Volatile Organic Compounds of the Glandular Trichomes of Ocimum basilicum and Artifacts during the Distillation of the Leaves

: Focusing on volatile organic compounds (VOC) of Ocimum basilicum , this study aims to determine the chemical composition of VOC in secretory trichomes and compare it with that of essential oil obtained by hydrodistillation of leaves. The technique of extracting the content of glandular trichomes refers to the microneedle shuttle analysis. Hydrodistillation of fresh leaves was done with a Clevenger distiller (EO). The chemical compositions were determined by GC/FID and GC/MS. The head of the capitate trichomes does not contain volatile compounds. Fifty volatile compounds were detected in the EO, and twenty-four volatile compounds were detected in the VOC; the main components were eugenol (from 15.47% ± 1.05% to 41.89% ± 2.83%) and linalool (from 32.05% ± 2.57% to 28.99% ± 2.32%), respectively. During the distillation of the basil leaves 26 artifacts are formed. The composition of the essential oil of O. basilicum therefore depends not only on the plant but also on the method used to obtain it.


Introduction
Ocimum basilicum L. (sweet basil) belonging to the family Lamiaceae, the genus includes about 35 species of aromatic annual and perennial herbs and shrubs [1]. O. basilicum is a multipurpose herb characterized by its rich and aromatic essential oil content [2,3]. The aromatic leaves, flowers, and seeds are added to foods and beverages for flavor; extracted as active ingredient for use in perfumes, soaps, cosmetics, and dental products; they are included in traditional herbal medicines to treat fevers, headaches, kidney problems, gum ulcers, childbirths, rheumatoid arthritis, and menstrual irregularities [4][5][6]. Beside these traditional medical uses, recent scientific studies have demonstrated potent antioxidant [7], antiviral [8], and anti-proliferative activities [9] of some compounds occurring in O. basilicum leaf essential oil and extract [10].
Basil oil presents remarkable differences in composition, and some chemotypes from different geographical origins have been reported [1,2]. O. basilicum is rich in essential oils and have been the subject of numerous chemical studies [11,12]. Four major essential oil chemotypes in O. basilicum were recognized, each with a number of small variants: (1) methyl chavicol-rich; (2) linalool-rich; (3) methyl eugenol-rich; and (4) methyl cinnamaterich [13].
In basil, as in the other species of the Lamiaceae family, essential oil is stored in glandular trichomes [14]. Although the essential oil composition of a great number of Lamiaceae species is well known, little information is available on the secretion products of the various types of trichomes. Most of previous works concerned the morphology and structure of the glands and involved microscopical observations [15][16][17][18][19]. Only in a few cases the morphological study was accompanied and supported by chemical analyses of secreted oils: in Mentha piperita [20,21], Salvia officinalis [22][23][24], Thymus vulgaris [25], Rosmarinus officinalis [26,27], and Ocimum basilicum [28].
O. basilicum bears capitate and peltate glandular trichomes, distributed on both sides of the leaf. The subcuticular space of peltate trichomes appeared intensely colored with lipophilic stain, thus indicating the presence of VOC [28].
Volatile plant constituents have different ecological functions. They play an important role in the process of plant growth, such as plant-plant competition and cooperative co-evolution, in the attraction of pollinators, in defense against insects and against the attraction of herbivores [33][34][35].
In recent years, micro-extraction methods for determining the composition of VOCs from plant material have developed considerably. These techniques have high sensitivity, and can be applied to matrices of gas, liquid, and solid samples.
Furthermore, the absorption of the fibers depends on the chemical class of the products present in the matrix [36][37][38].
The aim of this work is to compare the VOCs content directly sampled in capitate and peltate trichomes with the composition of the VOCs determined by hydrodistillation (essential oil) of the leaves of O. basilicum. It has been chosen to take the contents of the VOCs from the glandular hairs with the method of microneedle shuttle analysis since the possibility of forming artifacts during the collection is minimal.

