Introduction

Plant kingdom has provided a variety of medicines throughout history. Plants with antimicrobial activity are also known to be numerous; yet prior to a decade ago, minimal research had been conducted in the area of antifungal medicinal plants [27]. Recently, there was an increased interest for this type of research, and a great portion of the scientific community dedicated to the investigation into the medicinal properties of plants. The studies have dealt with a wide range of criteria. Many focus on determining the antimicrobial activity of plant extracts found in folk medicine, essential oils, or isolated compounds, such as, alkaloids, flavonoids, sesquiterpene lactones, diterpenes, triterpenes or naphtoquinones, and others [24].

Fungi are ubiquitous in the environment, and infection due to fungal pathogens has become more frequent [10]. With the rise of HIV, opportunistic fungal pathogens have become a common cause of morbidity and mortality [12]. Furthermore, since the first human kidney was transplanted in 1954, the frequency of organ transplantation has increased and the number of immunocompromised hosts has grown steadily with the introduction of novel immunosuppressive agents and improved rates of survival. Thus, the incidence of opportunistic infections, including fungal infections, has also increased [9, 28]. Paecilomyces lilacinus is one of the most frequent opportunistic fungi that seriously infect immunosuppressed patients and cause severe cutaneous dermatosis [11, 15]. As a result, antifungal therapy is playing a greater role in health care, and the screening of traditional plants in search of novel antifungals is now more frequently performed [16, 22]. The increase in the incidence of fungal infections is due to the emergence of resistant pathogens and their nosocomial dissemination, especially among immunocompromised or neutropenic patients. Scientific efforts to discover new potential antifungal drugs are principally leaned towards synthetic and natural products of plant origin [14].

The reliance on plants as a source of medicines warrants scientific validation of their safety, efficacy, quality, and the appropriate dosage of the plant material used [19]. With increasing acceptance of herbal medicine as an alternative form of health care, the screening of medicinal plants for active compounds is becoming increasingly important [23]. The screening of plant extracts for antimicrobial activity has shown that a great number of these plants contain active compounds. The presence of antibacterial, antifungal, and other biological activities has been demonstrated in extracts of different plant species used in traditional medicine practices [20, 21].

Consequently, the aim of this study was to screen the extracts of some medicinal plants against most frequent dermatophytes to explore their effectiveness in dermatitis treatments. Chemical composition of the most active extract as well as possible mechanism(s) of action against the most sensitive fungal species was determined.

Materials and Methods

Plants Materials

Five wild plants were collected from Sinai region, Egypt, during the spring season of 2008. They were Artemisia judaica L., Ballota undulate (Sieb.ex Fres.) Benth., Cleome amblyocarpa Barratte and Murb., Peganum harmala L., and Teucrium polium L. Plants were identified according to their morphological characteristics and were deposited at Assiut University herbarium.

Preparation of Crude Extracts

To obtain the crude extract from the tested plants, the method described by Cakir et al. [5] was applied with modification. The powder of aerial parts (leaves, stems or fruits) of dried plant materials was extracted separately with ethanol, ethyl acetate, chloroform, and n-hexane (100 g/l) at room temperature. Extraction with each solvent was carried out three times for 48 h. After each time, the extract was filtered through filter paper (Wattman No. 1). After filtration, the extracts were collected and concentrated under low pressure at 40°C. Finally, the percentage yield for each extract was determined. Standard concentration was prepared in the same solvent used in the extraction procedure.

Tested Fungi

The microorganisms used for the biological evaluation were kindly obtained from Assiut University mycological centre (AUMC) and from culture collection of Assiut University, Faculty of science, Botany department (AUSB). Trichophyton rubrum AUMC 1804 and Trichophyton tonsurans AUMC 2362 were selected to represent the dermatophytes, and Candida albicans AUMC 3880, Chrysosporium tropicum AUMC 1808, Paecilomyces lilacinus AUSB 2678, Paecilomyces variotii AUSB 2645, and Scopulariopsis bervicaulis AUMC 3618 were selected as opportunistic organisms. The fungi were maintained on potato dextrose agar (PDA) at 27°C.

