Synergistic antibacterial combination of Lavandula latifolia Medik. essential oil with camphor

: Combination of various compounds and essential oils for pharmaceutical formulations withdraw attention. In this present study, it was aimed to evaluate the in vitro potential synergistic antibacterial effect of Lavandula latifolia (spike lavender) essential oil with camphor by using the checkerboard method against the human pathogens; Staph-ylococcusaureus and Listeriamonocytogenes. Pharmacopoeia quality L. latifolia essential oil and racemic camphor were analyzed and veri ﬁ ed by GC-FID and GC/MS,simultaneously. Invitro antibacterialactivityofessentialoilandcamphor(MIC range: 0.16 – 20 mg/mL) and standard antimicrobial clarithromycin (MIC range: 0.125 – 16 μ g/mL) were carried out by broth microdilution against S. aureus and L. monocytogenes standard strains, respectively. Resulting antibacterial effects were evaluated for their fractional inhibitory concentrations (FICs) as antagonistic, additive and synergistic effects. The analytical results showed that the major component of essential oil was linalool (45.2%) and 1,8-cineole (25.6%). Antibacterial effects of essential oil were determined as MIC 1.25 – 5 mg/mL. As a result of the experiments, L. latifolia essential oil – camphor combinations were identi ﬁ ed as “ synergistic (FIC ≤ 0.5), and additive (0.5 < FIC ≤ 1) ” in the respective combinations, suggesting further evaluation for formulations for potential antimicrobial applications in food and pharmaceuticals.


Introduction
Lavandula sp. (Lamiaceae) is a valuable essential oil plant crop, which is cultivated around the world mainly for cosmetics and pharmaceutical utilization since centuries [1,2]. Lavandula essential oils are especially used for nervous system stimulation, sedation, tranquilization and stress relieve. In addition, it has useful dermatological uses in the treatment of sunburn and skin rashes, as well as strong antiseptic, and antimicrobial effects [3]. There are 39 Lavandula species, which are mostly of Mediterranean origin. However, there are three commercially important species within the genus among other cultivars, producing lavender (Lavandula angustifolia Mill. = L. officinalis L. = L. vera DC), lavandin (L. intermedia Emeric ex Loisel. = L. hybrida L.) and Spike lavender (L. spica = Lavandula latifolia Medik.) [4,5].
The study material camphor is a white crystalline monoterpene, generally obtained from the tropical Cinnamonum camphora tree, if not from synthetic origin from turpentine. In addition, camphor is found in many essential oils including Lavandula sp., Artemisia sp., Rosmarinus officinalis etc. Camphor is traditionally used to treat colds, pain and inflammation. In vitro antiviral, anticancer, anti-inflammatory, analgesic, antispasmodic, nasal decongestant and rubefacient effects are also reported. The use of 3-11% as a topical pain reliever and anesthetic is approved by the FDA. Also, it is well known that the enantiomeric structure of essential oil components such as camphor affects the bioactivity results, each enantiomer may have different activity and effects. However, overdose may cause toxic effects, especially in children, which urges careful use [17,18].
The selected microorganisms of the present study; Staphylococcus aureus is among the human pathogens causing a wide spectrum of diseases such as respiratory tract infections, endocarditis, arthritis among others [19], whereas Listeria monocytogenes is a foodborne pathogen, and its infection may cause brain invasion, listeriosis resulting fewer, diarrhea and muscle aches etc. [20,21].
Side effects of drugs and antimicrobial resistance caused using excessive and unnecessary antibiotics require the development of new antimicrobials [22,23]. Recently, efforts to increase antimicrobial activity have gained importance with the combination of essential oils with natural ingredients and standard antibiotics [24,25].
The aim of this work was to compare the antimicrobial efficacy by using binary combinations of L. latifolia essential oil with racemic camphor, alone and in combination using the checkerboard method, against human pathogenic S. aureus and L. monocytogenes standard strains.

Analytical
2.2.1 Gas chromatography/mass spectrometry (GC/MS): The analyses were carried out using the Agilent 5975 GC-MSD system (Sem Ltd., Istanbul, Turkey). Innowax FSC column (60 m × 0.25 mm, 0.25 μm film thickness) was used with helium as carrier gas (0.8 mL/min). GC oven temperature was kept at 60°C for 10 min and programmed to 220°C at a rate of 4°C/min and kept constant at 220°C for 10 min and then programmed to 240°C at a rate of 1°C/min. Split ratio was adjusted at 40:1. The injector temperature was set at 250°C. Mass spectra were recorded at 70 eV, where the mass range was from m/z 35 to 450.

Gas chromatography-flame ionization detector (GC-FID):
The GC analyses were carried out using an Agilent 6890 N GC system (Sem Ltd., Istanbul, Turkey). Flame ionization detector (FID) temperature was set to 300°C. To obtain the same elution order with the GC/MS system, simultaneous auto-injection was done on a duplicate of the same column applying the same operational conditions. Relative percentages (%) of the separated compounds were calculated from FID chromatograms.

