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Journal de Mycologie Médicale (2016) xxx, xxx—xxx

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ORIGINAL ARTICLE/ARTICLE ORIGINAL

The anti-candidal activity of Satureja


khuzistanica ethanol extract against clinical
isolates of C. albicans
L’activité anti-Candida de l’extrait éthanolique du Satureja
khuzistanica contre des isolats cliniques de C. albicans

M. Mahboubi *, N. Kazempour

Department of Microbiology, Medicinal Plants Research Center of Barij, PO 3795191916, Kashan, Iran

Received 21 January 2015; received in revised form 21 November 2015; accepted 22 November 2015

KEYWORDS Summary Candida albicans is the common cause of some infectious diseases such as vaginal
Satureja khuzistanica; candidiasis or candidemia. Due to the emergence of drug resistant isolates of C. albicans,
Ethanol extract; finding a new anti-Candida agent is a new strategy for current treatments. This study
Candida albicans; evaluated the anti-candidal activity of Satureja khuzistanica ethanol extract against clinical
Anti-candidal agent; isolates of C. albicans. S. khuzistanica ethanol extract from aerial parts of plant at full
Synergistic effect flowering stage was evaluated against 30 clinical isolates and two ATCC reference strains of
C. albicans by disc diffusion and micro-broth dilution assay. Also, in this study we evaluated
the synergistic effects of amphotericin B, clotrimazole and ketoconazole with S. khuzistanica
ethanol extract. The means of MIC and MFC of S. khuzistanica ethanol extract against clinical
isolates were 299.4 and 722.6 (mg/mL), respectively. S. khuzistanica ethanol extract in-
creased the anti-candidal effect of amphotericin B and ketoconazole, while it had no
synergistic effect on clotrimazole against clinical isolates of C. albicans. Therefore,
S. khuzistanica ethanol extract can be introduced as a new source of anti-candidal agent
against clinical isolates of C. albicans.
# 2015 Elsevier Masson SAS. All rights reserved.

Résumé Candida albicans est la cause fréquente de certaines maladies infectieuses telles que
MOTS CLÉS la candidose vaginale ou la candidémie. En raison de l’émergence de souches résistantes aux
Satureja khuzistanica ; médicaments, la recherche des nouveaux agents anti-Candida est une nouvelle stratégie pour les

* Corresponding author.
E-mail addresses: mahboubi1357@yahoo.com, mahboubi@barijessence.com (M. Mahboubi).

http://dx.doi.org/10.1016/j.mycmed.2015.11.003
1156-5233/# 2015 Elsevier Masson SAS. All rights reserved.

Please cite this article in press as: Mahboubi M, Kazempour N. The anti-candidal activity of Satureja khuzistanica ethanol extract against
clinical isolates of C. albicans. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.11.003
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MYCMED-586; No. of Pages 5

2 M. Mahboubi, N. Kazempour

Extraction à l’éthanol ; traitements actuels. Cette étude a évalué l’activité anti-Candida de l’extrait alcoolique de
Candida albicans ; Satureja khuzistanica contre les isolats cliniques de C. albicans. L’extrait éthanolique de parties
Agent anti-Candida ; aériennes de S. khuzistanica au stade de pleine floraison a été évalué contre 30 isolats cliniques
Effet synergique et deux souches de référence ATCC de C. albicans par diffusion du disque en gélose et par dosage
en micro-dilution. En outre, nous avons évalué les effets synergiques de l’amphotéricine B, du
clotrimazole et du kétoconazole avec l’extrait éthanolique de S. khuzistanica. Les moyennes de
MIC et MFC de l’extrait éthanolique de S. khuzistanica contre les isolats cliniques étaient de
299,4 et 722,6 (mg/mL), respectivement. L’extrait éthanolique de S. khuzistanica augmentait
l’effet anti-Candida de l’amphotéricine B et du kétoconazole, alors qu’il n’a eu aucun effet
synergique sur le clotrimazole contre des isolats cliniques de C. albicans. Par conséquent,
l’extrait éthanolique de S. khuzistanica peut être considéré comme une nouvelle source d’agent
anti-Candida contre les isolats cliniques de C. albicans.
# 2015 Elsevier Masson SAS. Tous droits réservés.