Screening of Antifungal Effect

Antifungal activity of ethanol, ethyl acetate, chloroform, and n-hexane extracts of each plant spices was evaluated, separately by the paper disk-agar diffusion method [2]. Test plates (diameter: 7 cm) were prepared with PDA medium and inoculated in surface with a spore suspension in sterile dissolution of 0.9% saline. The concentration was adjusted to ≈104 CFU/ml. Sterile paper disks (diameter: 5 mm) impregnated with 60 μl of extract dilutions at a concentration of 175 mg/ml were applied over the test plates. The diameters of the growth inhibition zones around each disk were measured after incubation at 27°C for 4–6 days. For each extract, three replicate trials were conducted against each fungus.

Minimum Inhibitory Concentration (MIC)

The MIC was determined according to Kariba et al. [17]. Briefly, paper disks (diameter: 5 mm) were impregnated with 60 μl of the reconstituted samples at a concentration of 25–150 mg/ml. The disks were transferred aseptically into PDA plates inoculated with the test fungi. The MIC was regarded as the lowest concentration that produced a visible zone of inhibition. Experiments were triplicated. The plates were incubated at 27°C for 4–6 days.

Gas Chromatography–Mass Spectrometry (GC–MS) Analysis

An aliquot of 1 μl extract (ethanol extract) of Ballota undulate was injected into GC–MS (6890 N/5975B). The HP-5MS column was 30 m in length, 0.25 mm i.d., and 0.25 μm in thickness. The carrier gas was helium with average velocity 36 cm/sec, and flow 1 ml/min. The operating condition of GC oven temperature was maintained as follows: initial temperature 40°C for 9 min, 150°C for 8 min, at 15°C/min up to final temperature 310°C with isotherm for 3 min at 25°C/min. The injector and detector temperatures were set at 250 and 280°C, respectively, according to the standard method 8270 EPA [5].

Light Microscopy

A sample of mycelium of P. lilacinus was taken from the periphery of the colony grown on PDA with 0, 10, 20, and 25 mg/ml treatments of crude extract of Ballota undulate after 6 days of incubation. The samples were fixed in lactophenol-cotton blue and examined under light microscope (Olympus 40, Germany) to examine structural abnormalities. Samples from control plates without oil were also stained and observed. Microphotographs were taken using a microscope attached camera.

Scanning Electron Microscopy

Six-day-old P. lilacinus culture on PDA treated with 0, 10, 20, and 25 mg/ml concentrations of Ballota undulate extract was used for all scanning electron microscopic (SEM) observations. From the cultures growing on potato dextrose plates, 5 × 10 mm segments were cut and promptly placed in vials containing 3% glutaraldehyde in 0.05 M phosphate buffer (pH 6.8) at 4°C. Samples were kept in this solution for 48 h for fixation and then washed with distilled water three times for 20 min each. The samples were then dehydrated in an ethanol series (30, 50, 70, and 95%), for 20 min in each alcohol dilution and finally with absolute ethanol for 45 min. Samples were at critical point dried in liquid carbon dioxide. Fungal segments were placed in desiccators until further use. Following drying, samples prepared were mounted on standard 1/2 in JEOL JSM SEM stubs using double-stick adhesive tabs and coated with gold–palladium electroplating (60 s, 1.8 mA, 2.4 kV) in a Polaron SEM Coating System sputter coater. All samples were viewed in JEOL JSM 5300 scanning electron microscope, and the samples were examined at 15 kV and photographed.

