Chemical Composition and Antimicrobial Activity of Laurus nobilis L. Essential Oils from Bulgaria

Laurel, Laurus nobilis L. is an evergreen plant belonging to the Lauraceae family, native to Southern Europe and the Mediterranean area. This is the first report on the composition and bioactivity of laurel essential oil (EO) from Bulgaria. The oil yield was 0.78%, 0.80%, and 3.25% in the fruits, twigs, and leaves, respectively. The main constituents in the fruit EO were 1,8-cineole (33.3%), α-terpinyl acetate (10.3%), α-pinene (11.0%), β-elemene (7.5%), sabinene (6.3%), β-phellandrene (5.2%), bornyl acetate (4.4%), and camphene (4.3%); those in the twig EO were 1,8-cineole (48.5%), α-terpinyl acetate (13.1%), methyl eugenol (6.6%), β-linalool (3.8%), β-pinene (3.4%), sabinene (3.3%) and terpinene-4-ol (3.3%); and the ones in the leaf EO were 1,8-cineole (41.0%), α-terpinyl acetate (14.4%), sabinene (8.8%), methyl eugenole (6.0%), β-linalool (4.9%), and α-terpineol (3.1%). The antibacterial and antifungal properties of laurel EOs were examined according to the agar well diffusion method. The leaf EO showed antibacterial and antifungal activities against almost all strains of the microorganisms tested, whereas the twig EO was only able to inhibit Staphylococcus aureus. Pseudomonas aeruginosa ATCC 9027 and Escherichia coli ATCC 8739 were the bacterial strains that showed the highest resistance to the laurel EO. The results can benefit the EO industry and biopesticide development.


Introduction
Laurel or sweet bay (Laurus nobilis L.) of the Lauraceae family is a plant native to the southern Mediterranean region. It is grown as high-value spice crop in that region, and as an ornamental plant throughout Europe and America. It also grows as an escapee, naturalized in eastern Bulgaria, along the Black Sea coast.
Aqueous extracts of laurel fruits and leaves have been used in herbal medicine as an astringent agent and for the treatment of several neurological, dermatological, and urological disorders [1]. In addition, laurel essential oil (EO) is currently used in folk medicines for the treatment of different health problems, such as rheumatism and dermatitis [1,2].
In Bulgaria, laurel is found mainly as an ornamental plant or escapee in the wild in the southernmost regions. The majority of the laurel genotype pool in Bulgaria was probably introduced from Greece after the Second World War [35]. However, some may have been transferred from Middle Eastern countries during the 15th to 18th century, or the Ottoman period. Generally, laurel fruits are not harvested and, therefore, not used although they have a potential to provide an EO with unique characteristics and other novel bioactivities. The leaves have been used as a spice and preservative in the food industry for making various products, as well as in folk medicine. According to Georgiev and Lazarov [36], the laurel trees found in Bulgaria had a sufficient EO content, which was higher in plants growing in the warmer parts of the country. The leaves of the middle part of the shoots had a higher EO content (1.9-3.35%). The EO in the twigs (less than 0.4%) allowed them to be used, together with the waste from the processing of the leaves (crushed and non-standard leaves), as a raw material for EO production. However, the latter authors did not analyze the EO composition.
There has been a growing interest in biologically active substances from non-traditional and underexplored plant species. Bulgaria is one of the largest EO producers in Europe; its distillation facilities and network could easily accommodate a novel EO from naturalized plants produced locally. Despite the interest demonstrated by the industry, there is no previous study on the composition and antimicrobial activity of EOs of laurel found in Bulgaria.
This study aimed to determine the chemical composition, antibacterial, and antifungal activity of the EOs from different parts of laurel grown in Bulgaria as a possible source of constituents for use in perfumery, cosmetics, and pharmaceutical products.

