible mechanisms of this compound. This work suggests that naturally occurring essential oil component-cinnamaldehyde could be a promising drug after improved formulations and also advocates the determination of optimal concentrations for clinical applications, as an alternate to fluconazole therapy for the treatment and prevention of candidiasis. The excellence of this compound demands more insight studies into all of the possible mechanisms of this compound.
120 100
Mean % Viability
1019
80 60 40 20 0
Acknowledgements 20
40
80
160
320
640
Concentration (µg/ml) Control
FCZ
CD
Fig. 4. Percentage of viable cells after 48 h pre-treatment of H9c2 myoblasts with fluconazole (FCZ) and cinnamaldehyde (CD) evaluated by MTT assay.
cinnamaldehyde externally. We, therefore, explored the effect on H+ extrusion by plasma membrane H+-ATPase of various fluconazole-resistant Candida isolates. Candida isolates showing susceptibility to cinnamaldehyde also showed inhibition of H+-ATPase-mediated proton pumping suggesting that the two events are linked. It is to be noted that the inhibition of H+ATPase function was achieved at the MIC90 concentrations of the compound the decrease in H+-extrusion being less when cells were exposed to cinnamaldehyde in presence of glucose. Glucose induced acidification of the external medium by yeast cells is a convenient measure of H+-ATPase-mediated proton pumping [44]. The enzyme may be existing in a different conformational state in the two situations. It is thus possible that the cinnamaldehyde may be directly interacting with the enzyme, which serves as the primary reason for their antifungal activity. Ergosterol also contributes to proper functioning of membrane bound enzymes. Analysis of sterols obtained from all the fluconazole-resistant Candida strains showed no major differences in either the sterol contents or the sterol patterns of the untreated controls of the isolates. Growth of Candida in the presence of subinhibitory concentrations of cinnamaldehyde altered the sterol patterns of the fluconazole-resistant strains. This essential oil component completely blocks ergosterol synthesis at its MIC90 value. Studying the effect of this compound at various sub-inhibitory concentrations on the sterols of the fluconazole-resistant Candida isolates showed that this compound acts in a dose-dependent fashion to decrease ergosterol content. Thus, our data indicates that cinnamaldehyde has similar modes of action as that of fluconazole as it inhibits ergosterol biosynthesis in case of sensitive strains. To conclude decrease in H +-ATPase-mediated proton pumping and ergosterol content indicates that these organisms show trailing growth in the presence of cinnamaldehyde. Increased and parallel co-relation of decrease in both the membrane associated properties with MIC90 values for all organisms suggests that ergosterol biosynthesis and H +ATPase-pump is the primary target of cinnamaldehyde [45]. This work suggests that naturally occurring EO componentcinnamaldehyde could be a promising drug as an alternate to fluconazole therapy for the treatment and prevention of candidiasis. Fungicidal nature and negligible cytotoxity demand
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