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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2011, Article ID 659704,
8
pages
doi:10.1155/2011/659704
Research Article
InVitroand InVivoActivities of Essential Oil from the Seed of
AnethumgraveolensL. against Candida spp.
Hong Zeng,
1
Jun Tian,
2
Yuechen Zheng,
3
Xiaoquan Ban,
1
Jingsi Zeng,
3
Ye hon g Ma o ,
3
and Youwei Wang
2
1
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
2
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Institute of
TCM & Natural Products, Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China
3
Department of Dermatology, A
liated Union Hospital, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan 430022, China
Correspondence should be addressed to Youwei Wang,
wyw@whu.edu.cn
Received 10 January 2011; Accepted 6 March 2011
Copyright © 2011 Hong Zeng et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
cacy evaluation of essential oil in the
prophylaxis and treatment of experimental vaginal candidiasis was performed in immunosuppressed mice. The anti-
Candida
activity was analyzed by microbiological and histological techniques and was compared with that of fluconazole (FCZ). The
results showed essential oil was active
in vitro
against all tested strains, with MICs ranging from 0.312
µ
L/mL (for
C. tropicalis,
C. parapsilosis, and C. krusei
) to 0.625
cacious in
accelerating
C. albicans
09-1555 clearance from experimentally infected mice vagina by prophylaxis and therapeutic treatments. In
both therapeutic e
L/mL (for 6 isolated
C. albicans
strains). Essential oil (2% v/v) was highly e
cacy and prophylaxis studies, the histological findings confirmed the microbiological results. The experimental
results revealed that the tested essential oil is e
ff
ective against vulvovaginal candidiasis in immunosuppressed mice.
1. Introduction
antifungal drugs, these are still limited in number; hence,
the great demand for novel antifungal agents justifies the
intense search for new drugs that are more e
Candida albicans
is the most common cause of opportunistic
fungal disease in humans. In recent years, the nonalbicans
Candida
spp., such as
Candida tropicalis
,
Candida glabrata
,
Candida parapsilosis,
and
Candida krusei
, have also emerged
as significant pathogens [
1
,
2
]. Vulvovaginal candidosis
(VVC) is one of the most common clinical manifestations
of
Candida
spp., a
ective and less
toxic than those already in use [
7
]. The essential oils from
many plants are known to possess antiviral, insecticidal, and
antioxidant properties as well as antibacterial and antifungal
activities [
8
,
9
].
A. graveolens
, one of species of Umbelliferae, is a
traditional Chinese herb. The essential oil obtained by
steam distillation from their seeds were used early in the
last century to treat many pathological conditions, such
as disease of the uterus, cervical ectropion [
10
]. Some
researchers have reported that essential oil from the seed of
A. graveolens
, a material not native to Xingjiang (China),
possessed anti-
C. albicans
activity [
11
,
12
]. However, to the
best of our knowledge, the anti-
Candida
activity of essential
oil from the seed of
A. graveolens
has not been demonstrated
both
in vitro
and
in vivo.
Thus, the objective of the current
research is to evaluate its anti-
Candida
activity
in vitro
and
ff
ecting 70–75% of women at least once in
their lifetime [
3
]. There are several factors that can lead to the
development of candidosis; these include immunodeficiency,
endocrine disorders, and malignant diseases. Majority of the
clinically used antifungals su
er from various drawbacks
in terms of toxicity, drug-drug interactions, and lack of
fungicidal e
ff
cacy, high cost, and emergence of resistant
strains resulting from frequent usage [
4
,
5
]. For example,
amphotericin B is very toxic, while others, such as FCZ, are
limited because of the high rate of primary and secondary
resistance [
6
]. Despite the recent introduction of new
The essential oil produced from the seed of
Anethum graveolens
L. (Umbelliferae) was tested
in vitro
and
in vivo
anti-
Candida
activity. The microbroth dilution method was used in the minimal inhibitory concentration (MIC), according to M27-A3 of
the guidelines of the Clinical and Laboratory Standard Institute (CLSI). And then, e
µ
ff
2
Evidence-Based Complementary and Alternative Medicine
in vivo
in order to develop new antifungal agents from
natural products.
TABLE 1: Isolated positions of the
Candida
species.
Strain
Number
Isolating position
2. Materials and Methods
C. albican
09-1519
Coronary sulcus
C. albican
09-1522
Throat swab
2.1. Plant Materials.
The seed of
A. graveolens
were procured
from the Xinjiang Uighur Medical College, located in Hotan,
Xinjiang, China.
