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E3 Journal of Microbiology Research Vol. 2(1). pp.009-013, January, 2014
Available online @ http://www.e3journals.org
© E3 Journals 2014
Full length research paper
Comparing the effect of ozonized olive oil with
clotrimazole on three Candida species C. albiacans, C.
glabrata, C. krusei
Fariba Berenji1*,Omid Rajabi2,Maryam Azish3,Nazanin Minoochehr2
1. Department of Parasitology & Mycology, Medical Faculty, Mashhad University of Medical Sciences, Mashhad, Iran
2 Department of Medicinal Chemistry. Mashhad University of Medical Sciences, Mashhad, Iran
3 Department of Medical Mycology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Accepted January 22, 2014
One of the most important pathogenic fungi in immune compromised patients is Candida.spp. Ozone is an
allotropic form of oxygen with high oxidation power; in addition it has fungicidal effects. Considering highly
prevalence of Candida infections and drug resistance, it is important to find a low cost medicine with high
effects and with low adverse effects for treating these kinds of infections. In this study, the effect of ozonated
olive oil compared with clotrimazole on three species of candida (C. albicans, C. glabrata, and C. krusei) on
saboraud dextrose agar (SDA) media was evaluated.Different concentrations of ozonated olive oil (166.66, 200,
233.33, 266.66, 300 mg/ml) in culture media were prepared and poured in some plates separately. Plates without
ozonated olive oil were used as negative control. Plates containing different amount of clotrimazole (1, 2, 3, 6,
and 8µg/ml) were considered as positive control. After inoculation of different candida spp. in all media, the
plates were incubated at 37˚C for 72 hours and observed for fungal growth in their status every 24 hours. Our
study showed that the minimum inhibitory concentration (MIC) of ozonated olive oil for Candida krusei was
166.66, and for Candida albicans and Candida glaberata were 233.33, 200 mg/ml respectively. Clotrimazole
inhibited all candida species in concentration much lower than ozonated olive oil. Considering that the ability of
ozonated olive oil to inhibit candida growth in the media, authors hope that future researches will be performed
based on this study and it can be a new product for topical treatment of candidiasis.
Keywords: Candida albicans; Candida krusei; Candida glabrata; Olive oil; ozone; Clotrimazole.
Introduction
Candidiasis is a primary and secondary infection which is
caused by Candida species especially Candida albicans.
Its clinical symptoms are acute, sub acute or chronic and
sporadic. Infection may be limited to mouth, throat, skin,
vagina, fingers, nails, trachea, lung or digestion system. It
may cause septicemia, endocarditis, meningitis,
systemically. Since Candida species are endogenous the
resultant disease demonstrates as an opportunist
infection (Zeini et al., 2009). More than 90% individuals
with defect in immune system are affected to oral
candidiasis and almost 75% of women are affected at
least one time to vaginal candidiasis in her life.
*Corresponding Author Email: fberenji@yahoo.com
Approximately 60% of healthy individuals and more than
65% of children are affected to candida infections without
clinical symptoms (Ying et al., 2010). Clotrimazole is an
antifungal medicine from azole group, which blocks
ergostrol synthesis by suppressing fungus demetylase
cytochrome enzyme and causes growth stop with
effecting on its membrane (Mahmoudabadi, 2002).Ozone
is a very strong oxidization material and it is mainly
applied as a disinfectant in different fields. Therapeutic
ozone is presented in National Institutes of Health (NIH)
in the United States as a method in supplementary
medicine and also it is stated that excessive use for
ozone can be poisonous like other medicines (Viebahn,
1985). Different ozonized oils are used successfully
against different infections (Geveely, 2006). One of these
materials is olive oil that in many countries, ozonized
010 E3J Microbiol.Res.
olive oil is used because of therapeutic effects and
efficient antimicrobial activity against bacteria, virus and
fungi (Lezcano et al., 2000, Sechi et al., 2001). Ozone
with oxidization properly can convert olive oil to strong
oxidizing material. Alvarez and Gundarova have reported
safety of this material (Gundarova et al., 1996; Alvarez et
al., 1997). Generally, excessive use of antibiotics for
treating infectious diseases and appearing fungus strains
resistance to some medicines along with inefficiency and
side effects, driven the research towards the comparing
therapeutic effects of ozone with clotimazole. (Shaschova
, 1995). In this study antifungal effect of ozonized olive oil
studied on three Candida species, including Candida
albicans, Candida glabrata and Candida krusei on
sabouraud dextrose agar medium.
