Introduction

Aromatic and medicinal plants were used to prevent or cure infectious diseases. Previous studies demonstrated their richness in polyphenols such as tannins, alkaloids and flavonoids which shown antimicrobial activities (González-Lamothe et al. 2009; Habbal et al. 2011).

Teucrium polium L. is called in Tunisia “Al-Ja’adeh”, “Khayata” and “Gattaba” which means “cicatrisant”. The genus Teucrium belongs to the Lamiaceae family and is represented by more than 340 species, from these, only 19 species are found in Tunisia. T. polium is mainly found in the Mediterranean and Western Irano-Turanian regions (Tepe et al. 2011). It is widely distributed in Jordan and Palestine (Afifi et al. 2005) and well known in Arab traditional medicine for its anti-diabetic, anti-inflammatory, hypotensive, anorexic, antispasmodic, antiulcer, antipyretic and antinociceptive properties (Darabpour et al. 2010; Tourandokht et al. 2005; Nosrati et al. 2010). According to Le Floc’h (1983), T. polium is regarded as an excellent depurative and febrifuge, it is also employed like tonic and digestive in the treatments of gastralgies and enteritis. De Marino et al. (2012) announce that this species is used as stimulants, tonics, diaphoretics, diuretics and in the treatments of stomach pain, asthma, amenorrhea, leucorrhoea, chronic bronchitis and gout by local nomadic populations. On the other hand, it was reported that essential oils and alcoholic extracts of T. polium had an antibacterial activities against both Gram negative and Gram positive bacteria. In this context, Djabou et al. (2013) demonstrated that essential oils of T. polium inhibit the growth of toxi-infectious and foodborne bacteria. Moreover, ethanolic and methanolic extracts of T. polium showed antibacterial properties against some bacterial strains (Darabpour et al. 2010; Khaled-Khodja et al. 2014). This work aims to study the antibacterial and antifungal activities of ethanol and aqueous extracts and essential oils of T. polium.

Materials and methods

Plant material

Aerial parts of T. polium were collected from Zarroug Mountain, Gafsa (South-west of Tunisia) in May 2010. After collection, the plant material was dried for 7–10 days in the shade at room temperature. The dried matter was then ground and stored in cloth bags at 5 °C before being used in experiments to prepare the plant extracts.

Extracts preparation

Aqueous extracts

Dried matter (100 g) was stirred in 1 l distilled water for 15 min at 95 °C, followed by a rapid filtration through four layers of gauze and a second filtration through filter paper (Whatman No. 1). The percent (w/w) extraction yield was 9%. The resulting solution was freeze-dried and the powder was stored at −20 °C in a desiccant until required.

Ethanol extracts

Dried powder matter (100 g) was extracted by continuous mixing in 1 l ethanol (95%) during 24 h at room temperature. After filtration, ethanol was evaporated using a rotary vacuum evaporator (K IKA-WERKE). The extract was stored at −20 °C. The percent (w/w) extraction yield was 7% on a dry matter basis.

Extraction and chemical analyses of essential oils

Essential oils were extracted, from powdered plant, by hydrodistillation method using the apparatus described in the IXth edition of French Pharmacopoeia according to Okoh et al. (2010) (similar to a Clevenger-type apparatus). The percentage yield based on the dried plant was 0.29% (v/w). The essential oil was stored at +4 °C until tested and analysed.

Gas chromatography analysis

The oil was analysed on Hewlett Packard (HP) 6890 using the following experimental conditions: flame ionization detector (FID), HP-5 fused silica capillary column temperature 50°–250 °C at 5 °C/min; injector and detector temperature 250 and 280 °C, respectively; carrier gas: nitrogen 1.2 ml/min; volume injected: 1 µl of the oil diluted in hexane (10%).

