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MYCMED-468; No. of Pages 8 Journal de Mycologie Médicale (2014) xxx, xxx—xxx

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ORIGINAL ARTICLE/ARTICLE ORIGINAL

Antifungal activities and chemical composition of some medicinal plants ´ s antifongiques et composition chimique de quelques Activite ´ dicinales plantes me A. Mohammadi a, H. Nazari a, S. Imani b, H. Amrollahi b,* a b

Department of biochemistry, faculty of medicine, Semnan university of medical sciences, Semnan, Iran Department of microbiology, faculty of medicine, Semnan university of medical sciences, Semnan, Iran

Received 7 September 2013; received in revised form 22 December 2013; accepted 3 February 2014

KEYWORDS Antifungal; Essential oil; Stachys pubescens; Thymus kotschyanus; Thymus daenensis; Bupleurum falcatum; GC/MS

MOTS CLÉS Antifongiques ; Huiles essentielles ; Stachys pubescens ;

Summary The use of and search for drugs and dietary supplements derived from plants have accelerated in recent years. Ethnopharmacologists, botanists, microbiologists and naturalproducts scientists are combing the earth for phytochemicals and leads, which could be developed for treatment of infectious diseases. The aim of this study was to investigate the antifungal activities of the essential oils of some medicinal plants such as Stachys pubescens, Thymus kotschyanus, Thymus daenensis and Bupleurum falcatum against Fusarium oxysporum, Aspergillus flavus and Alternaria alternata. The essential oils were used to evaluate their MICs and MFCs compared to the amphotricin B as a standard drug. The essential oils were also analyzed by GC/MS. Essential oils isolated from the S. pubescens, T. kotschyanus and B. falcatum showed strong antifungal activities. The essential oil of T. daenensis exhibited a moderate activity against the selected fungi in comparison with the other plants’ essential oils. In addition, the results showed that 26, 23, 22 and 15 components were identified from the essential oils of T. kotschyanus, S. pubescens, T. daenensis and B. falcatum, respectively. These oils exhibited a noticeable antifungal activity against the selected fungi. Regarding obtained results and that natural antimicrobial substances are inexpensive and have fewer side effects, they convey potential for implementation in fungal pathogenic systems. # 2014 Elsevier Masson SAS. All rights reserved. Résumé L’utilisation et la recherche de médicaments et de compléments alimentaires dérivés de plantes se sont ont accélérées au cours des dernières années. Les éthnopharmacologistes, les botanistes, les microbiologistes et les produits naturels scientifiques explorent la terre à la recherche composés phytochimiques qui pourraient être développés pour le traitement des maladies infectieuses. Le but de cette étude était de déterminer les activités antifongiques

* Corresponding author. E-mail address: [email protected] (H. Amrollahi). 1156-5233/$ — see front matter # 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.mycmed.2014.02.006

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A. Mohammadi et al. Thymus kotschyanus ; Thymus daenensis ; Bupleurum falcatum ; GC/MS

des huiles essentielles de certaines plantes médicinales telles que Stachys pubescens, Thymus kotschyanus, Thymus daenensis et Bupleurum falcatum contre Fusarium oxysporum, Aspergillus flavus et Alternaria alternata. Les huiles essentielles ont été utilisées pour évaluer leurs CMIs et CFMs par rapport à l’amphotéricine B comme médicament de référence. Les huiles essentielles ont également été analysées par GC/MS. Les huiles essentielles isolées à partir des S. pubescens, T. kotschyanus et B. falcatum ont montré une forte activité antifongique. L’huile essentielle de T. daenensis a montré une activité modérée contre les champignons sélectionnés en comparaison avec les huiles essentielles des autres plantes. En outre, les résultats ont montré que 26, 23, 22 et 15 composés ont été identifiés à partir des huiles essentielles de T. kotschyanus, S. pubescens, T. daenensis et B. falcatum, respectivement. Ces huiles ont montré une activité antifongique notable contre les champignons sélectionnés. Les substances antimicrobiennes naturelles ne sont pas chères et ont moins d’effets secondaires. Elles ont donc le potentiel d’être utilisées dans les infections fongiques. # 2014 Elsevier Masson SAS. Tous droits réservés.

