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Antimicrobial Activity and Synergy Investigation of Hypericum scabrum Essential Oil with Antifungal Drugs

The chemical composition of Lebanese Hypericum scabrum essential oil (EO) was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GG-MS). Its antimicrobial activity was evaluated by determining its minimal inhibitory concentrations (MICs) against a Gram-negative and a Gram-positive bacterium, one yeast, and five dermatophytes. H. scabrum EO was most active on filamentous fungi (MIC values of 32–64 µg/mL). Synergy within the oil was investigated by testing each of the following major components on Trichophyton rubrum: α-pinene, limonene, myrcene, β-pinene and nonane, as well as a reconstructed EO. The antifungal activity of the natural oil could not be reached, meaning that its activity might be due, in part, to minor constituent(s). The interactions between H. scabrum EO and commercially available antifungals were assessed by the checkerboard test. A synergistic effect was revealed in the combination of the EO with amphotericin B.


Introduction
Antimicrobial resistance is jeopardizing public health, and is thus turning into an urgent global concern. Resistance occurrence is the result of random genetic events, such as mutations and horizontal gene transfers that enable microbial genomes to evolve [1], while its persistence and spread in the ecosystem is favored by the selective pressure of antimicrobial agents [2,3].
One approach to avoid the emergence of resistant strains is combination therapy that consists of associating several antimicrobial agents. The chance of having mutants that are resistant to agents with independent mechanisms of action is extremely small. Resistance is thus less likely to develop [4]. Such associations could also be used to improve the therapy efficiency when they result in synergistic effects. Another advantage of such combinations is that lower doses are used [5], reducing the side effects and cost of the treatment [6]. In this field, associations between plant essential oils and classic antimicrobials have proven their efficiency [7,8].
Plants belonging to the genus Hypericum are well known for their therapeutic benefits. The most popular species is Hypericum perforatum (St John's Wort). Its extract is one of the best-selling herbal medicines in the world. It is used to treat mild-to-moderate depression [9,10].
Hypericum scabrum L. (Hypericaceae) has traditionally been, in different parts of the world, the basis of the medical treatment of many ailments, such as hemorrhoids [11], body heat [12], stomach ache, diarrhea [13], sun burn, and skin lesions [14].
Although extracts and essential oils (EOs) of H. scabrum have been investigated and reported to have relevant biological activities [15][16][17][18], our Lebanese EO differs significantly from those obtained elsewhere. It was, therefore, interesting to investigate its chemical composition and to evaluate its antimicrobial activity against selected microorganisms responsible for skin infections in humans. In fact, in the topical route of application that is often used traditionally, the EOs are administered in direct contact with the pathogen, and the adverse effects that they could cause when administered systemically become limited. In the present work, we demonstrate that the very good antifungal activity of the Lebanese H. scabrum does not originate from its major constituents, either alone or combined. Since the strong antifungal potential was due to minor components, we embarked upon investigating the potential of this oil to exacerbate the activity of conventional antimicrobials.

Chemical Analysis
The hydrodistillation of the aerial parts of H. scabrum yielded 0.6% essential oil. Thirtyseven compounds, representing 84.2% of the whole oil, were identified (Table 1; Figure 1).   Figure 6). It is noteworthy that α-pinene was also the major compound of the previously studied H. scabrum essential oils from the following different regions of the world: Iran [19], Turkey [20], Tajikistan [21], and Uzbekistan [22]. This does not apply to the other major compounds, which were sometimes minorly present or even totally absent in these other samples.

Antimicrobial Activity
The results of the antimicrobial activity of H. scabrum EO are presented in Table 2. EOs with minimal inhibitory concentrations (MICs) of 128 µg/mL and below are generally considered active [23,24]. Reference compounds The oil was inactive on Pseudomonas aeruginosa and Staphylococcus aureus (MIC > 512 µg/mL). Conversely, H. scabrum EO presented significant antifungal activity against filamentous dermatophytic fungi (MIC values of 32-64 µg/mL, whereas Candida albicans was weakly sensitive (MIC of 512 µg/mL).
Dermatophytes were the most sensitive microorganisms, which is a result that can be explained by the functional role of volatile organic compounds (VOCs), since the majority of pathogens infecting plants are fungi, most of them being filamentous [25,26].
In an attempt to shed light on the origin of the antifungal activity, we individually tested the major components and a reconstructed EO against Trichophyton rubrum ( Table 3). The artificial EO was prepared using the major compounds in pure form, in the same proportions as in the EO, whereas the minor compounds were replaced by DMSO (38.6%). α-Pinene, limonene, myrcene, β-pinene, and nonane showed no significant activity (MIC of 512 µg/mL). The reconstructed EO exhibited moderate activity (256 µg/mL), which points to a probable synergy between the major compounds. It is, however, less active than the natural EO (64 µg/mL), indicating that minor constituents contribute to the overall activity of the EO.
The interaction between H. scabrum and antifungal drugs was tested by the checkerboard assay on T. rubrum, and the results are given in Table 4. A synergistic effect (FICI 0.5) was observed by combining the EO with amphotericin B. When the EO was added at a concentration of 32 µg/mL, the MIC of amphotericin B was lowered from 4 to 0.125 µg/mL, thus the combined use of Hypericum scabrum essential oil with amphotericin B could reduce the amount of amphotericin B by 32 for the same antifungal activity. On the other hand, the associations with fluconazole and griseofulvin were moderately synergistic (FICI 0.6).

