Printer Friendly

In vitro antifungal, anti-elastase and anti-keratinase activity of essential oils of Cinnamomum-, Syzygium- and Cymbopogon-species against Aspergillus fumigatus and Trichophyton rubrum.

ABSTRACT

This study was aimed to evaluate effects of certain essential oils namely Cinnomomum verum, Slyzygium aromaticum, Cymbopogon citratus, Cymbopogon martini and their major components cinnamaldehyde, eugenol, citral and geraniol respectively, on growth, hyphal infrastructure and virulence factors of Aspergillus fumigatus and Trichophyton rubntm. The antifungal activity of essential oils and their major constituents was in the order of cinnamaldehyde > eugenol > geraniol = C. verum > citral >S. aromaticum > c. citratus > C. martini, both in liquid and solid media against T. rubrum and A. fumigatus. Based on promising antifungal activity of eugenol and cinnamaldehyde, these oils were further tested for their inhibitory activity against ungerminated and germinated conidia in test fungi. Cinnamaldehyde was found to be more active than eugenol. To assess the possible mode of action of cinnamaldehyde, electron microscopic studies were conducted. The observations revealed multiple sites of action of cinnamaldehyde mainly on cell membranes and endomembranous structures of the fungal cell. Further, test oils were also tested for their anti-virulence activity. More than 70% reduction in elastase activity was recorded in A fumigatus by the oils of C. verum, C. martini, eugenol, cinnamaldehyde and geraniol. Similar reduction in keratinase activity in A niger was recorded for the oils of C. martini and geraniol. Maximum reduction (96.56%) in elastase activity was produced by cinnamaldehyde whereas; geraniol caused maximum inhibition (97.31%) of keratinase activity. Our findings highlight anti-elastase and anti-keratinase activity of above mentioned essential oils as a novel property to be exploited in controlling invasive and superficial mycoses.

[c] 2011 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords: Essential oils Growth inhibition Electron microscopy Virulence factors Elastase activity Kevatinase activity Cinnamomum-, Syzygium-, Cymbopogon-specoes

Introduction

Fungal infections caused by various pathogenic and opportunistic groups are on the rise in the different parts of the world. Invasive aspergillosis caused by Aspergillus is considered as a major cause of morbidity and mortality in immunocompromised hosts and mortality rates may range from 40 to 90% in high risk populations (Dagenais and Keller 2009). Other chronic infections associated with the immunocompromised patients are caused by dermatophytes mainly Trichophyton sp, and had shown increased incidence in recent years especially in the tropical countries (Venkatesan et al. 2007). With the increasing number of immunosuppressed patients at an unprecedented rate, the management of these fungal infections would be a definite challenge to mankind.

Current antifungal therapy for such fungal infections has been threatened by the development of drug resistant strains, host toxicity of available polyenes and fungistatic mode of action of azoies (Barker and Rogers 2006). Therefore, development of newer drugs with improved efficacy and safety or alternative mode of combating infections is needed. Recent developments in fungal genomics have provided unprecedented opportunities for identifying new antifungal drug targets and subsequently exploiting in disease control. Targeting virulence and pathogenicity are now considered as valuable anti-pathogenic approaches (Gauwerky et al. 2009). Establishment of infection by fungi depends on the host-cell interaction with complex interplay of secretion of virulence factors mainly proteinases including elastinases, keratinases, gelatinases, lipases and phospholipases. These extracellular enzymes are probably essential for these organisms to degrade structural barrier and to obtain nutrient and in establishing infections (Voltan et al. 2008). Plant products traditionally being used in ethnomedicine have been expected to deliver newer antifungal compounds. Antifungal activities of the essential oils or their major constituents against Aspergillus and Trichophyton spp. have been reported by several workers (Zacchino et al. 1999; Cavaleiro et al. 2006; Bajpai et al. 2009). The influence of essential oils on fungal virulence factors synthesis and activity are not yet explored or poorly known.

In lieu of this, the present study was aimed to determine in vitro growth inhibition of Aspergillus fumigatus and Trichophyton rubrurn by four plant essential oils such as Cinnamomum verum, Syzygium aromaticum, Cymbopogon citratus and Cymbopogon martini and their respective major ingredients namely cinnamaldehyde, eugenol, citral and geraniol. These major compounds were chosen to assess their role in contributing activities alone and in their respective oils. Further, possible target sites of most active agent on fungal cell and inhibition of elastase and keratinase enzymes was examined.

Materials and methods

Plant essential oils and drugs

Essential oils and active compounds were obtained from Himalaya Drug Co. (Cinnamomum verum, cinnamon); Himedia Ltd. (eugenol and cinnamaldehyde, 98% purity each). Aroma Sales Corporation (oils of Cymbopogon citratus, lemongrass; Cymbopogon martini, palmrosa; and citral and geraniol) and Dabur Co. Ltd. (Syzygium aromaticum, clove). The drug powder of fluconazole was purchased from Himedia Laboratories, Mumbai, India. Stock solution of fluconazole was prepared in dimethyl sulphoxide (DMSO) at a concentration of 25 mg [ml.sub.-1] and stored at -20[degree] C until used. The purity of oils and active compounds was determined by physico-chemical analyses such as specific gravity, refractive index, optical rotation and solubility in alcohol (data not shown) at Fragrance and Flavour Development Centre, Kannauj, India. Chemical composition of oils was determined by gas chromatography-mass spectrometry at Sophisticated Analytical Instrument Facility of Indian Institute of Technology, Mumbai, India (Khan and Ahmad 2011) and Advanced Instrumentation Research Facility, Jawaharlal Nehru University, New Delhi, India. Essential oils were diluted 10 times in 1% DMSO and used in assays.

