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MYCMED-586; No. of Pages 5

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

determined. and the extract was dried under vacuum.doi. fluconazole. Kazempour Extraction à l’éthanol . Par conséquent. khuzistanica au stade de pleine floraison a été évalué contre 30 isolats cliniques Effet synergique et deux souches de référence ATCC de C. but due to increasing resistance to azole drugs [17]. Fusarium sp. pain with intercourse.3] yeast carbohydrate and fermentation tests. The antiseptic effects of chlamydospore formation. it is This study was conducted on 30 clinical isolates of C. khuzistanica etha. Dried abnormal vaginal discharge. khuzistanica peut être considéré comme une nouvelle source d’agent anti-Candida contre les isolats cliniques de C. albicans. Tous droits réservés. Also. Treatment of rated by the tap of percolator and then was filtered through Candidia infections are performed by miconazole. its traditional applications. formed with ethanol: water (70:30.1024 g/mL) and then it Please cite this article in press as: Mahboubi M. The dried plant was ing of the vulva [2]. khuzistanica were confirmed against bacteria [1. C. The residue was rinsed with the same solvent mazole. albicans on the basis of some antinociceptive and analgesic effects [22] according to biochemical test’s results such as germ tube (positive).0 (1  106 CFU/mL).1016/j. khuzistanica ethanol extract was evaluated the anti-candidal activity of S.6 (mg/mL). Staphylococcus aureus [4] and yeast like Crypto. khuzistanica contre les isolats cliniques étaient de 299. C.. alors qu’il n’a eu aucun effet synergique sur le clotrimazole contre des isolats cliniques de C. L’extrait éthanolique de parties Agent anti-Candida . About 75% of women have one recurrence during their lives. albicans can cause candidemia mixed with solvent at the ratio of 1:10 (w/v) for 24 h at or disseminated infections. 4 8C until the analysis.19.2015. absorption of S. En outre. clotri. and Mucor sp. 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]. Journal De Mycologie Médicale (2016). albicans. tiaconazole. indigestion. l’extrait éthanolique de S. nous avons évalué les effets synergiques de l’amphotéricine B. echino. du clotrimazole et du kétoconazole avec l’extrait éthanolique de S. Peni. By using the methods of CLSI (NCCLS) (disc diffusion and candidal activity of S. The extraction was per- vagina. Listeria monocytoge. and were identified as C. The symptoms of candidiasis are specimen was authenticated under the number 168-1. albicans ATCC 10231 and ATCC plant is for its pain relieving and antiseptic effects 66506 also were used in this study.. but there is no study that evaluate the anti. 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. nausea. In this investigation. which are the significant cause ambient temperature in percolator. on Sabouraud dextrose agar medium with chloramphenicol logical activities such as anti-inflammatory effects [15.23]. The possible presence and filamentous fungi [6. N. L’extrait éthanolique de S. of Pages 5 2 M. The stock solution for extract was prepared these clinical isolates. Cette étude a évalué l’activité anti-Candida de l’extrait alcoolique de Candida albicans .25]. 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]. No.003 . aériennes de S. Mahboubi. # 2015 Elsevier Masson SAS. The mixture was sepa- of morbidity and mortality of patients [14]. [23]. http://dx. niger. coccus neoformans [6]. albicans used in folk medicine for the treatment of different ail. albicans. Rhizopus sp. Although. Alternaria sp. the sensitivity of C.. albicans. Candida albicans [4] and filamentous fungi such as Aspergillus flavus.mycmed. wounds of patients with AIDS (n = 6) and genital infections of diarrhea and infectious diseases but the reputation of this diabetic patients (n = 3). The anti-candidal activity of Satureja khuzistanica ethanol extract against clinical isolates of C. ChromAgar Candida Agar at 37 8C after 24 h and the absence nes [3]. The optical density of fungal suspension at nol extract against 30 clinical isolates and two ATCC strains 530 nm was 1. of growth at 42—45 8C. Antifungal activity evaluation cillium sp. The extraction candines and amphotericin B and some other azole drugs yield was 47%. traitements actuels. Satureja khuzistanica Jamzad (Marzeh- E-Khuzistani) is a member of Labiatae family.4 et 722. there are some of C. Although. A. albicans clinical isolates of C. albicans.11.20].27]. oral ments such as cramp. filter paper. khuzistanica. we clinical isolates to S. in dimethyl sulfoxide (DMSO) (0.+ Models MYCMED-586. albicans. Kazempour N. khuzistanica ethanol extract against micro-broth dilution assays). Les moyennes de MIC et MFC de l’extrait éthanolique de S. Candida albicans is a S.org/10. itching and burning of the aerial parts of plant were grinded. that were isolated from the vaginal samples (n = 21). The strains were cultured [5. painful urination and swell. Introduction Materials and methods Vaginal yeast infections or vaginal candidiasis are common Plant materials yeast infections in the women. muscle pains. Recent studies have been confirmed some bio. albicans par diffusion du disque en gélose et par dosage en micro-dilution. The dried extract was kept in refrigerator at [18]. v/v). respectivement. the scientists are interested in finding the new sources of antifungal agents among the prescriptions in traditional Microbial strains medicine [13. Satureja khuzistanica contre les isolats cliniques de C. butaconazole. Candidal suspensions of C. khuzistanica augmentait l’effet anti-Candida de l’amphotéricine B et du kétoconazole. color of colony.

