Advances in Bioscience and Biotechnology, 2011, 2, 103-107 ABB
doi:10.4236/abb.2011.22016 Published Online April 2011 (http://www.SciRP.org/journal/abb/).
Published Online April 2011 in SciRes. http://www.scirp.org/journal/ABB
Anti-yeast activities of Origanum oil against human
pathogenic yeasts
Amber Adams, Satyanshu Kumar, Marck Clauson, Shivendra Sahi*
Department of Biology, Western Kentucky University, Kentucky, USA.
Received 8 October 2010; revised 21 February 2011; accepted 1 March 2011.
ABSTRACT
Outbreak of autoimmune diseases by pathogenic
yeasts has led to a serious medical threat. As these
organisms evolve resistance to existing antifungal
drugs, the concern could be further compounded.
The realm of plant derived products offers a wide
spectrum of potentially valuable alternatives to the
existing synthetic fungicides. Essential oils from sev-
eral medicinal plants have been shown to exhibit
pharmacological attributes. In the present study,
anti-yeast properties of Oregano essential oil (OEO)
were examined in vitro against four human patho-
genic yeasts i.e., Candida albicans, Cryptococcus al-
bidus, Cryptococcus neoformans and Rhodotorula ru-
brum. OEO concentration of 200 μg/mL was found to
be growth inhibitory against all four yeasts examined,
thereby showing its potential to function as a natural
anti-yeast agent.
Keywords: Orig anum vulgare; Candida albicans;
Cryptococcu s neo formans; Rhodotorula rubrum;
Cryptococcus albidus
1. INTRODUCTION
Aromatic plants and spices have been used in traditional
medicine since ancient times due to different potent ac-
tivities of their components. Essential oils produced by
plants in defense against pathogens are derived from
terpenes, sesquiterpenes and their oxygenated com-
pounds. They are a rich source of bioactive compounds
possessing antimicrobial, spasmolytic, carminative, he-
patoprotective, anticancer and antiviral properties and
have been pharmacologically evaluated for treatment of
many infectious diseases [1]. However, there is only lim-
ited information available on the antifungal activities of
different essential oils toward human fungal pathogens.
Candida albicans, Cryptococcus albidus, Cryptococcus
neoformans and Rhodotorula rubrum are some of the
most common pathogens infeeting immunocompromised
patients. C. albicans, an opportunistic fungal pathogen,
resides commensally in the mucocutaneous cavities of
skin, vagina and intestine of humans [2]. It is one of the
most frequent causes of fungal infections under altered
physiological and pathological conditions like infancy,
pregnancy, diabetes, prolonged broad-spectrum antibi-
otic administration, steroidal chemotherapy and AIDS
[3]. Whereas, C. neoformans is a basidiomycetous fun-
gus infecting immunocompromised patients. Infection
starts from the lungs and migrates to the central nervous
system resulting in meningoencephalitis. Fluconazole, a
fungistatic agent, has been used successfully to treat
fungal infections caused by C. albicans and other related
Candida species. However, emergence of intrinsically
resistant species like Candida glabrata and Candida
krusei to azoles is a worrisome development [4]. Human
fungal infections mainly among immunocompromised
patients have increased at an alarming rate during recent
times [5], thereby warranting the need to identify and
develop potent anti-yeast agents with broad spectrum
activities.
Origanum, comprising a wide range of species and
subspecies, are valuable sources of spices and essential
oils with the latter possessing broad spectrum antimicro-
bial properties [6-8]. Gram negative and positive bacte-
ria and their antibiotic resistant strains have shown sus-
ceptibility to the Oregano essential oil (OEO) [9]. Fur-
thermore, the potency of OEO in inhibiting mycelial
growth and spore germination of Aspergillus niger and A.
flavous have also been demonstrated. Several other
studies also have reported the efficacy of OEO as an
anti-yeast agent both under in vitro and in vivo condi-
tions against a wide range of pathogenic yeasts [3,10]. In
this study, anti yeast properties of OEO were examined
in vitro against four human pathogenic yeasts namely
Candida albicans, Cryptococcus albidus, Cryptococcus
neoformans and Rhodotorula rubrum. Only a few stud-
ies (including the present one) have demonstrated the
time and concentration dependent efficacy of OEO as an
anti-yeast agent on human pathogenic yeasts such as C.

