One-Year Seasonal Variation in the Content of Volatile Compounds in Bay Laurel Leaves

Abstract

The composition of an essential oil (EO) depends on both the plant’s genetic constitution and environmental factors. In this study, the leaves of female bay laurel (Laurus nobilis L., family Lauraceae) plants were collected each month in the period from 15 January to 15 December 2022. Twelve obtained leaf samples were hydrodistilled in a Clevenger apparatus and analyzed using gas chromatography–mass spectrometry (GC-MS). A total of 44 compounds were detected in EO and 39 compounds were identified based on MS spectra and RIs (retention indices), accounting for 99.44–99.94% of the oil. The EO consisted almost entirely of monoterpenes (95.56–99.28%) and small quantities of phenylpropanoids, sesquiterpenes and other compounds. The major volatile compound was 1,8-cineole (49.79–64.94%), followed by α-terpinyl acetate (7.14–11.96%), sabinene (3.16–9.01%), linalool (1.77–8.03%), α-pinene (1.46–4.49%), β-pinene (1.55–3.69%) and α-terpineol (0.99–4.77%). The ANOVA indicated statistically significant changes in the composition of the EO over one year. The contents of eugenol, methyl eugenol and elemicin, which are responsible for the spicy aroma of the leaves, were highest during flowering (March) and at the time of fruit ripening (October, November). The harvest time of the leaves can be adjusted to obtain leaves rich in the desired compounds according to whether they are to be used as a spice, medicine or repellent.

1. Introduction

Bay laurel (Laurus nobilis L., family Lauraceae) plays an important role as an aromatic and medicinal plant that has been used in the coastal region of Croatia for many years. It is used as a spice in cooking and in traditional medicine, especially for the treatment of respiratory ailments. Bay laurel is a Mediterranean evergreen shrub or small tree that grows up to 20 m high. The leathery, glabrous, glossy leaves are aromatic due to the essential oil (EO) they contain. The greenish flowers are unisexual, and the plants are dioecious [1,2]. The natural distribution area of the bay laurel includes southern Europe, northern Africa, the Levant and Asia Minor. It has also been introduced to many other areas, including western and eastern Europe; the Azores; the Balearic Islands; South Africa; southwest, south, east and southeast Asia; Australia; the Pacific; New Zealand; and North, Central and South America [3,4,5].

It is also known that EO acts as a natural preservative and prevents food spoilage due to its antimicrobial activity [6]. Dried bay laurel leaves are therefore used in cooking as an aromatic spice and flavoring agent [7,8]. The leaves and fruits are also used in ethnomedicine in Mediterranean countries to treat loss of appetite, stomach cramps and fever. The leaves are also often used as a carminative and in the treatment of bronchitis, colds and digestive problems. Additionally, the fruits are used to treat earache, furunculosis and menstrual cramps and as a diuretic and anti-rheumatic agent. The flowers and bark are used to flavor food [9]. The EO and various extracts of bay laurel leaves and bark have the ability to suppress headaches, migraines, high blood sugar, bacterial and fungal infections, and gastric ulcers. They have anti-inflammatory and antioxidant properties. The EO of the leaves is also used in the treatment of rheumatism and skin rashes, as a wound healing agent and in the cosmetics industry [9,10,11,12]. Two sesquiterpene lactones contained in bay laurel (dehydrocostus lactone and costunolide) have shown potential anti-cancer effects in various types of cancer: liver cancer [13], ovarian cancer [14], breast cancer [15], bladder cancer [16], prostate cancer [17] and leukemia [18].

Numerous authors studied the EO content of bay laurel leaves and found that the major volatile compound was 1,8-cineole (eucalyptol) in EO from Croatia, Montenegro, Italy, Portugal, Tunisia, Palestine, Turkey, Iran, Pakistan and Brazil [6,19,20,21,22,23,24,25,26,27,28,29,30,31]. The chemical composition of the EO of bay laurel leaves is complex, and the specific odor is derived from several volatile compounds. Studies have shown that the EO of bay laurel contains about 80 different compounds [19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39]. It can be assumed that the EO content of bay laurel leaves changes throughout the year. Previous studies on EO content have mainly focused on the identification of volatile compounds in a single sample [19,20] or in samples from several locations [28,35,36,37]. However, there are relatively few data on how the EO content changes throughout the year.

Generally, variations in the yield and composition of the EO depend on the geographical origin, harvesting time, growth stage and growth conditions [31]. Previous studies showed seasonal variation in the amount and/or composition of bay laurel EO [26,27,28,29,30,31,32,33,34]. According to some authors, the flowering stage is the best time for harvesting bay laurel leaves because this is when the plant contains the highest percentage of essential oil [27]. However, the results obtained from studies of seasonal variation in bay laurel EO were different. Some authors who studied the oil content during all four seasons found a higher amount of oil in the vegetative period [30] and others during the dormancy period [31]. Previous research also showed little or no variability between bay laurel oil from different geographic areas [28,35,36,37]. Geographical variability is not pronounced and no chemotypes have been observed across ten different locations of bay laurel in Tunisia [28]. Additionally, no differences were revealed in the chemical composition of EO from Tunisia and Algeria [35]. Investigations of bay laurel EO in Turkey indicated that chemical compositions from different regions were similar according to qualitative and quantitative analysis [36], or only minor qualitative and major quantitative variations in some compounds occurred with respect to collection localities [37].

Since the content of EO in bay laurel leaves shows seasonal variation, it is worth finding the best time to harvest in order to obtain the maximum amount of oil and/or the desired oil compound. The presence of one or more desired compounds in a larger quantity can significantly increase the quality of the EO. In this way, the timing of the leaf collection can be planned with a view to its use. For example, if the leaf is to be used as a spice, it can be harvested at a time when it contains most of the compounds that will give it a more intense odor. If it is to be used as a repellent, the harvest should be planned at a time when it contains the most 1,8-cineole. The collection time is a key factor in EO characterization that may lead to differences in pharmacological activities [34].

In this article, the composition of twelve EO samples was analyzed to gain insight into the seasonal variation in EO content throughout the year. Leaf samples were taken consecutively in the middle of each month during a calendar year and analyzed using gas chromatography–mass spectrometry (GC-MS). The aim of this study is to investigate the seasonal variation in bay laurel EO on Mljet Island (Croatia) in order to determine the best harvest time for the highest oil yield and the maximum content of desired compounds. The study is limited to one year and one specific geographic region, but regardless of this, the seasonal variation of volatile compounds in bay laurel leaves can be expected.

