Antifungal activity of Satureja hortensis alcoholic extract against Aspergillus and Candida species

Pharmacological and biological investigations have justified the traditional application of Satureja hortensis as a natural source of compounds for food conservation, as well as in the treatment of ailments including inflammatory diseases, cramps, muscle pains, nausea, indigestion, diarrhea, and infectious diseases. Regarding to the low number of reports of Satureja hortensis antifungal activities, we focused on the inhibitory and fungicidal effects of the Satureja alcoholic extract on Candida and Aspergillus species. The subjects included patients with clinical isolates of Candida and Aspergillus obtained from UMSU University Hospital, Urmia, Iran. A total of 51 clinical isolates, including 31(60.8%) Candida albicans and 20(39.2%) Aspergillus species, respectively, were obtained. McFarland 0.5 solutions of the fungal isolates were treated with alcoholic (ethanol) extract of S. hortensis using 1tube serial dilutions (MIC) and 2agar serial dilutions (MFC). The Inhibitory range for Candida isolates was shown to be 1/20 to 1/320. Findings of MIC and MFC on Aspergillus isolates showed that the lowest effective concentration was detected at the dilution point of 1/80 (MIC=12.5 mg/ml). Also, we got the result of 24 hours as the best incubation time of treatment for all the Aspergillus isolates tested. Our findings showed that the alcoholic extract of S. hortensis is able to inhibit growth of some Candida and Aspergillus clinical isolates in vitro and also killed them in higher concentrations.


INTRODUCTION
Pharmacological and biological investigations have justified the traditional application of Satureja hortensis as a natural source of compounds for food conservation, as well as in the treatment of ailments including inflammatory diseases, cramps, muscle pains, nausea, indigestion, diarrhea, and infectious diseases due to its antispasmodic, anti-diarrheal, antioxidant, antibacterial and antifungal properties as reported in literature (Adiguzel et al., 2007;Dikbas et al., 2008;Güllüce et al., 2003;Hajhashemi et al., 2000;Heydari et al., 2011;Mihajilov-Krstev et al., 2010;Razzaghi-Abyaneh et al., 2008;Şahin et al., 2003).The antimicrobial activity of S. hortensis extract has also been reported in previous studies.
Some studies have evaluated the antimicrobial and antioxidant activities of the essential oil obtained, using a Clevenger distillation apparatus, water soluble (polar) and water insoluble (non polar) sub fractions of the methanol extracts from aerial parts of S. hortensis plants, and methanol extract from calli established from the seeds *Corresponding author.E-mail: kambiz37diba@gmail.comor kdiba@umsu.ac.ir.using Gamborg's B5 basal media supplemented with indole-3-butyric acid (1.0 ppm), 6-benzylaminopurine (N(6)-benzyladenine) (1.0 ppm), and sucrose (2.5%) (Dikbas et al., 2008).The antimicrobial test results showed that the essential oil of S. hortensis had great potential antimicrobial activities against all 23 bacteria and 15 fungi and yeast species tested (Güllüce et al., 2003).The essential oil and methanol extract of S. hortensis were also evaluated for antifungal activity against Aspergillus flavus in vitro on Petri plates and liquid culture, and under storage conditions.The oil showed strong antifungal activity based on the inhibition zone and minimal inhibitory concentration values against the pathogen on Petri plates assays (Dikbas et al., 2008).Other studies reveal a new biological activity for S. hortensis L. as strong inhibition of aflatoxin production by Aspergillus parasiticus.Carvacrol and thymol, and the effective constituents of S. hortensis L., may be useful in controlling aflatoxin contamination of susceptible crops in the field (Razzaghi-Abyaneh et al., 2008).
It is well known that the chemical composition and yield of essential oils are affected by exogenous factors, such as geographical position, altitude, and climate and soil composition (Mihajilov-Krstev et al., 2010).For this reason, in the present study, the growth inhibitory and fungicidal effect of the alcoholic extract of S. hortensis on some hospital isolates of Aspergillus niger, A. flavus, Aspergillus fumigatus and Candida albicans obtained from clinical sources in Iran were studied as a model system for fungi.

Collection and identification of plants
About 2000 g of the S. hortensis seeds (called Marzea in Persian word) were obtained from a private traditional plant farm in the North-west of Iran, and confirmed by the Plants Systematic Laboratory, Pharmacology Dep.UMSU, Iran.