C. albican
09-1502
Oral mucosa
C. albican
09-1634
Dejecta
C. albican
09-1555
Vagina
2.2. Essential Oil Isolation.
The essential oil was obtained by
steam distillation. The oil was dried over anhydrous Na
2
SO
4
and preserved in a sealed vial at 4
◦
C until further use. The
yield of essential oil from the seed of
A. graveolens
was 3.5%.
C. albican
09-1394
Coronary sulcus
C. krusei
09-1681
Vagina
C. tropicalis
032
Throat swab
C. parapsilosis
07-305
Hand
2.3. Animals.
Female BALB/c mice (22
±
2 g) were obtained
from the Laboratory Animal Center of Wuhan University
(Wuhan, China). The photoperiods were adjusted daily to
a cycle of 12 h of light and 12 h of darkness. The environ-
mental temperature and relative humidity was constantly
maintained at 21
g/mL, respectively. Then, the yeast colonies
were suspended in RPMI 1640 medium and adjusted 1
×
10
3
CFU/mL with haemocytometer (twice the final inoculum
size), 100
µ
2
◦
C and 50–70%, respectively. The mice
were fed
ad libitum
on a diet of standard pellets and water.
The study received clearance from the Institutional Animal
Ethical Committee (IAEC) of the Committee for the Purpose
of Control and Supervision of Experiments on Animals
(CPCSEA), Wuhan University, Wuhan, China.
±
L was added to each well of the 96-well plates.
The tests 96-well plates were incubated at 37
◦
Cfor48h
(
Candida
spp.), after which the MICs were determined.
MICs were defined as the lowest concentration of the test
substances that prevented visible growth of microorganisms.
All experiments were performed in triplicate.
µ
2.4. Microorganisms.
The 10 isolates of
Candida
studied in
this work include
C. albicans
(
n
=
6),
C. tropicalis
(
n
=
1),
2.6. In Vivo Activity of Essential Oil against Candida albicans
09-1555.
The mice were maintained under pseudoestrus by
giving them estradiol benzoate. These were then immuno-
suppressed by giving them dexamethasone, as previously
reported by Martinez et al. [
14
]. In brief, the mice received
estradiol benzoate on day 6 before inoculation (0.1mg/20 g,
once every two days, s.c.). They then received dexamethasone
on day 1 before inoculation and on day 3 after inoculation
(22.5
n
=
1). All
Candida
species were clinically isolated from infected patients in the
Department of Dermatology and Venereology of the Union
Hospital located in Wuhan, China. The isolated positions of
Candida
species are presented in
Ta b l e 1
. All clinical isolates
were identified according to morphology on corn meal agar,
followed by germ tube formation, thick-walled spores, yeast
spores, and assimilation-fermentation profiles in the API
20 system (bioMerieux, Marcy l’ Etoile, France). For the
in
vitro
experiment, all isolated strains were tested and yeast
colonies were adjusted density of 1
×
10
3
CFU/mL with
haemocytometer. For the
in vivo
experiment, the
C. albicans
09-1555 was used. It was isolated from vagina and adjusted
density of 6
×
10
6
CFU/mL with haemocytometer.
n
=
1), and
C. krusei
(
g/20 g, once a day, i.p.). On inoculation day, the
mice were inoculated intravaginally with 6
×
10
6
cells of
C.
albicans
09-1555 in 20
µ
µ
L.
2.7. Prophylactic Treatment.
Prior to the infection of the
animals, they were separated randomly into 10 prophylactic
groups (Groups P). Control (CK) (
n
=
10) had infected, untreated but
not immunosuppressed animals; this group served to study
the impact of immunosuppression on the development
of the infection. Group 2 (
n
=
2.5. In Vitro Susceptibility Tests.
The broth macrodilution
protocols based on the CLSI reference document M27-A3
[
13
] with modifications was used to determine minimal
inhibitory concentration (MIC) for yeasts.
Tests were performed in sterile 96-well plates, into which
10) was the positive
control group that had mice that were immunosuppressed
and inoculated intravaginally with
C. albicans
09-1555; this
group received 20
n
=
L of RPMI-1640 (without sodium bicarbonate and
L-glutamine at pH 7.0) were added to each well. Before
inoculum, 100
µ
L of excipient solution [1% sodium
carboxymethylcellulose (CMC-Na), including 0.01% Tween
20] twice a day. Group P1 (
µ
L of the essential oil was added to the first
well and serially diluted from the first well by taking 100
µ
10) consisted of treated
groups that had immunosuppressed and infected animals
that received FCZ (20
n
=
L
into the second. This twofold dilution was continued down
the plate, after which 100
µ
µ
L at 100
µ
g/mL). Groups P2, P3, and
L from the 10th column of the
plate was discarded. The 11th column of the plate was
reserved for negative control wells (without inoculation),
and the last column was reserved for the positive growth
control wells (without essential oil or FCZ). The essential
oil and FCZ concentrations ranged from 20–0.039
µ
10 in each group) were the treated groups that
had immunosuppressed and infected animals that received
essential oils (20
n
=
L at 2, 1, and 0.0625% v/v). This treatment
began 2 days before the inoculation of
C. albicans
09-1555 in
the vagina and continued 15 days thereafter at a dose of 20
µ
µ
L
µ
L/mL
twice a day by intravaginal route.
