Chemical Composition of the Essential Oils of Three Ocimum basilicum L . Cultivars from Serbia

Basil essential oil (BEO) contains a wide range of chemical compounds whose content may vary depending on chemotypes, environmental conditions, agronomic techniques and particularly the origin of the plant. In our present study, essential oils (EOs) were isolated by hydrodistillation method from dry herbs of three basil cultivars and analyzed by GC-FID and GC-MS. Two of the tested cultivars belong to sweet basil group (B1 and B2) while the third one was large leafed ʽGenovese’ basil (B3). EO content in the dry herb was 0.65%, 0.41% and 0.62% respectively. The main classes of compounds of B1EO and B3EO were sesquiterpene hydrocarbons (38.39% and 37.95%), oxygenated monoterpenes (25.44% and 28.04%) and phenylpropanoids (17.43% and 15.71%). The main constituents of both EOs were monoterpene alcohol linalool (13.68% and 15.38%), phenoyl derivate eugenol (10.83% and 8.97%) and sesquiterpene hydrocarbon α-bergamotene (8.12% and 9.25%). In both EOs, epi-bicyclosesquiphellandrene was detected in considerable amount (7.03% and 8.07%). The most abundant compound classes in B2EO were oxygenated monoterpenes (52.07%), sesquiterpene hydrocarbons (24.27%) and phenylpropanoids (10.95%). Linalool was the dominant compound (40.97%), followed by epi-bicyclosesquiphellandrene (8.70%) and methyl chavicol (7.92%). The results showed complex chemical composition of BEOs and pointed out the presence of biologically active compounds of importance for different branches of the pharmaceutical, chemical and food industry. Although there are differences in the chemical composition of the BEOs, the obtained results show that all of the tested cultivars are rich in compounds which are responsible for biological activities.


Introduction
The family Lamiaceae is widely distributed over the world.It comprises over 5,000 medicinal and aromatic plant species whose EOs have multiple applications (Sakkas and Papadopoulou, 2017;Piras et al., 2018).Basil (Ocimum basilicum L.) is a widely known member of Lamiaceae family.At present, this annual aromatic plant, native to Southeast Asia, is globally cultivated and has significant economic value (Varga et al., 2017).Basil has been grown traditionally as a decorative, medicinal, seasoning and ritual herb (Jelačić et al., 2011).Additionally, basil is mostly cultivated for its EO, which has broad pharmaceuticals and industrial uses (Shiwakoti et al., 2017).BEO has been used in the food industry, especially in vegetables, meat and dairy products.It has a been used as a flavouring as well as a natural agent for increasing the shelf life of food products (Riveros et al., 2015;Sharafati-Chaleshtori et al., 2015;Piras et al., 2018).It is also used in commercial fragrances, oral care products and aromatherapy (Labra et al., 2004).
The content and chemical composition of the BEO has been the subject of many studies.The yield from different plant parts varies between 0.2-1.9% with the main components being linalool, methyl chavicol, eugenol and methyl cinnamate, as well as 1,8-cineole, methyl eugenol, geraniol, geranial, neral and α-bergamotene (Marotti et al., 1996;Labra et al., 2004;Sakkas and Papadopoulou, 2017).Based on the distribution and abundances of the main compounds in EO composition, Marotti et al. (1996) described three chemotypes: (1) linalool, (2) linalool / methyl chavicol and (3) linalool/eugenol chemotypes.In recent study which comprised 85 accessions of O. basilicum, based on seven major compounds, Varga et al. (2017) Essential oil analysis Qualitative and quantitative analysis was carried out using GC-FID and GC-MS.For GC-MS analyses, an Agilent 7890N gas chromatograph with a HP5-MS capillary column (30 m length; 0.25 mm inner diameter; 0.25 μm film) was used.The following temperature program was employed: 60 ºC for 0 min; then 3 ºC min -1 to 280 ºC and then held for 20 min.Helium was used as the carrier gas with the flow rate 1 mL min -1 .The GC was coupled to a Hewlett-Packard 5972 MSD operated at 70 eV and scanning masses in the 40-550 range.The peaks were identified by comparison of their retention indices (calculated relative to n-alkanes) to the literature data (Adams, 2007;Babushok et al., 2011), and by comparison of their mass spectra to the mass spectra in the databases (NIST/EPA/NIH mass spectral library NIST2000, Wiley/NBS registry of mass spectral data, 7th ed., electronic versions).
For GC-FID analyses, Agilent 4890A gas chromatograph with a HP5-MS capillary column (30 m length; 0.25 mm inner diameter; 0.25 μm film) was used.The temperature program employed was the same as the one used for GC-MS analyses.Hydrogen was used as carrier gas (1 mL min -1 ).The GC was coupled to a FID detector operating at 300 ºC.

