Essential oil composition of five Basil cultivars (Ocimum basilicum) from Albania

Basil (Ocimum basilicum L., fam. Lamiaceae) is an important medicinal and aromatic plant with very wide range of uses. This paper presents qualitative and quantitative analyses of essential oils obtained from five types of Italian basil cultivars: Napolitan, Red basil, Fino Verde, Limonez, and Genoveze culltivated in Albania and collected during the summer 2012. The hydrodistilled BEO (Basil essential oil) content ranged from 0.11% to 3.40%. Within the total of 65 identified compounds with GC/FID/MS, nine were considered as predominant (1,8-cineole, linalool, cis-thujone, methyl chavicol, eugenol, trans-(E)-caryophyllene, trans-(α)-bergamotene, germacrene D, and epi-α-cadinol) representing 49.20 to 85.43% of the components in the analysed essential oils. In all cultivars, linalool was detected as the most abundant component (36.20-46.59%). BEO’s from all five cultivars differ in their chemical composition but generally conform to EO’s from Sweet Basil grown in the Medditeranean region. The Napolitan cultivar showed the largest similarity to Sweet Basil, not only for the morphology, also due to the essential oil composition that comprises Basil’s most dominant chemical components (linalool, methyl chavicol and eucalyptol) in particular percents.


Introduction
Common basil (Ocimum basilicum L.), a member of the Lamiaceae family is an annual herb which grows in several regions around the world. Among more than 150 species of the genus Ocimum, basil is the major crop which is cultivated commercially in many countries (Sajjadi, 2006). Different authors have grouped basil into a subgenus based on its chemical composition or morphol-ogy (Imeri et al., 2014). There are many cultivars of basil which vary in their leaf color (green or purple), flower color (white, red, purple) and aroma (Morales and Simon, 1996), but most of commercial basil cultivars available in the market belong to the species O. basilicum. Acoording to Darrah (1980), these cultivars are classified in seven types: (1) tall slender types, which include the sweet basil group; (2) large-leafed, robust types, including 'Lettuce Leaf' also called 'Italian' basil; (3) dwarf types, which are short and small leafed, such as 'Bush' basil; (4) compact types, also described, O. basilicum var. thyrsiflora, commonly called 'Thai' basil; (5) purpurascens, the purple-colored basil types with traditional sweet basil flavor; (6) purple types such as 'Dark Opal', a possible hybrid between O. basilicum and O. forskolei, whith lobed-leaves and a sweet basil plus clove-like aroma; and (7) citriodorum types, which include lemon-flavored basils. The most popular and commonly cultivated is Sweet Basil (Genovese, Large leaf, Lettuce Leaf, Mammoth).
Traditionally, basil has been extensively utilized in food, as a flavoring agent, as well as in perfumery and pharmaceutical or cosmetic industries (Telci et al., 2006). In folk medicine, leaves and flowering tops of the plant are perceived as carminatives, galactogogues, stomachics and antispasmodics (Sajjadi, 2006). However, recently the potential uses of basil essential oil (BEO), particularly as antimicrobial and antioxidant agents have also been investigated (Politeo et al., 2007;Sartoratotto et al., 2004;Wannissorn et al., 2005). The O. basilicum essential oils exhibited a wide and varying array of chemical compounds, depending on variations in chemotypes, leaf and flower colors, as well as aroma and origin of the plants (Sajjadi, 2006).
However, only a few articles have examined the yield and composition of essential oils of Basil cultivars, excpecialy from Albania (Cheliku et al., 2015;Keita et. al., 2000;Morales and Simon, 1996), therefore the aim of this study was chemical characterization of essential oils of Napolitan, Red basil, Fino Verde, Limonez, and Genoveze cultivars planted in Albania.

Plant collection
The over-ground parts of the flowering plants (20-25 cm from the top) were obtained from five types of Italian basil cultivars: Napolitan, Red basil, Fino Verde, Limonez, and Genoveze cultivated and collected in Albania, during the summer 2012. The plant material was air dried, packed in paper bags and kept in a dark and cool place until analysis. Voucher specimens were deposited at the Institute of Pharmacognosy, Faculty of Pharmacy, Skopje (Napolitan -Ob/Al-N/12, Red basil -Ob/Al-RB/12, Fino Verde Ob/ Al-F/12, Limonez -Ob/Al-L/12, and Genoveze -Ob/Al-G/12).