Gas chromatography and gas chromatography-moss spectrometiy analysis

The percentage composition of oil C citratus was determined by GC-FID and the compounds were identified by GC-MS. GC analysis was carried out on a Shimadzu 2010 Gas Chromatograph equipped with an FID and 25 m x 0.25 mm x 0.25 [micro]m WCOT column coated with diethylene glycol (AB-lnnowax, 7031428, Japan). Injector temperature was set at 270 [degree]C and detector at 280 [degree]C. Nitrogen was used as a carrier gas at a flow rate of 3.0 ml/min at a column pressure of 74.9 kPa. 0.2 [micro]l of sample were injected into column with a split ratio of 90.0. The linear temperature program of 60-230 [degree]C set at a rate of 3 [degree]C [min.sub.-1] with hold time at 230 [degree]C for 10 min. The samples were then analyzed on the same Shimadzu instrument fitted with the same column and following the same temperature program as above. MS parameters used were: ionisation voltage (EI) 70 eV, peak width 2 s, mass range 40-600 amu and detector voltage 1.5 V. Results were based on GC-FID. Peak identification was carried out by comparison of the mass spectra with database of NIST05 and Wiley 8 libraries. Identification of compounds was confirmed by comparison of their relative retention indices with literature values (Daviesl990).

Fungal strains

Aspergillus flavus NRRL501 was kindly provided from the fungal culture collection of the Agricultural Research Service, USA; Aspergillus fumigatus MTCC2550, Altemaria solani MTCC2101 and Trichophyton rubrum MTCC296 were purchased from Microbial Type Culture Collection, India; Aspergillus niger IOA-3 and Trichophyton rubrum IOA-9 were collected from Jawaharlal Nehru Medical College and Hospital, AMU, Aligarh, India and are maintained at the departmental culture collection.

Assays for determination of antifungal activity

Effect of essential oils against test fungi was determined in terms of inhibition of biomass in liquid medium and mycelial radial growth on solid medium as described below.

Inhibition of biomass production

Method of Shafique et al. (2011) with slight modification was adapted. Briefly, 50 ml Sabouraud dextrose broth (SDB) containing different concentrations of oils (0.005-0.32% v/v) was inoculated with 500 [micro]J of freshly prepared spore suspension (~1.5 x [10.sub.6] cfu/ml) of test fungi. In the corresponding control an equal amount of distilled water was added. Fluconazole in the concentration range of 1.25-200 [micro]g/ml was also tested as a positive control. The flasks were incubated at 28[+ or -]2[degree]C for 5 days. Thereafter, mycelial biomass from triplicate samples for each treatment was collected on pre-weighed Whatman filter paper no. 1. Mycelial yield was determined after drying the mycelial mat at 80 [degree]C for 24 h and percent loss in mycelial dry weight was calculated over untreated control.

Inhibition of mycelial radial growth

Method of Quiroga et al. (2004) with little modification was adapted. Briefly, a 5 mm diameter disc of inoculum of the test fungi was cut from the periphery of an actively growing culture and placed onto the SDA petriplates amended with test oils (0.005-0.32% v/v). Fluconazole amended plates (1.25-200 [micro]g/ml) were included as positive control. The SDA plates without oils and inoculated with corresponding fungi were served untreated controls. All the inoculated plates were incubated at 28[+ or -]2 [degree]C for 5 days. Three replicates for each combination of test fungi and oils concentrations were used. The mean diameter of the radial growth of the fungi was recorded at the end of the incubation period and percent growth inhibition was calculated over untreated control.

Antifungal activities of cinnamaldehyde and eugenol against ungerminated and germinated conidia

The assay comprising the activities against ungerminated and germinated conidia of test fungi employed the modified method of De Lucca et al. (1997). For ungerminated conidia susceptibility assay, 90 [micro]l of freshly obtained conidial suspension (~1.5 x [10.sup.6] cfu/ml) was added to Eppendorf tubes containing SDB (final volume 1 ml) with a range of concentration of test agent (0.005-0.16% v/v). Control sample contained no test agent. Mixed samples were incubated at 30 C for 30min. Viable count was enumerated by plating 100 [micro]xl onto SDA plates and incubating at 28[+ or -]2 [degree]C for 24 h. For germinated conidia susceptibility assay, freshly obtained conidial suspension was first allowed to incubate at 30 [degree]C for 8h (A fumigatus MTCC2550) and for lOh (T. rubrum 10A-9) at 120 rpm and then treated as for ungerminated conidia susceptibility assay. Each assay was carried out three times per isolate per agent concentrations and data are presented as mean [+ or -] SD.

Determination of effect of essential oils on hyphal morphology and ultrastructure

Light microscopy

The time (0, 2, 4, 8, 16, 24, 32, 48 h) and concentration (0.005-0.32% v/v) dependent toxicity of oils C. verum and S. aromaticum towards T. rubrum IOA-9 and A. fumigatus MTCC2550 was observed in terms of morphological alterations using standard method at 40 x magnification under Light microscope (Olympus BS60, Japan).

Scanning electron microscopy

Effect of sub-inhibitory concentration of cinnamaldehyde (0.04% v/v) towards hyphal morphology in A. fumigatus MTCC2550 was examined by processing the treated and untreated sample as per standard procedure and observing under a LE0435VP SEM at 15 kV.

Transmission electron microscopy

Ultrastructural changes produced by cinnamaldehyde at sub-inhibitory concentration (0.04% v/v) towards A. fumigatus MTCC2550 were evaluated. The treated and untreated mycelial biomass was processed using standard method and viewed under a Morgagni 268D transmission electron microscope at 80 kV.