Materials and Methods
Ozone apparatus
Ozone was produced by controlled oxygen flow in zone
generator device (made in the USA with production
power 13.5 g/h).
Ozoning olive oil
This procedure is elaborated by ozone generator device.
In this method, ozone is passed as bubbling through olive
oil for three weeks and olive oil is become ozonic cream
gel material. This material is maintainable in environment
temperature for three years and it is melted in higher
temperature and it is returned by dropping temperature
(Geveely, 2006).
Standardization of the preparation was carried out
according to the following parameters: olive oil Acid
Index determination
Base on definition, Acid Index, is value KOH in term of
gram which is necessary to neutralize free fatty acid in
one gram of fat. To determine the Acid Index of olive oil,
10 g of weighed olive oil transmit to an erlen containing
50 ml of alcohol and ether, then add 1 ml of
phenolphethalein this mixture and titrate it to normal
hydroxide potassium 0.1 until the mixture is become
colorless pink to stabilize for 30seconds, indicating
titration end. Acid Index with used alkali mass neutralize
free fatty acid in 10 g olive oil with this formula is
achieved:56.11V× N/W Where, N is hydroxide potassium
normality, and W is tested sample mass, and V is alkali
acid and 56.11 is the atomic mass of hydroxide
potassium.
Olive oil Iodine Index determination
Base on definition, Iodine Index, is value Iodine base on
gram which is absorbed by 100 g of fatty sample. This
index indicates dual links numbers in test sample. In fats
with non saturated fatty acid, oxidation occurs in dual
links, this cause to produce hydroperoxide and
consequently forming aldehyde. To determine olive oil
Iodine Index, 1 g of weighed olive oil was transferred to
flask 250 ml and it was solved by chloroform 19 ml, and
then 250 ml of normal iodobromide 0.2% was added to
mixture. After closing the cap of flask, it was put in dark
place for 30 minutes and the flask was shaken one time
per 10minutes. Then 30ml of potassium iodine 15% and
100 ml of water were added to the materials the mixture
was titrated by normal thiosulphate 0.1% when the color
changes to colorless pink, 3 ml of amylum solution was
added to the materials and titration was continued until
blue color was appeared. This work was also performed
simultaneously on blank sample then Iodine Index
achieved by this formula:[126.9 (Vb –Vs) N]/10W. Where,
126.9 is Iodine atomic weight, Vb, Vs are normal
thiosulphate sodium 0.1, N is thiosulphate sodium
normality and W is olive oil weight
Fungal strains
Three pathogenic fungi species, C. albicans (PTCC:
5027), C. glabrata (PTCC: 5297), C. krusei (PTCC: 5295)
were provide as lyophilized from Iranian Research
Organization for Science and Technology. These
isolations were tested for determining their sensitivities to
ozonized olive oil.
Preparing medium containing ozonized olive oil
65 g of medium solid powder (sabouraud dextrose agar)
was added in one liter of distilled water by heating. Then
3 ml of prepared medium was poured into each testing
tube, and the tubes were autoclaved in 121˚̊c for 15
minutes. After cooling tubes to 50˚c, 50 µl tween 80 was
added as emulsifier to each tube, and they were vertex
mixed to uniform. Then ozonized olive oil in different
values was added to each tube (500 mg, 600 mg, 700
mg, 800 mg, 900 mg), and therefore different
concentration of ozonized olive oil medium were
prepared: (166.66 mg/ml, 200 mg/ml, 233.33 mg/ml,
266.66 mg/ml, and 300 mg/ml ).