Introduction Attention and use of herbal medicinal in the world and especially in Asian countries, contributes significantly to primary health care. Researchers and pharmaceutical industries are considering medicinal plants as a good choice, because the medicinal plants as natural resources have ordinarily fewer side effects [7,69]. Nowadays, about 25% of the drugs are prescribed worldwide come from plants and 252 of them are considered as basic and essential by the World Health Organization (WHO). The WHO considers phytotherapy in its health programs and suggests basic procedures for the validation of drugs in developing countries. Infectious diseases are the second leading cause of death worldwide [17]. From the time of the ancient Iranian, the plants were considered to protect against diseases. In Iran, many plants are used in the form of oils and crude extracts, infusion or plaster to treat common infections without any scientific evidence of their efficacies. This country has a very honorable past in traditional medicine, which goes back to the time of Babylonian — Assyrian civilization. Pharmacological studies carried out on essential oils of some aromatic plants’ species that were obtained in central regions of Iran, have shown antimicrobial activity, which is coherent with the use of these plants in folk medicine that show in the following. One of the most significant ancient heritages is sophisticated experience of people who have tried over millennia to find useful plants for health improvement, with each generation adding its own experience to this tradition [40]. Based on literature search, 18% of the species are used for medicinal purposes in Iran [69]. The aromatic and medicinal properties of the genus Thymus have made it one of the most popular medicinal plants [42]. Treatment of infections continues to be a problem in modern time because of side effects of some drugs and growing resistance to antimicrobial agents. Therefore, investigate for novel, safer and more effective antimicrobials are a pressing need. Herbal medicines have received much attention as a source of new antimicrobial with low side effect and significant activity [16,17]. Among of Lamiaceae family, Thymus with about 215 species is a significant genus [68], with importance medicinal value and display antimicrobial properties [32,65,66]. A significant study interest on the way to the compositional

analysis of Thymus essential oil and extract [62]. It has been declared that essential oil yield and their constituents in plants are related to genetic [58], climate, elevation and topography [47,48] and genotype (G), growing conditions (E) and their interaction (G  E) [8]. Recent studies have shown that this family especially Thymus species have strong biological activity [4,9,10,14,18—24,28,30,35—38,42,45,46,49, 53,57,59—61] and food derived microbial strains [13]. The antifungal activity of Stachys pubescens and Bupleurum falcatum do not determined until now, and it carried out for the first time in this study. The common use of antibiotics in medicine is playing an important role in the appearance of fungi resistant [26]. Drug resistance has a high metabolic value [64], in pathogens for which this perception is related [67]. It is interesting to determine whether their traditional uses are supported by actual pharmacological effects or merely based on folklore. In the present study were selected the four medicinal plants which are widely used in the folk medicine in our region. All of them have been used in the treatment of infectious diseases with different geographical area [11,16,51]. The aim of this study was to evaluate the antifungal potential of the essential oils derived from Thymus kotschyanus, Thymus daenensis, S. pubescens and B. falcatum which growth of the wild in the central part of Iran against standard fungal strains. The selected strains; Fusarium oxysporum (PTCC: 5115), Aspergillus flavus (PTCC: 5006) and Alternaria alternata (PTCC: 5224) purchased from Iranian Research Organization for Science and Technology (IROST). The antifungal potential was performed by Broth Microdilution Method (BMD) to determine the Minimum Inhibitory Concentrations (MICs) and Minimum Fungicidal Concentrations (MFCs).

Materials and methods Collection of plant materials and essential oil extraction The plants were collected from their wild habitat in Semnan region, the central part of Iran between April and June 2011, which are shown geographical and environmental conditions in Table 1. All plants were identified by experts of the University of Applied Science and Technology Education

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Antifungal activities and chemical composition of some medicinal plants

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Table 1 Geographical and environmental conditions. ´ ographiques et environnementales. Conditions ge No.

Plant

Region

Altitude (m asl1)

Latitude

Longitude

1 2 3 4

Thymus kotschyanus Bupleurum falcatum Thymus daenensis Stachys pubescens

Shahmirzad, Semnan Semnan, Fullad mahaleh Shahmirzad, Semnan Shahrood, Semnan

2100 2650 2650 2315

35.432670 35.78527 35.63567 36.32415

53.256050 53.28145 53.32405 54.35316

Center (UAST), Semnan, Iran. The flowers of B. falcatum and the leaves of S. pubescens, T. kotschyanus and T. daenensis were collected to determine their antifungal activity. A voucher specimen for each plant has been deposited in the herbarium of this center. Air-drying of plant material was performed in a shady place at room temperature for 4 days. Grinding and dried aerial parts of plants (100 g) were subjected to hydro-distillation for 3 h, using a Clevengertype apparatus. The distillated oils were dried over anhydrous sodium sulfate and stored in tightly closed dark vials at 4 8C until analysis.