Preparation of alcoholic extract
Air-drying of S. hortensis seeds was performed in a dark place at room temperature for 48 h.The dried seeds (1000 g) were ground and subjected to alcoholic (ethanol) extraction using a dehydrator apparatus.In brief, the Marzea powder was added to 3200 ml of ethanol 96%, shaken for 2 h and incubated over night at room temperature to remove alcoholic solvent from the crude extract.Again, the same amount of solvent was added to the crude extract and the process was repeated for five times.The alcoholic solvent was separated completely by a vacuum filtering system and the final product was collected in a 250 ml container and stored at 4°C until use.

Microorganisms
The main subjects were Candida and Aspergillus species (pathogenic opportunistic fungi) isolated from patients with symptomatic infections.The species used in this study included; C. albicans, A. fumigatus, A. flavus and A. niger, and some other Candida species with low frequency of isolations.All organisms were identified in the Department of Medical Mycology, Urmia University of Medical sciences, Urmia, Iran.Candida isolates were identified in the level of species using differential media CHROMagar Candida and the restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) method included C. albicans, Candida glabrata, Candida kefyre and Candida parapsilosis.The identification of Aspergillus species was performed using morphological characteristics, and also confirmed by the molecular method as earlier mentioned so that three species of A. fumigatus, A. flavus and A. niger were identified and used in the study.
We also used standard control strains, including Candida tropicalis (PTCC 5028), C. albicans 10231) and C. glabrata (UMSU C152), for the evaluation of the findings of clinical fungal isolates.The fungal suspensions were prepared from 48 -72 h Sabouraud broth cultures at room temperature and then adjusted to the McFarland 0.5 standard dilution (corresponding to 10 3 -10 4 spore/ml for fungal strains consensus standard by the NCCLS M38 [ISBN 1-56238-480-8).

Serial dilutions in tubes
A small volume (1 ml) of Sabouraud broth in capped tubes was used as substrate.Briefly, 1 ml of 10% solution of the crude extract in sterile water was added into the substrate and mixed thoroughly.Then, 1 ml of the solution was transferred into the next tube containing 1 ml of substrate, mixed and transferred into the next tube in same way up to 6 th tube, so that six decreasing titers of S. hortensis alcoholic extract including: 1/20, 1/40, 1/80, 1/160, 1/320, 1/640 were made.

Serial dilutions in agar media
The Sabouraud glucose agar media (SGA) was boiled, autoclaved and aliquated 10 ml in plates (8 cm in diameter).Then 1, 0.5, 0.25, 0.125 and 0.0625 ml of a prepared 1/10 dilution were added to the plates 1 to 5, respectively.

Treatment of fungal suspension with diluted extracts
an aseptic condition, 1 ml of Candida suspension was added to serial diluted tubes.Negative and positive controls were prepared by adding 1 ml to ½ diluted extract and Candida suspension, respectively.Agar dilution method was used only for Aspergillus isolates, using Sabouraud glucose agar media that was mixed in advance with serial diluted Satureja alcoholic extract.The spore suspensions (0.02% agar and 0.05% Tween 20) inoculated on the mixed media except the control negative plate and control positive included agar media free of extract.All treatments were incubated room temperature (RT), respectively.

Minimum inhibitory concentration (MIC)
After 24 -48 h of incubation at RT, the tubes of reactions were observed for Candida growth turbidity.This was performed by comparing the growth turbidity of test tubes with that of McFarland standard 0.5.The tubes without any turbidity were considered as positive and the other as negative.MIC was determined at the point of negative turbidity.Also, the first SGA plate in the serial agar dilution, without growth of Aspergilli showed MIC of Satureja on Aspergillus isolates.

Minimum fungicidal concentration (MFC)
All tubes without turbidity (negative tubes) were tested for fungicidal effect of Satureja alcoholic extract.MFC resulted from growth of the isolate on Sabouraud glucose agar media after 24 -72 h of incubation at RT.After the incubation time, all SGA plates were tested for the growth of Candida colonies.The plates with fungal growth were excluded, while the first plate without fungal growth was the cutoff point, and this showed the fungicidal activity of S. hortensis alcoholic extract.

RESULTS
In total, 100 fungal isolates including C. albicans (mostly), C. parapsilosis, C. kefyre, C. glabrata (rarely) and 3 Aspergillus species were tested for MIC and MFC.The results of broth micro dilution assay (Table 1) showed that the alcoholic extract was active against all the yeasts tested in the following range of concentrations: MIC = 0.312 to 1.25 mg/ml; MIC: 0.312 mg/ml for C. glabrata as the highest and MIC: 1.25 mg/ml for C. albicans as the lowest inhibitory effect of S. hortensis the alcoholic extract.Minimum fungicidal concentration of the alcoholic extract on Candida isolates showed a range of 0.312 to 2.5 mg/ml.From our results, the highest fungicidal activity of the extract included MFC: 0.625 mg/ml for C. glabrata, and 2.5 mg/ml was the lowest MFC for C. albicans.Furthermore, the S. hortensis alcoholic extract was active against the tested Aspergillus species in the range of MIC/MBC = 0.05-0.39/0.05-0.78μL/ml, which exhibited higher activity for A. niger and A. fumigatus compared to A. flavus.In fact, A. flavus cultures treated by S. hortensis extract resulted only in morphologic alterations of macroscopic features, including sporulation rate, color and size of colonies.There was no microscopic variation considerable for Aspergillus species.
It is clear that 24 h treatment at fungal suspension with S. hortensis is much more effective than 2 and 6 h treatment.