2. Materials and Methods

2.1. Plant Material

Leaves of Laurus nobilis were collected on Mljet Island, South Dalmatia, Croatia (GPS coordinates 42°47′06.7″ N; 17°23′39.5″ E; 25 m a.s.l.). The leaf samples were collected on the 15th calendar day of the month in the period from 15 January to December 2022 The leaf samples were identified by Dr. Dario Kremer. From each of the five female trees, 50–100 mature leaves were collected from the middle of the canopy and mixed to obtain a representative sample. After collection, the leaves were air-dried for 20 days and protected from direct sunlight in a well-ventilated room at room temperature (22 °C) and approximately 60% relative humidity. The dried samples were stored in a dry and cool place for about three weeks before distillation. A sample of the plant material was deposited in the herbarium “Fran Kušan” of the University of Zagreb Faculty of Pharmacy and Biochemistry, Croatia (voucher number HFK-HR-526321).

2.2. Study Site

Like other Adriatic islands, Mljet is an unsubmerged remnant of the Dinaric reef. Several chains of hills extend parallel to the island, between which fertile karst fields have formed. The highest peak on Mljet Island is Veliki Grad with an altitude of 514 m. From a geological point of view, Mljet Island consists of carbonate sediments—limestones and dolomites from the Jurassic and Cretaceous periods, with well-defined stratification and monoclines inclined to the north-northeast [40,41]. There is no impermeable geological base on the island, so there are no watercourses, but there are several sources of drinking water and four puddles of brackish water. The southern coast is exposed to strong waves and southerly winds, which have shaped the coast into steep cliffs and reefs with numerous small bays. The northern coast is milder and more accessible, which is why harbors have also been built on this side [42].

According to the Köppen classification, the climate of Mljet Island belongs to the Mediterranean climate zone of the Csa designation. This climate is characterized by long, hot and dry summers with a large number of sunny days. Winters are short, mild and rainy. The average annual air temperature (based on the period 1981–2014) is 16.7 °C, making the island one of the warmest regions in Croatia. Spring is colder than the relatively warm autumn, which is due to the colder sea. The average monthly air temperature is 8.8 °C in the coldest months of January and February. On the other hand, the average monthly air temperatures in the warmest months of July and August are 26.0 °C and 25.8 °C, respectively. The average multi-year precipitation is 815 mm (Goveđari Station). The slightly higher amount of precipitation on Mljet Island is mainly due to its relative proximity to the mainland and the cyclones that pass over the southern Adriatic and Ionian Sea and bring rain to Mljet. Snow is rare on the island and remains on the ground about once every ten years. The annual average relative humidity is 71% and changes slightly throughout the year. The main winds here are Jugo (SE), Bura (NE) and Tramontana (N) [41,42]. Climate indicators for Mljet Island for the year 2022, recorded at the island’s meteorological station Goveđari, are presented in

Table 1. Climate indicators for Mljet Island for the year 2022, recorded at the island’s meteorological station Goveđari.

I	II	III	IV	V	VI	VII	VIII	IX	X	XI	XII
Average monthly air temperature (°C)												Mean
8.9	10.6	10.1	14.3	20.9	26.6	27.8	27.3	22.5	19.6	15.2	13.2	18.1
Number of hot days (Tmax ≥ 30 °C)												Sum
0	0	0	0	8	29	30	29	7	0	0	0	103
Average monthly precipitation (mm)												Sum
3.3	11.7	10.5	37.6	11.3	61.7	5.1	3.5	43.1	8.2	131.5	76.7	404.2
Average monthly relative humidity (%)												Mean
56	64	54	65	61	57	52	56	64	69	72	83	63

.

The predominant forest trees on Mljet Island are holm oak (Quercus ilex L.) and Aleppo pine (Pinus halepensis L.). Most of Mljet Island is covered with maquis. It is a degraded Mediterranean forest consisting of a large number of evergreen species that have emerged from the degradation of holm oak forests. The most common species growing on Mljet Island as part of the maquis are Arbutus unedo L., Phillyrea latifolia L., Erica arborea L., Pistacia lentiscus L., Myrtus communis L., Viburnum tinus L., Juniperus oxycedrus L., J. macrocarpa Sm. and Juniperus phoenicea L. Olea europaea L. subsp. europaea, Ceratonia siliqua and Laurus nobilis can also be found in the maquis [41,42].

2.3. Essential Oil Extraction

An amount of 100 g of leaves from each sample was taken and hydrodistilled in a Clevenger apparatus for 3 h. The obtained EO was collected in a pentane/diethyl ether mixture (VWR, Radnor, PA, USA) and dried over anhydrous sodium sulfate. The oil extracts were stored in a closed dark glass bottle in a refrigerator at −20 °C until analysis.

2.4. Gas Chromatography–Mass Spectrometry (GC-MS) Analyses and Compounds Identification

Each leaf sample (100 g) was hydrodistilled in a Clevenger apparatus for 3 h and the EO obtained was collected in a pentane/diethyl ether mixture. The EO samples obtained from the leaves were analyzed using the QP 2010 Plus GC-MS (Shimadzu, Kyoto, Japan). A capillary column ZB-5ms 60 m × 0.32 mm, with a film thickness of 0.25 μm (Phenomenex, Torrance, USA), was used for the analysis. The composition of the stationary phase was 95% methyl silicone and 5% phenyl. The temperature program of the column was 60 °C for 1 min, with an increase to 250 °C at a rate of 4 °C/min and finally holding on 250 °C for 2 min. The temperature of the split/splitless injector was 260 °C, while the split ratio was set to 1:10. Helium was used as the carrier gas at a flow rate of 2.2 mL/min. The detector voltage was 1.2 kV and the transfer line temperature was set to 270 °C for GC-MS analysis. Prior to analysis, the samples were diluted with pentane at a ratio of 1:100 and injected manually (1 μL). The mass range was set to 43–350 m/z. Lab Solutions software version 2.72 (Shimadzu, Kyoto, Japan) was used for processing the results and for quantification.

The identity of the volatile compounds was determined based on the retention time (RT) of the components and from the GC-MS spectra and retention indices (RIs) obtained in relation to the C₈–C₂₀ n-alkanes. AMDIS software version 2.62 was used to process the GC-MS data using NIST library version 2.0 (both programs were developed by the National Institute of Standards and Technology, U.S. Department of Commerce, Gaithersburg, MD, USA). The obtained retention indices and spectra were compared with the literature [21,22,43].