Results

Antifungal Screening and Minimum Inhibitory Concentration (MIC)

The results of screening the antifungal activity of the crude extracts obtained from Artemisia judaica, Ballota undulate, Cleome amblyocarpa, Peganum harmala, and Teucrium polium are shown in Table 1. The extracts generally showed inhibition effects on the growth of the tested fungi. The most effective extracts were obtained from Ballota undulate, where ethanol extract exhibited the highest inhibitory effect on all tested fungi in most cases. Paecilomyces lilacinus was the most sensitive fungus to this extract, with an inhibition zone diameter of 25.3 mm. This fungus was also inhibited by chloroform and n-hexane extracts approximately at the same extent (25.7 and 25.0 mm, respectively). Paecilomyces variotii was inhibited greatly by n-hexane extract (inhibition zone: 20 mm). Candida albicans showed great sensitivity to ethanol extract of Ballota undulate. The inhibition zone diameter produced as a result of application of this extract against this fungus was 18.3 mm. Chloroform extract showed moderate effect on C. albicans (11.0 mm), while both ethyl acetate and n-hexane extracts showed relatively low inhibitory effect, and the reduced inhibition zones were 6.7 and 5.7 mm, respectively. Peganum harmala extract were considered the second most effective extracts against the majority of tested fungi. P. lilacinus and P. variotii showed the highest sensitivity to the ethanol extract. C. albicans and Trichophyton tonsurans were inhibited moderately as a result of application of this extract, and the inhibition zone was measured as 7.0 mm for both organisms.

Table 1 Antifungal activity of crude extract of the tested plant against dermatophytes and emerging fungi (inhibition zone in mm)
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The results of minimum inhibitory concentration (MIC) test are presented in Table 2. The results indicated that Ballota undulate ethanol extract was the most active against C. albicans, P. lilacinus, P. variotii fungi with MIC value of 25 mg/ml, while the MIC value for Chrysosporium tropicum, Scopulariopsis bervicaulis, Trichophyton rubrum was 50 mg/ml, and for T. tonsurans was 100 mg/ml. Ethyl acetate, chloroform, and n-hexane extracts were found to be effective against P. lilacinus with MIC value of 25 mg/ml. The extracts of other plant species showed lower inhibitory effect against most of the tested fungi with MIC values of 50–150 mg/ml with some exceptions in case of P. lilacinus and P. variotii, where MIC was 25 mg/ml like ethanol and ethyl acetate extract of Cleome amblyocarpa, ethanol extract of Peganum harmala, and chloroform and n-hexane extract of Teucrium polium.

Table 2 Minimum inhibitory concentration of crude extract of the tested plant against dermatophytes and emerging fungi (concentration in mg/ml)
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GC–MS Analysis

GC–MS analysis of Ballota undulate ethanol extract led to the identification of 35 different components. The identified compounds are listed in Table 3. It was found that the extract contained a complex mixture consisting of aliphatic and aromatic hydrocarbons, sesquiterpene hydrocarbon along with some other essential phytochemicals. The major components in the extract detected were 3-Hydroxy-(5-Beta)Androst-2-en-17-one (24.58%), 4-(1,1-Dimethyl)-1,2-Benzenediol (10.34%), Desoxy-dihydro-isosteviol (9.24%), 2,5-Dimethoxy ethyl benzene (8.39%), 2-methyl-4,6-dipropyl-pyridine (5.24%), 1-Methyl-4-(1-metylethyl)-1,4cyclohexadiene (5.04%), 2-isopropyl-5-methyl-9-methylene-bicyclo[4,4,0]dec-1-ene (3.25%), Vitamin E (3.15%), Caryophyllene Oxide (2.70%), 5,6-Dihydro-5,5-dimethyl-benzo[d] pyrazdo[3,4-b] azepin-3(2H)-one (2.70%), 3-Methoxy-2,5,6-Trimethyl phenol (2.55%), and Octadecane (2.20%). The other compounds (no. 13–35) were found as trace or minor components (1.85–0.05%).