Antimicrobial Activity
The results of the antimicrobial assay are presented in Table 2. The laurel fruit EO in this study showed low inhibitory activity against Gram-positive bacteria Staphylococcus aureus and Kocuria rhizophila, Gram-negative bacterium Salmonella abony, yeast Saccharomyces cerevisiae, and fungus Aspergillus brasiliensis. However, the fruit EO did not show inhibitory activity against Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa.
The laurel leaf EO in this study possessed low antimicrobial potential against Gram-positive bacteria Staphylococcus aureus, Kocuria rhizophila, and Bacillus subtilis, Gram-negative bacterium Salmonella abony, and fungus Aspergillus brasiliensis. Candida albicans was sensitive, while Saccharomyces cerevisiae showed high sensitivity to the inhibitory effect of the leaf EO. Escherichia coli and Pseudomonas aeruginosa were resistant to the inhibitory activity of the leaf EO. The laurel twigs EO showed weak action against Staphylococcus aureus but not against the other test microorganisms in this study.

Discussion
The differences between the Bulgarian laurel EO composition in this study and that from other countries reported in the literature are probably due to the different genotypes, climatic conditions in the respective locality where the plants were grown, and also to the plant parts processed and extracted.
The observed high concentration of 1,8-cineole in the twig EO in this study was the basis for suggesting that in the industrial production of laurel EO, both the leaves and the twigs should be used if a high 1,8-cineole concentration was desirable.
The differences in the quantitative and qualitative composition of the EOs, and in their composition determined in this and previous reports could be due to a number of factors, such as the collection location, soil characteristics, climatic conditions, harvest time, possible differences in the plant genotypes, postharvest processing, and the EO extraction method.
In this study, the oxygenated monoterpenes and monoterpene hydrocarbons were the dominant groups of chemical constituents in the EOs from the three plant parts, followed by phenyl propanoids.
Fruit and twig EO have a lower content of the linalool and eugenol allergens than that of leaf EO. The European Cosmetic Directive prohibits the use of these allergens unless they are a natural constituent of plant EO or other natural flavoring (as in the case of laurel EO). These allergens must not exceed the permissible concentration of 0.01% in shower gels and rinse-off products and must not be higher than 0.001% in body oils, massage oils, and creams [37]. Due to their opulent chemical composition and characterization, the inclusion of oils in different cosmetic products will be a subject of our next research.
Linalool ( Figure 1) is a monoterpene alcohol which occurs as one of its enantiomers in many EOs, where it is often the main constituent. For example, (−)-linalool occurs at a concentration of 80 to 85% in Cinnamomnum camphora oil, and rosewood oil contains around 80%. (+)-Linalool makes up 60 to 70% of coriander oil. Eugenol (Figure 1) belongs to the chemical group of phenyl propanoids and is the main constituent of several EOs; e.g., clove oil and cinnamon leaf oil may contain up to 90% eugenol [38]. Laurel EO has been reported to inhibit a broad spectrum of microorganisms. Overall, the results from this study are in line with previous literature reports [13,[39][40][41]. Laurel EO has shown significant antibacterial properties and greater effectiveness against some microorganisms than tetracycline antibiotics [39]. Our results are in good agreement with the ones reported by Caputo et al. [11]. A possible explanation for the antibacterial activity of laurel EO is the fact that plant EOs disrupt cellular membranes and increase membrane permeability; they may alter membrane-embedded proteins and subsequently disrupt membrane transport. It was previously demonstrated that terpenes are the constituents responsible for the antibacterial activity of laurel EO [40].
There are several different methods for testing the antimicrobial activity of plants and their constituents, which may significantly influence the observed levels of inhibition. Additionally, various other factors, such as seasonality, variability the plant material, and the EO composition within a plant species, may cause differences in the antimicrobial activity outcomes. This study showed that laurel EOs were effective against all tested Gram-positive bacterial strains, while two of the Gram-negatives were completely resistant to the tested EOs. Gram-positive bacteria are generally more susceptible to the action of the oils compared with the Gram-negative ones. This is due to the presence of an additional outer membrane in Gram-negative bacteria, which may better protect the cytoplasmic membrane from the antimicrobial compounds, such as EOs [42]. The main constituent of laurel EO in this and in some previous studies was 1,8-cineole, that has shown antimicrobial activity against several microorganisms [13,29]. Each EO included an admixture of a number of constituents that may have contributed to the extended spectrum of antimicrobial activity. No assays with pure 1,8-cineole were conducted in this study though. The inhibition strength of laurel EO on microbial growth in this study was probably due to the synergistic or antagonistic effect of 1,8-cineole with oxygenated terpenes of the oil [29,[43][44][45].
The inhibitory effects of laurel fruit, leaf and twig EOs against selected target microorganisms are shown in Figure 2.