Identification of the components:
Identification of the essential oil components was carried out by comparison of their relative retention times (RT) with those of authentic samples or by comparison of their relative retention index (RRI) to that of a series of n-alkanes. Computer matching against commercial (Wiley GC/MS Library, MassFinder 4 Library) and in-house "Başer Library of Essential Oil Constituents" libraries built up by genuine compounds and components of known oils as well as MS literature data was used [25,27]. Results are given in Table 1

RRI
Compounds

In vitro antibacterial activity by microdilution method:
The lavender essential oil and camphor were evaluated for their potential antibacterial activity by using the microdilution broth susceptibility assay [27,28,30]. Essential oil and camphor dilution series were prepared in 96-well microtiter plates at a concentration range of 0.16-20 mg/mL. About 100 μL of 1:100 diluted bacterial suspensions were then added to each well. The last row containing medium with microorganism was used as negative control and the well containing only medium served as a positive growth control. Clarithromycin (0.125-16 μg/mL) was used for standard antimicrobial. The microplates were covered with airtight films just before they were placed in the incubator. After incubation at 37°C for 24 h, for staining of viable microorganisms, 20 μL of resazurin of 0.01% was added to all the plates. The first blue well was determined as the minimal inhibitory concentration (MIC, mg/mL). All experiments were repeated in triplicate, and average MICs were reported in Table 2. The results were interpreted as synergistic (FIC < 0.5), additive (0.5 ≤ FIC ≤ 1), indifference (1 < FIC ≤ 4) or antagonism (FIC > 4) [25,31], as shown in Table 2.

Chemical components of L. latifolia essential oil
For the verification of the pharmacopoeia quality, the essential oil was analyzed by both GC/FID and GC-MS, simultaneously. A total of 24 of the main components were detected in L. latifolia essential oil, with a ratio of 97.9%. In this present study, the major compounds of the spike lavender essential oil were identified as linalool (45.2%), 1,8-cineole (25.6%) and camphor (12.2%) confirming the quality, which confirms the Ph. Eur. 9.0 [26] and are listed in Table 1.

Antimicrobial activity
Initially, antibacterial activity of the essential oil, camphor (MIC range: 0.16-20 mg/mL) and standard antimicrobial  Table 2. Accordingly, in this method, essential oil and camphor were effective at 2.5 and 5 mg/mL against S. aureus and 1.25 and 2.5 mg/mL against L. monocytogenes pathogen, respectively. The antimicrobial effect of the lavender essential oil was previously reported, where the agar dilution results showed MIC values 0.2 and >0.8 % (vol/vol) against S. aureus and L. monocytogenes strains, respectively [36]. The inhibitory activity of L. latifolia essential oil against S. aureus and L. monocytogenes strains was reported in the concentration range of 0.5-2 μL/mL [37]. In another publication, the effect of L. latifolia essential oil vapor against S. aureus showed no effect at a test concentration of 500 μL/L [38].
Overall, 64 different essential oil + camphor combinations were evaluated against S. aureus, and L. monocytogenes using the checkerboard microdilution method. Results of combination tests with the checkerboard assay are shown in Table 2. Synergistic effects were observed from 0.16 + 0.62 and 0.08 + 0.62 (mg/mL) essential oil + camphor combinations against L. monocytogenes and 0.31 + 1.25 (mg/mL) against S. aureus. In addition to the synergistic effects, additive effects were observed against the particular studied microorganisms.
De Rapper and coworkers (2013) analyzed the antimicrobial activity of L. angustifolia essential oil in combination with other 45 essential oils against pathogenic microorganisms like S. aureus, Pseudomonas aeruginosa and Candida albicans, and reported that the combinations showed 26.7% synergistic, and 48.9% additive effects [39]. Magi et al. 2015 evaluated the in vitro antibacterial activity of L. angustifolia, Origanum vulgare, Thymus vulgaris, Mentha piperita, and Melaleuca alternifolia essential oils; as well as carvacrol, and synergy of carvacrol and erythromycin, against 32 clinical, erythromycin-resistant groups of Streptococci [40]. Stevic et al. 2014 determined antifungal synergistic effects, with lavender-rose oil and lavender-oregano oil against Fusarium subglutinans and F. semitectum [41].
Camphor was present in the tested L. latifolia essential oil, in relatively high amounts. The combination of L. latifolia essential oil and racemic camphor indeed increases the relative amount, which may have an impact to the antimicrobial effects. To confirm the combination effects of camphor on an essential oil, more detailed work is needed such as using an essential oil without camphor content.
As a result, additive and synergistic effects of L. latifolia essential oil and the monoterpene camphor were demonstrated for the first time, with an overall success rate of 23.4 + 4.7%, respectively. The results also suggested that approx. 72% of the random mixtures lead to "indifferent activity" outcomes. Essential oil + camphor combinations were identified as "synergistic (FIC≤0.5) and additive (0.5 < FIC≤1)" in the respective combinations, suggesting a positive impact on antimicrobial activity holding promise for future in-depth formulation studies.

Conclusion
One of the current approaches toward synergic antimicrobial combination evaluation is the in vitro checkerboard method used in this present study. As observed in the present study, L. latifolia and camphor combinations were relatively more inhibitory against the pathogens L. monocytogenes and S. aureus, respectively. For safe future utilization in formulations, more detailed in vitro and in vivo toxicological evaluations are suggested and needed.