Gas chromatography/mass spectrometry (GC/MS) analysis The essential oils were analyzed on an Agilent Technologies 7890A GC system coupled to a 5975C VLMSD mass spectrometer with an injector 7683B series device. A fused silica capillary column DB-5 (30 mm, 0.25 mm i.d, film thickness 0.25 mm) and a flame ionization detector (FID) were used for the separation. Helium was used as a carrier gas at a flow rate of 1 mL/min. The oven temperature was programmed at 60 8C (4 min), and then rising to 300 8C at 4 8C/min. The injector and detector temperature were kept at 250 8C and 300 8C, respectively. The mass spectrometer was operated in electron-impact ionization (EI) mode with 70 eV energy with MS transfer line at temperature of 300 8C was used. Ion source and interface temperatures were 200 8C and 250 8C, respectively. The split ratio was 1:50. The percentage compositions were obtained from electronic integration measurements using flame ionization detector (FID), set at 250 8C. The column was programmed as follows: 60 8C for 2 min and then increased by 3 8C/min up to 300 8C. Volume of injected samples was 0.5 ml. Identification of components in the oil was based on GC retention indices relative to n-alkanes and computer matching with the Wiley 275 L library as well as by comparison of the fragmentation patterns of mass spectra with those reported in the literature [1,33]. The relative percentage of the oil constituents was calculated from the GC peak areas (Table 3).

Microorganisms, inoculums and antifungal assay Microorganisms and cultural methods In the present study, three standard fungal strains: F. oxysporum (PTCC: 5115), A. flavus (PTCC: 5006) and A. alternata (PTCC: 5224) were obtained from IROST Center in 2011. The tested organisms were selected according to their ease of availability and pathogenicity to human, animals and plants. The isolates of organisms were subculture once onto potato dextrose agar (PDA) (Merck, Darmstadt, Germany) and incubated for 48 to 72 h at 35 8C.

Inocula preparation Inocula were prepared by growing the fungi on PDA for 48 to 72 h at 35 8C and then until 7th day at 25 8C as described by the reference method M38-A2 recommended by NCCLS guidelines [12]. The Inocula were prepared by flooded colonies with approximately 5 mL of sterile 0.85% saline. Tween 20 (0.01 mL) was added to facilitate the preparation of A. flavus inocula. The resulting mixture is transferred to a sterile tube. After the settling of the larger and heavy particles for 4 to 5 minutes, the upper homogeneous suspension is transferred to a sterile tube and mixed with a vortex mixer for 15 seconds. These suspensions were diluted 1:50 in the RPMI medium. The suspensions were mixed for 15 second to ensure homogeneity and subsequently diluted to adjust the turbidity of a 0.5 McFarland standard (0.4  10.4 to 5  10.4 CFU/ml). This density were read using a spectrophotometer (UV-VIS 1650 Shimatzu, Japan) and matched to an optical density (OD) for each strain. Assay for antifungal activity The MICs of each oil was determined using broth microdilution method as described by the NCCLS guidelines for fungal strains (NCCLS, 2008) in flat-bottomed 96-well (Cellstar, Greiner Bio-One, Germany) clear plastic tissue-cultured plates composed of eight series identified from A to H, each one with twelve wells. The RPMI 1640 medium with glutamine and morpholinepropanesulfonic acid (MOPS) buffered to pH 7.0, 0.165 mol/l was used. Stock solutions of the essential oils and the positive control drug, amphotricin B (Sigma-Aldrich Co.) were prepared in dimethylsulphoxide (DMSO) and further diluted (1:50) in the RPMI medium. Amphotericin B was used with concentrations ranging from 16 to 0.125 mg/mL. The final concentration of oils was prepared from 8 to 0.125 mg/mL. The final solvent concentration (1%) DMSO, without oil or the standard drug was used in the test as a negative control. A growth control well together with the RPMI medium without antifungal agents was used for each fungi tested (RPMI + fungi) and the tests were performed simultaneously for sterility control (RPMI + oil). The diluted oils solutions plus RPMI were transferred in 96-wells plates (100 mL/well). Briefly, MIC parameters were determined in triplicate by inoculating 0.1 mL of fungal suspensions. The 96-well microtiter plates were used for twofold serial dilution with the RPMI. The final Volume of each well was 200 mL/well. The cultures were incubated at 35 8C for 72 h. The minimum inhibitory concentration (MIC) was the lowest concentration of oil that completely inhibited the growth of microorganisms in the microdilution wells, as can be detected by the unaided eye. In this study, the MICs were recorded by spectrophotometrically at 530 nm using SpectraMaxplus384 after 72 h