Introduction Materials and methods

Vaginal yeast infections or vaginal candidiasis are common Plant materials


yeast infections in the women. About 75% of women have
one recurrence during their lives. Candida albicans is a S. khuzistanica aerial parts at the full flowering stage were
common type of yeast that causes candidiasis in the vagina collected from Lorestan Province in June 2014 and a voucher
of infected women [8]. The symptoms of candidiasis are specimen was authenticated under the number 168-1. Dried
abnormal vaginal discharge, itching and burning of the aerial parts of plant were grinded. The extraction was per-
vagina, pain with intercourse, painful urination and swell- formed with ethanol: water (70:30, v/v). The dried plant was
ing of the vulva [2]. Also, C. albicans can cause candidemia mixed with solvent at the ratio of 1:10 (w/v) for 24 h at
or disseminated infections, which are the significant cause ambient temperature in percolator. The mixture was sepa-
of morbidity and mortality of patients [14]. Treatment of rated by the tap of percolator and then was filtered through
Candidia infections are performed by miconazole, clotri- filter paper. The residue was rinsed with the same solvent
mazole, tiaconazole, butaconazole, fluconazole, echino- and the extract was dried under vacuum. The extraction
candines and amphotericin B and some other azole drugs yield was 47%. The dried extract was kept in refrigerator at
[18], but due to increasing resistance to azole drugs [17], 4 8C until the analysis.
the scientists are interested in finding the new sources of
antifungal agents among the prescriptions in traditional Microbial strains
medicine [13,20]. Satureja khuzistanica Jamzad (Marzeh-
E-Khuzistani) is a member of Labiatae family. Although, it is This study was conducted on 30 clinical isolates of C. albicans
used in folk medicine for the treatment of different ail- that were isolated from the vaginal samples (n = 21), oral
ments such as cramp, muscle pains, nausea, indigestion, wounds of patients with AIDS (n = 6) and genital infections of
diarrhea and infectious diseases but the reputation of this diabetic patients (n = 3). C. albicans ATCC 10231 and ATCC
plant is for its pain relieving and antiseptic effects 66506 also were used in this study. The strains were cultured
[5,19,27]. Recent studies have been confirmed some bio- on Sabouraud dextrose agar medium with chloramphenicol
logical activities such as anti-inflammatory effects [15,25], and were identified as C. albicans on the basis of some
antinociceptive and analgesic effects [22] according to biochemical test’s results such as germ tube (positive),
its traditional applications. The antiseptic effects of chlamydospore formation, color of colony, absorption of
S. khuzistanica were confirmed against bacteria [1,3] yeast carbohydrate and fermentation tests. The possible presence
and filamentous fungi [6,23]. Although, there are some of C. dubliniensis in the clinical isolate panel was considered
investigations in the regards of its antiseptic activities by removing the strains with dark green color colonies on
against Pseudomonas aeruginosa [1], Listeria monocytoge- ChromAgar Candida Agar at 37 8C after 24 h and the absence
nes [3], Staphylococcus aureus [4] and yeast like Crypto- of growth at 42—45 8C.
coccus neoformans [6], Candida albicans [4] and
filamentous fungi such as Aspergillus flavus, A. niger, Peni- Antifungal activity evaluation
cillium sp., Fusarium sp., Alternaria sp., Rhizopus sp. and
Mucor sp. [23], but there is no study that evaluate the anti- By using the methods of CLSI (NCCLS) (disc diffusion and
candidal activity of S. khuzistanica ethanol extract against micro-broth dilution assays), the sensitivity of C. albicans
clinical isolates of C. albicans. In this investigation, we clinical isolates to S. khuzistanica ethanol extract was
evaluated the anti-candidal activity of S. khuzistanica etha- determined. The optical density of fungal suspension at
nol extract against 30 clinical isolates and two ATCC strains 530 nm was 1.0 (1  106 CFU/mL). Candidal suspensions
of C. albicans. Also this study evaluated the synergistic were cultured on Sabouraud dextrose agar using a sterile
effects of some antifungal agents and this extract against cotton swab. The stock solution for extract was prepared
these clinical isolates. in dimethyl sulfoxide (DMSO) (0.1024 g/mL) and then it

Please cite this article in press as: Mahboubi M, Kazempour N. The anti-candidal activity of Satureja khuzistanica ethanol extract against
clinical isolates of C. albicans. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.11.003
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MYCMED-586; No. of Pages 5

The anti-candidal activity of Satureja khuzistanica ethanol extract 3

was filtered. Subsequently, filter discs (6 mm in diameter)


