Chemical Composition of the Essential Oils in Portuguese Daucus Carota Subspecies

. This study focused on five different subspecies of Portuguese Daucus carota L., to identify the main compounds in the essential oils of umbels from each subspecies. The isolation of the essential oils was performed by hydrodistillation methods followed by further composition analysis by gas chromatography-mass spectrometry, ultimately demonstrating that each subspecies had different major compounds. This study concluded that the main compounds found in Daucus carota subsp. maritimus were geranyl acetate, followed by trans-methyl-isoeugenol, α-pinene, cis-asarone and elemicin. Daucus carota subsp. hispidus presented with the major compounds as geranyl acetate, caryophyllene oxide, trans-methyl-isoeugenol and sabinene. For Daucus carota subsp. maximus , the major compounds were cis-asarone, geranyl acetate and elemicin, which was similar to Daucus carota subsp. carota which presented with the major compounds as geranyl acetate, cis-asarone, trans-methyl-isoeugenol and α-pinene. Finally, the major components of Daucus carota subsp. sativus were carotol and daucol.


Introduction
Essential oils from aromatic plants are used for hundreds of years ago, as they are composed by volatile secondary metabolites that rapidly are absorbed through the skin and further provide notorious therapeutic benefits of high importance in Human life [1].
Essential oils are complex mixtures and researchers aim to know their composition, to allow practical application and understand which beneficial and adverse effects these oils might have [2]. The Apiaceae family is one of the most studied related to essential oils, not only for being one of the most consumed worldwide, but also because they

KnE Materials Science
FibEnTech21 are used as food, flavoring agents, medical purposes and for being nutraceutical plants [1,2]. One of the species of this family is Daucus carota, or as it is commonly called, carrot. D. carota is a biannual plant with its flowering seasons from June to August. The umbels appear white and round in full blossom. Before the domestication, the carrot root appeared white, but after intensive breeding, orange carrot became carotenoidrich existing still other root colours like purple [3]. This specie has some subspecies like Daucus carota supbsp. sativus and Daucus carota subsp. carota that are presented worldwide, being the first one the only cultivated and assumed to be originated by selective breeding of the ancestral wild form of the second one [4,5]. According with Heywood study in 1968, there were recognized eleven wild subspecies of Daucus carota. In previous studies, a great variety of compounds with different proportions were described in essential oils from umbels. In Tunisia, Daucus carota subsp. maritimus presented as major components elemicin, geranyl acetate, α-pinene, myrcene and cubenol [1]. Daucus carota subsp. hispidus, presented as major components αcadinol, trans-methyl-isoeugenol, premnaspirodiene and carotol [6]. As in the chemical analysis of Daucus carota subsp. maximus, in Italy, it appears that the major compounds are carotol and β-bisabolene, however, a previous Portuguese study presented as major components α-pinene, geranyl acetate, cis-asarone, trans-methyl-isoeugenol and elemicin [7,5]. For Daucus carota subsp. carota, a German study compared Polish, French and Moroccan essential oils and it has reported as main component in all of them carotol. The one collected in Poland had higher amounts of verbenol, caryophyllene oxide, α-pinene and trans-pinocarveol. French oil presented α-pinene, caryophyllene oxide and caryophyllene. Finally, Moroccan oil presented caryophyllene oxide and βbisabolene as major components [8]. Accordingly with the same Portuguese study α-pinene and geranyl acetate are the higher percentage components [5], which differs from the ones observed in Daucus carota subsp. sativus, in India, where the major components are carotol α-pinene, myrcene and limonene [9].

Plant Material
Plant material was provided and maintained in ex situ by the Banco Português de Germoplasma Vegetal (BPGV). Flowering and mature umbels were preserved at dry place and room temperature. DOI

Essential oil isolation
All the subspecies were submitted to hydrodistillation, where essential oils from the plant umbels were extracted. First 10 g of plant materials are packed in a distillation flask and then is added 200 mL of water and brought to boil and maintain for 2 h [10].
For this method, we used a Clevenger apparatus.

Essential oil analysis and chemical composition
Essential oils analysis and chemical composition were proceeded by GC-MS chromatography using an Agilent 7890A GC coupled with an Agilent 5975C inert XL mass selective detector. For the separation of the volatile compounds was used an DB-5 J&W GC capillary column (5% phenylmethylpolysiloxane, 30m length, 0,25mm diameter and 0,25μm film), being helium the mobile phase, at 1mL/min. The components identification were obtained by comparison of the retention times and mass spectra, which were also compared with the ones at the data system library NIST and Wiley.

Results and discussion
The yields obtained in our hydro-distillation extractions are present in table 1 and they are very small in all subspecies.
After the oil extraction, we proceeded to analyse the compounds present in our oils.
Many outcomes indicate these variations in the percentage of the major components depend on the location, if they are wild or cultivated and stage of flowering, and for that reason, some diversity was observed to other studies. As stated for Snene [6], due to its nutritional and therapeutic values, the essential oil combination from Daucus carota subspecies from different countries all over the world show considerable quantitative and qualitative variability [6]. It is also considerable as a possible variation reason the harvesting stage. The extraction method can also be one of the causes of variety in these components, as we can see in Madjdoub study, different percentage of the components were presented, depending on the extraction method used [1].
The percentage of some of the most prominent compounds and its distribution per each subspecies are present as visual representation in Figure 1 and in Figure 2 their structures.

Conclusions
Essential oils obtained by hydrodistillation of umbels from five subspecies of Portuguese Daucus carota L. were analysed by gas chromatography-mass spectrometry (GC-MS).
A total of 38 compounds were identified. For three subspecies (maritimus; hispidus and carota), high levels of geranyl acetate are found. Daucus carota subsp. maximus presented cis-asarone as main compound but geranyl acetate is the second main component and elemicin the third. Daucus carota subsp. sativus has more than 50% of carotol and daucol as second main compound. Knowing the amount of geranyl acetate present in these oils, and considering the therapeutic value of monoterpenes like it, we could use these oils due to its pharmacological properties, in cases off inflammation, pain-related and antioxidant effects [11]. Previous studies also demonstrated that both carotol and daucol present high antifungal activity, probably due to its polar groups, which can be a good indicator that Daucus carota subsp. sativus essential oil might be a good antifungal oil [12].