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incubation. The minimum fungicidal concentrations (MFCs) were determined by sub-culturing the negative wells on a Sabouraud Dextrose Agar (Oxoid) plates. MFC was defined as the lowest concentration resulting in no growth on subculture. For determined of MFC, after the MIC for each strain was determined, the microtiter plates were shaken and 20 ml suspensions from each well showing complete inhibition (100% or an optically clear well) and from the growth control (drug-free medium) was subcultured onto Sabouraud dextrose agar plates. The plates were incubated at between 28 and 30 8C until growth was seen in the growth control subculture. The MFC was the lowest drug concentration that resulted in either no growth or fewer than three colonies after 48 h (99.9% killing).

Results All essential oils showed effective antifungal activities on the selected saprophytic, pathogenic and phytopathogenic fungi. Antifungal activities of essential oils were investigated by broth macrodilution method. The MICs and MFCs of the selected oils on the fungi are shown in Table 2. The results showed that essential oil of the plants were active against all the pathogenic bacteria species with different degree in the following range of concentrations: Essential oil of T. kotschyanus and S. pubescens had a best antibacterial effect and its MIC values was between 0.5 to 1 mg/mL. B. falcatum is second degree with MIC values between 0.5 to 2 mg/mL. While, T. daenensis had a lowest antibacterial effect comparison to above essences and its MIC values were 1 to 4 mg/mL. Amphotericin B used as positive control as well as DMSO as a negative control, which did not show any inhibition against the pathogens fungi. The MIC range of standard antibiotics ‘‘amphotericin B’’ was 0.5—2 mg/mL. Even at low concentrations, the plant’s species showed antifungal activity more or nearly equal to the commercial fungicidal agent (amphotericin B). A. alternata with the highest MIC and MFC was persistent strain but A. flavus and F. oxysporum were sensitive with the lowest MIC and MFC against the oils. In comparison to the amphotericin B, these data showed that antifungal value of T. Kotschyanus and S. pubescens had the highest activity; B. falcatum had the lower activity but with the lowest different, while the different properties of T. daenensis was more. Briefly, the best antifungal activities were seen in T. kotschyanus and S. pubescens oils, whilst B. falcatum displayed a moderate response against the fungal species and T. daenensis displayed less susceptibility against all of

the fungi. All of the oils had a potent antifungal activity except T. daenensis that had not antifungal activity on the A. alternata. The weakest activity was observed against A. alternata with the highest MIC and MFC and A. flavus was displayed more sensitivity against oils and did not show any resistance. The results of the chemical analyses using GC/MS of the essential oils were listed in Table 3. The number of identified constituents in T. kotschyanus, S. pubescens, T. daenensis and B. falcatum were 26, 23, 22 and 15 respectively. Also, analysis of data with creditable library shows that, the main components of S. pubescens were: Germacrene (22.4%), d-Cadinene (19.7%), 2,6-Octadien (11.5%), Linalool (9.7%); and in B. falcatum were: Torilenol (39.1%), Spathulenol (19.6) and a-Cubebene (8.1%), in T. kotschyanus were: Thymol (46.72%), Benzene (6.880%), Carvacrol (3.73%), g-Terpinene (3.58%), trans-Caryophyllene (3.39%) and T. daenensis was include: Carvacrol (31.46%), a-Terpineol (22.95%), Thymol (20.20%), Bicycloheptane (6.27%), 2,6-Octadien (2.2%). The results of this study showed that essential oils of plants have a very broad spectrum of antifungal activities. However, it is necessary to determine the toxicity of the active constituents, their side effects and pharmaco-kinetic properties. Concerning the antifungal properties of T. kotschyanus and T. daenensis, other studies confirmed the inhibition effects of these plants on the some pathogen fungi such as Aspergillus niger, Aspergillus fumigatus, Aspergillus parasiticus, Pythium ultimum, Rhizoctonia solani, Gaeumannomyces graminis and Candida albicans [5,18,25,28,34,39,52,59]. The results confirmed the antimicrobial potency of these plants, especially about the S. pubescens and B. falcatum that evaluated for the first time. In this assay the composition of the plants were determined by GC/MS, which are different from other regions in the world. Concerning the S. pubescens many studies have not been conducted so far; Iranian researcher reported; (Z)-b-Ocimene, Germacrene D and Bicyclogermacrene as main components [6]. Previous studies related to the chemical composition of B. falcatum, a-pinene reported as main component, which are not similar to our result [56]. Since, B. falcatum display the high antimicrobial activity, therefore, we concluded that the most main components as torilenol and spathulenol have an antimicrobial activity alone or mix to other components. Similar to our result, in previous studies on Thymus species showed that the main components of the oils and extracts were carvacrol and thymol [3,4,35,36,41,42,50,54,55,58]. The percentage of carvacrol in the T. daenensis was 31.46%. As a result it showed carvacrol is the first main component in T. daenensis. In the other reported carvacrol was as the first