2.5. Statistical Analysis

All analyses were performed in triplicate and the results were expressed as the mean ± SD (standard deviation); n = 3. Significant differences between the samples for the relative percentage of compounds were determined using one-way ANOVA followed by Tukey’s HSD post hoc test (p < 0.05). Principal Component Analysis (PCA) was performed for the ten most abundant volatile compounds (1,8-cineole, α-terpinyl acetate, sabinene, linalool, α-pinene, β-pinene, α-terpineol, terpinen-4-ol, limonene, methyl eugenol). The statistical analysis was conducted using Statistica software version 7 (StatSoft Inc., Tulsa, OK, USA).

3. Results and Discussion

GC-MS Profile

The content of EO in twelve samples of bay laurel leaves ranged from 0.47% (February) to 0.90% (May) of the dry leaf mass (

Table 2. Chemical composition of the essential oil of Laurus nobilis leaf determined via GC-MS.

			I	II	III	IV	V	VI
			Essential oil content (%) ± SD
			0.50 ± 0.01	0.47 ± 0.01	0.85 ± 0.01	0.80 ± 0.01	0.90 ± 0.01	0.80 ± 0.01
Compound	RT (min)	RI	Compound content (%) ± SD
3-Heksen-1-ol	7.49	852	–	–	–	tr	0.08 ± 0.01	tr
Isobutyl isobutanoate	8.57	915	tr	–	0.05 ± 0.01 a	–	tr	0.07 ± 0.01 b
α-Thujene	8.72	931	0.24 ± 0.01 a	0.16 ± 0.01 b	0.11 ± 0.01 c	0.17 ± 0.01 bd	0.30 ± 0.01 e	0.21 ± 0.01 f
α-Pinene	8.94	940	3.73 ± 0.11 a	3.13 ± 0.09 b	2.29 ± 0.07 c	3.81 ± 0.11 ad	3.38 ± 0.10 be	4.19 ± 0.13 f
Camphene	9.35	956	0.05 ± 0.01 a	0.18 ± 0.01 b	–	0.05 ± 0.01 a	0.10 ± 0.01 c	0.42 ± 0.01 d
Sabinene	9.95	978	7.62 ± 0.23 a	6.62 ± 0.20 b	7.65 ± 0.23 ac	9.01 ± 0.27 d	8.03 ± 0.24 ae	8.08 ± 0.24 aef
β-Pinene	10.15	983	3.16 ± 0.09 a	2.81 ± 0.08 b	2.32 ± 0.07 c	3.29 ± 0.10 ad	2.92 ± 0.09 abe	3.44 ± 0.10 df
Myrcene	10.38	991	0.22 ± 0.01 a	0.40 ± 0.01 b	0.42 ± 0.01 bc	0.41 ± 0.01 bcd	0.71 ± 0.02 e	0.76 ± 0.02 f
2,3-Dehidro-1,8-cineole	11.59	996	0.05 ± 0.01 a	tr	–	–	0.05 ± 0.01 a	tr
2-Methylbutyl isobutanoat	10.49	1002	–	–	0.05 ± 0.01 a	–	0.06 ± 0.01 ab	0.07 ± 0.01 b
β-Phellandrene	10.56	1008	0.07 ± 0.01 a	tr	tr	–	0.05 ± 0.01 a	tr
Δ-3-Carene	10.63	1015	tr	tr	tr	0.06 ± 0.01 a	0.12 ± 0.01 a	0.33 ± 0.01 ab
α-Terpinene	11.17	1021	0.13 ± 0.01 a	0.09 ± 0.01 b	tr	tr	0.22 ± 0.01 c	0.15 ± 0.01 ad
o-Cymene	11.41	1026	–	–	–	–	tr	0.05 ± 0.01 a
p-Cymene	11.65	1029	0.20 ± 0.01 a	0.20 ± 0.01 a	0.07 ± 0.01 b	0.14 ± 0.01 c	0.16 ± 0.01 cd	0.08 ± 0.01 be
Limonene	11.82	1034	1.89 ± 0.06 a	1.35 ± 0.04 b	1.35 ± 0.04 bc	1.39 ± 0.04 bcd	1.64 ± 0.05 e	1.93 ± 0.06 af
1,8-Cineole	11.94	1038	60.41 ± 1.81 a	63.74 ± 1.91 ab	64.14 ± 1.92 abc	62.61 ± 1.88 abd	55.61 ± 1.67 ae	54.01 ± 1.62 ef
γ-Terpinen	12.22	1063	0.44 ± 0.01 a	0.26 ± 0.01 b	0.15 ± 0.01 c	0.14 ± 0.01 cd	0.50 ± 0.01 e	0.43 ± 0.01 af
cis-Sabinene hydrate	12.64	1072	0.30± 0.01 a	0.22 ± 0.01 b	0.19 ± 0.01 c	0.09 ± 0.01 cd	0.39 ± 0.01 e	0.37 ± 0.01 ef
Terpinolene	12.84	1092	0.05 ± 0.01 a	tr	tr	tr	0.14 ± 0.01 b	0.07 ± 0.01 ac
Linalool	13.52	1099	2.08 ± 0.06 a	6.56 ± 0.20 b	1.77 ± 0.05 ac	3.80 ± 0.11 d	5.66 ± 0.17 e	5.67 ± 0.17 f
trans-Sabinene hydrate	13.65	1101	0.32 ± 0.01 a	0.22 ± 0.01 b	0.21 ± 0.01 bc	0.12 ± 0.01 d	0.24 ± 0.01 be	0.37 ± 0.01 f
1-Octen-3-ol acetate	15.52	1127	tr	–	–	–	0.05 ± 0.01 a	–
Un	16.80	1145	–	–	–	–	–	tr
d-Terpineol + borneol	16.48	1173	0.17 ± 0.01 a	0.24 ± 0.01 b	0.18 ± 0.01 ac	0.06 ± 0.01 d	0.32 ± 0.01 e	0.32 ± 0.01 ef
Terpinen-4-ol	16.84	1183	2.44 ± 0.07 a	1.81 ± 0.05 b	1.64 ± 0.05 bc	1.20 ± 0.04 d	2.41 ± 0.07 ae	1.89 ± 0.06 bf
α-Terpineol	17.32	1195	2.46 ± 0.07 a	3.86 ± 0.12 b	2.87 ± 0.09 c	3.07 ± 0.09 cd	2.52 ± 0.08 ae	2.79 ± 0.08 cef
Nerol	18.25	1231	tr	–	tr	–	0.16 ± 0.01 a	0.07 ± 0.01 b
Linalyl acetate	20.72	1259	–	–	–	tr	0.12 ± 0.01 a	0.16 ± 0.01 a
Bornyl acetate	21.57	1291	–	0.12 ± 0.01 a	2.76 ± 0.08 b	–	0.05 ± 0.01 c	0.38 ± 0.01 d
Terpinen-4-ol acetate	21.61	1293	–	–	–	–	0.08 ± 0.01	–
Acetate	22.09	1322	0.24 ± 0.01 a	0.05 ± 0.01 b	0.14 ± 0.01 c	0.06 ± 0.01 bd	0.48 ± 0.01 e	0.33 ± 0.01 f
α-Terpinyl acetate	22.65	1355	11.96 ± 0.36 a	7.26 ± 0.22 b	10.99 ± 0.33 c	8.63 ± 0.26 d	10.90 ± 0.33 ce	10.61 ± 0.32 cef
Eugenol	22.77	1362	0.32 ±0.01 a	0.44 ± 0.01 b	0.59 ± 0.02 c	1.83 ± 0.05 d	0.56 ± 0.02 ce	0.97 ± 0.03 f
β-Elemene	24.21	1401	–	tr	–	–	–	tr
Methyl eugenol	24.42	1404	1.46 ± 0.04 a	0.07 ± 0.01 b	0.05 ± 0.01 c	0.06 ± 0.01 cd	2.00 ± 0.06 e	1.67 ± 0.05 f
β-Caryophyllene	25.38	1434	–	–	tr	–	–	–
Methyl isoeugenol	26.72	1498	–	–	tr	–	–	–
Sq	27.62	1510	–	0.23 ± 0.01	–	–	–	–
Elemicin	29.14	1559	–	–	–	–	–	–
Spathulenol	30.44	1591	–	tr	tr	–	tr	–
Caryophyllene oxide	30.69	1598	–	–	–	tr	tr	–
Sqol 1	31.89	1666	–	–	–	–	tr	0.12 ± 0.01 a
Sqol 2	32.07	1670	–	tr	tr	–	–	–
Total identified			99.76	99.72	99.86	99.94	99.52	99.55
Monoterpene			17.79	15.20	14.36	18.47	18.27	20.14