Plant Material
Aerial parts of Hypericum scabrum were collected in June 2013 from Mzaar Kferdebian, Mount Lebanon (33 • 59 38" N 35 • 50 5" E) at an altitude of 1818 m. The plant identification was based on 'Nouvelle flore du Liban et de la Syrie' by Paul Mouterde [27]. Air drying of the plant materiel was performed in a shady place for two weeks at room temperature.

Essential Oil Extraction
The extraction of the essential oil was carried out by hydrodistillation for three hours using a Clevenger-type apparatus according to the European Pharmacopoeia, 1997 [28].

GC Analyses
Analytical gas chromatography was performed on a Thermo Electron Corporation gas chromatograph fitted with a flame ionization detector (FID), a DB-5 MS capillary column (30 m × 0.25 mm) with 0.1 µm film thickness or a fused silica HP Innowax polyethylene glycol capillary column (50 m × 0.20 mm, film thickness 0.20 µm). Helium was the carrier gas (0.7 mL/min). The column temperature was initially set to 35 • C before being gradually increased to 85 • C at 5 • C/min, held for 20 min at 85 • C, raised to 300 • C at 10 • C/min and finally held for 5 min at 300 • C. Diluted 1 µL samples (1/100, v/v) were injected at 250 • C manually and in the splitless mode. Flame ionization detection (FID) was performed at 310 • C.

GC-MS Analyses
The GC-MS analyses were performed using an Agilent 6890 gas chromatograph coupled with 5975 mass detector. The 7683 B auto sampler injected 1 µL of each diluted oil sample (1/100, v/v). A fused silica capillary column DB-5 MS (30 m × 0.25 mm internal diameter, film thickness 0.1 µm) or a fused silica HP Innowax polyethylene glycol capillary column (50 m × 0.20 mm, film thickness 0.20 µm) was used. Helium was the carrier gas (0.7 mL/min). The oven temperature program was identical to that described above (c.f. 3.3 GC Analysis). The mass spectra were recorded at 70 eV with an ion source temperature of 310 • C and a transfer line heated to 320 • C. The acquisition was recorded in full scan mode (50-400 m/z).

Qualitative and Quantitative Analysis
Most constituents were identified by gas chromatography by comparing their retention indices (RI) with those from the literature [29,30] or with those of authentic compounds obtained from Sigma-Aldrich (Beirut-Lebanon, and Paris-France). The retention indices were determined relatively to a homologous series of n-alkanes (C 8 to C 24 ) analyzed under the same operating conditions. Concomitantly, their mass spectra on both columns were compared with those provided in the NIST and Wiley 275 libraries, which are our homemade libraries constructed with pure compounds and EOs of known composition or with mass spectra from the literature [29,31]. The relative concentration of each component was calculated based on the GC peak areas without using correction factors.
The antimicrobial activity of the EOs was measured using a broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [33][34][35][36][37]. The essential oil and its major components were diluted in DMSO and were tested at concentrations ranging from 16 to 512 µg/mL. The microplates were incubated at 37 • C for 24 h for bacteria, 48 h for yeasts, and 5 days for dermatophytes. The minimal inhibitory concentrations (MIC) refer to the lowest concentrations that prevent visible microbial growth (Tables 2 and 3). Oxacillin and gentamicin (0.03-16 µg/mL) were used as reference antibiotics, while itraconazole (0.03-16 µg/mL) and fluconazole (0.125-64 µg/mL) were used as positive controls for the antifungal assays. The antimicrobial standards were purchased from Molekula-Dorset, UK and the pure terpenes from Sigma-Aldrich, France.

Synergy Test by Microdilution Checkerboard Technique
This test is performed in a 96-well microplate and is based on the microdilution method. In particular, two-fold dilution series of the essential oil are made in the vertical direction on the plate while those of the antimicrobial agent are made in the horizontal direction of the same plate, in order to have a fixed amount of the first agent in each row and column and a decreasing amount of the second agent [38,39]. The essential oil was tested at concentrations ranging from 256 to 8 µg/mL. Antimicrobial agents were tested at concentrations ranging from 16 to 0.03 µg/mL for griseofulvin and amphotericin B, and from 64 to 0.125 µg/mL for fluconazole.
The quantitative assessment of the interactions was based on the calculation of fractional inhibitory concentrations (FICs) defined as the ratio of the MIC of the combinations of the two components to the MIC of the essential oil or the drug alone, and the FIC index (FICI), which is the sum of the two FICs. The results were interpreted as follows: FICI ≤ 0.5; synergistic effect > 0.5 and ≤ 2.0; additive effect > 2; antagonistic effect as proposed by Shin and Lim (2004) [40].

Conclusions
Our results showed that H. scabrum EO exhibited interesting antifungal activity. This activity could not be attributed to one major constituent of the oil. It clearly resulted from the interaction of several constituents, including minor ones. In addition, its synergistic interaction with amphotericin B, and the moderately synergistic association with fluconazole and griseofulvin, suggested that combinations between this EO and conventional antifungal agents may lead to new, more potent formulations with less dose-related toxicity.