Anti-fungal activities of Origanum oil against Candida albicans

Abstract

The antimicrobial properties of volatile aromatic oils from medicinal as well as other edible plants has been recognized since antiquity. Origanum oil, which is used as a food flavoring agent, possesses a broad spectrum of in vitro antimicrobial activities attributed to the high content of phenolic derivatives such as carvacrol and thymol. In the present study, antifungal properties of origanum oil were examined both in vitro and in vivo. Using Candida albicans in broth cultures and a micro dilution method, comparative efficacy of origanum oil, carvacrol, nystatin and amphotericin B were examined in vitro. Origanum oil at 0.25 mg/ml was found to completely inhibit the growth of C. albicans in culture. Growth inhibitions of 75% and >50% were observed at 0.125 mg/ml and 0.0625 mg/ml level, respectively. In addition, both the germination and the mycelial growth of C. albicans were found to be inhibited by origanum oil and carvacrol in a dose-dependent manner. Furthermore, the therapeutic efficacy of origanum oil was examined in an experimental murine systemic candidiasis model. Groups of mice (n = 6) infected with C. albicans (5 LD50) were fed varying amounts of origanum oil in a final vol. of 0.1 ml of olive oil (vehicle). The daily administration of 8.6 mg of origanum oil in 100 l of olive oil/kg body weight for 30 days resulted in 80% survivability, with no renal burden of C. albicans as opposed to the group of mice fed olive oil alone, who died within 10 days. Similar results were obtained with carvacrol. However, mice fed origanum oil exhibited cosmetically better clinical appearance compared to those cured with carvacrol. The results from our study encourage examination of the efficacy of origanum oil in other forms of systemic and superficial fungal infections and exploration of its broad spectrum effect against other pathogenic manifestations including malignancy.

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Molecular and Cellular Biochemistry 228: 111–117, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
Antifungal activities of origanum oil against
Candida albicans
Vijaya Manohar,1 Cass Ingram,2 Judy Gray,2 Nadeem A. Talpur,1 Bobby
W. Echard,1 Debasis Bagchi3 and Harry G. Preuss1
1Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, DC; 2North American
Herbs and Spices, Waukeegan, IL; 3Department of Pharmacy Sciences, Creighton University School of Pharmacy and AHP,
Omaha, NE, USA
Received 27 June 2001; accepted 14 September 2001
Abstract
The antimicrobial properties of volatile aromatic oils from medicinal as well as other edible plants has been recognized since
antiquity. Origanum oil, which is used as a food flavoring agent, possesses a broad spectrum of in vitro antimicrobial activities
attributed to the high content of phenolic derivatives such as carvacrol and thymol. In the present study, antifungal properties
of origanum oil were examined both in vitro and in vivo. Using Candida albicans in broth cultures and a micro dilution method,
comparative efficacy of origanum oil, carvacrol, nystatin and amphotericin B were examined in vitro. Origanum oil at 0.25
mg/ml was found to completely inhibit the growth of C. albicans in culture. Growth inhibitions of 75% and >50% were ob-
served at 0.125 mg/ml and 0.0625 mg/ml level, respectively. In addition, both the germination and the mycelial growth of C.