Statistical analysis
The analysis of variance (ANOVA) was carried out using a significance level of p ≤ 0.05.The least significant difference (LSD) test, if necessary, was used to determine the significant differences between tested basil cultivars.

Results
The yield of BEOs All cultivars analyzed in the present research yielded light yellow EOs.The contents of B1EO, B2EO and B3EO were 0.65% (0.65 mL EO/100 g of dry plant material), 0.41% and 0.62% respectively.Comparing the obtained yields of BEO using LSD test, it can be concluded that there is a statistically significant difference between the observed cultivars in their content of the EOs.
Content of EO in basil was investigated by different authors.Jelačić et al. (2011) found that the yield of EO from aerial parts of 10 tested basil populations varied from 0.87% to 1.84%.Božin et al. (2006) and Elgndi et al. (2017) reported the yield of BEO from Serbia to be 0.37% and 0.67%, respectively.Beatović et al. (2015) reported the yield of EO from twelve Ocimum species varied from 0.65% to 1.90%.Literature data of Romanian basil cultivars show the EO level from 0.2% to 0.6% (Benedec et al., 2009).The yield of obtained BEO in Republika Srpska (Bosnia and Herzegovina) was 0.4% (Stanojević et al., 2017).A study by Wesołowska et al. (2012) showed that the content of EO in herbs of three tested basil cultivars ranged from 0.38% to 0.55%.Content of the EO of basil cultivars from Albania was ranged from 0.11% to 3.40%, while content of the EO obtained from aerial parts of 10 Italian basil cultivars was in the range from 0.3% to 0.8% (Marotti et al., 1996;Cheliku et al., 2015).
It can be concluded that the content of BEO proposed intraspecific characterization into five chemotypes: (A) linalool, (B) linalool/trans-α-bergamotene, (C) linalool/methyl chavicol, (D) linalool/trans-methyl cinnamate and (E) methyl chavicol.Chemotypes A and C can be considered to be European chemotypes.Chemotype D is a Tropical chemotype, while Chemotype E is described as a Reunion chemotype (Varga et al., 2017).The composition of BEO is highly dependent on the chemotype, phenological stage of the plant as well as on other factors (growing and agroclimatic conditions, drying and distillation method) and thus also affects the biological activites (Marotti et al., 1996;Klimánková et al., 2008;Taie et al., 2010;Wesolowska et al., 2012;Shiwakoti et al., 2017).
The content and composition of EO are very important parameters for assessing the quality of basil and its application as raw material for different branches of pharmaceutical, food and chemical industries (Jelačić et al., 2011).Therefore, the aim of the present study was to investigate the chemical constituents of the BEOs from three cultivars planted in the Republic of Serbia.

Plant material
Dried basil herb B1 was obtained from "Bilje Borča" Belgrade while B2 material was obtained from Institute for Medicinal Plant Research "Dr Josif Pančić" Belgrade in 2016.The materials were identified and representative herbarium specimens were deposited at "Bilje Borča" (serial No. L018-05-15) and the Institute for Medicinal Plant Research "Dr Josif Pančić" (serial No. 02740216).The B3 material was ʽGenovese' basil from the collection of the Institute for Crop Sciences of the Faculty of Agriculture in Belgrade and the Plant Gene Bank of Serbia where it is deposited under DB code (DB-01).

Isolation of the essential oils
The plant material was milled in a laboratory electric mill immediately before extraction and 20 g was subjected to hydrodistillation for 2 h using a Clevenger type apparatus according to the procedure IV of the Yugoslavian Pharmacopoeia (1984).The EOs were dried over anhydrous sodium sulphate and stored in sealed vials at 4 ºC until further analysis.The yield of EO was calculated based on dry weight of plant material and expressed as % (mL EO/100g of dry plant material).determined in our present research is in good agreement with the literature data.