Gas chromatography–mass spectrometry analysis

The samples were assayed by GC/MS using a HP 5972 gas chromatograph equipped with a fused silica capillary column (HP-5, 25 m × 0.2 mm, film thickness 0.25 mm). The GC/MS was operated under the following operating conditions: injector temperature: 250 °C; oven temperature: programmed from 50 to 280 °C at 5 °C/min; carrier gas: He at 20 psi: 1 µl of 1/dilution solution in 100 hexane. The oil components were identified by comparison of their retention indices [relatives to C26–C28 alkanes on the BP-5 column] and mass spectra with those of authentic standards of library searches of the oil library LIBR Tped using the Finnegan library search routine based on fit and purity of mass spectra (Hossain et al. 2012).

Antimicrobial activities

Antibacterial activity

Bacterial strains

Both extracts (aqueous and ethanol) and essential oil were used against a range of bacteria, namely Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Citrobacter freundei and Proteus mirabilis. Microorganisms were obtained from the culture collection of the Laboratory of transmissible diseases and biologically active substances, Tunisia.

Susceptibility tests

Susceptibility of the bacterial strains to the extracts and essential oils was investigated using the disk diffusion method (Teixeira et al. 2012) and by comparing their antibiogram inhibition zones to those reported by the National Committee for Clinical Laboratory Standards. Organisms were maintained on Muller-Hinton agar (MH). Inoculate were prepared by diluting overnight (24 h at 37 °C) cultures in Muller-Hinton Broth medium to approximately 106 CFU/ml. Whatman disks No. 3 of 6 mm diameter were impregnated with 10 µl of oil or aqueous and ethanol extracts at 20 mg/ml and then placed on to the surface of inoculated plates (90 mm). The diameters of inhibition zones were measured after incubation at 37 °C for 24 h and compared with positive control disk of Gentamycin 10 µg/ml. All experiments were performed in triplicate. The susceptibility of the strains to the extracts and essential oils was further evaluated by a microdilution method (Zander et al. 2010). This method aims to determine the minimum inhibitory concentration (MIC), which was applied only to the extracts that have shown inhibition zones ≥8 mm. The initial concentration was 10 mg/ml. Essential oils and ethanol extract were dissolved in 10% of ethanol 99% (v/v). This method was applied using 96-well plate with round bottom according to Marzouk et al. (2010). Column 2–11, wells were filled with 100 µl of Muller Hinton Browth (MHB). The wells of column 12, containing only solvent were taken as control for sterility. The first column contains only extracts used as sterility control. The wells in column 10 were taken as control bacterial and column eleven’s wells containing only MHB. The final volume in each well was 200 µl. The dilutions were made in 1/2. Gentamycin at the concentration range of 1 mg was prepared in MHB and used as standard drug for positive control. The agar plates were prepared and incubated at 37 °C for 24 h. MIC was defined as the concentration at which no colony was observed after incubation. The assays were performed in triplicate.

Antifungal activity

Microbial strains

The extracts and essential oils were tested against seven strains of fungi which can be classified into three groups; two opportunist pathogenic yeast (Candida albicans and Cryptococcus neoformans), three dermatophytes (Trichophyton rubrum, T. soudanense and Microsporum canis) and two hyphamycets (Aspergillus fumigatus and Scopulariopsis brevicaulis). The microorganisms were preserved and maintained in the Laboratory of transmissible diseases and biologically active substances, Tunisia.

Antifungal testing

Susceptibility of the fungi strains to the extracts and essential oils was investigated using the agar incorporation method (dilution in a solid medium). These phytopathogens were cultivated on sabouraud-chloramphenicol agar (SCA) in 6 cm Petri dishes. The essential oils, the ethanol extract or the aqueous extract was mixed with 100 ml of SCA to give final concentrations of 0.5–1 mg/ml. The ethanol extract and essential oils were dissolved in 10% of ethanol 99%, and this solvent was used as a negative control. After cooling and solidification, the medium was inoculated with 5 mm in diameter of a 7-day old mycelium culture of the tested dermatophytes, a 3-day culture suspension adjusted to 105 conidies/ml for A. fumigatus and a 3-day yeast culture suspended in sterile distilled water and adjusted to 105 spores/ml. The Petri dishes were then incubated at 24 °C for 7 days for dermatophytes, 24 h at 37 °C for C. albicans and A. fumigatus, and 48 h at 37 °C for C. neoformans. Three replicates for each concentration and microorganisms were carried out.