Table 2 MIC and MFC (mg/mL) values for essential oil of plants. Concentrations minimales inhibitrices (CMI) et fongicides (CMF) (mg/mL) des huiles essentielles des plantes. Microorganisms

Aspergillus flavus Alternaria alternata Fusarium oxysporum

Amphotericin B

TK

SP

BF

TD

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

1 2 0.5

2 4 1

0.5 1 0/5

1 2 0/5

0/5 1 1

0/5 2 1

0/5 2 2

1 4 2

1 — 4

2 — 8

MIC: minimum inhibitory concentrations; MBC: minimum fungicidal concentrations; SP: Stachys pubescens; TK: Thymus kotschyanus; TD: Thymus daenensis; BF: Bupleurum falcatum; —: no grows inhibition.

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Thymus kotschyanus

Component

PA (%)

RT

Component

1 2 3 4 5 6 7 8 9 10 11

b-myrcene Benzene Limonene 1,8-cineole g-terpinene Trans-sabinene hydrate Linalool Borneol 3-cyclohexen-1-ol a-terpineol dl-limonene

1.25 1.27 0.37 0.51 1.11 0.56 0.78 2.17 0.44 22.95 1.76

6.308 6.875 6.943 7.012 7.407 7.561 8.008 9.106 9.261 9.461 9.884

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Total

Camphene Cyclohexanon 2,6-octadien 2,6-octadienal Thymol Carvacrol b-caryophyllene b-cubebene Bicyclogermacrene b-bisabolene CIS-a-bisabolemne — — — —

6.27 2.10 2.22 0.33 20.20 31.46 1.59 0.86 0.58 0.47 0.74 — — — — 99.99

9.925 10.182 10.302 10.565 10.817 10.977 12.705 13.478 13.667 13.712 14.113 — — — —

a-terpinene Benzene 1,8-cineole g-terpinene Terpineol 2-butoxyethyl acetate Linalool Borneol 3-cyclohexen-1-ol a-terpineol Benzene (1-methoxy-4(2-ropenyl-)) Isopropyl Carvacrol methyl ether Octadien-1-ol Phenol Thyme camphor Thymol m-thymol Carvacrol Nerol Trans-caryophyllene Naphthalene Delta-cadinene Nerolidol Caryophyllene oxide Germacrene

Bupleurum falcatum PA (%)

RT

0.58 6.88 0.92 3.58 1.07 1.70 2.88 2.51 0.65 1.07 0.65

6.755 6.871 7.012 7.413 7.567 7.848 8.013 9.106 9.437 9.450 9.541

2.07 1.51 1.34 2.61 1.58 46.72 0.61 3.73 0.48 3.39 0.50 0.69 0.92 0.78 0.60 90.02

10.033 10.176 10.297 10.634 10.714 10.817 10.874 10.955 12.042 12.705 13.861 13.947 14.336 14.760 15.360

Stachys pubescens

Component

PA (%)

RT

Component

PA (%)

RT

a-pinene Pinocarvone a-cubebene Pinocamphone Heptanal cis-verbenol Myrtenal Thyopsene Trans-pinocarveol Trans-verbenol Cuparene