albicans were found to be inhibited by origanum oil and carvacrol in a dose-dependent manner. Furthermore, the therapeutic
efficacy of origanum oil was examined in an experimental murine systemic candidiasis model. Groups of mice (n = 6) infected
with C. albicans (5 × LD50) were fed varying amounts of origanum oil in a final vol. of 0.1 ml of olive oil (vehicle). The daily
administration of 8.6 mg of origanum oil in 100 µl of olive oil/kg body weight for 30 days resulted in 80% survivability, with
no renal burden of C. albicans as opposed to the group of mice fed olive oil alone, who died within 10 days. Similar results
were obtained with carvacrol. However, mice fed origanum oil exhibited cosmetically better clinical appearance compared to
those cured with carvacrol. The results from our study encourage examination of the efficacy of origanum oil in other forms
of systemic and superficial fungal infections and exploration of its broad spectrum effect against other pathogenic manifesta-
tions including malignancy. (Mol Cell Biochem 228: 111–117, 2001
Key words: antifungal activity, in vitro, in vivo, female BALB/c mice, origanum oil, Candida albicans, carvacrol, nystatin,
amphotericin B
Introduction
The antimicrobial properties of volatile aromatic oils from
edible plants have been recognized since antiquity. Origa-
num oil, which is used as a food flavoring agent, has been
shown to possess a broad spectrum of antimicrobial activ-
ity due to its high content of phenolic derivatives such as
carvacrol and thymol [1–3]. Earlier studies have demon-
strated the ability of origanum oil to retard and inhibit the
growth of various food spoiling organisms including the
species of Aspergillus (mycotoxinogenic filamentous fungi)
and Hansenula (industrial yeasts) [4–7]. While the oil and
many of its constituents have been demonstrated to be an-
tifungal in vitro against non-pathogenic yeasts [8, 9], a few
studies have suggested a potential therapeutic effect against
experimental infections in rats due to Trichophyton rubrum,
a human dermatophytic filamentous fungus) [10]. Never-
theless, very little information is available on its compara-
Address for offprints: H.G. Preuss, Department of Physiology and Biophysics, Georgetown University Medical Center, Med-Dent Building, SE103, 3900
Reservoir Road, NW, Washington, DC 20007, USA (E-mail: preusshg@georgetown.edu)

112
tive antifungal activity on the growth and physiology of
human pathogenic yeasts or filamentous fungi either in vitro
or in vivo. Furthermore, its direct therapeutic use either in
superficial or systemic infections due to bacteria or fungi has
not been clearly established. In the present study, we exam-
ined the antifungal properties of origanum oil and its major
chemical constituent carvacrol against Candida albicans, a di-
morphic yeast-like fungus. C. albicans which resides as com-
mensal in the mucocutaneous cavities of skin, vagina and
intestine of humans [11], can cause infections under altered
physiological and pathological conditions such as infancy,
pregnancy, diabetes, prolonged broad spectrum antibiotic ad-
ministration, steroidal chemotherapy as well as AIDS [12–
18]. We have demonstrated that origanum oil inhibits the
growth of C. albicans in vitro as well as in vivo. We conclude
that the daily oral administration of origanum oil may be highly
effective in the prevention and treatment of candidiasis.
Materials and methods
Animals and treatment
Female BALB/c mice (15–18 g) were obtained from Taconic
Farms (Germantown, NY, USA). The animals were main-
tained in a controlled environment at 25°C with a 12 h light
and 12 h dark cycle, and were acclimatized for 3–5 days
before use. The mice were housed in groups of five, fed com-
mercial rodent pellets and given water ad libitum through-
out the experiments. The protocol for the entire investigation
was approved by the Animal Welfare Board at Georgetown
University Medical Center.
Plant oils and chemicals
Origanum (P73 OreganolTM) and olive oil were provided by
North American Herbs and Spices, Waukegan, IL, USA. Car-
vacrol, nystatin and amphotericin B were purchased from
Sigma Chemical Co. (St. Louis, MO, USA). Sabouraud’s glu-
cose (S.g) broth and agar media were purchased from Difco
Laboratories (Detroit, MI, USA). All other chemicals used
in this study were obtained from Sigma Chemical Co. (St
Louis, MO, USA) and were of analytical grade or the high-
est commercial grade available.
Organisms
Standard strain of Candida albicans (ATCC No. 48274) ob-
tained from ATCC, Fairfax, VA, USA, was grown and main-
tained on S.g. agar slants.
Susceptibility testing
A micro-broth dilution technique was employed to determine
the susceptibility of the strains of C. albicans to oil of origa-
num and carvacrol [19, 20]. Susceptibility was expressed as
minimum inhibitory concentration (MIC) and minimum fun-
gicidal concentration (MFC). The stock solutions of origa-
num oil, olive oil and carvacrol (63% v/v adjusted in olive
oil) were dissolved in ethanol-Tween 80 solvent. Nystatin and
amphotericin B were dissolved in 50% ethanol, and used as
positive controls. Solvent and media controls were also in-
cluded for reference. The Sabouraud’s glucose (S.g) broth
containing varying amounts (logarithmic, serially and 2-fold
diluted) of origanum oil and carvacrol and the various con-
trols were inoculated with actively dividing C. albicans cells.