Plant Material
The laurel fruit, twigs, and leaves were harvested from wild-grown trees in 2018 in the vicinity of the town of Nesebar, eastern Bulgaria, along the Black See coast, a region characterized by a temperate continental climate. The plant species was identified as Laurus nobilis L. by the Department of Botany and Methods of Biology Teaching, Faculty of Biology, Paisii Hilendarski University of Plovdiv in Plovdiv, Bulgaria, according to the morphological features of the plant described in the European Pharmacopoeia and the Flora Europaea.

Isolation of the Essential Oil
The air-dried fruits were ground in a laboratory mill to a size of 0.7 to 1 cm, and the twigs and leaves were cut to a size of 1 cm. The EO was isolated by hydrodistillation for 3 h in a laboratory glass apparatus of the British Pharmacopoeia modified by Balinova and Diakov [47]. The oil obtained was dried over anhydrous sodium sulfate and stored in tightly closed dark vials at 4 • C until analysis. The EO yields are represented on absolute dry weight basis.

Gas Chromatographic (GC) Mass Spectroscopy (MS) Analyses of the Essential Oil
The gas chromatographic (GC) analyses of all samples of laurel EO from Bulgaria were performed using a GC Agilent 7890A, an HP-5 ms column (30 m × 250 µm × 0.25 µm), temperature: 35 • C/3 min, 5 • C/min to 250 • C for 3 min, total: 49 min; helium as a carrier gas at 1mL/min constant speed, and 30:1 split ratio. The GC/MS analysis was carried out on an Agilent 5975C mass spectrometer, using helium as a carrier gas, and the same column and temperature as in the GC analysis. The identification of chemical compounds was made by comparison to their relative retention time and library data. The identified constituents were arranged in order of retention time and quantity in percentage.

Antimicrobial Activity of the Essential Oil
The antibacterial activity of laurel fruit, twig, and leaf EOs was tested against test microorganisms provided by the National Bank for Industrial Microorganisms and Cell Cultures in Sofia, Bulgaria: Gram-positive bacteria: Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633, Kocuria rhizophila ATCC 9341; Gram-negative bacteria: Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Salmonella abony NTCC 6017; yeast: Saccharomyces cerevisiae ATCC 2601, Candida albicans ATCC 10231; and fungal strain: Aspergillus brasiliensis ATCC 16404.
The antimicrobial activity was determined by the agar well diffusion method with a well size of 8 mm. The growth media were Tryptic soy agar (Merck) for the tested bacterial strains and Sabouraud-Dextrose-Agar (Merck) for the yeast and fungi. The media were inoculated with a 24-h suspension of the bacterial species with a density of approximately 10 7 cfu (colony forming units)/mL (turbidity: 0.5 McFarland standards). Media melted and cooled to 50 • C were inoculated with the tested microorganisms and then equally dispensed into Petry dishes. Next, a hole with a diameter of 8 mm was punched aseptically with a sterile cork borer, and a volume (50 µL) of the antimicrobial agent was introduced into the well. After that, the agar plates were incubated at 37 • C or 28 • C for 24 or 72 h according to the microbial species. After cultivation, the distinct zone of growth inhibition around the wells was measured using a digital caliper. The diameter of the zones, including the diameter of the well, was recorded in mm, for instance, up to 15 mm the microbial culture was poorly sensitive, from 15 to 25 mm it was considered sensitive, and over 25 mm it was considered very sensitive. The tests were performed in parallel with solvent controls [48].

Statistics
All the analyses and measurements were done in triplicate. The results are presented as the mean value of the individual measurements with the corresponding standard deviation (SD).

Conclusions
This is the first study on the EO composition and bioactivity of laurel fruits, leaves, and twigs from Bulgaria. The laurel EOs from Bulgaria was characterized by a higher content of 1,8-cineole and α-terpinyl acetate with a characteristic odor. The oils produced demonstrated antimicrobial activity against some of the highly susceptible strains of pathogenic and spoilage bacteria and yeasts. Laurel fruit and leaves, along with the branches that carry them, can be used as a non-traditional material for the production of EOs to be used as an additive in the cosmetic industry. Currently, the studies on the application of laurel EOs in cosmetic and food products are in progress.