A. Adams et al. / Advances in Bioscience and Biotechnology 2 (2011) 103-107 104
neoformans. To the best of our knowledge, this is the
first report on the potency of OEO on the growth inhibi-
tion of C. albidus. Since the effects of OEO an anti-yeast
agent against Candida albicans have been demonstrated
in earlier studies, we also included this strain in the pre-
sent study to compare our results with those reported
elsewhere.
2. MATERIALS AND METHODS
2.1. Plant Material and Extraction of Oil
Fresh oregano plant material was purchased from Shen-
andoah Growers in Harrisonburg, VA, USA. Stem and
leaves were cut into small pieces, air-dried, powdered
using a plant grinder and 100 g used for isolation of oil
using hydro-distillation.
2.2. Yeast Culture
Cultures of C. albicans and C. neoformans were ob-
tained from Presque Island Cultures in Erie, PA, USA. C.
albidus and R. rubrum were obtained from Department
of Biology, Western Kentucky University, KY, USA.
Initially culture were incubated at 37ºC for 3 days and
subsequently transferred to room temperature (25ºC). All
the yeast cultures were routinely sub-cultured for culture
maintenance and inhibition test.
2.3. Culture Media
Sabourad Dextrose Agar (DIFCO) and buffered yeast
extract-peptone-dextrose (YPD) was used for yeast cul-
tivation [11,12]. Initially all the yeast strains were main-
tained on Sabourad Dextrose Agar (DIFCO) and later
cultured in YPD (ATCC medium 1245) for solid me-
dium growth inhibition test. The YPD broth (ATCC me-
dium 1245) was also used for liquid medium yeast
growth inhibition test experiments.
2.4. Liquid Medium Test
Pre-cultures of C. albicans, C. neoformans, C. albidus
and R. rubrum were maintained by inoculating 10 mL of
YEPD broth with an appropriate yeast strain and incu-
bating for 12 - 15 hr at 37ºC to ensure that yeast cells
were actively dividing [3]. The pre-cultures were then
used for inoculating the tubes in the test group. Test
groups were prepared with 5 mL of medium containing
4.5 mL of YPD broth and 0.5 mL of yeast pre-cultures.
Five and 10 μL of OEO corresponding to concentrations
of 200 μg/mL and 400 μg/mL, respectively were added
to the culture tubes containing medium in the test groups.
Test tubes were incubated at 37ºC on a rotary shaker and
growth measurements were taken over a period of one
week by measuring the optical density using UV-Visible
spectrophotometer at 600 nm (DU 530, Beckman Coul-
ter). Blank and ethanol control groups with 200 μg/mL
and 400 μg/mL of OEO in 5 mL YPD broth were also
monitored.
2.5. Solid Medium Inhibition Test for Anti Yeast
Activity
C. albicans and C. neoformans were assessed for OEO
susceptibility using solid medium growth inhibition test
by employing the disk diffusion method [5]. Aliquot
(100 μL from 10 mg/mL OEO in ethanol (equivalent to
1000 µg OEO) was added drop wise to a 7 mm sterile
paper disk under sterile condition. A control was main-
tained by adding 90% ethanol to each sterile disk. All
sterile disks were then allowed to air-dry in a sterile
hood for a minimum of one hour or until they were
ready for application on to the agar plate. Yeast strains
were spread on the Sabourd’s dextrose agar plates to
create a lawn pattern holding 0.5 McFarland standards
liquid inoculants. Disks containing 1000 μg of OEO
were placed to the freshly inoculated plates with each
plate containing three disks. Ethanol control disks were
also set up in a similar fashion. Plates were then incu-
bated for 7 days at 37ºC and zones of inhibition were
measured. Disk diffusion test for C. neoformans was
conducted on YEPD agar plates using a slightly different
method of inoculation. Plates were inoculated with 1 mL
of C. neoformans cell suspension standardized to
McFarland standards [13]. One mL inoculum was spread
across the plate under sterile conditions. Plates were
allowed to dry at room temperature in a sterile hood.