The cultures were incubated for 24 and 48 h at 30°C on a
metabolic rotary shaker (200 rev/min), and the growth was
monitored both visually and colorimetrically (at 540 nM).
The minimum inhibitory concentration (MIC) was defined
as the lowest concentration required to arrest the growth of
the fungi at the end of 24 h of incubation. Minimum fungi-
cidal concentration (MFC) was determined by sub-culturing
a 0.01 ml aliquot of the medium drawn from the culture tubes
showing no macroscopic growth at the end of 48 h of cul-
ture on S.g. agar plates and incubated further for the appear-
ance of yeast-like growth. The plates were scored for growth
of the yeast colonies. The lowest concentration of the anti-
fungal agent from which negative growth or fewer than 3
colonies were recorded was considered as minimum fungi-
cidal concentration (MFC).
Effect on yeast and mycelial forms of C. albicans
The effect of origanum oil on the formation of germ tubes
(filament initiation) by C. albicans was examined, by incu-
bating the blastospores with various concentrations of origa-
num oil, carvacrol and other agents in 20% egg albumin at
37°C [21]. The comparative percentage inhibition of germ
tube formation was determined by microscopic examination.
The effect on the mycelial formation (filament elongation) was
carried out by incubating the previously germinated blasto-
spores of C. albicans in the presence of varying amounts of
antifungal agents at 37°C for an additional 24 h and 48 h. Both
the germination and the filament elongation were microscopi-
cally monitored. The MIC and MFC were determined as above.
Toxicity tests with mice
Since origanum oil is known to be pungent and corrosive, a
titration of the doses of origanum oil was conducted to de-
termine the tolerable dose to be given orally. Both the neat

113
oil and the dilutions in olive oil (vehicle) were orally admin-
istered to the mice according to the body weight using a ball
tip gavage needle (Harvard Apparatus, South Natick, MA,
USA). Higher concentrations of the oil were given as single
bolus dose, while the lower concentrations were given daily
for 7 days. The overall health of the animals such as coat
color, body weight, other side effects such as scruffiness, and
death were recorded for 14 days. At the end of the experi-
ment, mice were sacrificed to visually examine the internal
organs for any abnormalities.
Effect on experimental murine systemic candidiasis
A murine systemic candidiasis model developed previously
[22], was employed to evaluate the in vivo anti fungal activi-
ties of origanum oil and carvacrol. Initially, the concentra-
tions of C. albicans needed to achieve 50% mortality of the
animals (LD50) were determined. Mice were injected i.v. with
varying doses of actively growing C. albicans in a final vol.
of 0.1 ml. Mortality of mice was monitored for 30 days. The
death of the mice due to C. albicans was confirmed by ana-
tomical evidence for organ involvement, i.e. the histological
presence of yeasts and/or pseudomycelia. Furthermore, re-
nal burden of C. albicans was assessed by culturing an ali-
quot of the kidney homogenate on S.g. agar plates. The dose
titration revealed that 2.5 × 106 C. albicans cells were needed
to kill 50% of the mice. Subsequently, batches of mice in-
jected with 5 × LD50 dose of C. albicans were used to assess
therapeutic activity.
Treatment protocol
In two separate experiments, groups of mice (6 each) in-
fected with C. albicans (5 × LD50) were gavaged, daily with
origanum oil or carvacrol in 0.1 ml of olive oil for 8 days
and 30 days. The amount of antifungal agents administered
was calculated based on the body weight of the mice. Con-
trol mice received either olive oil orally alone (negative
control), or olive oil orally plus amphotericin B 25 µg i.p.
(positive control). The experiments were terminated at the
end of 30 days. The body weights, the disease status and
the overall health of the mice during the experiment were
recorded. The pathological status of the mice was deter-
mined by visual examination of the internal organs after
their death or sacrifice at the completion of the experiment.
For histology, kidney smears were examined microscopi-
cally after staining with 1% methylene blue for the presence
of fungal elements. Renal burden of C. albicans was fur-
ther tested by culturing aliquots of kidney homogenates on
S.g. agar plates.