DISCUSSION
S. hortensis is considered from ancient time to date as a plant with drug properties.Some compounds of this plant have anti-parasite and anti-microbial effects.The essential oil of S. montana have a broad-spectrum of antimicrobial activity against 46 species of yeasts.This high and diffused activity could be used to control potential pathogenic and spoilage yeasts (Ciani et al., 2000).
In the present study, the anti-fungal activity of S hortensis on some species of Candida and Aspergillus was tested in vitro, respectively.The inhibitory effects of alcoholic extract of Peganum harmala have been measured and indicated by MIC and MFC parameters (Diba et al., 2011).The activity of this extract against Candida spp. was in the range of: MIC=0.312 to 1.25 mg/ml.
The least MIC has indicated in C. albicans and the most MIC in C. glabrata.The least measured MIC in C. albicans can be explained by the resistance of most C. albicans strains to antifungal drug.Its MIC/MBC values are very low (lower than the referent antibiotics) and in most cases they are at the same concentration.The results of bacterial studies indicate that S. hortensis alcoholic extract, showed various activities in relation studies.
The activity of this extract against Candida spp. was in the range of MIC/MBC=0.39/0.78μL/ml, while the oil collected from Yusufli in Turkey did not show any effect against this strain.Also, it exhibited higher activity against two strains of S. aureus (MIC=0.20 and 0.39 µL/ml, respectively) in relation to the oil from Turkey (MIC=15.62-62.50μL/ml, respectively) (Adiguzel et al., 2007).The result of Pseudomonas aeruginosa susceptibility is also in accordance with the effect of the Turkish S. hortensis essential oil against this strain (MIC=31.25,62.50 and 125 μL/ml, respectively) (Adiguzel et al., 2007).
In previous studies, the alcoholic extract of S. hortensis showed antifungal activity against phytopathogenic fungi (Boyraz and Ozcan, 2006) and against food spoilage fungi (Adiguzel et al., 2007).The dominant component of this oil, carvacrol, is capable of inhibiting aflatoxin production by A. parasiticus (Razzaghi-Abyaneh et al., 2008) and A. flavus in a liquid medium and tomato paste (Dikbas et al., 2008;Omidbaigi et al., 2005).The same authors suggested that carvacrol could be useful in controlling aflatoxin contamination of susceptible crops.In the present study, the extract exhibited a partial antifungal activity against A. niger (MIC/MBC= 0.78 μL/ml), Saccharomyces cerevisiae (MIC/MBC = 0.39/0.20 μL/ml) and C. albicans (MIC/MBC = 0.20 μL/ml).Against C. albicans, however, the oil from Turkey did not show any effect (Adiguzel et al., 2007;Mihajilov-Krstev et al., 2010).Moreover, incubation time at 2 h did not show fungicidal effects on Candida and Aspergillus isolates, although there was a fungicidal result for 2 of 14 Candida tested isolates (Figure 1).Nevertheless, we got results on 24 h incubation for all the tested Aspergillus isolates.
The essential oil of S. hortensis L. collected in Serbia yielded 2.05%, which was higher than previously reported (4).The oil exhibited very high antibacterial and antifungal activity, owing to high content of monoterpene carvacrol, which is a well known antimicrobial compound.Active concentrations were much lower than those of the referent antibiotics.These values, together with high yield and lack of toxicity economically justify the use of essential oil derived from S. hortensis L. for many purposes: food conservation, treatment of different human diseases, and also for the treatment of phytopathogens that infect economically important plants (Adiguzel et al., 2007;Stammati et al., 1999).
To our knowledge, the Satureja alcoholic extract is active against opportunistic and food borne fungi (Candida spp).The present study clearly demonstrates that the alcoholic extract of S. hortensis contains compounds possessing antifungal properties.Therefore, on the basis of our results in present and previous studies, S. hortensis can be added as a protective agent to various food products (Razzaghi-Abyaneh et al., 2008).

Table 1 .
The results on the effects of Satureja hortensis alcoholic assay on the tested Candida and Aspergillus isolates in broth micro dilution assay.