InVitro and InVivo Activities of Essential Oil fromthe.PDF

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

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

ﬀ

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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

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.

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

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

L of excipient solution [1% sodium

carboxymethylcellulose (CMC-Na), including 0.01% Tween

20] twice a day. Group P1 (

L of the essential oil was added to the ﬁrst

well and serially diluted from the ﬁrst well by taking 100

10) consisted of treated

groups that had immunosuppressed and infected animals

that received FCZ (20

n =

into the second. This twofold dilution was continued down

the plate, after which 100

L at 100

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

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

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 (

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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

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

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.

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

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

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

L/mL) values were similar. For the 6 isolated

C. albicans strains, MIC (0.625

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

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% (

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∗

∗∗

∗

∗∗

(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. (

L at 100

n = 6). ∗∗ : compared with Group 2 (

P<.

01); ∗ : compared with Group 2 (

P<.

05).

∗

∗∗

∗

∗∗

(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 (

L at 100

P<.

01); ∗ : compared with Group 2 (

P<.

05).

FIGURE 1: Microbiological study of the prophylactic e

FIGURE 2: Microbiological study of the therapeutic e

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Postinfection

day 2 (A)

Postinfection

day 8 (B)

Postinfection

day 15 (C)

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

L at 100

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

(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

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

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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