OEO (1000 μg) containing disks were placed on the
plates as described above.
All the experiments were conducted in triplicate and
liquid growth medium inhibition tests were duplicated
for C. albicans, C. albidus, C. neoformans, and R. ru-
brum.
3. RESULTS AND DISCUSSION
To determine the efficacy of OEO in inhibiting the
growth of the yeasts, Candida albicans was grown in
liquid medium supplemented with 200 and 400 µg/mL
of OEO for 21, 40, 60 and 85 hr (Figure 1(a)). There
was an appreciable increase in the percent growth inhi-
bition of this strain with an increase in the incubation
period at both OEO concentrations. Although no signifi-
cant difference was observed in percent growth inhibi-
tion of C. albicans at both concentrations of OEO (200
and 400 µg/mL) at 40 hr incubation, at other time inter-
vals the percent growth inhibition of the yeast strain at
400 µg/mL of OEO was relatively higher compared to
those grown at 200 µg/mL of OEO. The inhibitory effect
of OEO on the growth of this yeast strain was further
corroborated by the disk diffusion method (Table 1, Fig-
ure 2(a)). A zone of inhibition of 27 mm in diameter was

A. Adams et al. / Advances in Bioscience and Biotechnology 2 (2011) 103-107 105
Figure 1. Temporal effects of 200 (black bar) and 400
(white bar) μg/mL of OEO on the percent growth inhi-
bition of (a) Candida albicans, (b) Cryptococcus al-
bidus, (c) Candida neoformans, and (d) Rhodotorula
rubrum grown in liquid medium.
recorded in response to this OEO treatment. Zone of
inhibition was not observed in the presence of the con-
trol disk. Further, the temporal growth response of C.
albidus in the presence of 200 and 400 µg/mL of OEO in
liquid medium incubated for 6, 95, 115 and 140 hr was
evaluated (Figure 1(b)). The effect of OEO on the per-
Table 1. Anti-yeast activity of Oregano essential oil (OEO)
measured as zone of inhibition using solid disk diffusion
method for C. albicans and C. neoformans (size of the disk is
included in the measurement).
Species OEO concentration
(μg)
Diameter of inhibi-
tion zone
(mm)
C. albicans 0 7
1000 27
C. neoformans0 7
1000 14.5
cent growth inhibition of C. albidus was evident at both
concentrations tested at different time intervals. Higher
concentration of OEO (400 µg/mL) and longer duration of
incubation resulted in higher percent growth inhibition of
this strain. Interestingly, OEO treatment at 200 μg/mL
resulted in more than 90% inhibition of C. neoformans
growth after incubation for different time intervals (40,
60 and 85 hr). Whereas, at 400 µg/mL of OEO there was
a complete inhibition in the growth of C. neoforman
within 40 hr of incubation period (Figure 1(c)).
Disk diffusion experiment also showed a smaller size
zone of inhibition (14.5 mm diameter) for C. neoformans
at 1000 μg/mL OEO concentration following seven days
of inoculation (Table 1 and Figure 2(b)). Although the
percent growth inhibition of R. rubrum at 200 μg/mL
OEO was about 50% after 40 hr incubation, the values
increased significantly (> 90%) during longer periods of
incubation (60 and 85 hr). Whereas, almost complete
growth inhibition of this yeast strain could be docu-
mented within 40 hr of incubation treatment at 400 µg/mL
of OEO (Figure 1(d)). Time dependent cell growth
measurement (data not shown) revealed that OEO ap-
parently reduced the length of time spent by C. albicans
for undergoing rapid growth, but did not inhibit the ex-
ponential growth phase. This observation is in contrast
to the growth responses of C. albidus, C. neoformans
and R. rubrum to OEO. Inherent genetic variability of C.
albicans and its temperature sensitivity could possibly
be the plausible explanation to account for its indifferent
response to OEO.