Monoterpene alcohols			7.77	12.89	6.86	8.34	11.69	11.47
Monoterpene oxides			60.46	63.81	64.14	62.67	55.66	54.01
Monoterpene esters			11.96	7.38	13.75	8.63	11.15	11.15
Total monoterpene			97.98	99.28	99.12	98.11	96.77	96.77
Phenylpropanoids			1.78	0.44	0.64	1.83	2.56	2.64
Total sesquiterpene			0.00	0.00	0.00	0.00	0.00	0.00
Other compounds			0.00	0.00	0.10	0.00	0.19	0.14
			VII	VIII	IX	X	XI	XII
				Essential oil content (%) ± SD
			0.85 ± 0.01	0.82 ± 0.02	0.80 ± 0.01	0.70 ± 0.01	0.70 ± 0.01	0.6 ± 0.01
Compound	RT (min)	RI	Compound content (%) ± SD
3-Heksen-1-ol	7.49	852	–	tr	–	–	–	0.05 ± 0.01 b
Isobutyl isobutanoate	8.57	915	tr	0.08 ± 0.01 b	–	0.05 ± 0.01 a	–	–
α-Thujene	8.72	931	0.19 ± 0.01 dfg	0.24 ± 0.01 ah	0.05 ± 0.01 i	0.41 ± 0.01 j	0.16 ± 0.01 bdk	0.21 ± 0.01 mfg
α-Pinene	8.94	940	3.34 ± 0.10 beg	4.49 ± 0.15 h	1.46 ± 0.04 i	4.22 ± 0.13 fhj	2.80 ± 0.08 k	2.35 ± 0.07 cm
Camphene	9.35	956	tr	0.49 ± 0.01 e	tr	0.15 ± 0.01 bf	0.14 ± 0.01 cf	0.23 ± 0.01 g
Sabinene	9.95	978	8.50 ± 0.25 defg	8.55 ± 0.26 defgh	3.16 ± 0.09 i	8.22 ± 0.25 abefghj	5.23 ± 0.16 k	6.10 ± 0.18 cm
β-Pinene	10.15	983	3.04 ± 0.09 abeg	3.69 ± 0.11 h	1.55 ± 0.05 i	3.53 ± 0.11 dhj	2.68 ± 0.08 bek	2.17 ± 0.06 cm
Myrcene	10.38	991	0.27 ± 0.01 ag	0.82 ± 0.02 h	0.10 ± 0.01 i	0.75 ± 0.02 efj	0.49 ± 0.01 k	0.31 ± 0.01 gm
2,3-Dehidro-1,8-cineole	11.59	996	–	tr	–	0.07 ± 0.01 a	–	–
2-Methylbutyl isobutanoat	10.49	1002	0.06 ± 0.01 ab	0.07 ± 0.01 bc	–	0.08 ± 0.01 c	–	–
β-Phellandrene	10.56	1008	tr	tr	–	0.07 ± 0.01 a	tr	tr
Δ-3-Carene	10.63	1015	tr	0.39 ± 0.01 b	tr	0.06 ± 0.01 a	0.05 ± 0.01 a	0.05 ± 0.01 a
α-Terpinene	11.17	1021	0.10 ± 0.01 abe	0.18 ± 0.01 f	tr	0.38 ± 0.01 g	0.25 ± 0.01 h	0.08 ± 0.01 bei
o-Cymene	11.41	1026	–	0.05 ± 0.01 a	tr	–	tr	–
p-Cymene	11.65	1029	0.13 ± 0.01 cf	0.09 ± 0.01 beg	0.44 ± 0.01 h	0.26 ± 0.01 i	0.16 ± 0.01 cdj	0.41 ± 0.01 k
Limonene	11.82	1034	1.87 ± 0.06 afg	1.94 ± 0.06 afgh	0.73 ± 0.02 i	2.07 ± 0.06 fhj	1.54 ± 0.05 dek	1.15 ± 0.03 m
1,8-Cineole	11.94	1038	64.94 ± 1.95 abdg	53.87 ± 1.62 efh	63.03 ± 1.89 abdgi	49.79 ± 1.49 fhj	58.36 ± 1.75 adefhik	56.98 ± 1.71 aefhkm
γ-Terpinen	12.22	1063	0.35 ± 0.01 g	0.48 ± 0.01 eh	tr	0.79 ± 0.02 i	0.58± 0.02 j	0.17 ± 0.01 cdk
cis-Sabinene hydrate	12.64	1072	0.31 ± 0.01 ag	0.43 ± 0.01 h	tr	0.48 ± 0.01 i	0.26 ± 0.01 j	0.40 ± 0.01 ek
Terpinolene	12.84	1092	tr	0.08 ± 0.01 ac	tr	0.19 ± 0.01 c	0.08 ± 0.01 ac	0.05 ± 0.01 ac
Linalool	13.52	1099	2.47 ± 0.07 ag	5.88 ± 0.18 h	7.82 ± 0.23 i	4.82 ± 0.14 j	7.67 ± 0.23 k	8.03 ± 0.24 l
trans-Sabinene hydrate	13.65	1101	0.33 ± 0.01 ag	0.39 ± 0.01 fh	0.13 ± 0.01 id	0.35 ± 0.01 fgj	0.26 ± 0.01 ek	0.24 ± 0.01 bekm
1-Octen-3-ol acetate	15.52	1127	tr	0.05 ± 0.01 a	–	0.08 ± 0.01 b	tr	–
Un	16.80	1145	tr	–	–	0.05 ± 0.01	tr	–
d-Terpineol + borneol	16.48	1173	0.22 ± 0.01 bg	0.39 ± 0.01 h	tr	0.50 ± 0.02 i	0.31 ± 0.01 efj	0.67 ± 0.02 k
Terpinen-4-ol	16.84	1183	2.14 ± 0.06 g	2.02 ± 0.06 fgh	2.05 ± 0.06 fgi	3.04 ± 0.09 ij	2.82 ± 0.08 k	2.50 ± 0.08 aem
α-terpineol	17.32	1195	2.62 ± 0.08 acefg	2.89 ± 0.09 cdfgh	0.99 ± 0.03 i	3.45 ± 0.01 j	4.43 ± 0.13 k	4.77 ± 0.14 m
Nerol	18.25	1231	tr	0.09 ± 0.01 b	–	0.20 ± 0.01 a	–	0.06 ± 0.01 b
Linalyl acetate	20.72	1259	–	–	tr	–	–	tr
Bornyl acetate	21.57	1291	–	0.43 ± 0.01 de	8.50 ± 0.25 f	0.09 ± 0.01 acg	0.18 ± 0.01 acdg	0.32 ± 0.01 adeg
Terpinen-4-ol acetate	21.61	1293	–	–	–	tr	–	tr
Acetate	22.09	1322	0.24 ± 0.01 ag	0.24 ± 0.11 agh	0.11 ± 0.01 i	0.45 ± 0.01 j	0.11 ± 0.01 ik	0.16 ± 0.01 cm
α-Terpinyl acetate	22.65	1355	7.14 ± 0.21 bg	9.10 ± 0.27 dh	8.50 ± 0.26 dhi	11.63 ± 0.35 caej	9.92 ± 0.30 fhk	9.02 ± 0.27 him
Eugenol	22.77	1362	0.34 ± 0.01 ag	0.71 ± 0.02 h	0.05 ± 0.01 i	0.94 ± 0.03 fj	0.83 ± 0.02 k	1.78 ± 0.05 dm
β-Elemene	24.21	1401	tr	tr	–	0.10 ± 0.01 a	–	0.09 ± 0.01 a
Methyl eugenol	24.42	1404	1.39 ± 0.04 ag	1.67 ± 0.05 fh	1.32 ± 0.04 agi	2.59 ± 0.08 j	0.42 ± 0.01 k	0.55 ± 0.02 bm
β-Caryophyllene	25.38	1434	–	–	–	tr	–	–
Methyl isoeugenol	26.72	1498	–	–	–	–	–	0.16 ± 0.01
Sq	27.62	1510	–	–	tr	–	–	–
Elemicin	29.14	1559	–	0.05 ± 0.01	–	tr	–	–
Spathulenol	30.44	1591	–	–	tr	–	tr	0.24 ± 0.01
Caryophyllene oxide	30.69	1598	tr	–	–	0.05 ± 0.01 a	tr	0.47 ± 0.01 b
Sqol 1	31.89	1666	–	0.14 ± 0.01 a	tr	0.05 ± 0.01 b	0.17 ± 0.01 c	0.27 ± 0.01 d
Sqol 2	32.07	1670	tr	–	–	–	0.13 ± 0.01	–
Total identified			99.76	99.62	99.89	99.44	99.60	99.57
Monoterpene			17.79	21.50	7.50	21.11	14.15	13.26
Monoterpene alcohols			8.09	12.08	10.99	12.85	15.75	16.66
Monoterpene oxides			64.94	53.87	63.03	49.86	58.36	56.98
Monoterpene esters			7,14	9.53	17.01	11.72	10.10	9.33
Total monoterpene			97.97	96.99	98.52	95.56	98.35	96.23
Phenylpropanoids			1.73	2.43	1.37	3.52	1.25	2.49
Total sesquiterpene			0.00	0.00	0.00	0.15	0.00	0.80
Other compounds			0.06	0.20	0.00	0.21	0.00	0.05