Sorbitol protection assay

Sorbitol assay was performed as described by Frost et al. (1995) with some modifications for filamentous fungi using the broth macrodilution procedure. Briefly, duplicate tubes containing 1 ml SDB were prepared; one containing two-fold dilutions of test oils and the other containing test agents plus 0.8 M sorbitol as an osmoprotectant. MIC was determined as the lowest concentration of test agents inhibiting the visible growth and evaluated after 5 and 10 days of incubation. Each experiment was repeated three times and mean values were calculated for MICs.

Detection of elastase activity

The method of Kothary et al. (1984) was adapted to detect the production of elastase among test isolates and elastase activity was determined by colorimetric assay of Sachar et al. (1955) with some modifications employing elastin congo red (Sigma) as a substrate. Briefly, 250 [micro]l each of 50 mM sodium borate buffer (pH 8.5) and elastin congo red (20 mg [ml.sup.-1] in 50 mM sodium borate buffer, pH 8.5) was mixed and vortexed at room temperature for 5 min. Next, 250 |xl of test sample was added and incubated at 37 C for 3 h at 180 rpm. Test sample was replaced by 250 [micro]l of buffer as control. After incubation, 750 [micro]l of 10% trichloro acetic acid was added to stop the reaction and kept for 30 min on ice. Insoluble material in the assay mixture was removed by centrifugation at 5000 rpm, 30 min and the absorbance was read at 495 nm using a double beam UV-vis spectrophotometer (UV5704SS, India).

Effect of essential oils on elastase activity

Inhibition of elastase activity by test agents (oils at 1:10 dilution) was evaluated using the modified method of Okumura et al. (2007). The known inhibitors of proteinases namely, phenyl methyl sulphonyl fluoride (PMSF), ImM; ethylene diamine tetra acetic acid (EDTA), 5 mM; and metal ion [Mg.sup.2+] (Mg[SO.sub.4]), 1 mM; were also used in the study. In the assay, 200 (xl of test sample was mixed with 50 (xl of inhibitor solution and incubated at 37 [degree]C for 45 min before adding to elastin congo red substrate mixture. Further, procedure was followed as performed for elastase activity. Test sample without exposure to inhibitor was run as control. All the experiments were done in triplicate and the mean test absorbance value was subtracted from the mean untreated control absorbance value to obtain the percent reduction in elastase activity.

Detection ofkeratinase activity

Estimation of keratinase activity in test fungi was performed using the modified method of Muhsin and Aubaid (2000). In brief, 500 [micro]l of cell free supernatant was mixed with 50 mg of guinea pig hair in 5 ml of 0.03 M phosphate buffer (pH 7.8) and incubated at 37 [degree]C for 3 h, 150rpm. Test sample was replaced by buffer to run the control. Reaction mixture was stopped by adding 5 ml of 10% TCA and kept on ice for 30 min and centrifuged at 5000 rpm for 30 min. The hair was removed by filtration and the absorbance was read at 280 nm using a double beam UV-vis spectrophotometer (UVS504SS, India).

Effect of essential oils on keratinase activity

Inhibitory effect of test agents was evaluated by the method of Okumura et al. (2007) with some modifications, and employing known inhibitors of proteinases as used in elastase inhibition assays. In the assay, 400 [micro]l of test sample was mixed with 100 [micro]l of inhibitor solution and incubated at 37 [degree]C for 45min. The control was run without treating with inhibitors. Each experiment was performed in triplicate and inhibition of keratinase activity was calculated in terms of percent reduction in mean absorbance value of test sample compared to untreated control.

Results and discussion

All essential oils tested and their components exerted concentration dependent inhibitory effects on the production of fungal biomass (Table 1) and mycelial radial growth (Table 2) and were highly active even at lower concentrations (0.02-0.04% v/v). On the basis of ability to test fungi cinnamaldehyde was most inhibitory resulting in 95.30% reduction in the biomass and 98.11% reduction in the radial growth in I rubrum IOA-9 at 0.04% v/v. Similarly, 76.27% reduction in biomass and 80.74% in radial growth was recorded against A. fumigatus MTCC2550 at 0.08% v/v of cinnamaldehyde. The other test oils exerted antifungal activity in the order of eugenol > geraniol = C verum >citral>S. aromaticum > C citratus > C martini, against both the test fungi. Similar, antifungal activities of these oils and compounds are also reported by several other workers (Saikia et al. 2001; Wang et al. 2005; Cheng et al. 2008). Reference drug fluconazole also exhibited concentration dependent inhibition in biomass and radial growth of test fungi up to 84.89% and 87.08%, respectively at 200 [micro]g/ml (Table 3). A similar pattern of reduction in biomass and radial growth was also observed for other test fungi including A niger IOA-3, A flavus NRRL501, A solani MTCC2101 and Z rubrum MTCC296 (data not shown). Concentration of oils exhibiting > 90% reduction in biomass was comparable to minimum inhibitory concentration of these oils as determined by broth macrodilution method and described elsewhere (Khan and Ahmad 2011). These test oils were subjected to GC-MS analysis to determine the presence of major ingredients. As reported elsewhere (Khan and Ahmad 2011), cinnamaldehyde was the predominant component (79.10%) of C. verum. Geran-iol was the prominent constituent (50.74%) of C martini oil with geraniol acetate (19.21%) as the other major ingredient. Oil of S. aromaticum was predominately composed of eugenol (74.32%) and caryophyllenes (27.97%). In this study, GC and GC-MS analysis of C citratus revealed the presence of [alpha]-citral (48.0%) and [beta]-citraI (32%) as major active ingredients. It was observed that cinnamaldehyde and eugenol inhibited significantly (p = 0.05) both ungerminated and germinated conidia of A. fumigatus MTCC2550 and T. rubrurn IOA-9 at a concentration range from 0.005 to 0.08% v/v, as depicted in Fig. la and b. Cinnamaldehyde exhibited edge over eugenol in activity against both ungerminated and germinated conidia of these fungi at sub-inhibitory concentrations. Our findings are comparable with antifungal drugs such as amphotericin B, itraconazole and voriconazole inhibiting both ungerminated and germinated conidia in clinical isolates of A. fumigatus as reported by Manavathu et al.(1999).
Table 1

Concentration dependent inhibition of dry bio mass production in
A. fumigatus MTCC2550 and Z vubvum IOA-9 by essential oils and
active compounds.