In the present study we demonstrated an inhibitory
effect of OEO at 200 and 400 μg/mL concentrations on
the growth of C. albicans. Our data is consistent with the
earlier studies reporting the susceptibility of this yeast
strain to OEO and its fungistatic and fungicidal behavior
[3,8,10]. Twenty μg/mL OEO was found to be the mini-
mal inhibitory concentration (MIC) required for C. albi-
can [8]. Whereas, concentrations of OEO in the range of
250 - 500 μg/mL facilitated complete growth inhibition
of this strain [3,10]. Although the effective concentra-
tions (200 and 400 μg/mL) of OEO tested against C.
albicans in this study lies in the range of reported effect-
(a)
(c)
(d)
(b)

A. Adams et al. / Advances in Bioscience and Biotechnology 2 (2011) 103-107 106
Figure 2. Typical zone of inhibition of OEO (1000 μg/mL)
against (a) Candida albicans, and (b) Cryptocococcus neofor-
mans.
tive OEO concentrations, differences in the exact values
of MIC of OEO required for C. albicans growth inhibi-
tion may possibly be due to the presence of different
chemotypes, variable growth conditions, harvesting and
extraction methods of Origanum. A few studies have
also demonstrated the time-and concentration-dependent
efficacy of OEO as an anti-yeast agent on other human
pathogenic yeasts such as C. neoformans, R. rubrum and
Trichophyton beigelii [14,15]. However, to the best of
our knowledge, there is no information available on the
effect of OEO on the growth performance of C. albidus.
Thus, our study provides the first report on the potency
of OEO on the growth inhibition of this yeast strain.
Thymol, a major constituent of OEO, has been shown
to stimulate deformities in the envelope of the yeast
(Saccharomyces cerevisiae) cells, and was presumed to
influence the exponential growth phase of the yeast by
inducing cracking of non-dividing cells, thereby, affect-
ing the budding process [16]. This could possibly ex-
plain the differential effects of OEO on four different
yeast strains in the present study. In the faster growing
strains (C. albicans and C. albidus) OEO was compara-
tively less effective to halt cell division completely. Low
concentrations of growth inhibiting molecules in OEO or
rapid cell division of these two yeast strains in the liquid
medium could be attributed as possible reasons. On the
contrary, R. rubrum and C. neoformans took longer time
to begin their exponential growth phases, thereby allow-
ing OEO sufficient time to affect their dividing cells,
resulting either in the complete inhibition or lengthy
delay of the exponential growth phase.
Origanum vulgare showed great potential as a natural
anti-yeast agent, in addition to its already reported bac-
tericidal and fungicidal activities. Different yeast strains
responded differentially to OEO. C. albidus, C. neofor-
mans and R. rubrum showed higher susceptibility to
Oregano oils as compared to C. albicans. Present and
earlier studies indicate that OEO hold great potential for
combating many fungal and yeast pathogens. Further
investigations are warranted to decipher as how the in-
oculums size and length of lag phase affect the action of
OEO on yeast growth. It is also imperative that the ef-
fects of different growing/harvesting methods upon oil
content and antifungal and anti-yeast action be deter-
mined. With further experimentation and in vivo testing,
essential oil from Origanum vulgare could serve as a
highly effective anti-yeast agent in pharmacology.
5. ACKNOWLEDGEMENTS
Financial support from WKU Honors College, Biology Department
and Applied Research and Technology Program of Ogden College of
Science and Engineering are gratefully acknowledged. We also thank
Drs. Mohd Israr and Ajay Jain for their technical help and critical
review of this manuscript, respectively.
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