Chemical Composition, Antioxidant Activity, and Antifungal Effects of Essential Oil from Laurus nobilis L. Flowers Growing in Morocco

In this study, the chemical composition and the antioxidant and antifungal activities of essential oil from Laurus nobilis flowers were examined. ,e essential oil was prepared using steam distillation in a modified Clevenger-type apparatus. ,e chemical composition of the obtained essential oil and chemotypes was determined using gas chromatography coupled with mass spectrometry (GC/MS) and gas chromatography with flame ionization detection (GC-FID). Twenty-five volatile compounds were identified, which made up 92.07% of the total essential oil content. ,e essential oil yield was 1.06% and the most abundant compounds were 1.8-cineole (45.01%), α-caryophyllene (7.54%), germacradienol (6.13%), limonene (4.69%), α-pinene (3.04%), and germacrene D (3.14%). ,e antifungal activity of the obtained essential oil was tested against seven fungal strains: Aspergillus clavatus, A. niger, Chaetomium globosum, Cladosporium cladosporioides, Myrothecium verrucaria, Penicillium citrinum, and Trichoderma viride. ,e results indicated that essential oil from L. nobilis flowers exhibited significant antifungal activity against the tested fungal strains with minimum inhibitory concentrations (MICs) ranging from 0.05 to 0.46mg/mL. ,e essential oil of L. nobilis also exhibited strong total antioxidant capacity (TAC) as indicated by its ability to scavenge free radical DPPH. Taken together, this study indicates that the essential oil from L. nobilis flowers possesses significant antifungal and antioxidant activities, possibly due to the high level of 1,8-cineole.


Introduction
Herbs and spices have been used for generations, not simply as food ingredients but also to treat a plethora of ailments and, in recent times, scientific data are accumulating that demonstrate, for many spices and related essential oils, medicinal properties useful in the prevention of diseases or the relief of their symptoms. In fact, many natural compounds extracted from plants have demonstrated biological activities, notably antibacterial, antifungal, and antioxidant properties. e antimicrobial properties of plant volatile oils and their constituents from a wide variety of plants have been documented [1,2]. e Lauraceae comprises 32 genera and about 2.500 species. Laurel (Laurus nobilis L.) is a plant native to the southern Mediterranean region. It is an evergreen tree cultivated in many warm regions of the world, particularly in the Mediterranean countries (Turkey, Greece, Spain, Portugal, and Morocco). In Europe and the USA, L. nobilis is widely cultivated as an ornamental plant [3].
Essential oils are valuable natural products used as raw materials in perfumes, cosmetics, aromatherapy, phytotherapy, spices, and nutrition [4]. e leaves of L. nobilis have been used to treat Neuralgia and Parkinsonism [5], while the essential oil obtained from the leaves of this plant has been used for relieving hemorrhoid and rheumatic pains and has been used to treat epilepsy [6]. e chemical composition of the volatile fraction of L. nobilis as well as the composition and bioactivities of the alcoholic and nonpolar extracts has been studied extensively [7][8][9]. Intensive research has been conducted on this species [10][11][12]. e antifungal activity of essential oil extracted from L. nobilis has been documented [13,14], the oil presents a low minimum inhibitory concentration and is rich in monoterpenes and sesquiterpenes. Essential oils have been shown to possess antibacterial, antifungal, antiviral, insecticidal, antidiarrheal, and antioxidant properties [15][16][17][18][19]. ere is currently an increased interest in looking at antimicrobial properties of extracts from aromatic plants particularly essential oils [20]. Essential oils are a rich source of biologically active compounds; they have been used in cancer treatment [21] and food preservation [22]. e chemical composition and antimicrobial properties of essential oils extracted from diverse plant species have been demonstrated using a variety of experimental methods [23,24]. However, to the best of our knowledge, the chemical composition, antioxidant activity, and antifungal effects of essential oil from L. nobilis flowers in Morocco have not been studied. e objective of this study was to identify the chemical compounds and examine the antioxidant and antifungal activities of essential oil from L. nobilis flowers.

Chemicals and Standards.
All solvents used in this study were of analytical grade unless otherwise specified. Hexane solution, methanol, sulfuric acid, anhydrous sodium sulfate, series of alkanes (C 4 -C 28 ) standards, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were purchased from Sigma-Aldrich (St. Louis, MI, USA). e fungal strains were obtained from the Microbiology Laboratory, Faculty of Medicine and Pharmacy, Fez, Morocco.

Collection and Preparation of the Plant Material.
e flowers of L. nobilis were collected in July 2019, from the Middle Atlas mountains in Morocco, a region where people frequently use this plant for therapeutic uses [25]. e region is located 15 km southeast of Boulemane city (latitude: 25°31′11″; longitude: 5°22′21″; altitude: 2100 m); it is characterised by semidesertic climate with strong continental influence, and an annual average temperature of 20°C. e collected flowers were air-dried for sixteen days and stored in the laboratory, at the Faculty of Sciences, University Sidi Mohamed Ben Abdellah, Fez, Morocco.