and 100–0.18
C. parapsilosis
(
10) was the
negative control group that had neither infected nor treated
animals. Group 1 (
100
P4 (
Evidence-Based Complementary and Alternative Medicine
3
n
=
10) were separated randomly into 4 groups: Groups
T1, T2, T3, and T4 (
TABLE 2: Antifungal activities of essential oil from the seed of
A.
graveolens
against
Candida
spp.
10 in each group), all of which
received therapeutic treatment with FCZ (100
n
=
Fluconazole
(
µ
g/mL) MIC
Essential oil
(
µ
L/mL) MIC
µ
g/mL) and
Strain
L at 2, 1, and 0.0625% v/v), respectively;
all groups also received the same concentrations as the
prophylaxis. This treatment began 4 day after the inoculation
and continued for 15 days thereafter at a dose of 20
µ
C. albicans
09-1519
3.125
0.625
C. albicans
09-1522
3.125
0.625
Ltwice
a day by intravaginal route. These CK, Group 1 and Group
2 served as controls for both prophylactic and therapeutic
treatments.
µ
C. albicans
09-1502
1.56
0.625
C. albicans
09-1634
1.56
0.625
C. albicans
09-1555
3.125
0.625
C. albicans
09-1394
3.125
0.625
2.9. The Microbiological Test.
For the prophylactic treatment,
the evaluation of vaginal burden was performed on samples
washed with 1mL of sterile saline bu
C. krusei
09-1681
25
0.312
C. parapsilosis
07-305
0.78
0.312
er. The obtained cells
were harvested by centrifugation at 3200
ff
C. tropicals
032
3.125
0.312
g for 15min. This
operation was repeated on days 2, 4, 8, 10, 12, and 15 after
inoculation to observe the course of infection. Determina-
tion of the number of
Candida
organisms was conducted in
duplicate after performing serial 10-fold dilution of washing
fluid and plating on Sabouraud glucose agar containing
0.05% of chloramphenicol. All plates were incubated at 37
◦
C
for 24 h for each series of dilutions. For the therapeutic
treatments, the evaluation of vaginal burden was carried out
on days 4, 8, 10, 12, and 15 after-infection to observe the
course of infection.
×
candidiasis model was used, and the anti-
Candida
activity of
essential oil
in vivo
was evaluated.
In the prophylactic treatment (
Figure 1
), prior to the
initiation of the experiments, individual vaginal cavity
cultures were performed and no
Candida
organisms were
found. Essential oil and FCZ exerted a marked acceleration
of the clearance of the yeast, as demonstrated by a statistically
significant decrease in cfu counts 15 days after the vaginal
challenge, compared with control Group 2. The clearance
values with di
erent essential oil concentrations suggest a
substantial essential oil dose dependence of fungus clearance.
As with all dose regimens, the infection was decreased in 15
days, whereas the untreated control mice remained infected
(approximately log 5.16
±
2.36
C. albicans
cfu/mL of the
vaginal fluid). In comparative terms, the acceleration of
Candida
clearance in mice treated with FCZ (100
ff
2.10. The Histological Data.
Vaginas were removed and
longitudinally opened. These were then fixed in 10%
formaldehyde solution for at least 48 h, stained using the
periodic acid-Schi
(PAS) stain for fungal visualization. For
the prophylactic treatment, the vaginas were removed on
days 2, 8, and 15 after inoculation to observe the course of
infection. For the therapeutic treatments, the vaginas were
removed on days 4, 8, and 15 after-infection to observe the
course of infection.
ff
g/mL)
solution substantially overlapped the activity of a 1% v/v
solution of essential oil. On the other hand, no e
µ
ect on the
rate of fungal clearance was observed in mice treated with
essential oil-untreated animals, given the 1% CMC solution,
including 0.01% Tween 20. The mice in CK (negative control
group: neither infected nor treated animals) showed negative
culture results throughout the experiment. Essential oil
showed stronger anti-
Candida
activity than FCZ, similar to
the results obtained
in vitro
.