the anti- S. the significant (additive effect).3  0. The significant level was 0. After shaking. of Pages 5 The anti-candidal activity of Satureja khuzistanica ethanol extract 3 was filtered.05. while the synthetic antifungal agents (20. 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. activity of S. The plates were incubated at 37 8C for 48 h. In this investigation. clotrimazole and ketoco- dextrose without extract. The anti-candidal activity of Satureja khuzistanica ethanol extract against clinical isolates of C. albicans cultivated plant was investigated. Synergistic activity Synergistic or antagonistic effects of After determining the MIC values for clinical isolates.4  54. khuzistanica methanol extract [4]. one isolate of C. Therefore. khuzistanica ethanol extract with amphotericin B and ketoconazole. khuzistanica methanol extract (concentration revealed that the mean of inhibition zone diameter (mm) 2.1%) than the etha- In disc diffusion method.3  2.1—0.5. The antifungal activity of S. the methanol extract from wild and extract against clinical isolates of C. The results of increasing in ethanol extract showed that there was a synergistic effect (P < 0.003 . khuzistanica ethanol extract against clinical Please cite this article in press as: Mahboubi M.85  1. albicans by disc diffusion assay. khuzistanica methanol extract against 0. fluco- nazole (10 mg) and amphotericin B (10 U/disc) (Rosco. conazole with 150 mg/mL of S.2015.11. MICs were defined as the lowest Figure 1 The antifungal activity of S. growth after 48 h.6 mm were for amphotericin B. No. we evaluated the diameter of inhibition zone was measured by dial caliper and synergistic effects of amphotericin B. Subsequently.36.7—3. MFC values were the first well L’activité antifongique de l’extrait de S.5 and 722.36 (mg/mL) for amphotericin B. albicans exhibited no microbial suspensions (1  106 CFU/mL) by sterile swabs and significant synergistic effects. 100 ml of two-fold serially diluted compounds (3. Tehran.1  2. http://dx. the antifungal activity of nol extraction from our study (47%). khuzistanica metha- nol extract was confirmed against one isolate of C. The means of MIC and MFC from our study was close to the MIC value of wild type values were 299. Iran) were saturated with 15 and 20 ml of diluted extract. khuzistanica ethanol extract while these amounts were candidal activity of S.53  0. At this concentration.+ Models MYCMED-586. 100 ml of diluted microbial suspensions (105 CFU/mL) was added to each well and incubated at 35  2 8C. filter discs (6 mm in diameter) (Padtan Teb Co. clotrimazole and keto- reported [7]. khuzistanica ethanol extract against clinical isolates luation of S. Then. while this extract had no effect on clo- The data were analyzed in Graph Pad prism and the diffe- trimazole activity (Table 1. S. khuzistanica extraire showing no growth on Sabouraud dextrose agar with 2% les isolats cliniques de C. albicans exhibited that of extracts (1. albicans was similar to the anti-candidal respectively (Fig.doi. respectively). MIC values were 4 and 1 mg/mL for wild and cultivated 24.6  53. the the antibiotic discs (clotrimazole [10 mg/disc]. Plates were inoculated with the nazole against clinical isolates of C. The anti-candidal eva- S. albicans. Synergistic activity: S. albicans par dosage en diffusion par la dextrose [9]. khuzistanica ethanol [4]. 26. The amount of MIC value ketoconazole. méthode des disques. khuzistanica methanol extract. albicans The antifungal activity of S.44 (mg/mL) for S.org/10. Fig. Diagnostica A/S.98  0. the MIC S.mycmed. khuzistanica extract concentration of extract that inhibited the fungal against clinical isolates of C. 1). Discussion Medicinal plants are new sources for finding the antimicro- Results bial agents. khuzistanica ethanol extract The control group was the Sabouraud dextrose agar with 2% (75 mg/mL) with amphotericin B.5 mg/disc = 11. 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.05) bet- Statistical analysis ween S. Kazempour N.1016/j. khuzistanica ethanol extract (1/2 MIC90). Journal De Mycologie Médicale (2016). Taastrupgaardsvej 30 DK-2630 Taastrup) were used as controls. clotrimazole. The yield of extraction by methanol solvent was much lower (2. The inhibition zone diameters were measured by dial caliper and were recorded in millimeter  Standard Deviation (mm  SD) [10].19 and 0. fluconazole inhibition zone diameters (mm) of amphotericin B.7 mg/mL) against one isolate of C. Clotrimazole (10 mg/disc).0025 mg/mL) in MOPS-buffered RPMI 1640 were poured in 96-well micro- titer plates. 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. Therefore.62 mm) was lower than the inhibition zone diameters were 15 and 16 mm. rences between the results and its significance were esti- mated by Anova One-Way in GraphPad Prism. 2).