Conc.(%v/v)    Percent inhibition in
               dry biomass over control

                 S.                    C.                C
             aromaticum               verum            martinl

                 AF          TR        AF       TR        AF

0.005        14.87 [+ or  23.56 [+  20.56 [+  19.25  12.89 [+
                 -] 1.37     or -]     or -]  [+ or     or -]
                              2.24      1.73     -]      1.11
                                               1.09

0.01         29.49 [+ or  31.86 [+  35.89 [+  37.25  27.32 [+
                 -] 2.56     or -]     or -]  [+ or     or -]
                              1.21      1.22     -]      1.21
                                               1.66

0.02         40.90 [+ or  44.54 [+  45.96 [+  49.12  42.29 [+
                 -] 2.54     or -]     or -]  [+ or     or -]
                              4.81      2.72     -]      4.86
                                               4.42

0.04          5831 [+ or  63.33 [+  69.02 [+  64.95  64.80 [+
                 -] 6.13     or -]     or -]  [+ or     or -]
                              2.71      5.39     -]      2.78
                                               4.04

0.08         72.62 [+ or  75.70 [+  97.50 [+  99.75  75.12 [+
                 -] 3.47     or -]     or -]  [+ or     or -]
                              2.80      6.92     -]      1.82
                                               3.25

0.16         87.63 [+ or  97.71 [+         -      -  85.47 [+
                 -] 1.90     or -]                      or -]
                              5.19                       1.22

0/32         99.32 [+ or         -         -      -  99.45 [+
                  -]0.47                                or -]
                                                         0.70

Conc.(%v/v)

                        C              Eugenol
                    martini

               TR        AF     TR        AF     TR

0.005        17.37  16.58 [+  12.83     12.91  19.47
             [+ or     or -]  [+ or      1.50  [+ or
                -]      1.07     -]               -]
              1.42             0.99             0.41

0.01         27.72  22.59 [+  23.53  29.39 [+  35.75
             [+ or     or -]  [+ or     or -]  [+ or
                -]      2.69     -]      2.51     -]
              1.53             2.35             3.47

0.02         42.54  35.28 [+  36.18  40.56 [+  52.23
             [+ or     or -]  [+ or     or -]  [+ or
                -]      3.21     -]      3.16     -]
              4.03             2.62             4.43

0.04         62.66  52.27 [+  56.73  55.78 [+  72.37
             [+ or     or -]  [+ or     or -]  [+ or
                -]      2.18     -]      1.18     -]
              5.64             5.18             5.19

0.08         75.27  72.04 [+  76.85  68.75 [+  94.65
             [+ or     or -]  [+ or     or -]  [+ or
                -]      4.06     -]      6.49     -]
              3.30             3.36             2.50

0.16         94.03  95.90 [+  94.97  81.42 [+    100
             [+ or     or -]  [+ or     or -]
                -]      2.47     -]      1.76
              3.55             2.07

0/32             -         -      -  99.24 [+      -
                                        or -]
                                         0.64

Conc.(%v/v)

             Cinnamaldehyde         Citral         Geraniol

                   AF          TR       AF    TR       AF       TR

0.005        31.29 [+ or -]  23.30   24.51  17.90  26.65 [+  14.07
                       2.96  [+ or   [+ or  [+ or     or -]  [+ or
                                -]      -]     -]      1.98     -]
                              1.62    2.45   1.27             1.27

0.01         48-21 [+ or -]  46.77   35.88  33.55  36.45 [+   31.7
                       3.90  [+ or   [+ or  [+ or     or -]  [+ or
                                -]      -]     -]      1.83     -]
                              4.10    2.19   3.36             1.10

0.02         60.43 [+ or -]  78.95   47.54  54.44  48.13 [+  43.08
                       4.50  [+ or   [+ or  [+ or     or -]  [+ or
                                -]      -]     -]      4.34     -]
                              5.90    2.08   4.28             2.81

0.04         76.27 [+ or -]  95.30   59.37  69.02  61.71 [+  67.53
                       3.59  [+ or   [+ or  [+ or     or -]  [+ or
                                -]      -]     -]      6.45     -]
                              3.99    4.68   5.50             4.07

0.08         98.35 [+ or -]      -   71.79  82.27  77.62 [+  94.12
                       2.98          [+ or  [+ or     or -]  [+ or
                                        -]     -]      5.52     -]
                                      2.29   2.02             3.11

0.16                      -      -   86.02    100       100      -
                                     [+ or
                                        -]
                                      1.40

0/32                      -      -   98.42      -         -      -
                                     [+ or
                                        -]
                                      1.40

All the experiments were performed in triplicate and data are
presented as mean [+ or -] SD.

AF - A. fumigatus MTCC2550.

TR - T. rubrum IOA-9.

(-) No biomass.
Table 2

Concentration dependent inhibition of mycelial radial growth in
A. fumigatus MTCC2550 and T, rubntm IOA-9 by essential oils and
active compounds.