I–XII, month during one year; RT, retention time; RI, retention indices; C, content; SD, standard deviation; Un, unidentified compound; Acetat, unidentified acetate; Sq, unidentified sesquiterpene; Sqol 1–Sqol 2, unidentified sesquiterpene alcohols; tr, compound present in trace amounts; superscript letters indicate a statistically significant difference between fraction at p < 0.05.

).

All samples were analyzed via GC-MS to detect changes in EO composition during a period of one year. The chromatogram is shown in Figure 1. In total, 44 compounds were detected in the twelve analyzed samples of the bay laurel EO, and 39 compounds were identified. Identification was based on MS spectra and RI, accounting for 99.44–99.94% of the oil (Table 2). All unidentified compounds were categorized into groups based on the characteristic ions found in the spectrum. The chemical composition of the twelve samples of bay laurel EO is shown in Table 2. Comparison with the literature data showed that 25 compounds were identified in the EO of bay laurel leaves from Central Dalmatia, Croatia (accounting for 98.5% of the total EO) [19]. Ivanović et al. [20] detected and identified 34 compounds (accounting for 98.5% of the total EO) in leaves collected in Montenegro. In the EO from southern Italy, 55 compounds were identified (accounting for 91.6% of the total EO) [21]. In addition, a total of 78 compounds were identified in EO isolated from leaves collected from seven locations in central–southern Italy [22]. A smaller number of compounds (31) were identified in EO from fresh bay laurel leaves collected in Palestine [24]. Finally, Di Leo Lira et al. [8] analyzed EO from stems and leaves collected in Argentina and identified 33 volatile compounds.