Plant Materials
Commercial plants of O. basilicum var. Italia were bought at flower market and cultivated in pots at the Botanical Garden of Urbino. Fresh mature leaves were collected, before blossom, for chemical analyses. Voucher specimens were deposited in the Herbarium Urbinate (Botany Institute, Urbino University), under the acquisition number OB2234/20.

Sampling from Capitate and Peltate Trichomes
The trichome secretion was sampled by perforating with microneedles peltate and capitate trichomes as reported before [41]; the secretion was accumulated in microvials containing n-hexane and stored in sealed vials under refrigeration prior to analysis.
Samples were taken from three plants by sampling about 500 trichomes of both types per plant.

Isolation of the Essential Oil
Fresh leaves were subjected to hydrodistillation using a Clevenger-type apparatus for 4-h yielding 0.5% ± 0.1% of a yellowish oil. The oils were dried over anhydrous sodium sulfate and stored in sealed vials under refrigeration prior to analysis.
The same three plants sampled with microneedles were hydrodistilled separately.

GC-FID e GC-MS Analysis
GC-FID analysis of the volatile components was carried out using an Agilent 4890D instrument coupled to an ionization flame detector (FID). Compounds were separated on a HP-5 capillary column (5% phenylmethylpolysiloxane, 25 m × 0.32 mm i.d.; 0.17 mm film thickness; J&W Scientific, Folsom, CA, USA), working with the following temperature program: 5 min at 60 • C, rising at 4 • C/min to 220 • C, then at 11 • C/min to 280 • C, then held for 15 min; injector and detector temperatures, 280 • C; carrier gas, helium (1.4 mL/min); injection volume, 1 µL; split ratio, 1:34. A mixture of aliphatic hydrocarbons (C8-C30; Sigma, Milan, Italy) in hexane was directly injected into the GC injector under the above temperature program in order to calculate the retention indices of each compound.
GC-MS analysis was performed using an Agilent 6890N gas chromatograph coupled to a 5973N mass spectrometer equipped with a HP-5MS capillary column (5% phenylmethylpolysiloxane, 30 m × 0.25 mm i.d., 0.1 mm film thickness; J&W Scientific). The same was programmed at 60 • C for 5 min, rising at 4 • C/min to 220 • C, then at 11 • C/min to 280 • C, then held for 15 min, and finally at 11 • C/min to 300 • C and held for 5 min; carrier gas, helium; flow rate, 1.0 mL/min; injector and transfer line temperatures, 280 • C; injection volume, 2 µL; split ratio, 1:50; scan time, 75 min; acquisition mass range, 29-400 m/z. All mass spectra were acquired in electron-impact (EI) mode with an ionization voltage of 70 eV.
The identification of volatile components was based on computer matching with the WILEY 275, NIST 05 and ADAMS libraries. A home-made library was used as well. Whenever possible, components were identified by comparing the retention times and mass spectra with those of authentic compounds using the program MSD Chemstation G1701 EA (Agilent).

Results
The head of the capitate trichomes does not contain volatile compounds and this in accordance with the numerous histochemical observations made on this type of trichomes [25,26].
The observed composition of the volatile compounds of O. basilicum hydrodistilled leaf oils (LEO) is presented in Table 1 and a chromatogram is shown in Figure 1. The compounds are listed in order of elution on the HP5 column. Fifty-nine volatile compounds were detected in the LEO and 50 were identified with peak weight percentages of 99.81 ± 0.11% (mean ± S.E.).
In addition, amounts of allylbenzenes such as eugenol (15.47 ± 1.05%) and oxygenated sesquiterpenes such as epi-alpha-cadinol (9.97 ± 0.85%) were detected in LEO, along with nine monoterpenes, one ketone, and one alcohol. The ketone and the alcohol were present at trace levels. The composition of LEO was compared with that of trichomes peltate heads (VOC).  Table 1.
The compounds are listed in order of elution on the HP5 column. Fifty-nine volatile compounds were detected in the LEO and 50 were identified with peak weight percentages of 99.81 ± 0.11% (mean ± S.E.).  Table 1.
The observed composition of VOC is presented in Table 1 and a chromatogram is shown in Figure 1.
Twenty-four volatile compounds were detected in the VOC and all were identified with peak weight percentages of 100 ± 0.01%.
Similarly, sesquiterpenes are about twice as abundant in LEO as in VOC.
During the distillation of the basil leaves 26 compounds are formed that are not present in the heads of the peltate trichomes. These compounds are present in modest quantities from 0.01% to 0.27% for monoterpenes, from 0.03 to 0.76 for oxygenated monoterpenes. Bornyl acetate is formed in appreciable quantities (2.77% ± 0.22%) and is the most abundant neo-formed compound.