In the therapeutic treatment (
Figure 2
), just before the
inoculation with
Candida
cells, the vaginal cavities of all the
mice were sampled and shown to be free from infection. In
order to verify the establishment of the infection 4 days after
inoculation, all the groups of animals were re-sampled. The
results showed that the vaginal swabs were positive for all
groups of animals.
After 15 consecutive days of therapeutic treatment (day
15), the viable
C. albicans
09-1555 cells for FCZ (100
ff
2.11. Statistical Analysis.
All experiments were done in
triplicate, and the results were reported as mean
±
S.E.M.
6). The data were analyzed by one-way ANOVA.
Statistically significant e
n
=
ects were further analyzed, and
means were compared using Duncan’s multiple range test.
Statistical significance was determined at
ff
P<.
01.
3. Results
3.1. Antifungal Susceptibility Test. In vitro
antifungal activity
of essential oil was investigated against 10 clinical strains of
yeasts. The MICs value is reported in
Ta b l e 2
. The results
showed that the essential oil was active against all the tested
strains. For
C. tropicalis, C. parapsilosis,
and
C. krusei
strains,
MIC (0.312
g/mL)
and essential oils (2, 1, and 0.0625% v/v) treatments showed
values of log 3.6
±
1.77 and log 2.24
±
1.93, 3.43
±
1.25
and 4.19
µ
L/mL) values were similar. For the 6 isolated
C. albicans
strains, MIC (0.625
µ
µ
L/mL) values were also the
same.
3 cfu/mL, respectively, indicating a significant
reduction of
Candida
organisms compared with Group 2
with a value of 5.16
±
2.36 cfu/mL (
±
3.2. Microbiological Results of Prophylactic and Therapeutic
Trea tment .
After establishing activity
in vitro
,lowvalues
of MICs were obtained, and we examined the activity of
essential oil
in vivo
. The experimental model of the vaginal
P<.
01). However, there
was no significant di
ff
erence between FCZ (100
µ
g/mL) and
05). CK showed negative culture results
throughout the experiment. The infected animals in Group 1
P>.
2.8. Therapeutic Treatment.
Prior to inoculation, animals
(
essential oils (20
(
essential oil 1% (
4
Evidence-Based Complementary and Alternative Medicine
6
5
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
4
∗∗
∗∗
∗∗
∗
∗
3
∗∗
2
∗∗
∗∗
1
0
2
4
8
10
12
15
(day)
Group 1
Group P2
Group P3
Group P4
Group 2
Group P1
cacy of essential oil versus FCZ against vaginal candidiasis in mice. Outcome
of vaginal infection by
C. albicans
09-1555 in immunosuppressed BALB/c mice inoculated intravaginally with essential oil and FCZ.
CK: negative control group (no
Candida
was found in the vaginal lumina). Group 1: infected, untreated but not immunosuppressed
animals. Group 2: positive control group, immunosuppressed inoculated intravaginally with
C. albicans
09-1555, received excipient. Group
P1: treated groups: immunosuppressed, infected animals that received FCZ (20
g/mL). Groups P2, P3, P4: treated groups:
immunosuppressed, infected animals that received essential oils (20
µ
L at 2, 1, and 0.0625% v/v). The value log cfu was showed by the
mean
±
S.E.M. (
µ
L at 100
µ
n
=
6).
∗∗
: compared with Group 2 (
P<.
01);
∗
: compared with Group 2 (
P<.
05).
6
5
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
∗
4
∗
∗∗
∗
∗
3
∗∗
∗∗
2
∗∗
1
0
4
8
10
12
15
(day)
Group 1
Group T2
Group T3
Group T4
Group 2
Group T1
cacy of essential oil versus FCZ against vaginal candidiasis in mice. Outcome of vaginal
infection by
C. albicans
09-1555 in immunosuppressed BALB/c mice inoculated intravaginally with essential oil and FCZ. CK: negative
control group (no
Candida
was found in the vaginal lumina). Group 1: infected, untreated but not immunosuppressed animals. Group 2:
positive control group, immunosuppressed, inoculated intravaginally with
C. albicans
09-1555, received excipient. Group T1: treated groups:
immunosuppressed, infected animals that received FCZ (20
g/mL). Groups T2, T3, T4: treated groups: immunosuppressed,
infected animals that received essential oils (20
µ
L at 2, 1, and 0.0625% v/v). The value log cfu was showed by the mean
±
S.E.M. (
n
=
6).