Essential Oil Extraction.
e essential oil was extracted by hydrodistillation using an apparatus of Clevenger type as previously described [26,27]; briefly, 200 g of L. nobilis flowers were mixed in 1.4 ml of distilled water for 2.5 hours. e yellowish oil (0.5 ml) was dissolved in hexane and then dried over anhydrous sodium sulfate. After filtration, the solvent was eliminated by pressure distillation reduced in a rotary evaporator at 35°C, and the extracted oil was stored at 4°C in obscurity until analysis. e amount of oil obtained was calculated as follows: oil(% v/w) � observed volume of oil(ml) weight of the sample(g) × 100. e column temperature was programmed from 40°C to 280°C for 5°C/min. e temperature of the injector was fixed to 250°C and one of the detectors (FID) to 260°C. e debit of the gas vector (nitrogen) was fixed to 1 ml/min. e volume of the injected specimens was 0.5 μl of diluted oil in hexane solution (10%). e percentage of each constituent in the oil was determined by area peaks. e essential oil was subjected to GC-FID analysis (Polaris Q, ermo Fischer, France) using the same type of column used for GC-MS (the constituents of essential oil were identified in comparison with their Kovats index, calculated concerning the retention time of a series of linear alkanes (C 4 -C 28 ) with those of reference products and in comparison with their Kovats index with those of the chemical components gathered in [28], in comparison with their specters of mass with those gathered in a library of (NIST-MS) type.

Fungal Strains.
e antifungal activity of the essential oil prepared from L. nobilis flowers was evaluated by measuring the zone of inhibition and the minimal inhibition concentration (MIC). Values were calculated according to published procedures [29,30] with minor modifications.
Antifungal tests were then carried out by the disc diffusion method [31], using 50 μl of suspension containing 52 spores/ml of fungi spread on potato dextrose agar (PDA). e discs (6 mm in diameter) were impregnated with 10 μl of essential oil and placed on the inoculated agar. Negative controls were prepared using the same solvents used to dissolve the plant extract. Ofloxacin (20 μg per disc) and sulbactam (30 μg) + cefoperazone (70 μg) (100 μg/disc) were used as positive reference standards to determine the sensitivity of one strain/isolate in each microbial species tested. e inoculated plates were incubated at 27°C for 72 h. e fungal strains tested were Aspergillus clavatus, A. niger, Chaetomium globosum, Cladosporium cladosporioides, Myrothecium verrucaria, Penicillium citrinum, and Trichoderma viride., and assays were performed in duplicate.

DPPH Radical Scavenging Activity.
e ability of the essential oil to scavenge the DPPH radical was tested as previously described [32]; 100 μl of different concentrations of the essential oil was added to 750 μl of an ethanolic solution containing 0.1 mmol of DPPH (2,2-diphenyl-1 picrylhydrazyl). Mixtures were incubated at room temperature for 30 min in dark. e absorbance of the mixture was then measured at 517 nm using a spectrophotometer (UV-2005. J.P. SELECTA. s.a) and the percentage of inhibition (PI) was calculated using the following equation: where A c is the absorbance of the negative control and A s is the absorbance of the sample. Butylated hydroxytoluene (BHT) and ascorbic acid served as positive controls for maximal radical quenching. e IC 50 values were calculated as the concentration causing 50% inhibition of DPPH radical.

Total Antioxidant Capacity (TAC).
Total antioxidant capacity was measured as previously described [33]. Different volumes (100 μl, 50 μl, and 25 μl) of the essential oil were mixed in 1 ml of reactive solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate.) After incubation at 95°C for 90 minutes, the optical density at 695 nm was measured using a spectrophotometer, with a blank containing methanol instead of the essential oil. e antioxidant capacity was expressed in milligrams of ascorbic acid equivalent per gram of extracts (Mg EAA/g extracts). Ascorbic acid was used to make a standard curve.

Antifungal Activity.
e antifungal activity of the essential oil extracted from the flowers of L. nobilis was tested against Aspergillus clavatus, A. niger, Chaetomium globosum, Cladosporium cladosporioides, Myrothecium verrucaria, Penicillium citrinum, and Trichoderma viride. e obtained results are presented in (Table 2). e disc diffusion assay indicated that the inhibition zone of the essential oil ranged between 6.05 mm and 19.25 mm, depending on the fungal strain tested. e strongest inhibition zone was observed for A. clavatus followed by A. niger and C. globosum, indicating the sensitivity of these strains to the essential oil from L. nobilis flowers. e MIC values obtained for these strains were 0.05, 0.14, and 0.18 mg/mL, respectively. e other fungal strains tested were also sensitive to the essential but to a lesser extent.