Table 3 GC–MS analysis of ethanol extract of Ballota undulate
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Light Microscopy

To explore the possible mechanisms of Ballota undulate ethanol extract to inhibit the growth of fungi, P. lilacinus was selected to be examined under light microscope at 400–1,000 × magnification, after treated with different concentrations (0, 10, and 25 mg/ml), and the findings are presented in Fig. 1a-f. In addition to the conspicuous inhibition of growth, mycelia grown in the presence of the extract seemed to exhibit distinct morphological changes when compared to control. The variations included lack of sporulation, visible loss of pigmentation, and aberrant development of conidiophores. Untreated mycelia had homogenous, clear cytoplasm, and profuse conidiation on conidial heads, metullae, and phialides bearing conidia was clearly visible (Figs. 1a, b), while treatment with 10 mg/ml concentration clearly showed reduction in conidial heads, poorly developed metullae with less or absence of conidiation and conidiophores becoming distorted (Figs. 1c, d). At 25 mg/ml concentration of the extract, there were clear alterations in hyphal structures, which started with budding of hyphal tip, anomalous structures such as swelling, localized along the hyphae (Fig. 1e). There was visible decrease in cytoplasmic content, with clear separation of cytoplasm from cell wall in hyphae. Eventually, retraction of cytoplasm from hyphae and ultimately hyphae without cytoplasm were found (Fig. 1f).

Fig. 1
figure 1

Microphotographs of Paecilomyces lilacinus mycelium grown on PDA with or without Ballota undulate crude extract during 6 days of incubation at 27°C. a Paecilomyces lilacinus control mycelium, structure is homogenous, well-developed mycelia, condiophores and conidia (× 400). b Control conidial head of Paecilomyces lilacinus, clear development of vesicle on conidiophore, metullae and phialides bearing conidia in chains are clearly visible (× 1000). c & d Mycelia and conidial head modifications induced by 10 mg/ml of Ballota undulate crude extract, then malformed mycelia, clear absence of conidia and conidiophore distorted (× 400). e & f Treatment with 25 mg/ml of crude extract, the apical hyphal tip become distorted, budding of hyphal tip and unusual structures clearly visible (e), hyphae with greater anomalous structures, budded apical tip, with cytoplasmic granulations, hyphae showing clear separation of cytoplasm from cell wall, showing clear decrease in cytoplasmic content and note the cytoplasmic retraction and empty hyphae (e) (× 1000)

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Scanning Electron Microscopy

The effect of Ballota undulate ethanol extract on the morphology of P. lilacinus examined by SEM is shown in Fig. 2. The P. lilacinus mycelium grown on PDA medium without treatment (control) showed the characteristic morphology with stretched, regular, homogenous hyphae of constant diameter with smooth external surface (Fig. 1a) and well-developed conidiophores bearing obvious metullae (Fig. 1b). After 6 days of incubation in the treatment of 10 mg/ml, fungal mycelium showed alterations in the morphology of the hyphae. There was clear showing of distorted mycelium, squashed and flattened conidiophores bearing damaged metullae (Figs. 2c, d). At 25 mg/ml of concentration, highly papillated and flattened empty hyphae were clearly visible. On the hyphae, some punctured portions, complete cell wall disruption and squashed hyphae, were observed (Figs. 2e, f).

Fig. 2
figure 2

Scanning electron micrographs of Paecilomyces lilacinus mycelium grown on PDA with or without Ballota undulate crude extract during 6 days of incubation at 27°C. a Control, magnification of well-developed hyphae and conidia. b Control, single conidiophores bearing clearly seen metullae. c & d Treatment with crude extract at 10 mg/ml, showing distorted mycelium, squashed and flattened conidiphore bearing damaged metullae and collapsed hyphae. e & f Effect of 25 mg/ml crude extract, the flattened hyphae clearly visible, note the presence of cell wall disruption, squashed and degrading hyphae