Our study shows that bay laurel EO consists mainly of monoterpenes, the content of which ranged from 95.56% (October) to 99.28% (February). The content of other groups of compounds was very low (Table 2). Other studies have also shown that bay laurel EO consists mainly of monoterpenes [19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39]. For example, Caputo et al. [21] found that oxygenated monoterpenes represented 48.6% of the oil, followed by monoterpene hydrocarbons (34.0%), sesquiterpene hydrocarbons (3.2%), oxygenated sesquiterpenes (0.2%) and phenolic compounds (5.6%). In the EO from Palestine, oxygenated monoterpenes represent 74.55%, followed by monoterpene hydrocarbons (24.20%) and sesquiterpenes (0.37%) [24].

Our results also show that the major volatile compound in the EO of bay laurel leaves is 1,8-cineole. The percentage ranged from 49.79% (October) to 64.94% (July) during the study period of one year (Table 2). 1,8-Cineole is followed by α-terpinyl acetate with a range of 7.14% (July) to 11.96% (January). The third most abundant compound was sabinene, with a content of between 3.16% (September) and 9.01% (April). The fourth compound was linalool with a percentage range of 1.77% (March) to 8.03% (December).

Comparison with other literature data showed that 1,8-cineole was also the main volatile compound of EO from Central Dalmatia in Croatia, accounting for 45.5% [19]. The other important compounds in that study were methyl eugenol (10.0%), α-terpinyl acetate (9.1%), linalool (8.5%) and sabinene (5.7%) [19]. Interestingly, methyl eugenol was weakly represented in our samples. Its content ranged from traces (January, April) to 2.56% (October). The main compounds in the EO from the geographically close area of Montenegro were 1,8-cineole (33.4%), linalool (16.0%), α-terpinyl acetate (13.8%), sabinene (6.91%), methyl eugenol (5.32%), α-pinene (4.39%) and β-pinene (3.52%) [20]. In the EO from southern Italy, which was also relatively close geographically, 1,8-cineole (31.9%) was the most abundant compound. Among the other compounds, sabinene (12.2%) and linalool (10.2%) were predominant [21]. In a study of EO from seven locations in central–southern Italy, Fantasma et al. [22] also found that 1,8-cineole (eucalyptol) was the predominant compound in all samples, containing between 43.52% and 31.31%. This was followed by methyl eugenol (14.96–4.07%), α-terpinyl acetate (13.00–8.51%), linalool (11.72–1.08%) and sabinene (10.57–4.85%) [22]. 1,8-cineole was also the main compound in the EO from geographically more distant areas. For example, this compound was also identified as the most abundant (27.2%) in EO from Portugal, followed by α-terpinenyl acetate (10.2%), linalool (8.4%), methyl eugenol (5.4%), sabinene (4.0%) and carvacrol (3.2%) [23]. In the EO isolated from fresh leaves collected in Palestine, the major compound was, again, 1,8-cineole (48.54%), followed by α-terpinyl acetate (13.46%) and α-terpinyl (3.84%) [24]. 1,8-cineole was the major compound in EO from Turkey, containing between 24.2 and 32.1%, followed by β-pinene (3.9–5.0%), α-pinene (3.0–3.8%), sabinene (7.1–7.6%), α-terpinyl acetate (4.8–6.5%), α-terpineol (1.3–1.8%), linalool (trace—1.5%), eugenol (1.6–0.1%) and β-elemene (1.4–1.8%) [25]. The major compounds detected in EO from Iran were 1,8-cineole or eucalyptol (34.4–50.0%), α-terpinyl acetate (14.9–18.8%), terpinene-4-ol (4.7–6.0%) and sabinene (4.9–5.9%) [38]. In the EO from Egypt, the main constituents were 1,8-cineole (50.38%), α-terpinyl acetate (19.97%) and 4-terpinol (6.48%) [44]. The main compounds in the EO from Morocco were 1,8-cineole (52.43%), α-terpinyl acetate (8.96%) and sabinene (6.13%) [45]. In our study, the 1,8-cinole content was higher than in most other studies. While there are no data from other studies, the samples analyzed in our study were taken from female plants, which could also partly explain the high content of 1,8-cineole. Indeed, Yahyaa et al. [46] found that the leaf stage and gender of the plant significantly affected the composition of volatiles in the leaf. In general, the organs of female plants contained more terpenes than the corresponding organs of male plants. In summary, all male plant parts had a consistently lower concentration of 1,8-cineole than female plants. In contrast, the leaves and flowers of the male plants contained significantly more elemene than the corresponding organs of female plants [46].

Based on our study and the literature data, it can be seen that the main compound of the EO isolated from the bay laurel leaves is 1,8-cineole. Generally, a considerable content of 1,8-cineole was found in samples collected in geographically very distant areas, spanning from Portugal to Croatia and from Italy to Iran and South America. Interestingly, 1,8-cineole was also strongly represented in the oil isolated from bay laurel flowers and berries [47,48]. The content of the other main compounds was also similar in the EO of leaves collected in different parts of the world. These compounds include α-terpinyl acetate, sabinene, linalool, methyl eugenol, α-pinene and β-pinene [19,20,21,22,23,24,25,38,39].

Our study showed seasonal variations in both the yield and composition of the EO. The seasonal variations were to be expected as all plants go through different phases of activity during the year. The flowering period is a particular challenge for the plants, and during this time, many of them have a high concentration of EO. This is one of the reasons why many medicinal plants are collected for medicinal purposes during the flowering period. In our study, the maximum oil yield was recorded from March to September and ranged between 0.8% and 0.9% of the dry leaf mass (Table 2). Thereafter, the oil yield constantly decreased and reached its minimum values in January (0.50%) and February (0.47%). Seasonal variation in the amount of bay laurel oil isolated from the leaves was also observed by several other authors. According to Bahmanzadegan et al. [32], the EO content in southern Iran was highest in winter (January, 2.1%), while the lowest was found in spring (May, 0.87%). From this, the authors concluded that the best harvest time to obtain high oil content is winter [32]. Marzouki et al. [28] analyzed leaves collected in Tunisia during four growing stages: seed production (October), dormancy (January), flowering (April) and vegetative activity (July). The highest EO yield was obtained in nine out of ten samples in July (vegetative activity) and ranged from 0.9% to 2.2%. Only one sample had a slightly higher oil yield in October (1.6%) than in July (1.3%) [28]. Similarly, Verdian-rizi and Hadjiakhoondi [27] analyzed leaves collected in northwestern Iran at four different developmental stages: vegetative plants (May), before anthesis (August), fully flowering plants (September) and seed-bearing plants (November). They found that the EO yield ranged from 0.6% to 1.1% in November and September, respectively [27].