The antifungal activity was evaluated using two methods:

  1. 1.

    By calculating the percentage inhibition (I  %) from the diameter values of the colonies in the control plate (dC) and the colonies in the plate added with the assayed extracts (dE), I% = (dC − dE)/dC, according to the method of Singh et al. (1993).

  2. 2.

    By determining the minimal inhibitory concentration (MIC): the lowest concentration which inhibits the visible growth of fungi during the incubation period adapted to each species (Mahlo et al. 2010).

Results

Chemical composition of essential oils

Pale yellow essential oils were extracted with yield of 0.29% for aerial parts of T. polium (v/w) on a dry matter basis. The oils were analyzed by GC and GC/MS and the qualitative and quantitative composition are presented in Table 1. The GC/MS analyses showed that the essential oils of T. polium were mainly represented by β-pinene (35.97%), α-pinene (13.32%), α-thujene (8.46%), p-cymene (5.25%) and Verbenone (5.03%).

Table 1 Components of the essential oil of Teucrium polium L
Full size table

Antibacterial activity

As shown in Table 2, all extracts exhibited an antibacterial activity against both Gram-negative and Gram-positive bacteria at different levels. P. aeruginosa was the most resistant (9–13 mm), whereas C. freundei (13–25 mm) and S. aureus (10–17 mm) were the most sensitive. These results were confirmed by the agar dilution method (Table 2), (0.156–0.625, 0.078–0.156 and 0.078–0.625 mg/ml, respectively, against P. aeruginosa, C. freundei and S. aureus).

Table 2 Antibacterial activity of the essential oil, ethanol and aqueous extracts of Teucrium polium L. using percentage inhibition (I%) of bacterial growth and minimal inhibitory concentration (MIC, mg/ml)
Full size table

Antifungal activity

The essential oils and both extracts (aqueous and ethanol) of T. polium had interesting activities against fungi with a percentage of inhibition varying from 0 to 100% (Table 3). All extracts had no adverse effects on A. fumigatus growth. For the three tested dermatophytes, M. canis was markedly inhibited (48.88–100%), whereas S. brevicaulis was slightly inhibited (8.6–58%). For the two opportunist pathogenic yeasts, C. albicans was weakly inhibited (2.03–9.63%), in contrast C. neoformans was only inhibited by the essential oils.

Table 3 Antifungal activity of the ethanol and aqueous extracts, and essential oil of Teucrium polium L. using percentage inhibition (I%) of microorganisms and minimal inhibitory concentration (MIC, mg/ml)
Full size table

Discussion

The unavoidable appearance every now and then of new antibiotic-resistant strains explains the perpetual demand on more efficient medicines. Under this conjuncture, medicinal plants represent a major part in the drug research programs. They can be found either as a natural drug component like in many developing countries, or as a raw material for pure chemical derivatives processing like in industrialized countries.