Essential oil isolation
Essential oil isolation from basil was performed by hydrodistillation in all-glass Clevenger apparatus for 2.5 h, according to the method described in the European Pharmacopoeia (Ph. Eu. 8.0., 2014).

Analysis of essential oil's chemical composition
EO samples in hexane (1:1000) were analyzed on Agilent 7890А Gas Chromatography system equipped with FID detector and Agilent 5975C mass spectrometer. For this purpose, HP-5 ms capillary column (30 m x 0.25 mm, film thickness 0.25 μm) was used. Adam's analytical conditions were as follows: oven temperature at 60 °C (0 min), 3 °C/min to 240 °C (1 min) and at the end increased to 280 °C at a rate of 10 °C/min (1 min) (Adams, 2007); helium, as carrier gas, at a flow rate of 1 ml/min; injector temperature 220 °C and that of the FID detector 270 °C. One ml of each sample was injected at a split ratio of 1:1. The mass spectrometry conditions were: ionization voltage 70 eV, ion source temperature 230 °C, transfer line temperature 280 °C and mass range from 50 to 550 Da. The MS was operated in scan mode.

Identification of the components
The components were identified according to the aveliable literature considering their retention times (Adams, 2007), as well as Kovat's (retention) indices determined using a homologous mixture of normal alkanes (C9 -C25) analysed under Automated Mass Spectral Deconvolution and Identification System (AMDIS) conditions (AMDIS ver.2.1.).
Confirmation was done by comparing the mass spectra of components present in the BEOs with the reference spectra obtained from Nist, Wiley and Adams' mass spectra libraries. Quantification of the BEOs components was performed using the normalisation method of the GC/FID peak areas without any correction factors.

Essential oil yield and composition
The BEO yield of five basil cultivars from Albania ranged from 1.10 ml/kg to 34.00 ml/kg (0.11-3.40 %) (Table 1). The highest yield was recorded in one Fino Verde cutivar (No.1) while the lowest was found in one Napolitan cultivar (No.2).
Regarding gas chromatography analyses, it is evident that the examined five basil cultivars had variances in the chemical composition of the essential oil (Fig. 1). The Napolitan cultivars often known as wide leaf cultivars, were reach in methyl chavicol (23.70-33.05%), which is second abundand compound in the BEO thus it differs from all the other investigated cultivars. Red or purple basil is the only cultivar where trans-thujone (up to 6.56%) and trans-(α)bergamotene (up to 12.58%) were identified in significant amount. On the other hand, trans-(E)-caryophyllene was present in both, the Red basil and Limones cultivars. Also, characteristic for the Limonese cultivars was α-bisabolene, which was absent in the other types of basil. In the composition of the BEO's of Fino Verde and Genovese samples, eugenol was found in significant amount (5.37-13.73%) but was absent from the other examined cultivars.
High contents of linalool and methyl chavicol in Napolitan basil indicated that this cultivar could be considered as European originated chemotype with some influences of the North African (the Egyptian) chemotypes wich are with high methyl chavicol contents (Telci et al., 2005). Alike, geraniol and methyl chavicol were the main components in the oil of O. basilicum L. cv. purple from Turkey and Iran (Özcan and Chalchat, 2002;Sajjadi, 2006). Although the EO's of some Turkish and Bulgarian green basil were characterized by a high content of citral (neral and geranial) (46.10%) and methyl cinnamate (Sajjadi, 2006), these were barely detected in our BEO's.
BEO's from all five investigated cultivars differ in their chemical composition but generally conform to EO's from Sweet Basil grown in the Medditeranean region. The Napolitan cultivar showed the largest similarity to Sweet Basil, not only for the morphology, also due to the essential oil composition that comprises Basil's most dominant chemical components (linalool, methyl chavicol and eucalyptol) in particular percents.