Cone.     Percent reduction in
(%v/v)    mycelial radial
          growth over control

            S.                 C.                 C.
        aromaticum           ventm             citratus

            AF         TR      AF       TR        AF       TR

0.005   18.90 [+ or   16.26   15.37  16.34 [+  11.00 [+   13.20
            -] 1.35   [+ or   [+ or     or -]     or -]   [+ or
                         -]      -]      1.75      1.58      -]
                       1.95    1.34                        1.69

0.01    26.71 [+ or   26.66   26.23  34.44 [+  18.75 [+   24.92
            -] 1.18   [+ or   [+ or        or     or -]   [+ or
                         -]      -]    -]3.20      1.27      -]
                       2.47    3.22                        1.07

0.02    36.89 [+ or   33.66   36.07  49.29 [+  33.04 [+   39.03
            -] 1.94   [+ or   [+ or        or     or -]   [+ or
                         -]  -]2.11    -]3.68      2.86      -]
                       1.83                                2.93

0.04    51.70 [+ or   49.06   44.37  64.73 [+  46.39 [+   53.38
            -] 4.14   [+ or   [+ or        or     or -]   [+ or
                         -]      -]    -]4.09      4.92      -]
                       4.00    2.00                        1.58

0.08    66.38 [+ or   66.10   85.04  81.33 [+  66.47 [+   67.87
            -] 2.81   [+ or   [+ or        or     or -]   [+ or
                         -]      -]    -]2.72      5.53  -]2.18
                       3.04    2.18

0.16    80.41 [+ or   77.73   97.10   91.10[+  81.04 [+   80.67
            -] 1.16   [+ or   [+ or        or     or -]   [+ or
                     -]1.31      -]    -]2.07      1.57      -]
                               1.63                        3.60

0.32    90.78 [+ or   97.48       -       100  87.97 [+   97.00
            -] 1.90   [+ or                       or -]   [+ or
                         -]                        2.98      -]
                       1.77                                2.94

Cone.
(%v/v)

            C              F.ugenol         Cinnamaldehyde
        martini
           AF       TR        AF     TR           AF        TR

0.005   13.03 [+  13.46  16.26 [+  18.44  21.71 [+ or -]  21.42
              or  [+ or  or -].63  [+ or            1.18  [+ or
          -]2.12     -]               -]                     -]
                   1.65             1.17                   1.18

0.01    21.00 [+  22.13  28.60 [+  31.55  39.76 [+ or -]  34.04
           or -]  [+ or     or -]  [+ or            1-66  [+ or
            1.59     -]      0.99     -]                     -]
                   2.03             1.83                   3.52

0.02    33.40 [+  32.67  36.07 [+  50.38  53.39 [+ or -]  55.00
           or -]  [+ or     or -]  [+ or            3.60  [+ or
            2.40     -]      2.09     -]                     -]
                   2.39             4.11                   4.08

0.04    45.20 [+  56.41  46.40 [+  76.70  69.42 [+ or -]  82.38
           or -]  [+ or     or -]  [+ or            1.22  [+ or
            3.47     -]      1.97     -]                     -]
                   2.55             4.83                   3.38

0.08    62.39 [+  75.13  61.27 [+  91.11  80.74 [+ or -]  98.11
           or -]  [+ or     or -]  [+ or            3.21  [+ or
            5.39     -]      2.65     -]                     -]
                   3.30             2.23                   2.23

0.16    86.44 [+  86.41  78.04 [+    100  99.23 [+ or -]      -
           or -]  [+ or     or -]                   1.08
            5.82     -]      1.49
                   2.35

0.32    98.26 [+  98.36  90.67 [+      -               -      -
           or -]  [+ or     or -]
            2.53     -]      1.18
                   1.21

Cone.
(%v/v)

        Citral          Geraniol
          AF      TR       AF        TR

0.005    15.89   15.16  21.73 [+   12.04[+
         [+ or   [+ or     or -]     or -]
        -]0.79      -]      1.15      1.37
                  1.13

0.01     27.96   28.28  31.70 [+  22.89 [+
         [+ or   [+ or        or     or -]
            -]      -]    -]2.57      1.34
          3.58    1.06

0.02     43.04   54.86  40.79 [+  36.70 [+
         [+ or   [+ or     or -]     or -]
            -]      -]      2.20      2.33
          2.79    4.26

0.04     53.08   72.03  51.42 [+  64.70 [+
         [+ or   [+ or     or -]        or
            -]  -]4.12      4.38    -]4.10
          3.49

0.08     67.09   85.35  65.26 [+  85.07 [+
         [+ or   [+ or     or -]     or -]
            -]      -]      2.94      3.64
          2.89    3.57

0.16     76.06   98.33  82.74 [+  98.33 [+
         [+ or   [+ or     or -]     or -]
            -]      -]      3.21      1.69
          2.10    1.69

0.32     88.08    --98.66 [+         -
         [+ or             or -]
            -]              1.24
          3.16

All the experiments were performed in triplicate and data are
presented as mean [+ or -]SD.

AF - A. fumigatus MTCC2550.

TR - T. nibmtn IOA-9.

(-) No radial growth.

Table 3

Concentration dependent inhibition of fungal biomass production and
radial growth in A. fumigutus MTCC2550and T. nihntm IOA-9 by
fluconazole.

Conc.([micro]g/ml)  Percent inhibition in           Percent
                      dry biomass over             reduction
                           control                in mycelial
                                                 radial growth
                                                  over control

                        A. fumigatus         T.        A.         T.
                          MTCC2550         rubrum  fumigatus  rubntm
                                           IOA-9   MTCC2550   IOA-9

1.25                  12.32 [+ or -] 1.81   11.41   10.56 [+   12.34
                                            [+ or  or -]1.81   [+ or
                                               -]                 -]
                                             3.24               2.69

2.5                   21.29 [+ or -] 2.56   21.26   17.25 [+   19.92
                                            [+ or      or -]   [+ or
                                               -]       1.27      -]
                                             1.41               3.41

5                     30.90 [+ or -] 2.54   44.54   23.04 [+   29.03
                                            [+ or      or -]   [+ or
                                               -]       2.86      -]
                                             2.81               4.93

25                    54.31 [+ or -] 6.13   53.33   36.39 [+   53.38
                                            [+ or      or -]   [+ or
                                               -]       2.92      -]
                                             3.41               4.58

50                    66.62 [+ or -] 3.47   65.70   66.47 [+   61.87
                                            [+ or      or -]   [+ or
                                               -]       4.53      -]
                                             1.81               6.18

100                   77.63 [+ or -] 1.90   77.71  71.04  [+   70.67
                                            [+ or      or -]   [+ or
                                           -]4.16       3.57      -]
                                                                5.60

200                   85.32 [+ or -] 0.47   84.89   87.08 [+   86.00
                                            [+ or      or -]   [+ or
                                               -]       2.98      -]
                                             3.19               4.64

All the experiments were performed in triplicate and data are
presented as mean [+ or -] SD.