∗∗
: compared with Group 2 (
µ
L at 100
µ
P<.
01);
∗
: compared with Group 2 (
P<.
05).
FIGURE 1: Microbiological study of the prophylactic e
FIGURE 2: Microbiological study of the therapeutic e
Evidence-Based Complementary and Alternative Medicine
5
Postinfection
day 2 (A)
Postinfection
day 8 (B)
Postinfection
day 15 (C)
CK
FIGURE 3: Microscopic observation of the prophylactic ecacy of essential oil versus FCZ against vaginal candidiasis in BALB/c mice on
days 2, 8, and 15 after-infection (periodic acid-Schi
Group 1
Group 2
Group P1
Group P2
Group P3
Group P4
staining, 400x). CK: neither infected nor treated animals. Group 1: infected, untreated
but not immunosuppressed animals. Group 2: immunosuppressed and inoculated intravaginally with
C. albicans
09-1555, and animals that
received excipient. Group P1: treated groups: immunosuppressed, infected animals that received FCZ (20
ff
µ
L at 100
µ
g/mL). Groups P2, P3,
P4: treated groups: immunosuppressed, infected animals that received essential oils (20
L at 2, 1, and 0.0625% v/v).The bar is 10
µ
m.
(nonimmunosuppressed infected and untreated group) had
no
Candida
cell.
Therapeutic treatment: vaginal sections of all the animals
were also studied by light microscopy. Infected and untreated
mice demonstrated the presence of
C. albicans
09-1555 at the
surface of the epithelium with desquamation of superficial
layers, whereas in CK (neither infected nor treated animals),
no
Candida
was found in the vaginal lumina (
Figure 4
).
On day 4 after inoculation, the infected animals had great
amounts of budding yeast and pseudohyphae in the vaginal
lumina (Groups T1–T4 and Group 2) (
Figure 4
).
On day 8 after administration, therapeutic treatment
with FCZ and essential oils (Groups T1–T4) signifi-
cantly reduced the fungal burden compared with Group 2
(immunosuppressed infected and treated with excipient) in
the vaginal luminal (
Figure 4
).
On the 15th day after administration, Group T2,
(immunosuppressed infected and treated with essential
oil 2% v/v) completely eradicated the vaginal
Can-
dida
(
Figure 4
). Regarding Group T1 (immunosuppressed
infected and treated with FCZ) (
Figure 4
), only a few
Candida
was found in the vaginal lumina similar to Group
T3 (immunosuppressed infected and treated with essential
oil 1% v/v).Vaginal sections in CK and Group1 presented the
same aspect that no
Candida
was found in the vaginal lumina
(
Figure 4
).
3.3. Histological Test.
Prophylactic treatment: in all animals,
the presence of yeast was found on sections of vaginas stained
with PAS. In infected untreated mice, both budding yeast and
the pseudohyphae form of
C. albicans
09-1555—dark with
PAS stain—were found in the luminal vagina. At the surface
of the epithelium, we also noticed keratin debris, whereas in
the noninfected control group, no
Candida
was observed in
the luminal vagina.
The infected mice from Groups P1–P4 (immunosup-
pressed and then infected and treated with FCZ and essential
oil) all showed budding yeast and pseudohyphae in the
vaginal luminal, on day 2 after inoculation (
Figure 3
).
However, those from Group 2 (immunosuppressed infected
and treated with excipient) showed large amounts of yeast
and pseudohyphae in the vaginal luminal (
Figure 3
). Other
groups showed yeast and pseudohyphae in the vaginal
luminal dose dependently. Samples from Groups P1, P2, P3,
andP4receivedFCZ(100
µ
4. Discussions
A. graveolens
is found in many places, such as India, Europe,
United States, Turkey, and China. It has been used for
cooking and in Uygur medicine since ancient times in
China. Aromatic herbal oils used for cooking and flavoring
cover a broad spectrum of antimicrobial activities. The main
µ
g/mL) and essential oils (2, 1,
0.0625% v/v) on day 8 after inoculation, less than that for
Candida
in the vaginal luminal (
Figure 3
), compared with
Group 2 (
Figure 3
). On the 15th day after inoculation, Group
P2 showed no
Candida
organisms detected by histological
techniques (
Figure 3
) and it showed the same aspect as CK
(neither infected nor treated animals), which was better than
GroupP1(infectedgroupstreatedwithFCZ).
In Group 1 (nonimmunosuppressed infected and un-
treated group), the infected animals had few pseudohyphae
in the vaginal lumen on day 2 after inoculation, and no
Candida
was observed in the vaginal lumina on day 15.
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