Chemical composition of BEOs
The chemical composition of examined BEOs is presented in Table 1.Overall, 60 volatile constituents in the three BEOs were identified: 38 in B1EO (94.99% of the total oil), 42 in B2EO (90.85% of the total oil) and 30 compounds in B3EO (95.45% of the total oil).Statistical analysis of the results obtained by GC-MS analysis was conducted for all constituents with the content higher than 1% of the EO.(Adams, 2007;Babushok et al., 2011;NIST database, electronic version).RI b -Relative retention indices experimentally determined and calculated against n-alkanes (C8-C32) on the HP-5MS column.
Based on statistical analysis of the selected constituents, it can be concluded that there were no statistically significant differences between the examined BEOs.
As B1EO and B3EO showed similarity in chemical composition, the major difference between these BEOs and B2EO was in the content of eugenol.B2EO had the lowest content of this phenoyl derivate (0.62%) in which the two other cultivars were rich (10.83% and 8.97%).Politeo et al. (2007) found that the antioxidant activity of BEO highly depends on the content of this compound.Accordingly, it can be expected that EOs from these two basil cultivars will be more effective antioxidants compared to B2EO.Furthermore, B1EO and B3EO had higher concentration of eucalyptol (5.98% and 6.20%) relative to B2EO (2.12%).Monoterpene alcohol geraniol was present in considerable amount in B2EO (4.80%) while in two other EOs it was in trace.D-germacrene and α-muurolene were sesquiterpene hydrocarbons identified only in B2EO while β-cubebene was identified in significant amount in B1EO and it was absent in EOs of other investigated cultivars.
The differences in the chemical composition of the EOs analyzed can be explained as a consequence of differences in growing and agroclimatic conditions.
Many studies reported linalool as the main compound of investigated BEOs (Marotti et al., 1996;Juliani and Simon, 2002;Wesołowska et al., 2012;Cheliku et al., 2015;Riveros et al., 2015;Silva et al., 2015;Elgndi et al., 2017).In study of Beatović et al. (2015), which included twelve basil cultivars, the oxigenated monoterpenes were predominant in eight cultivars with linalool as the main constituent.Shirazi et al. (2014) and Sharafati-Chaleshtori et al. (2015) reported methyl chavicol as the main compound of BEO while eugenol was detected as compound with highest abundance by Piras et al. (2018).Presence of the sesquiterpene hydrocarbon epi-bicyclosesquiephellandrene as a compound with higher abundance is comparable with investigation of Benedec et al. (2009).The chemical composition of BEO 'Genovese' has been well described in the literature and the composition and abudances of major components in our study is in accordance with earlier studies (Marotti et al., 1996;Juliani and Simon, 2002;Labra et al., 2004;Carović-Stanko et al., 2010;Beatović et al., 2013;Stefan et al., 2013).
It can be concluded that the compositions of the BEOs investigated in our present research are in good agreement with the literature data.

Conclusions
In this study, the chemical composition of the EOs from three basil cultivars from Serbia was investigated.The contents of BEOs were in good agreement with the literature data.The statistical analysis demonstrated a statistically significant difference between the observed cultivars in their contents of the EOs.
It was concluded that the compositions of the BEOs investigated were in good agreement with the literature data.Some differences in abundances and distribution of their constituents were observed.However, the statistical analysis of the selected constituents did not reveal any statistically significant differences between the examined BEOs.The differences in the chemical composition of the EOs analyzed were explained as a consequence of differences in growing and agroclimatic conditions.Although there were differences between the tested BEOs, the obtained results showed that all of them were rich in compounds which are responsible for biological activities.Further studies of examined cultivars are required to determine their biological activities and applicability as food additives.

Table 1 .
Chemical composition of Ocimum basilicum L. essential oils from three tested cultivars 1 '-' compound not detected.RI a -Relative retention indices on a column with dimethylsilicone stationary phase with 5% phenyl groups reported in literature

Table 2 .
The relative abundances of the main classes of compounds in tested BEOs