[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Furthermore, the concentration and time dependent toxic effects of oils C verum and S. aromaticum were evaluated against the test fungi under light microscopy. As shown in Fig. 2a-d, the treatment with 0.02% v/v C verum resulted in the formation of chlamydoconidia and also, autolysis of the hyphal cytoplasm in 20 h in T. rubrum IOA-9. Formation of chlamydospores is considered as an indicator of stress conditions produced in the presence of oils. Several morphological alterations such as hyphal shrinkage, autolysis of cytoplasm and necrosis occurred in 24 h with the treatment of 0.08% v/v S. aromaticum. Similar morphological effects by these oils were also observed for A. fumigatus MTCC2550 (data not shown).

Further, to explore the possible mechanism of interaction of essential oils with fungal cell wall, membrane and cellular content, the strains of A. fumigatus MTCC2550 was subjected to electron microscopic studies after treatment with sub-inhibitory concentration (0.04% v/v) of cinnamaldehyde. As clearly evident from SEM analysis, the healthy and continuous hyphae are produced in control sample (Fig. 3a), whereas treated sample exhibited unusual pattern of hyphal growth as well as alterations in cell shapes and sizes such as blistering and necrosis of hyphae (Fig. 3b-d). Severely collapsed and squashed hyphae were evident due to the lack of cytoplasm. Similar effects of essential oils on the hyphal morphology of the plant pathogenic fungi have also been reported by other authors (De Billerbeck et al., 2001; Soylu et al. 2006). These changes in hyphal morphology could be related to the loss of integrity of cell wall and, therefore, it could be postulated that oils are interfering with the synthesis of cell wall directly or indirectly.

[FIGURE 3 OMITTED]

Additionally, the ultrastructural alterations in fungal cells were observed under TEM. In untreated sample, organelles such as nuclei, mitochondria and nucleus are appeared to be normal (Fig. 4a). Treated sample exhibited several changes including stretching of cell membrane, expansion of endoplasmic reticulum (Fig. 4b), leakage of cell wall and cell membrane (Fig. 4c). Due to the abnormal distribution of polysaccharides deterioration of cytoplasmic contents was also observed (Fig. 4d). These observations find support from the findings of ultrastructural changes as reported in T. rubrum by onion extracts (Ghahfarokhi et al. 2004) and in A. niger by oil of Thymus sp. (Rasooli et al. 2006). It is believed that lipophilic properties of oils may assist in penetration of cell membrane and, accumulation of polysaccharides in stress condition may lead to rupture of plasmalema in fungal cells. It is speculated that such kinds of adverse effects by oils on fungal hypha or cell are responsible for a decrease in the rate of conidial and mycelial growth, biomass production and morphogenesis. The ultrastructural analysis has highlighted the multiple sites of action of oils in fungal cells including damages to the cell walls, cell membranes and cytoplasmic contents.

[FIGURE 4 OMITTED]

A distinctive feature of compounds acting on the fungal cell wall is that the antifungal effect can be reversed in a medium containing an osmotic stabilizer such as sorbitol. Therefore, MIC of cinnamaklehycle, eugenol and fluconazole was evaluated against A fumigatus MTCC2550 in the presence of sorbitol. MIC of eugenol and cinnamaldehyde was increased up to two and four-folds, respectively (Table 4). This increase in MIC is not enough to conclude the cell wall as target site by these compounds. Therefore, it may be suggested that primary target of these compounds would not be the cell walls. However, the morphological changes observed in the treated fungi lead to suggest that fungal membrane could be the target for them. Nevertheless, further experiments such as absorbance of cytoplasmic content leakage, effect on ergosterol biosynthesis or activity are needed to ensure the mode of action of these compounds.
Table 4

MIC of cinnamaidehyde. eugenol and fluconazole against A.
fumimtits MTCC2550 in the absence and presence of sorbitol.

Tesi agents          5 days                 10 days

                -Sorbitol  +Sorbitol  -Sorbitol  +Sorbitol

Cinnamaidehyde     0.08       0.08        0.08       0.32
Eugenol            0.32        032        0.32       0.64
Fluconazole         200        200         200        400

MICs of oils and fluconazole are expressed in % v/v and
[mu]g/ml. respectively.