3.5 4 8.1 1.7 4.2 1.5 2.9 3.1 4.1 0.3 2.8

5.240 5.285 5.432 6.641 7.405 7.910 10.470 10.958 11.690 11.825 11.931

b-pinene 1,4-cyclohexadiene Myrcene a-terpinene Benzene Limonene (E)-b-ocimene g-terpinene 3-cyclohexen-1-ol Linalool 2,6-octadien

1.8 0.4 0.9 2.7 0.9 6.3 2.8 1.2 1.5 9.7 11.5

4.840 5.412 6.124 6.529 6.790 6.893 7.115 7.430 9.280 9.987 10.420

Torilenol Spathulenol a-calacorene Pentacosane — — — — — — — — — — —

39.1 19.6 2.4 1.5 — — — — — — — — — — — 98.8

11.965 12.560 12.940 13.850 — — — — — — — — — — —

Octen-1-ol acetate 2,6-octadienal Linalyl acetate d-elemene b-bourbonene d-cadinene Naphthalene b-gurjunene Bicyclogermacrene Caryophyllene oxide Spathulenol Germacrene — — —

1.6 2.1 1.2 5.4 0.2 19.7 1.2 0.3 1.8 1.3 0.8 22.4 — — — 97.7

10.560 10.602 10.742 11.124 12.247 13.905 13.926 13.945 14.364 14.821 14.834 15.248 — — —

RT: retention time; PA: peak area.

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Antifungal activities and chemical composition of some medicinal plants

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Table 3 Chemical analyses constituents of essential oils. Analyses chimiques des constituants des huiles essentielles des plantes.

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major constituent [4], which agrees to our data for T. daenansis. In T. kotschyanus and T. daenensis, the percentage of thymol was 46.72% and 20% respectively while the thymol was only identified as the first main components in T. kotschyanus. In the other words, the thymol was not the first main components in T. daenensis. Since T. kotschyanus and S. pubescens showed the highest antibacterial activities, then it was suggested the thymol and Germacrene may be play a major antibacterial role, while the rest of its components were less than 7%. Another study concerning the T. kotschyanus showed the major components were carvacrol, g-terpinene, thymol, myrcene, p-cymene, bcaryophyllene and borneol, a-phellandrene, [4,29] as can be seen, the carvacrol reported as first main component, whereas, the percentage of thymol was lower than the carvacrol in them while at the present study the thymol was first main component. Agreement to our data, the important point in the previous studies is presented the carvacrol and thymol, which are two medically important constituents [2,15,27,43,44], as main components with different percentages except for a few examples. It was suggested these differences in components could be due to the variety of the ecotype system that reported by other scientists and references [4]. Since the essential oils are complex mixtures of several compounds, it is difficult to attribute their biological activity to a particular constituent. Usually, major compounds are the ones responsible for the antimicrobial activity of the essential oils. However, some studies showed that minor components may have a crucial role in the biological activity of the oils [31].

Discussion Our result confirmed the potential antifungal activity of selected plants on the A. flavus, F. oxysporum and A. alternata. The above finding supports their traditional usage of these spp. as antiseptics [5,52]. These plants could safely be used as organic preservatives to replace synthetic fungicides in the prevention and cure of some human and animal mycoses disease as well as food industrial preservatives. These data, together with high yield and nontoxicity [63], justify their usage for those purposes. However, the mechanism of inhibitory effects of these plant’s oils against infectious fungi is still unclear. Further investigations regarding the in vitro and in vivo should be conducted in order to clear mechanisms pathway and develop such products. These data suggest that the essential oils of T. kotschyanus, S. pubescens, T. daenensis and B. falcatum could be a possible source to obtain new and effective herbal medicines to treat infections caused by multi-drug resistant strains of microorganisms and also in the search for novel antifungal agents with the potential application of some major or minor constituents alone for the treatment and prevention of pathologies associated with multiresistant fungi. Finally, it was suggested it would be useful to carry out more study on the synergistic effects plants oil in combination with synthetic antibiotics. In conclusion, we suggest the more study for determined the antifungal mechanism of selected plants especially on the Aspergillus sp., F. oxysporum and A. alternata which have spread to other parts of the world.

Statistical analysis Comparison of data was performed using the one way ANOVA or the unpaired Student’s t-test and is presented as mean  standard deviation. Comparison of MIC and MFC values, tests were made in triplicate for quantification. Values of P < 0.05 were considered significant.

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