Environmental factors such as sunshine duration, temperature, soil, ground water, availability of nutrients are known to affect the presence and/or the concentrations of the active principles, including the volatile oils of various medicinal herbs. Sampling site and the time of collection of plant material are additional factors that may influence the phytochemical composition (Robbers et al. 1996). In Jordan, Aburjai et al. (2006), working on T. polium showed that the major components of its aerial part essential oils are 8-cedren-13-ol, β-caryophyllene, germacrene D and sabinene with a percentage of 24.8, 8.7, 6.8 and 5.2% respectively. For the same species, it was shown (Afifi et al. 2009), that there are differences in the chemical composition and concentration of volatile constituents between collected from different regions in Jordan. Our data are different from those found by Boulila et al. (2008) on Tunisian T. polium. These authors showed that main volatile components are myrcene (15.3%), germacrene D (9.0%), α-pinene (6.6%), β-pinene (5.8%), and α-cadinol (5.1%). In addition, Turkish T. polium essential oils obtained from the aerial parts have shown 37 components with β-pinene (18.0%), β-caryophyllene (17.8%), α-pinene (12.0%), caryophyllene oxide (10.0%), myrcene (6.8%), germacrene D (5.3%), limonene (3.5%) and spathulenol (3.3%) were the major identified components (Çakir et al. 1998). Working on the same species originated from Saudi Arabia, Hassan et al. (1979) affirmed that T. polium essential oil rich in terpenoid compounds including β-pinene, limonene, α-cadinene, α-phellandrene and the alcohols linalool, terpinen-4-ol, guaiol, cedrenol and cedrol. In addition, it was free from esters. Egyptian T. polium mainly contains Myrcene, OC-pinene, menthofuran, ocimene, pulegone and menthone (Wassel and Ahmed 1974). In Spain, Velasco-Negueruela and Perez-Alonso (1990) reported that the major compounds of T. polium were α-pinene (15.8%), β-pinene (11.7%), sabinene (7.2%), trans-pinocarveol (4.3%), terpinen-4-ol (4.5%) and β-bisabolene (2.5%).

In the present study, the essential oils of T. polium were rich in α-pinene (13.32%), β-pinene (35.97%), α-thujene (8.46%), p-cymene (5.25%) and Verbenone (5.03%). This difference in essential oil compositions of T. polium, may be explained by the difference of the origin of plants (Bourgou et al. 2010). α-pinene has been previously known for its antibacterial activity (Kabouche et al. 2005). Moreover, Ultee et al. (2002) demonstrated that p-cymene had a very low antibacterial activity, however could induce a bacterial membrane swelling. By this mechanism p-cymene probably facilitates the transport of α-pinene and β-pinene into the cell. The mechanism of action of terpenes which were active against bacteria is still unknown, but probably some lipophilic compounds caused a membrane interruption (Cowan 1999). The oil antimicrobial activity can be explained by the presence of borneol, linalool and terpinen-4-ol, which were proven to have bacteriostatic activity against many microorganisms (Barel et al. 1991, Cosentino et al. 1999). At lower concentrations, these components can also have synergic interactions with other active compounds (Marino et al. 2001).

Previous studies on the composition of T. polium showed the presence of phenolic compounds (Djabou et al. 2012). Our data show interesting antibacterial and antifungal activities of the ethanol extract from T. polium, this is probably due to polyphenol substances present in the extract. Furthermore, many authors have focused in their studies on the effect of phenolic compounds on cellular membranes. In this context, Tsuchiya et al. (1996) demonstrated the interaction of phenolic compounds and extracellular proteins with the cell membrane. Indeed, phenolics leak out intracellular constituents by altering cells walls and membranes, which increase cells permeability. Critical membrane functions regulating cells exchange and metabolite synthesis are also affected by these alterations. Consequently, the multilevel cellular effect of the phenolic compounds may block the bacterial activity. Tassou et al. (2000) noticed a loss of intracellular material from E. coli and S. aureus which was related to phenol concentration. Ethanol extract of T. polium may be considered a well inhibitory of Trichophyton rubrum which is the main cause of athlete’s foot and onichomycoses in human beings.

As a result of the variability in chemical composition of T. polium, there was variability in antimicrobial activity. According to Darabpour et al. (2010), Bacillus anthracis was the most sensitive species when treated by the ethanol extract (50–400 mg/ml), while Escherichia coli and Proteus mirabilis were more resistant. These authors reported that the MIC of Staphylococcus aureus and Salmonella typhi was 40 mg/ml; while in Bordetella bronchiseptica and Bacillus anthracis was 10 mg/ml.

The effect of ethanol extract against S. aureus in this work (11 mm inhibition zone) was similar to that found by Sarac and Ugur (2007) (9 mm).

Overall, the contemporary presence of antibacterial and antifungal activities in the essential oil and ethanol extract from T. polium proves that this plant contains bioactive compounds and may be utilized for several activities.