(Padtan Teb Co, Tehran, Iran) were saturated with 15 and
20 ml of diluted extract. Clotrimazole (10 mg/disc), fluco-
nazole (10 mg) and amphotericin B (10 U/disc) (Rosco,
Diagnostica A/S, Taastrupgaardsvej 30 DK-2630 Taastrup)
were used as controls. The plates were incubated at 37 8C
for 48 h. The inhibition zone diameters were measured by
dial caliper and were recorded in millimeter  Standard
Deviation (mm  SD) [10]. The minimum inhibitory
concentration (MIC) and minimum fungicidal concentra-
tion (MFC) values of ethanol extract and amphotericin
B were determined by micro-broth dilution assay. 100 ml
of two-fold serially diluted compounds (3.1—0.0025 mg/mL)
in MOPS-buffered RPMI 1640 were poured in 96-well micro-
titer plates. After shaking, 100 ml of diluted microbial
suspensions (105 CFU/mL) was added to each well and
incubated at 35  2 8C. MICs were defined as the lowest Figure 1 The antifungal activity of S. khuzistanica extract
concentration of extract that inhibited the fungal against clinical isolates of C. albicans by disc diffusion assay.
growth after 48 h. MFC values were the first well L’activité antifongique de l’extrait de S. khuzistanica extraire
showing no growth on Sabouraud dextrose agar with 2% les isolats cliniques de C. albicans par dosage en diffusion par la
dextrose [9]. méthode des disques.

Synergistic activity
Synergistic or antagonistic effects of
After determining the MIC values for clinical isolates, the MIC S. khuzistanica ethanol extract with antifungal
value for 90% of isolates (MIC90) were determined and the drugs
sub-inhibitory concentration of extract (1/4 or 1/2 of MIC90)
was inserted to Sabouraud dextrose agar with 2% dextrose. Synergistic activity: S. khuzistanica ethanol extract
The control group was the Sabouraud dextrose agar with 2% (75 mg/mL) with amphotericin B, clotrimazole and ketoco-
dextrose without extract. Plates were inoculated with the nazole against clinical isolates of C. albicans exhibited no
microbial suspensions (1  106 CFU/mL) by sterile swabs and significant synergistic effects. At this concentration, the
the antibiotic discs (clotrimazole [10 mg/disc], fluconazole inhibition zone diameters (mm) of amphotericin B, and
[10 mg] and amphotericin B [10 U/disc]) were put on the clotrimazole were increased but these increases were not
plates and were incubated at 35  2 8C for 48 h. Then, the significant (additive effect). Therefore, we evaluated the
diameter of inhibition zone was measured by dial caliper and synergistic effects of amphotericin B, clotrimazole and keto-
reported [7]. conazole with 150 mg/mL of S. khuzistanica ethanol extract
(1/2 MIC90). The results of increasing in ethanol extract
showed that there was a synergistic effect (P < 0.05) bet-
Statistical analysis
ween S. khuzistanica ethanol extract with amphotericin B
and ketoconazole, while this extract had no effect on clo-
The data were analyzed in Graph Pad prism and the diffe-
trimazole activity (Table 1; Fig. 2).
rences between the results and its significance were esti-
mated by Anova One-Way in GraphPad Prism. The significant
level was 0.05. Discussion

Medicinal plants are new sources for finding the antimicro-


Results bial agents. The antifungal activity of S. khuzistanica metha-
nol extract was confirmed against one isolate of C. albicans
The antifungal activity of S. khuzistanica ethanol [4]. In this investigation, the methanol extract from wild and
extract against clinical isolates of C. albicans cultivated plant was investigated. The yield of extraction by
methanol solvent was much lower (2.7—3.1%) than the etha-
In disc diffusion method, the antifungal activity of nol extraction from our study (47%). The anti-candidal eva-
S. khuzistanica ethanol extract against clinical isolates luation of S. khuzistanica methanol extract (concentration
revealed that the mean of inhibition zone diameter (mm) 2.7 mg/mL) against one isolate of C. albicans exhibited that
of extracts (1.5 mg/disc = 11.3  0.62 mm) was lower than the inhibition zone diameters were 15 and 16 mm, while the
synthetic antifungal agents (20.85  1.36, 26.3  2.5, MIC values were 4 and 1 mg/mL for wild and cultivated
24.1  2.6 mm were for amphotericin B, clotrimazole, S. khuzistanica methanol extract. The amount of MIC value
ketoconazole, respectively). The means of MIC and MFC from our study was close to the MIC value of wild type
values were 299.4  54.5 and 722.6  53.44 (mg/mL) for S. khuzistanica methanol extract [4]. Therefore, the anti-
S. khuzistanica ethanol extract while these amounts were candidal activity of S. khuzistanica methanol extract against
0.53  0.19 and 0.98  0.36 (mg/mL) for amphotericin B, one isolate of C. albicans was similar to the anti-candidal
respectively (Fig. 1). activity of S. khuzistanica ethanol extract against clinical