Preparing fungi, determining species, and controls
0.5ml of prepared sabouraud dextrose agar medium was
added to ampoule containing lyophilized powder of three
fungi species and prepared fungal suspension, some of
this suspension was cultured on sabouraud
Fariba et al. 011
Table 1: Different ozonized olive oil concentrations effects at mg/ml on C. albiacans, C. glabrata, C.
krusei in 24, 48 and 72 hours after culture
Ozonized olive
oil mg/ml
C.albicans
24h 48h 72h
C.glabrata
24h 48h 72h
C.krusei
24h 48h 72h
166.66
+ + +
+
+ +
- -
200
+ + +
- - - - - -
233.33
-
-
-
-
-
-
- - -
266.66
-
-
-
-
-
-
- - -
300
-
-
-
- - - - - -
Table 2: Different clotrimazole concentrations effects at mg/ml on C. albacans, C. glabrata, C. krusei in
24, 48, and 72 hours after culture
Clotrimazole
C.albicans
24h 48h 72h
C.glabrata
24h 48h 72h
C.krusei
24h 48h 72h
1 µg/ml
+ + +
+ + +
+ + +
2 µg/ml
+ +
+
-
+ +
- - -
3 µg/ml
+ +
+
- - - - - -
6 µg/ml
+ + + - - - - - -
8 µg/ml - - - - - - - -
dextrose agar with chloramphenicol (SC) and the results
is cream color colonies after 48-72 hours after incubation
at 37˚c, which indicates fungi growth. To confirm the
presence of Candida species one colony was removed
from SD medium and put on glass slide and a drop of
KOH15% was added to it, and it covered with glass slide
and examined under microscope for pseudohypha and
blastoconidia that indicate the presence of Candida
species. For determining species, tests used were germ
tube test, Chlamydoconidium production, and culture on
Chrom agar medium and Corn meal agar medium. Germ
tube test and Chlamydoconidium production were used to
identity Candida albicans.
For determining species, one colony was removed from
SC medium and it was cultured on chrom agar plate
(CHROM agar Candida, France) and after incubation at
37 ̊c for 48-72 hours, the results were read based on
changing the color of medium. Changing the color of
medium to green indicates Candida albicans, changing to
white: Candida krusei and changing to pink means
Candida glabrata.
MIC determination
MIC determination was performed by agar dilution
(Geveely, 2006). Clotrimazole was used as positive
control and the antifungal efficacy of it compared with
ozonated olive oil. A serial of concentrations 1, 2, 3, 6,
8µg/ml was produced from vaginal cream clotrimazole
1%. Then three Candida species from standard species
were cultured on medium. The plates were incubated at
37˚c and the growth or non growth results were read after
24, 48 and 72hours. As negative control concentrations
166.66 mg/ml, 200 mg/ml, 233.33 mg/ml, 266.66 mg/ml
and 300mg/ml olive oil non- ozonized medium were
produced.
Results
After 24, 48, and 72 hours incubation at 37˚c, the results
were observed and recorded for all Candida species. In
addition the results of the control plates were examined.
The presence of the white convex colonies indicates
fungus growth. The condition was uniform when the
experiments were performed and the experiment was
repeated three times to accuracy confirmation condition
in each concentration. To equalize inoculate amount in
each plate, the culture was performed in two places. After
24hour reading was done for plates without fungi growth
and growth inhibition confirmed against ozone and/or
clotrimazole. From Table 1: C. albicans and C. glabrata
growth in concentration 166.66 mg/ml ozonated olive oil
was observed. C. albicans growth in concentration 200
mg/ml of ozonated olive oil was observed. In
concentration 233.33, 266.66 and 300 mg/ml ozonated
olive oil, There was not any growth for all species.
Minimum ozone concentration (MIC) was 233.33 mg/ml
for C. albicans, 200 mg/ml for C. glabrata and 166.66
mg/ml for C.krusei to suppress their growth (Table
1).From Table 2: The minimum clotrimazole
concentration for suppressing growth (MIC) was 8 µg/ml
for C. albicans, 3 µg/ml for C. glabrata and 2 µg/ml for C.
krusei.