Results
Origanum oil is fungicidal to C. albicans in vitro
In the present study, we have examined the antifungal prop-
erties of origanum oil and one of its major constituents, car-
vacrol and thymol, both in vitro and in vivo. Using Candida
albicans in broth cultures and a micro-dilution method, com-
parative efficacies of origanum oil, carvacrol, nystatin and
amphotericin B were examined in vitro. Origanum oil at 0.25
mg/ml was found to completely inhibit the growth of C. al-
bicans in culture. Growth inhibitions of 75% and >50% were
observed at 0.125 mg/ml and 0.0625 mg/ml level, respec-
tively (Table 1). Figure 1 provides a pictorial presentation of
the inhibitory effect. A 2-fold higher concentration of carvac-
rol was required to be fungicidal.
Both the germination and mycelial elongation of C.
albicans are inhibited in vitro
Origanum oil and carvacrol both inhibit germ-tube formation
by the blastospores of C. albicans (Table 2). The production
of germ tubes and subsequent mycelial formation when ex-
posed to either serum or hen’s egg albumen is an in vitro
correlate of the in vivo tissue invasive capabilities of the
pathogenic strains of C. albicans. Origanum oil inhibited
Table1. Effect of origanum oil on the growth of Candida albicans
Agent 24 h MIC 48 h MFC
(mg/ml) (mg/ml)
Origanum oil 0.125 0.25
Carvacrol 0.25 0.5
Amphotericin B 0. 0015 0.0015
Nystatin 0. 005 0.005
Olive oil No effect No effect
MIC = minimum inhibitory concentration; MFC = minimum fungicidal
concentration.
Table 2. Effect of origanum oil on germ tube formation and filament elon-
gation
Agent Germ tube formation Filament elongation
MIC MFC MIC MFC
mg/ml mg/ml mg/ml mg/ml
Origanum oil 0. 062 0.125 0.125 0.125
Carvacrol 0. 125 0.125 0.125 0.25
Olive oil NE NE NE NE
NE = no effect; MIC and MFC for nystatin and amphotericin B = 2.5 and
5.0 µg/ml respectively.

114
germ-tube production by blastospores of C. albicans in a dose-
dependent manner. The MIC required to inhibit the production
of germ tube formation was 0.062 mg/ml (24 h) and the fun-
gicidal effect (at 48 h) required double the concentration
(MFC = 0.125 mg/ml). Carvacrol exerted effects similar to
origanum oil at twice the concentrations. Origanum oil and
carvacrol were also found to affect filament elongation. The
mycelial forms of C. albicans were also inhibited by origa-
num oil at 0.125 mg/ml (MIC) and 0.125 mg/ml (MFC) lev-
els, and by carvacrol at 0.125 mg/ml (MIC) and 0.25 mg/ml
(MFC) levels, respectively.
Origanum oil can protect mice from systemic candidiasis
The therapeutic efficacy of origanum oil was examined in an
experimental murine systemic candidiasis model. Toxicity
studies to determine the least toxic dose of origanum oil that
can be fed safely to mice were conducted (Table 3). A single
bolus dose of 650 mg/kg body weight caused uniform mor-
tality. However, daily administration of smaller doses of ori-
ganum oil mixed with olive oil was well tolerated, showing
no apparent clinical abnormalities (Table 3).
In the first in vivo study, the therapeutic ability of origa-
num oil given only for 8 days to prevent the mortality of mice
with systemic infection due to C. albicans was tested (Table
4). Groups of mice (n = 6) infected with C. albicans (5 × LD50)
were gavaged with varying amounts of origanum oil in a fi-
nal vol. of 0.1 ml. An 8-day treatment with 162.5 mg and 325
mg per kg body weight was found to rescue the mice from
mortality with 100% survival rate over the 30 days of obser-
vation, similar to mice that received amphotericin B (1 mg/
Fig. 1. In vitro antifungal activity of origanum oil against Candida albicans. Exponentially growing C. albicans cells were cultured with varying concentra-
tions of antifungal agents. Aliquots from the cultures drawn at the end of 48 h of culturing were plated on to Sabouraud’s glucose agar. The plates were
further incubated for an additional 48 h for the appearance of yeast like colonies.