The results from Turkey reported by Muéller-Riebau et al. [31] showed that the oil yield was highest in September (10.2 mL/100 g of dried leaves) and lowest in August (4.1 mL/100 g). Unfortunately, the authors only analyzed the samples from February to September, so data from the winter period are almost completely missing. Roque [30] pointed to the end of August as the optimal harvest time with the highest oil yield. The comparative study by Riaz et al. [29] showed that the yield was highest at the end of September (0.36%) and constantly decreased thereafter until it reached the minimum in March (0.13%). Our results of the highest oil yield in the period of vegetative activity (May, 0.9%) are in line with the results of most studies. Finally, the study by Rodilla et al. [49] showed that the oil content in the leaves of the closely related species Laurus novocanariensis Rivas Mart., Lousã, Fern. Prieto, E. Días, J. C. Costa & C. Aguiar varies between 0.3% and 0.4% in the leaves collected in spring and fall, respectively.

Significant seasonal variation was also found between most of the individual compounds (Table 2). In our study, the content of 1,8-cineole varied throughout the year and was lowest from October (49.79%) to December (56.98%). On the other hand, it was highest in the period before flowering (February, 63.74%) and during flowering (March, 64.14%). After flowering, the 1,8-cineole content decreased in May (55.61%) and June (54.01%) and then increased again until it reached a maximum in July (64.94%). 1,8-cineole performs essential ecological functions, such as repelling insects and deterring herbivores [50,51]. Considering that 1,8-cineole acts as a repellent, it is possible that the concentration of 1,8-cineole in the EO increases before flowering. After flowering, the concentration decreases and then increases again, perhaps due to the potential protection provided by the berries developing.

The seasonal variations in the ten most abundant compounds of bay laurel EO from Mljet Island are shown in Figure 2.

Perhaps the most interesting month is September, when the content of several compounds (α-pinene, sabinene, β-pinene, limonene, α-terpineol, α-terpinyl acetate, methyl eugenol) suddenly decreased sharply. On the other hand, the content of linalool and 1,8-cineole was increased. This indicates a possible environmental influence. After three months of high air temperatures, the average temperature in September was actually almost 5 degrees lower than in July and August. In addition, after two months with very little precipitation (5.1 mm in July and 3.5 mm in August), a larger amount of precipitation (43.1 mm) fell in September (Table 1). It is possible that this climatic factor also had an influence on the higher content of linalool and 1,8-cineole. Linalool is also interesting because of the significant differences between the months within the individual seasons. For example, the linalool content was relatively low in January (2.08%) and March (1.77%), while it was significantly higher in February (6.56%). A similar situation was observed a few months later, when the linalool content was higher in June (5.67%) and August (5.88%), while it was significantly lower in July (2.47%). The same variation was observed in the period between September and November (Table 2, Figure 2). A comparable study was presented by Di Leo Lira et al. [8], who analyzed EO from leaves collected in Argentina from February 2006 to May 2007. However, they did not take samples in every month of the one-year study period. The linalool content in Argentina varied between 9.7% (March 2006) and 15.9% (June 2006). Although there were seasonal variations, the linalool content was much more consistent [8].

PCAs were carried out for the ten most abundant compounds. Principal component (PC) 1 and PC 2 explained 69.48% of the variance and distinguished several clusters. The first cluster consists of samples collected in January, May, June and August (Figure 3a). All these samples are characterized by a higher content of limonene, α-pinene, β-pinene, sabinene and methyl eugenol. The sample from October differs slightly from the first cluster and is characterized by a higher content of terpinen-4-ol and metyleugenol. The second cluster consists of samples collected in February, March, April and July. These samples are characterized by a higher content of 1,8-cineole. The third cluster consists of samples from September, November and December and is characterized by a high content of linalool. The PCA loading plots of the volatile compounds from the first and second principal components are shown in Figure 3b.

Other authors have also observed seasonal variations in the qualitative characteristics of bay laurel EO. The study by Bahmanzadegan et al. [32] is somewhat different from other studies conducted on materials from the wider Mediterranean area and is worth discussing. In this study, the samples were collected in southern Iran (Shiraz) in all four seasons, and winter (January) was found to be the best harvesting season. The main compound in this study was 1,8-cineole, whose content was 5.7%, 37.5%, 20.3% and 42.6% in spring (May), summer (August), fall (November) and winter (January), respectively. Interestingly, the content of 1,8-cineole was more than seven times higher in winter than in spring [32]. In some other studies, the differences between the highest and lowest values were not as pronounced. Verdian-rizi and Hadjiakhoondi [27] analyzed leaf samples collected in northwestern Iran (Tabriz) during four different developmental stages of the plant. The 1,8-cineole content showed relatively little seasonal variation and amounted to 35.7% (May), 34.9% (August), 31.4% (September) and 35.7% (November). A comparison of these data with the results of Bahmanzadegan et al. [32], shows that only the maximum values of 1,8-cineole content are comparable. The minimum values are inexplicably low in the study by Bahmanzadegan et al. [32]. Seasonal variations in EO from southern Iran (Isfahan) were also investigated by Shokoohinia et al. [34]. In this study, the content of 1,8-cineole showed higher seasonal variations than in the study by Verdian-rizi and Hadjiakhoondi [27] but significantly lower variations than in the study by Bahmanzadegan et al. [32]. The 1,8-cineole content was 34.29% (March), 40.25% (June), 37.32% (September) and 30.80% (December) [34]. The slightly greater seasonal variation may be partly explained by the time of harvest, as the time of collection with the lowest 1,8-cineole content was later, in December, than in the study by Verdian-rizi and Hadjiakhoondi [27]. Another reason could be the different geographical origins of the samples examined. However, geographical origin as a cause of variability in bay laurel EO should be treated with caution, as most authors have found little or no variability between different geographical areas [28,35,36,37,50]. Some of these studies cover a relatively large area. For example, the oils from Tunisia, Algeria and Morocco showed quantitative rather than qualitative differences in their chemical composition [52].

In the samples from Tunisia analyzed by Marzouki et al. [28], the seasonal variations in the content of 1,8-cineole as the main compound were negligible. The 1,8-cineole content was 26.2% (seed production, October), 27.1% (dormancy, January), 29.7% (flowering, April) and 28.0% (vegetative activity, July) [28]. Seasonal variation was also observed in leaves collected at five different periods of the year in Portugal. Although the author did not specify in which periods of the year the leaves were collected, she reported that the l,8-cineole content varied between 40.00% and 48.50% [30].

