الفهرس 
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Abstract
The present study aimed to evaluate In vitro antifungal activities of
some essential oils against some clinically important filamentous fungi
and to explore the mechanism of action of most active essential oil
ingredients on the morphological changes of the most susceptible
filamentous fungus using transmission electron microscope.
Invasive fungal infections treated mostly with antifungals such as
Fluconazole, Itraconazole and Voriconazole, but the long use of these
antifungals can cause liver dysfunction, toxicity and increase the
resistance to fungi due to the extensive empirical use of antifungals.
These emerging fungi are characterized by resistance or lower
susceptibility to standard antifungal agents. Many researchers
recommended the use of plants essential oil as a valuable potential
source of antimicrobials of natural origin. Essential oils have been
reported to have antibacterial, antifungal, antiviral and ant parasitic. as a
potential valuable source of antimicrobials of natural origin.
The pathogenic fungal strains were kindly provided by Ain Shams
University Hospitals. Aspergillus flavus (AY373848), Alternaria
alternate (MH594503), Penicillium chrysogeum (AY373926)
Aspergillus terreus (HQ219673), Aspergillus niger (AY585554) and
Aspergillus fumigatus (KF201647).
Essential oils were purchased from Sekem national company they
include twelve essential oils, Melaleuca alternifolia (Tea tree oil),
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Pogostemon cablin (Patchouli oil), Eugenia caryophyllus (Clove oil),
Cymbopogon nardus (Citronella oil), Lavandula officinalis (Lavender
oil), Salvia officinalis (Sage oil), Cymbopogon citratus (Lemongrass
oil), Ocimum basilicum L. (Basil oil), Thumus vulgaris (Thyme oil),
Rosmarinus officinalis (Rosemary oil), Cinnamomum zeylanicum
(Cinnamon oil) and Mentha piperita (Peppermint oil).
Three synthetic antifungal drugs were used in this study
(Fluconazole 150 mg capsule, Itraconazole 100 mg capsule and
Voriconazole 200 mg tablet), were purchased from pharmacy.
The results of this work can be summarized in the following points:
1) Results of well diffusion assay showed that, all essential oils have
variable antifungal effect against all of the tested fungal strains
except sag oil showed no activity against all strains.
2) At concentration 100%, thyme, cinnamon and clove essential oils
completely inhibited the growth of all 6 fungal strains, while the
rest of essential oils such as tea tree, lavender, citronella, basil,
peppermint oil showed variable antifungal effect against the
selected fungal strains.
3) Regarding 75% concentration of essential oils, also thyme,
cinnamon and clove essential oils completely inhibit the growth of
all strains, while the rest other essential oils such as tea tree,
lavender, citronella, basil, peppermint oil showed good antifungal
effect against selected strains especially against Alternaria
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alternata. However, the rest essential oils such as, rosemary,
lemongrass and patchouli exhibited low antifungal effect
4) The results of 50% concentration of essential oils revealed that,
thyme, cinnamon and clove oils showed high antifungal activities
against all 6 selected strains. While the rest of essential oils showed
variable antifungal effect against the tested fungal strains.
5) At 25% concentration, all tested essential oils lost their antifungal
activity.
6) Regarding the synthetic antifungal drugs, the result revealed that
fluconazole showed resistance against all 6 selected fungal strains.
7) Itraconazole showed high antifungal activity against the 6 selected
fungal strains at concentration 20 and 15 µg/ml while at
concentrations, 10 and 5 µg/ml showed no antifungal effect against
all selected strains.
8) Voriconazole showed high antifungal effect against the 6 selected
fungal strains at concentrations, 20, 15, 10 µg/ml while at 5 µg/ml
concentrations, it showed no antifungal effect against the selected
fungal strains.
9) Thyme was the most effective essential oils against all tested fungal
strains especially against Aspergillus flavus (AY373848) with
inhibition zone diameter of (39mm) at 50% concentration of thyme
oil.
10) characterization of the active chemical compounds of thyme oil
using Gas chromatography- Mass Spectrometry
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(GC/Mass), revealed that thyme oil comprises 30 active
ingredients, The major components of thyme oil were
a: Thymol, the most abundant ingredient with area percent
(47.53%) at retention time 19.59 minutes. Followed by,
b: γ-terpinene with area percent (19.1%) at retention time 13.3
minutes. Followed by,
c: P-Cymene with area percent (13.12%) at retention time 14.12
minutes. Followed by,
d: Linalool with area percent (11.3%) at retention time 18.51
minutes. And,
e: Carvacrol with area percent (3.12%) at retention time 21.91
minutes.
11) Fractionation of the active compounds of thyme oil by thin layer
chromatography TLC gave five bands with different Rf (retention
factor) readings as follow, γ-terpinene, linalool, thymol, carvacrol
and p-cymene with retention time of (0.97), (0.88), (0.65), (0.33)
and (0.25) respectively.
12) The antifungal activity of the 5 fractions against the most sensitive
strain, Aspergillus flavus (AY373848), revealed that thymol gave the
highest antifungal activity with inhibition zone (44mm), followed by
p-cymene with inhibition zone (11mm) and followed by p-cymene
with inhibition zone (7mm), while γ-terpinene and linalool showed
inhibition zone (6mm) negative antifungal activity.
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13) The chemical characterization of thymol using gas chromatographymass spectrometry verified its purity with the presence of one peak
of retention time 19.24. It was identified as (2-isopropyl-5-
methylphenol), with chemical formula (C10H14O), molar mass of
(150)
14) Results of broth microdilution assay of thymol, voriconazole and
thyme oil against A. flavus (AY373848), revealed that MIC value
of 0.125 µg/ml of thymol was lower than voriconazole and thyme
oil with MIC value of (0.25 µg/ml)
15) The result of transmission electron microscope revealed that,
thymol affected greatly A. flavus (AY373848) cell morphology
which became irregular with dissymmetric distribution of
cytoplasm, extensive vacuolization of the cytoplasmic matrix
and structural damage of mitochondria, nucleus and other cell
organelles Also, the plasma membrane become thinner and more
damaged leading to fungal cell death.