Table 3. Toxicity of origanum oil to BALB/c mice
Concentration of oil* No. of mice
(mg/kg) (tested/survived)
Bolus dose 2600 5/0
1300 5/0
650 5/0
325.0 5/5
162.5 5/5
Daily dose for 1 week 26 6/6
52 6/6
78 6/6
104 6/6
130 6/6
*Oil was mixed with 100 µl olive oil and gavaged.
Table 4. Therapeutic efficacy of origanum oil: 8-day treatment
Agent Conc. @ per No. of mice No. of mice with
kg body wt. (mg) tested/survived renal burden#
Origanum oil 325. 5 6/6 4/6
162. 5 6/6 6/6
Amphotericin B 1. 0 6/6 2/6
Olive oil (vehicle) 6/0* –
*Died within 10 days. #Renal burden+ = kidney smear+ culture+.

115
kg body weight). In contrast, the control group of mice who
received 0.1 ml of olive oil alone died within 10 days. At the
end of 30 days, the surviving animals appeared to be clinically
healthy, without scruffiness and with regain of normal coat
luster. Internally, no abnormalities of organs were noted. Fur-
thermore, the kidney smears failed to demonstrate the pres-
ence of either yeasts or pseudomycelia. However, the culture
of their kidney homogenates on S.g. agar revealed the pres-
ence of C. albicans. (Table 4).
The results from the second experiment to determine the
optimal dose over a 30-day treatment course required for
clinical cure and to completely eliminate the renal burden of
C. albicans are shown in Table 5. A daily administration of
origanum oil as low as 8.66 mg/kg body weight (equivalent
of 1 µl per mouse) was found to cure 80% of the infected
mice. Initially all groups of infected mice, exhibited scruffi-
ness, loss of body weight, and lack of coat luster. In the ve-
hicle-control mice, the average body weight was found to
decrease 7 days after injection from approximately 16–11 g.
In contrast, mice receiving treatment had a gradual increase
in body weight after injection from 15–17 to 16–19 g by the
end of 30 days (Fig. 2). The daily administration of equiva-
lent amounts (8.66 and 17.33 mg/kg body weight) of carva-
crol was found to confer similar therapeutic properties (data
not shown). However, the overall clinical appearance of the
mice receiving origanum oil was cosmetically better than
those receiving carvacrol, as evidenced by the improved coat
luster and minimal scruffiness.
Discussion
Aromatic herbal oils used for cooking and flavoring are in-
creasingly claimed to have broad spectrum antimicrobial
activities. Origanum oil has been suggested to have potent
antimicrobial activity, including anthelmintic properties, due
to its phenolic, alcoholic and terpenoid constituents [4]. The
objective of the present study was to assess the antifungal
properties of origanum oil against C. albicans in both in vitro
and in vivo. Furthermore, the comparative efficacy of origa-
num oil, carvacrol, nystatin and amphotericin B was also
examined. The results clearly demonstrate that the origanum
oil can act as a potent antifungal agent against C. albicans,
and can function similar to antifungal antibiotics such as
nystatin or amphotericin B.
C . albicans is a harmless commensal yeast-like fungus in
healthy humans, which can cause superficial as well as life-
threatening systemic infections under immune compromised
situations. C .albicans can colonize or infect virtually all body
sites because of its high adaptability to different host niches
by the activation of appropriate sets of genes in response to
complex environmental signals [21–25]. Thus, our objec-
tive was to assess the possible therapeutic potential of the
commonly used origanum oil against this human dimorphic
commensal, which can become a facultative pathogen un-
der altered physiological situations.
Previous studies to assess the inhibitory effect of origanum
oil against twenty-five different genera of microorganisms
including animal and plant pathogens, food poisoning and
spoilage bacteria, have demonstrated the growth inhibition
properties by origanum oil [26]. Essential oils of oregano
(Origanum vulgare) were found to inhibit the growth and
production of ochratoxin A by Aspergillus ochraceus NRRL
3174 up to 21 days in culture [27].