Finally, Di Leo Lira et al. [8] analyzed EO from leaves collected in Argentina and found that the content of 1,8-cineole as the main compound ranged from 34.8% (March 2006) to 48.6% (May 2007). This was followed by linalool with a content of 9.7% (March 2006) to 15.9% (June 2006). Di Leo Lira et al. [8] found that although there is seasonal variability, it is not statistically significant. When making a comparison with samples from Argentina, it should be noted that March and May are the autumn and winter months, respectively, in the southern hemisphere. Aside from the study by Bahmanzadegan et al. [32], this is the only study that showed a higher content of 1,8-cineole in the winter, i.e., 46.8% in May 2006 and 48.6% in May 2007 [8]. Such results indicate the importance of investigating seasonal variation in each geographical area in order to determine the optimal period for leaf harvest.

The content of 1,8-cineole in our study was highest in the second part of winter (the only major similarity with the samples from Iran) and in early spring, i.e., from February to April. In summer, the content was lower but still high. It also reached its maximum in July. The lowest content of 1,8-cineole was found in the fall and early winter (from October to December). Our results, demonstrating the highest 1,8-cineole content in the vegetative period (July), are similar to most other studies. Similarly, the 1,8-cineole content in L. novocanariensis was 15.8% and 17.0% in spring and fall, respectively [49].

The phenylpropene derivatives eugenol, methyl eugenol and elemicin are responsible for the spicy aroma of bay laurel leaves and are important factors that determine their sensory quality [53]. In our study, the content of eugenol was highest in April (1.83%) and December (1.78%), while the content of methyl eugenol was highest in May (2.00%) and October (2.59%) (Table 2). It may therefore be advisable to collect the leaves in the months with a higher content of these compounds if they are to be used as a spice.

According to Bahmanzadegan et al. [32], the content of eugenol in southern Iran was highest in fall (6.9% in November), followed by winter (3.4% in January), summer (0.8% in August) and spring (0.1% in May). Shokoohinia et al. [34] also investigated the seasonal variation in southern Iran and found that the content of eugenol was highest in March (2.88%), followed by September (2.74%), June (2.25%) and December (2.03%). Samples from the northwest of Iran showed that the content of eugenol was 5.5% (September), 4.8% (May), 4.3% (November) and 3.8% (August) [27]. A study conducted in Pakistan showed relatively low seasonal variation in eugenol content, which was higher in spring (16.70% in March) than in summer (16.41% in July) and fall (15.16% in November) [29]. A study of seasonal variations in Tunisia revealed the highest content in October (5.0%). In January and April, the content was the same (3.6%), while it was the lowest in July [28]. It is also worth mentioning results from Argentina, where the highest eugenol content was found in April 2007 (1.0%), followed by March 2007 and August 2006 (0.9%). The lowest eugenol content was recorded in May 2007 (0.3%) [8]. We must not forget that March and May are the fall and winter months in Argentina, respectively. In general, fall seems to be the season with the highest eugenol content in most geographical areas.

The content of another compound, methyl eugenol, which is responsible for the spicy aroma of bay laurel leaves, should also be discussed. Three studies in Iran showed the following results. The first study showed that the methyl eugenol content was higher in fall (5.5%) and summer (3.3%) than in winter (2.7%) and spring (0.5%) [32]. According to the second study from southern Iran, the content was highest in March (5.18%), followed by June (5.05%), September (4.28%) and December (4.17%) [34]. The third study, using samples from the northwest of Iran, showed that the content of methyl eugenol was 9.4% (September), 8.1% (August), 7.9% (November) and 6.8% (May) [27]. Bay laurel from Pakistan showed little seasonal variation, as the methyl eugenol content was 2.48% (November), 2.46% (July) and 2.44% (March) [29]. A study on seasonal variation in Tunisia showed that the content of methyl eugenol was highest in October (15.8%), followed by April (14.3%), January (13.7%) and July (12.7%) [28]. In Argentina, the content of methyl eugenol ranged from 1.2 (March 2006) to 5.4% (March 2007) [8]. Of course, March is a fall month in Argentina. Interestingly, in this study, the minimum and maximum content were found in the same month (season) but in different years. This indicates an influence of some environmental factors on the production of methyl eugenol in these two years. A comparison of the seasonal variations in methyl eugenol with those of eugenol shows that the methyl eugenol content is less variable than eugenol in most studies. In our studies, the methyl eugenol content was significantly lower in the colder season, i.e., from November to April (with the exception of January), ranging from 0.05% in March to 0.55% in December and 1.46% in January. In the warmer months of the year, i.e., from April to October, it ranged from 1.32% in September to 2.59% in October (Table 2).

The content of elemicin, the third compound responsible for the spicy aroma of bay laurel, is very low and exhibits much greater variability than the content of eugenol and methyl eugenol. When it has been detected, its content has been found to vary throughout the year between traces and 0.7% in Iran [27,32] and from 0.4% to 0.5% in Tunisia [28]. In our study, elemicin was only detected in August (0.05%). Elemicin was not detected in bay laurel leaves from Argentina [8]. It is therefore difficult to draw conclusions about its seasonal variability. Based on the content of eugenol and methyl eugenol, the optimal season for harvesting leaves with spicy aroma in most geographical areas is autumn.

4. Conclusions

GC-MS analysis of the EO extracted from the leaves of female Laurus nobilis plants revealed that it consisted almost exclusively of monoterpenes, while phenylpropanoids, sesquiterpenes and other compounds were present in small amounts. The major volatile compound in the EO was 1,8-cineole, followed by α-terpinyl acetate, sabinene and linalool. Significant differences were found between the twelve samples collected every month for one year. The content of 1,8-cineole, known as an insect repellent and herbivore deterrent, was highest before and during flowering (March) as well as before and during fruit ripening (October, November). The content of phenylpropene derivatives (eugenol, methyl eugenol, elemicin), which are responsible for the spicy aroma of the leaves, was highest during flowering, immediately after flowering and at the time of fruit ripening. The collection time can therefore be adjusted according to the intended use of the leaves (spice, medicine or repellent) in order to obtain leaves rich with the desired EO compounds. Although the study was conducted in one specific year and in one specific geographic region, the seasonal variation of volatile compounds in bay laurel leaves was observed. Further studies on the seasonal variation of bay laurel EO should be continued and include multiple locations and a comparison between male and female plants of each population. Such studies will give more reliable conclusions about the seasonal variation in the content of volatile compounds in bay laurel leaves.