khuzistanica ethanol extract had synergistic effects with amphotericin B and ketocona- zole.1  0. reja khuzistanica essential oil against standard and isolated Please cite this article in press as: Mahboubi M. due to the Our results showed potent synergistic effects of high yield of extraction for S. albicans from this study. Ghaemi N. khuzistanica ethanol tract.8 22. References [1] Abbasi A.4 C. isolates of C.doi. Journal De Mycologie Médicale (2016).18 18.1  0.7  0. Kazempour Table 1 The synergistic activity of S.9 et 149 mg/mL) et des antibiotiques. Acknowledgements This study is supported by Medicinal Plants. infections. Kashan. Furthermore. Mahbubi A.4 28.32 20. khuzistanica inhibits AP-Extract 22.8 efficacy of S.2015.3 So.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 21.5 33. khuzistanica as a treatment can help reducing the pain and inflammation in women’s Means  SE Lower Upper vagina. anti-inflam- AP-Extract 20.23:253—73.12. No. albicans infections. S. khuzistanica ethanol extract by its analgesic.3:614—8.7 S. Therefore. Keto-Control 27.36 23.6 28. The studies would be using of lower amount of harmful anti- high sensitivity of C.5 31. S. Int J Curr Microbiol App Sci 2014. khuzistanica extrait de l’éthanol Due to the pain and inflammation as a result of C. N. S.3 18.4 matory [15.8  0. albicans. toxico- (75 mg/mL) logical and clinical studies are required to confirm the Clo-Extract 25. Fries BC. L’évaluation synergique de l’extrait éthanolique de Shokri-Fashtali S.4 The synergistic effect of plant extracts and antibiotics has been the subject of many other studies [7. khuzistanica were confirmed in clinics.mycmed. abyssinica spp. Bahador A.5 34.7 tion and increases the immune system of patients. Issazadeh K.32 18. The antinociceptive. Amiri M. If methanol residue for application in human uses.22]. it can be an alternative source for treatment of Clo-Extract 23.4 36. Disclosure of interest The authors declare that they have no competing interest.2  0. The study of inhibitory effects of Satureja khuzistanica against MDR isolates of Pseudomonas aeruginosa. albicans related diseases. S. Therefore. khuzistanica (74.: 9323). khuzistanica ethanol extract S. albicans to other species of Satureja biotics or decreasing the time of treatment in patients that sp is reported [24.25  0. Clo-Control 24. http://dx. [2] Achkar JM.12 17. Candida infections of the genitourinary Figure 2 The synergistic evaluation of S. paradoxa and S.2  0.23 18.26]. Evaluation of antibacterial activity of Satu- S. khuzistanica ethanol extract and amphotericin B and rather than its methanol extract and the adverse effects of ketoconazole as treatment for C. analgesic [11. [3] Akbari-Shahabi S. khuzistanica were confirmed. Assmar M. albicans. There- (75 mg/mL) fore. synergistic effects of itraconazole and fluconazole with Echinophora platyloba ethanol extract were confirmed [7]. Mahboubi. khuzistanica ethanol extract against (150 mg/mL) C. albicans.21].7 28. Abyssinica. More pharmacological. hortensis Conclusion This study introduces S. khuzistanica ethanol extract as a new antifungal candidate against clinical isolates of C. Iran (Grant No. Research Center of Barij.org/10.25] and immune-stimulatory effects of (75 mg/mL) S.1016/j. the advantage of these kinds of ethanol extract as antifungal agent against C. khuzistanica ethanol extract economical to do further investigations on S. isolates of C. Kazempour N. The AP: amphotericin B.19 9 25.9  0. albicans infections. Esmaeili D. extract (74. Massih A.35 21.003 . khuzistanica ethanol had the acceptable anti-candidal activity against clinical extract and antibiotics (mm). Ket: ketoconazole. albicans. L’activité synergique de S.1 lysosyme [16] and finally decreases the pain and inflamma- Keto-Extract 27. Clin Microbiol Rev 2010.+ Models MYCMED-586. Amiri M. of Pages 5 4 M. albicans infected patients may benefit from Keto-Extract 31. it is more the application and safety of S. The anti-candidal activity of Satureja khuzistanica ethanol extract against clinical isolates of C. Clo: clotrimazole. C. khuzistanica like finally leads to decreasing the cost of treatment. albicans et des antibiotiques (mm). the application of S.9  0. anti-inflam- (150 mg/mL) matory and anti-candidal effects.11.9 and 149 mg/mL) and antibiotics. S.

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