Origanum oil has been shown to delay or inhibit the growth
of saprophytic food spoiling fungi such as Aspergillus fla-
vus (mycotoxogenic) and industrial yeasts such as Hansenula
Table 5. Therapeutic efficacy of origanum oil: 30-day treatment
Agent Conc. @ per No. of mice No. of mice with
kg body wt. (mg) tested/survived renal burden#
Origanum oil 52. 00 6/6 0/6
43. 33 6/6 0/6
31. 00 6/6 0/6
26. 00 6/6 0/6
17. 33 6/6 0/6
8. 66 6/5 0/5
Carvacrol 17. 33 6/6 0/6
8. 66 6/5 0/5
Amphotericin B 1. 0 6/6 0/6
Olive oil 6/0* 6/6
*Died within 10 days. #Renal burden+ = kidney smear+ culture+. One mouse
treated with 8.66 mg origanum oil/kg b.w. died on day 15, while one mouse
treated with 8.66 mg carvacrol/kg b.w. died on day 13. Fig. 2. In vivo effect of origanum oil in experimental murine candidiasis.
Infected mice were fed orally with varying amounts of origanum oil in 0.1
ml vol. of olive oil (vehicle). The control animals received either olive oil
alone or olive oil plus amphotericin B (i.p.). The body weights and the
overall health of the mice were recorded daily.

116
anoma [8]. Furthermore, origanum oil, and carvacrol have
been reported to have some promising effect on rats infected
with a human dermatophytic fungus, Trichophyton rubrum
[10]. However, the essential oils including origanum oil at
100 ppm had no effect on the pseudomycelial formation by
Candida lipolytica [10,11].
In this paper we have demonstrated, that origanum oil ef-
fectively inhibits the in vitro growth of C. albicans, a human
yeast-like fungus which can cause both systemic and super-
ficial infections in debilitated individuals. In addition we have
shown that origanum oil directly inhibits germination and
filament formation (the two phases required for tissue inva-
sion) by C. albicans. Nevertheless, the novel fungicidal prop-
erty of origanum oil was of superior efficacy as compared to
carvacrol against the germination and mycelial elongation of
pathogenic strain of C. albicans in vitro. Furthermore, selected
concentrations of origanum oil demonstrated therapeutic ef-
ficacy comparable to the effective examined dose for am-
photericin B (1.0 mg) in protecting the mice from systemic
candidiasis. The overall clinical appearance of the mice re-
ceiving origanum oil was cosmetically better than those re-
ceiving carvacrol (data not shown), although this difference
in appearance could possibly be attributed to the presence of
additional constituent phenols such as thymol, terpenes and
flavonoids in origanum oil [4]. It has been demonstrated that
some of these components of origanum oil have antispas-
modic and antioxidant activities, in addition to their antimi-
crobial potentials [23, 28, 29]. A comparative study of these
components may shed more light not only on the antifungal
potentials, but also on the pharmacological effects of each of
these components. It is interesting to speculate that secondary
infections due to C. albicans in debilitated individuals such as
those having diabetes and HIV infection may be prophy-
lactically controlled by the daily intake of small amounts of
oregano oil, either alone or added to a food.
In summary, the results presented in this paper conclusively
demonstrate the antifungal potential of edible origanum oil.
Origanum oil is shown to be both fungistatic and fungicidal
to C. albicans, the human pathogenic yeast. Both the germi-
nation and the pseudomycelial phase of the yeast are inhib-
ited in vitro. Further, the daily oral administration of as little
1.0 µl of oil for 30 days completely freed 80% of the labora-
tory mice from experimental systemic candidiasis, which is
fatal if not treated. Thus, daily oral administration of origanum
oil may be highly effective in the prevention and treatment of
candidiasis. The results from our study not only encourage
examination of the efficacy of origanum oil in other forms
of systemic and superficial fungal infections, but also to ex-
plore its broad spectrum effect against other pathogenic
manifestations including malignancies.
Acknowledgement
This work was supported by a generous grant from North
American Herb and Spice located in Waukeegan, IL, USA.
Oreganum oil was a generous gift from the North American
Herb and Spice, Waukeegan, IL, USA.
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