012 E3J Microbiol.Res.
Discussion
Diseases resulted from genus Candida yeasts have a
wide spectrum and generally have greater prevalence in
immunocompromised patients .These fungi has various
species, including C. albicans, C. glabrata, C. krusei, C.
tropicalis, C. luisitaniae, C. guilliermondii and C.
dubliensis (Zeini et al., 2009). Candida species are
among the most important opportunistic fungi that in
recent decades, systemic and local infections as well as
new Candida species have been increased (Rani et al.,
2002). Among Candida species, C. albicans has been
more prevalent as an etiologic agent of candidiasis. This
fungus habituates in digestion system, mouth and vagina
and human acquires it on birth time while passing from
vagina. Other Candida species are part of natural fungi
dermis and mucosa, some exist in nature, in soil and
different materials which have much less pathogenic
power and they enter to body through external sources
and under particular conditions they can be pathogenic
with dominating natural defensive mechanisms of host
(Shadzi, 2009). Generally, there are many factors which
disorder natural equilibrium between Candida and host
causing pathologic symptoms. These factors are age,
physiologic changes, continuous use of antibiotics,
breaking natural defensive barrier, disabling diseases,
job, overweight, and alcoholism and avitaminosis for
vitamins A, B, C (Zeini et al., 2009).Considering high
prevalence of Candida infections and recent performed
studies and also reported drug resistances in these fungi,
it is important to find a low cost medicine with high effects
and without any consequences for treating fungal
infections (Ying et al., 2010).
In this study, we surveyed antifungal ozonated olive oil
effects on Candida species. Ozone has strong fungicide
effects. Geveely in a study showed the fungicide effect of
the ozonized olive oil on Dermatophyts, Candida albicans
and Aspergillus fumigates (Geveely, 2006). In addition,
Menendez used successfully ozonized olive oil for
treating ring worm of the foot (Menendez et al., 2008).
Since ozone doesn’t exist in atmosphere, yet, fungus
resistance to this material hasn’t been reported (Geveely,
2006). Ozone reacts exclusively with dual bands in
nonsaturation olive oil fatty acids, thus this reaction
results in different toxic products. These compounds
include hydroperoxidases, ozonides, aldehydes,
peroxides, diperoxides and polyperoxides, have high
oxidizing power.In this study obtained results showed that
all three studied species were sensitive to ozonized olive
oil and sensitivity to species was different and it
depended on concentration. In this study, ozonized olive
oil had better suppression effect on Candida krusei than
Candida glabrata and Candida albicans. Also according
to the results, inhibitory effect of clotrimazole on Candida
krusei has been better than Candida glabrata and
Candida albicans. The amplitude of MIC clotrimazole and
ozonized olive oil has been determined 2-8 µg/ml and
166.66-233.33 mg/ml respectively. These results show
that ozonized olive oil and clotrimazole are able to inhibit
growth of these three Candida species.
As Tara and colleagues in 2012 performed a clinical
study on patients with vaginal candidiasis, ozonized olive
oil and clotrimazole was effective in improving these
patients (Tara et al., 2012). In studies of other
researchers, observations based on ozonized olive oil
effect on these species were observed. Riechart in a
study in 2007 showed that ozonized olive oil has a more
suppressing effect on Candida krusei than Candida
albicans (Reichart, 2007). The results of this study are in
agreed with our study results. Generally there are few
studies on olive oil effect on Candida species for growth
suppression, but based on current studies and other
performed studies by other researchers (Geveely, 2006),
we recommend that ozonized olive oil has a suppressing
effect on these three species of Candida and its effect
depends on concentration, which was observed in higher
concentration in short time.
Conclusion
According to the results of this study and comparing it
with negative control results, it was proofed antifungal
ozone effects. The minimal inhibitory concentration (MIC)
for every species was revealed. Also it was observed that
Candida krusei has a more sensitivity to ozonized olive
oil and clotrimazole than Candida albicans and Candida
glabrata. Considering obtained results in this study and
mentioned clinical studies, ozonized olive oil is an
effective medicine to treat Candida infections (e.g. dermal
and mucosal infections). Authors hope that future
researches will be performed based on this study.