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Discussion

The antifungal activity screening of the crude extracts of the five tested plants showed some inhibitory effects on the growth of both dermatophytes and emerging fungi. However, the ethanol extract of Ballota undulate was the most effective against all fungi. P. lilacinus, P. variotii, and C. albicans were the most sensitive organisms. The minimum inhibitory concentration (MIC) test indicated that Ballota undulate ethanol extract had a lethal effect on C. albicans, P. lilacinus, P. variotii with values of 25 mg/ml, on Chrysosporium tropicum, Scopulariopsis bervicaulis, Trichophyton rubrum with value of 50 mg/ml, and against T. tonsurans with of 100 mg/ml. Extracts of the other plant species Artemisia judaica, Cleome amblyocarpa, Peganum harmala, and Teucrium polium were less effective in inhibiting fungi. Based on the literature review, no previous work has been done on antimicrobial effect of Ballota undulate, Cleome amblyocarpa, and Teucrium polium. To the best of the author’s knowledge, this is the first report on antifungal effect of Ballota undulate. The antifungal activity obtained in this study is supported by previous scientific studies as reported by Brinker [3] and Chitwood [6] who mentioned that a great variety of plants have a broad spectrum of bioactive organic compounds, such as, isocyanate, glucocynolate, glycoside, lipid, and phenol with fungicide and fungistatic properties. Medicinal plants are potentially rich sources of antibacterial and antifungal agents [4]. GC–MS analyses of Ballota undulate ethanol extract led to the identification of 35 aliphatic and aromatic hydrocarbons, sesquiterpene hydrocarbon along with some other essential oils (Table 3). 3-Hydroxy-(5-Beta)Androst-2-en-17-one was one of the detected compounds in large amount. It is a steroidal compound plays an important role in cellular metabolism. Among the detected compounds is caryophyllene oxide, which playing a critical role in plant defense [26] and exhibited fungitoxic activity against some fungi [5]. Vitamin E is an antioxidant compound plays a critical role in the cell. Regarding other detected compounds, they could be involved singly or in combination with others in the inhibition of fungal growth. It was mentioned that phenolic compounds, terpenoids, and alkaloids are very important components in antimicrobial or antioxidant effects [24]. The more effectiveness of ethanol extract comparing to other extracts is due to the ability of alcohol to provide a more complete extraction, including less polar compounds, and many of these extracts have been found to possess antifungal properties [1].

Light and scanning electron microscopies have proved that Ballota undulate ethanol extract has a fungicidal effect on P. lilacinus through alterations in hyphal structures, which started with budding of hyphal tip, anomalous structures such as swelling, localized along the hyphae, decrease in cytoplasmic content, with clear separation of cytoplasm from cell wall in hyphae. SEM clearly showed distorted mycelium, squashed and flattened conidiophores bearing damaged metullae. Eventually, by increasing the concentration of the extract mycelia had become papillated, flattened, and empty. Puncturing and squashing of hyphae as well as complete cell wall disruption were clear signs on the complete death of hyphae. These results were concomitant with those obtained by Billerbeck et al. [7] using Cymbopogon nardus essential oil against A. niger and by Mares et al. [18] using Tagetes patula extract on Pythium ultimum. The mode of antifungal activity of plant essential oil could be due to cell wall attack and retraction of cytoplasm in the hyphae and ultimately death of the mycelium [25]. Such modifications induced by essential oil may be related to the interference of essential oil components with enzymatic reactions of wall synthesis, which affects fungal morphogenesis and growth. It has been demonstrated that monoterpene hydrocarbons and oxygenated monoterpenes in the flower essential oils are able to destroy cellular integrity and thereby inhibit respiration and ion transport processes [8, 13].

Conclusion

From the results obtained here, it was clear that ethanol extract of Ballota undulate effectively inhibited both dermatophytes and opportunistic fungi (MIC = 25–100 mg/ml). The most sensitive fungi were Paecilomyces lilacinus, P. variotii, and Candida albicans. GC–MS analysis approved that ethanol extract of Ballota undulate is composed of complex mixture of 35 of aliphatic and aromatic hydrocarbons, sesquiterpene hydrocarbon along with some other essential oils. Among the detected compounds is caryophyllene oxide, which could play a critical role fungitoxic activity against the tested fungi. Light and electron microscopies have proved the fungicidal effect of ethanol extract of Ballota undulate on P. lilacinus through alterations in hyphal structures stating with budding of hyphal tip and swelling and ending by death of hyphae.

The result recommends intensive pharmaceutical and clinical investigation to apply extract of Ballota undulate as a natural antifungal drug to treat the skin infections specially those caused by opportunistic fungi.