Recent trends in screening or plant products tor their anti-virulent activity against fungi prompted us to evaluate these test essential oils for their anti-pathogenic efficacy. Therefore, inhibition of elastase and keratinase activities in A. fumigatus and T, rubrum was determined by test oils. Production of elastases and keratinases are reported to aid in the pathogenesis of Aspergillus sp. and Trichpohyton sp. (Okumura et al. 2007; Vermout et at. 2008) and, therefore proteinase inhibitors are explored as potential antifungal agents. Test fungi A. fumigatus MTCC2550, A. flavus NRRL501, A. solani MTCC2101 A. niger IOA-3, T. rubrum IOA-9 and T. rubrum MTCC296 were assessed for the production of elastase and keratinase enzymes. Strains A. niger IOA-3 and T rubrum IOA-9 showed relatively higher production of elastase and keratinase, respectively in the detection assays. Therefore, oils were tested for their anti-elastase and anti-keratinase activity against these two strains only. As shown in Table 5, cinnamaldehyde showed highest reduction (95.56%) over untreated control in elastase activity followed by C. martini (94.56%) and C. verum (93.67%). Highest inhibition (97.31 %) over untreated control in keratinase activity was recorded for geraniol followed by C martini (77.46%) and citral (57.18%). Among the known inhibitors tested. Mg[S0.sub.4] showed 94.10% and 39.77% inhibition in elastase and keratinase activities, respectively. Similarly. EDTA exhibited inhibition by 72.63% and 54.48% and PMSF by 70.06% and 5.70%. Since proteinases contribute to fungal virulence by destroying host tissues and digesting immunologically important proteins such as antibodies and complement factors (Santos et al. 2007), the inhibition of these enzyme activities may reduce the pathogenesis of fungi. Therefore, the broad spectrum anti-proteinase activities of the test oils in addition to growth inhibition are indicative of their efficacy as potential antifungal drugs.
Table 5

Effect of essential oils and active compounds on elastase and keratinase
activities in A. tliger IOA-3 and t mbtvm IOA 9. respectively.

LTest agents    % reduction in OD at 495 nm  % reduction in OD At
                 over control for elastase   280mil over control
                          activity                for keratinase
                                                     activity

Essential
oils/active
compounds

S. arotnaticnm          68.52 [+ or -] 2.08    3.97 [+ or -] 1.16

C ventm                 93.67 [+ or -] 1.90    5.37 [+ or -] 2.18

C Citrfflm              56.24 [+ or -] 1.99   13.76 [+ or -] 1.01

C. martini              94.56 [+ or -] 0.84   77.46 [+ or -] 1.34

Eugeno!                 77.95 [+ or -] 2.20    9.67 [+ or -] 1.61

Cinnamaldehyde          96.56 [+ or -] 1.09   11.90 [+ or -] 2.22

Citral                  29.59 [+ or -] 0.50   57.18 [+ or -] 1.74

Geraniol                84.54 [+ or -] 2.63   97.31 [+ or -] 0.59
Inhibitors

PMSF                    70.06 [+ or -] 1.61    5.70 [+ or -] 0.95

EDTA                    72.63 [+ or -] 1.26   54.48 [+ or -] 2.20

Mg[SO.sub.4]            94.10 [+ or -] 1.63   39.77 [+ or -] 1.06

All (lie experiments were performed in triplicate and data are
presented as mean [+ or -] SD.


Conclusion

Many plant products have been used as antifungal agents in eth-nomedicine but their mode of action and anti-pathogenic effects arc yet to be explored. Present report draws attention about the in vitro antifungal activity of eight oils especially eugenol and cin-namaldehyde against A. fumigatus and T. rubrum. Further, site of action of cinnamaldehydc primarily appears not to be the fungal cell wall. However, the fungal malformations observed in treated fungi suggest that the fungal membrane could be the target for this compound although new evidences are needed to corroborate this mode of action. In addition, inhibition of elastase and keratinase activities in A. fumigatus and T. rubrwv by test oils indicates their potential utilization as antifungal agent in the treatment of aspergillosis and dermatophytosis. However, In vivo evaluation is needed to develop these oils as suitable antifungal drugs for combating such mycoses.

Acknowledgements

We are grateful to electron microscopy division at Sophisticated Analytical Instrument Facility, All India institute of Medical Sciences, New Delhi, for assisting in SEM and TEM analysis of test fungi. We are also thankful to University Grants Commission and also Indian Council of Medical Research, New Delhi, for financial assistance.

0944-71 13/$ - see front matter [C] 2011 Elsevier GmbH. All rights reserved. doi: 10.1016/j.phy med.2011.07.005

doi: 10.1016/j.phy med.2011.07.005

References

Bajpai, V.K.. Yoon, J.I.. Kang, S.C, 2009. Antifungal potential of essential oil and various organic extracts of Nandina domestica'Yhunb against skin infectious fungal pathogens. Appl. Microbiol. Biotechnol. 83, 1127-1133.

Barker, K.S., Rogers, P.O., 2006. Recent insights into the mechanisms of antifungal resistance. Cure Infect. Dis. Rep. 8, 2816-2823.

Cavaleiro. C, Pinto, E., Goncalves, M.J., Salgueiro. L. 2006. Antifungal activity of Juniperus essential oils against dermatophyte. Aspergillus and Candida strains. J. Appl. Microbiol. 100, 1333-1338.

Cheng, S., Iiu, J., Chang, L. Chang. S., 2008. Antifungal activity of cinnamaldehyde and eugenol congeners against wood-rot fungi. Bioresour. Technol. 99, 5145-5149.

Dagenais, T.R.T.. Keller. N.P., 2009. Pathogenesis of Aspergillus fumigatus in invasive aspergillosis. Clin. Microbiol. Rev. 22, 447-465.

Davies, N.W.. 1990. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone and carbowax 20 M phases. J. Chromatogr. 503, 1-24.

De Billerbeck, V.G.. Roques. C.G.. Bessiere. J.M.. Fonvieille, J.L, Dargent. R.. 2001. Effect of Cymbopogon aardus (L) W. Watson essential oil on the growth and morphogenesis of Aspergillus niger. Can. J. Microbiol. 47, 9-17.

De Lucca, A.J., Bland. J.M., Jacks, T.J., Grimm, C. Cleveland, T.E, Walsh, T.J.. 1997. Fungicidal activity of cccropin A. Antimicrob. Agents Chemother. 41, 481 -483.