Please cite this article in press as: Mahboubi M, Kazempour N. The anti-candidal activity of Satureja khuzistanica ethanol extract against
clinical isolates of C. albicans. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.11.003
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MYCMED-586; No. of Pages 5

4 M. Mahboubi, N. Kazempour

Table 1 The synergistic activity of S. khuzistanica ethanol had the acceptable anti-candidal activity against clinical
extract and antibiotics (mm). isolates of C. albicans.
L’activité synergique de S. khuzistanica extrait de l’éthanol Due to the pain and inflammation as a result of C. albicans
et des antibiotiques (mm). infections, the application of S. khuzistanica as a treatment
can help reducing the pain and inflammation in women’s
Means  SE Lower Upper
vagina. The antinociceptive, analgesic [11,22], anti-inflam-
AP-Extract 20.8  0.12 17.8 22.4 matory [15,25] and immune-stimulatory effects of
(75 mg/mL) S. khuzistanica were confirmed. S. khuzistanica inhibits
AP-Extract 22.2  0.18 18.7 28.5 the expression of iNos gene in macrophages [15] and the
(150 mg/mL) activation of spinal glial cell [11] and activation of the serum
AP-Control 21.1  0.19 9 25.1 lysosyme [16] and finally decreases the pain and inflamma-
Keto-Extract 27.1  0.35 21.5 33.7 tion and increases the immune system of patients. There-
(75 mg/mL) fore, C. albicans infected patients may benefit from
Keto-Extract 31.25  0.36 23.5 34.7 S. khuzistanica ethanol extract by its analgesic, anti-inflam-
(150 mg/mL) matory and anti-candidal effects.
Keto-Control 27.9  0.32 18.4 36.3 So, it can be an alternative source for treatment of
Clo-Extract 23.9  0.23 18.4 28.4 C. albicans related diseases. More pharmacological, toxico-
(75 mg/mL) logical and clinical studies are required to confirm the
Clo-Extract 25.2  0.32 20.6 28.8 efficacy of S. khuzistanica ethanol extract against
(150 mg/mL) C. albicans infections.
Clo-Control 24.7  0.3 18.5 31.4 The synergistic effect of plant extracts and antibiotics has
been the subject of many other studies [7,12,21]. The
AP: amphotericin B; Ket: ketoconazole; Clo: clotrimazole.
synergistic effects of itraconazole and fluconazole with
Echinophora platyloba ethanol extract were confirmed [7].
isolates of C. albicans from this study. Therefore, due to the Our results showed potent synergistic effects of
high yield of extraction for S. khuzistanica ethanol extract S. khuzistanica ethanol extract and amphotericin B and
rather than its methanol extract and the adverse effects of ketoconazole as treatment for C. albicans infections. If
methanol residue for application in human uses, it is more the application and safety of S. khuzistanica ethanol extract
economical to do further investigations on S. khuzistanica were confirmed in clinics, the advantage of these kinds of
ethanol extract as antifungal agent against C. albicans. The studies would be using of lower amount of harmful anti-
high sensitivity of C. albicans to other species of Satureja biotics or decreasing the time of treatment in patients that
sp is reported [24,26]. Therefore, S. khuzistanica like finally leads to decreasing the cost of treatment.
S. abyssinica spp. Abyssinica, S. paradoxa and S. hortensis
Conclusion

This study introduces S. khuzistanica ethanol extract as a


new antifungal candidate against clinical isolates of
C. albicans. Furthermore, S. khuzistanica ethanol extract
had synergistic effects with amphotericin B and ketocona-
zole.

Disclosure of interest

The authors declare that they have no competing interest.

Acknowledgements

This study is supported by Medicinal Plants, Research Center


of Barij, Kashan, Iran (Grant No.: 9323).

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Please cite this article in press as: Mahboubi M, Kazempour N. The anti-candidal activity of Satureja khuzistanica ethanol extract against
clinical isolates of C. albicans. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.11.003
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Please cite this article in press as: Mahboubi M, Kazempour N. The anti-candidal activity of Satureja khuzistanica ethanol extract against
clinical isolates of C. albicans. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.11.003