Eventually Ozonized olive oil could be a substitute
product for treating fungal infections and can be
suggested as a proper replacement for antibiotics and it
can be a solution for overuse of antibiotics in the
treatment of fungal disease and overcoming of multi-drug
resistant of candida strains.
Acknowledgement
The authors greatly acknowledge the Research Council
of Mashhad University of Medical Sciences (MUMS),
Mashhad, Iran for their financial grant and Mr. Majid
Ganjbakhsh for his kind efforts during this project. The
results presented in this work have been taken from Dr.
Nazanin Minoochehr’s thesis, with the ID number "1328".
Reference
Alvarez R, Mendez S, P eguera M, Turrent T (1997).Treatment of
primary pioderma with ozonized sunflower oil. In Second
international symposium on ozone application. Cuba: National
Center of Scientific Research; 76.
Geveely NSI ( 2006). Antifungal activity of ozonized olive oil (oleozone).
International Journal of Agriculture and Biology, 8(5): 670 675.
Gundarova RA, Khoroshilova-Maslova IP, Bordiugova GG, Ilatovskaia
LV, Lapina IM (1996). Experimental validation of using ozonized
physiological solutions in intraocular infection, vestnik oftalmologii,
112(5): 9-11.
Lezcano I, Nunez N, Espino M, Gomes M (2000). Antibacterial activity
of ozonized sunflower oil, oleozon, against Staphylococcus aureus
and Staphylococcus epidermidis. Ozone: Sci ence & Engineering: The
Journal of the International Ozone Association, 22(2): 207-214.
Mahmoudabadi AZ (2002). Antifungal drugs, Iran: Ahvaz Jundishapur
University of Medical Sciences Press.
Menendez S, Re L, F alcon L, Argote MB, Mendez I, and Fernandez D,
Elias-Calle B, Valero M (2008). Safety of topical OLEOZON® in the
treatment of tineapedis: phase IV clinical trial. Intern ational Journal of
Ozone Therapy, 7(1): 55-59.
Rani R, Mohapatra NP, Mehta G, Randhawa VS (2002). Changing
trends of Candida species in neonatal septicaemia in a tertiary north
indian hospital. Indian Journal of Medical Microbiolo gy, 20(1): 42-44.
Reichart PA, Samaranayake LP, Bendick C, Schmidt-Westhausen AM,
Jayatilake JAMS (2007). Prevalence of oral Candida species in
leprosy patients from Cambodia and Thailand. Journal of Oral
Pathology & Medicine, 36(6): 342-346.
Sechi LA, Lezcano I, Nunez N, Espim M, D upre I, Pinna A, Molicotti P,
Fadda G, Z anetti S (2001). Antibacterial activity of ozonized
sunflower oil (Oleozon). Journal of Applied Microbiology, 90: 279-
284.
Shadzi S (2009). Medical mycology, identify laboratory methods and
treatment. 11 Ed. Iran: Isfahan ACECR Press.
Shaschova NM, Kachalina T S, Nevmjatullin AL (1995). Application of
ozone therapy in complex treatment of inner female genital in
flammatory disease. Russia: Medical Academy N. Novgorod.
Tara F, Zand-Kargar Z, Rajabi O, Berenji F, A zizi H (2012): Comparing
effect of ozonated olive oil to clotrimazole cream in the treatment of
vulvovaginal candidiasis. BMC Complementary and Alternative
Medicine, 12(Suppl 1): 1 96.
Fariba et al. 013
Viebahn R (1985). The biochemical processes underlying ozone
therapy. Ozone: Science & Engineering: Th e Journal of the
International Ozone Association, 4: 275-285.
Ying S, Qin g S, Chunyang L (2010). The effect of gentian violet on
virulent properties of Candida albicans. Mycopathologia, 169(4): 279-
285.
Zeini F, Mahbod ASA, Emami M (2009). Comprehensive medical
mycology. Iran: University of Tehran Press.