Frost, D.J., Brandt. K.D.. Cugier, D.. Goldman, R.. 1995. A whole-cell Candida albicans assay for the detection of inhibitors towards fungal cell wall synthesis, and assembly. J. Antibiot. 48, 306-310.

Gauwerky, K., Borelli, C, Korting, H.C., 2009, Targeting virulence: a new paradigm for antifungals. Drug Discov. Today 14, 214-222.

Ghahfarokhi, M.S., Goodarzi, M., Abyaneh, M.R., Al-Tiraihi, T., Seyedipour, G., 2004. Morphological evidences for onion-induced growth inhibition of Trichophyton rubrum and Trichophyton mentagrophytcs. Fkoterapia 75, 645-655.

Khan, M.S.A., Ahmad, I., 2011. Antifungal activity of essential oils and their synergy with fluconazole against drug-resistant strains of Aspergillus fumigatus and Trichophyton rubrum. Appl. Microbiol. Biotechnol. 90,1083-1094.

Kothary, M.H., Chase Jr., T., Macmillan, J.D., 1984. Correlation of elastase production by some strains of Aspergillus fumigatus with ability to cause pulmonary invasive aspergillosis in mice. Infect, lmmun. 43, 320-325.

Manavathu, EX, Cutright, J., Chandrasekar, P.H.. 1999. Comparative study of susceptibilities of germinated and ungerminated conidia of Aspergillus fumigatus to various antifungal agents. J. Clin. Microbiol. 37, 858-861.

Muhsin, T.M., Aubaid, A.H., 2000. Partial purification and some biochemical characteristics of exocellular keratinase from Trichophyton mentagrophytcs var. erinacei. Mycopathologia 150, 121-125.

Okumura. Y., Ogawa, IC, Uchgiya, K., Nikai, T., 2007. Characterization and primary structure of elastase inhibitor, AFLEI from Aspergillus /tovus. Jpn. J. Med. Mycol. 18, 13-18.

Quiroga, E.N., Sampietro, A.R., Vattuone, M.A., 2004. In vitro fungitoxic activity of Larrea divaricata cav. extracts. Lett. Appl. Microbiol. 39, 7-12.

Rasooli, I., Rezaei, M.B., Allameh, A., 2006. Growth inhibition and morphological alterations of Aspergillus niger by essential oils from Thymus erioca/yx and Thymus x-porlock. Food Control 17, 359-364.

Sachar, LA, Winter, KX, Sicher, N., Frankel, S., 1955. Photometric method for estimation of elastase activity. Proc. Soc. Exp. Biol. Med. 90, 323-326.

Saikia, D.r Khanuja, S.P.S., Kahol, A.P., Gupta, S.C., Kumar, SM 2001. Comparative antifungal activity of essential oils and constituents from three distinct genotypes of Cymbopogon spp. Curr. Sci. 80, 1264-1266.

Santos, A.L.S., Palmeira, V.F., Rozental, S., Kneipp, L.F., Nimrichter, L, Alviano, D.S., Rodrigues, M.L, Alviano, C.S., 2007. Biology and pathogenesis of Fonsecaeaa pedrosoi, the major etiologic agent of Chromoblastomycosis. FEMS Microbiol. Rev. 31, 570-591.

Shafique, S., Shafique, S., Bajwa, R., Akhtar, N., Hanif, S.f 2011. Fungitoxic activity of aqueous and organic solvent extracts ofTagetes erectus on phyioparhogenic fungus - Ascochyta rabid Pak. J. Bot. 43, 59-64.

Soylu, M.E., Soylu, S., Kurt, S., 2006. Antimicrobial activities of the essential oils of various plants against late blight disease agent P/iyrop/irhora infestans. Mycopathologia 161, 119-128.

Venkatesan, G., Singh, AJAR., Murugesan, A.G., Janaki, C, Shankar, S.G., 2007. Trichophyton mbivm - the predominant etiological agent in human dermatophytosis in Chennai, India. Afr.J. Microbiol. Res. 1, 9-12.

Vermont S., Tabart, J.t Baldo, A., Mathy, A., Losson, B., Nignon, B., 2008. Pathogenesis of dermatophytosis. Mycopathologia 166, 267-275.

Voltan, A.R., Donofrio, F., Miranda, E.T., Moraes, R.A., Mendes-Giannini, M.J.S., 2008. Induction and secretion of eiascfnoiytic and proteolytic activity in cultures of Paracoccidioides brasiliensis.], Basic Appl. Pharm. Sci. 29, 97-106.

Wang, S., Chen, P., Chang, S., 2005. Antifungal activities of essential oils and their constituents from indigenous cinnamon (Cinnamomum osmophloeum) leaves against wood decay fungi. Biores. Techno). 96, 813-818.

Zacchino, S., Lopez, S., Pezzcnati, C, Furlan, R., Santecchia, C, Munoz, L, Giannini, F., Rodriguez, A., Enriz, R., 1999. In vitro evaluation of antifungal properties of phenylpropanoidsand related compounds acting against dermatophytes.J. Nat. Prod. 63, 1353-1357.

Mohd Sajjad Ahmad Khan, Iqbal Ahmad *

Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University. Aligarh 202002, India

* Corresponding author at: Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh 202002, India.

E-mail address: ahmacliqba18@yahoo.co.in (I Ahmad).
COPYRIGHT 2011 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2011 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Khan, Mohd Sajjad Ahmad; Ahmad, Iqbal
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Dec 15, 2011
Words:6596
Previous Article:Antifungal activity of Coriandrum Sativum essential oil, its mode of action against Candida species and potential synergism with amphotericin B.
Next Article:Comparison between allicin and fluconazole in Candida albicans biofilm inhibition and in suppression of HWP1 gene expression.
Topics:

Terms of use | Copyright © 2017 Farlex, Inc. | Feedback | For webmasters