DPPH Radical Scavenging Activity.
e antioxidant activity of the extracted essential oil was determined based on its ability to reduce the free radicals DPPH. e essential oil from L. nobilis flowers exhibited strong power of reducing DPPH (IC 50 � 82.01 ± 0.002 μg/ml), compared to BHT (IC 50 � 7.71 ± 0.0001) and ascorbic acid (IC 50 � 1.16 ± 0.0001 μg/ml) ( Table 3). A low inhibitory concentration is an indication of a high scavenging activity. A substance can be considered as an antioxidant if its IC 50 does not exceed 5 mg/ml [34]. In this study, the essential oil of L. nobilis has proven its ability as a strong antioxidant; this is accordance with previous reports [35,36]. In fact, the major compound of the essential oil, 1.8-cineole (45.01%), has been shown to have strong antioxidant capacity [37].

Total Antioxidant Activity.
e total antioxidant capacity was determined by the method of phosphomolybdenum, based on the reduction of Mo (VI) to Mo (V) by the antioxidant substance [38], and the values were expressed as ascorbic acid equivalents per gram (AAE/g) ( Figure 2). For the different volumes of the essential oil used (100 μl, 50 μl, and 25 μl), the total antioxidant activities obtained were 796.74 ± 3.53 AAE/g), (568.91 ± 96.16 AAE/g), and (440.91 ± 10.37 AAE/g), respectively, indicating a positive correlation between the volume of the essential oil used and the level of antioxidant activity.
e results indicate that the essential oil of L. nobilis flowers exhibits strong antifungal activity especially against A. clavatus, A. niger, and C. globosum. Previous studies have reported the antimicrobial activity of members of the genus Laurus. is activity might be due to 1.8-cineole and α-caryophyllene and their precursors [62][63][64][65]. In fact, 1.8-cineole alone has been shown to exhibit a better antifungal activity than the whole essential oil [66]. e antifungal activities, in general, have been mainly explained through terpenes with aromatic rings and phenolic hydroxyl groups able to form hydrogen bonds with active sites of the target enzymes, although other active terpenes, as well as alcohols, aldehydes, and esters can contribute to the overall antifungal effect of essential oils [67]. Pinene-type monoterpene hydrocarbons are wellknown chemicals having antimicrobial properties [68]. e difference in antifungal efficacy is a result of higher concentrations of the same chemical or a result of different chemical composition between plants. Several studies have been conducted to understand the mechanism of action of plant extracts and essential oils; however, it is still unclear [69]. It has been suggested that components of the essential oils and extracts cross the cell membrane interact with the enzymes and proteins of the membrane producing a flux of protons towards the cell exterior, causing cell death [66]. Pinene-type monoterpene hydrocarbons (α-pinene and β-pinene), limonene, and linalool are well-known chemicals having antimicrobial properties [67,70,71].
Furthermore, the antioxidant activity may be altered by synergistic and antagonistic effects between some components of the essential oil [72]. e synergistic activity of 1.8cineole and α-caryophyllene has been documented [73]. Our

Journal of Food Quality
results are in accordance with those found by other researchers [74][75][76][77], further confirming the antioxidant capacity of essential oil from L. nobilis and its potential as a natural preservative in food and pharmaceutical industries.

Total Antioxidant
Activity. e essential oil of L. nobilis flowers exhibited higher total antioxidant capacity compared to previous reports [72], and this can be explained by the variation in the chemical composition of the essential oil due to the difference of regions affected by environmental factors, and also by the redox power of this substance and its electron/hydrogen exchange capacity. ese results represent a basis for further studies that could lead to the utilisation of this essential oil as a natural antioxidant agent, in both food and pharmaceutical fields.

Conclusion
is study revealed the chemical composition of the essential oil of L. nobilis flowers originated from the Middle Atlas mountains in Morocco. e essential oil was characterised by GC-MS and GC-FID, and twenty-five volatile compounds were identified which made up 92.07% of the total essential oil. e essential oil yield of the studies was 1.06%, and the major constituents were 1.8-cineole, α-caryophyllene, and germacradienol (6.13%). e essential oil of L. nobilis flowers exhibited important antioxidant and antifungal activities especially against A. clavatus, A. niger, and C. globosum, suggesting its potential as a source of antifungal compounds identification. However, further studies should be conducted to evaluate its efficacy against phytopathogenic fungi under field conditions. ese results constitute an important step in the search for biologically active natural substances. Additional tests will be necessary to find formulations for the use of the essential oil from L. nobilis flowers in food, pharmaceutical, and cosmetic industries.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.