Discussion
Hydrodistillation at atmospheric pressure is the most frequently used method of essential oils isolation. The advantage with respect to other isolation methods, e.g., extraction by organic solvents and supercritical CO 2 , is that isolates by hydrodistillation do not include non-volatile compounds. The main disadvantage is the formation of artefacts, thermal degradation reactions and hydrolysis, especially for aromatic plants that contain unstable volatile compounds as the main constituents of their essential oils. During hydrodistillation, water as polar solvent accelerates many reactions, especially reactions via carbocations as intermediates. The pH can fall as low as 2.8 during such extraction of the oil [42].
Many of the trace components of essential oils that are detected could well arise during the isolation procedures. Such oils are steam distilled under conditions where organic acids can be liberated from the plant material, and cyclizations of aldehydes and other monoterpenes may occur [43].
The action of phosphatases must be inhibited. The latter cleave phosphate esters to give the free alcohols characteristic of isolated plant oils. The distribution of terpene alcohols between free and esterified forms in vivo is not known in any particular oil, but considerable amounts of the latter and other bonded forms (e.g., glucosides and esters) are probably present [44].
Oils that have been obtained from plant material which has been gathered and stored may also contain products of photolysis, oxidation, and other chemical modification. Such contaminants are often extremely important as regards odor and flavor for commercial use [45].
The distilled oil of Citrus deliciosa Tenore var. Caí was characterized by aromatic nuances making the oil less appreciated (inferior quality) than the cold-pressed oil. Probably as consequence of artifacts formation during the distillation process [46].
These compounds are present in very low concentrations from 0.01 to 0.76 except for bornyl acetate which was formed in significant quantities (2.77% ± 0.22%).
Some of these compounds have already been reported in the literature as artifacts produced during distillation or during the storage of essential oil.
During the distillation of Abies x arnoldiana Nitz., and A. veitchii Lindl. various artifacts, including a derivative of borneol, were formed [49].
Moreover, during the distillation time, acid-catalyzed hydrolysis of bornyl acetate takes place. This hydrolysis only takes place to a small extent during distillation. On the basis of hydrodiffusion, the oxygenated compounds are available for distillation much faster than the hydrocarbons. Thus, bornyl acetate is in contact with the acidic medium for a relatively short period, whereby extreme hydrolysis of this compound is prevented [49].
Distilled EO generally increase their proportion in oxygenated monoterpenes (alphaterpineol, 4-terpineol and sabinene hydrates) that could originate as a result of the process (artifacts).
Structural rearrangements of limonene, sabinene, gamma-terpinene, and beta-pinene influenced by heat and oxidation would lead to p-cymene and related compounds of the p-menthadiene skeleton, such as terpinolene and beta-phellandrene [46].
The bicyclogermacrene could be partially transformed to spathulenol. A mechanism for the transformation of bicyclogermacrene into spathulenol seems to be a possible reaction sequence which may explain the transformation by autoxidation [50][51][52].
Changes in 44 compounds of Citrus junos Sieb. ex Tanaka steam-distilled peel oil and possible artifacts that accrue during storage at 25 • C were investigated by Kashiwagi, et al. Total monoterpene hydrocarbons decreased markedly, with major losses of limonene and gamma-terpinene and notable increases in p-cymene, as well as alcohols [52].
The increase in the relative percentages of the sesquiterpene alcohols could to some extent be attributed to possible reactions with the germacrene hydrocarbons. It is suggested that the possible formation of the tricyclic sesquiterpene alcohols, globulol, viridiflorol, and spathulenol, as artifacts in the oil could be attributed to oxidation of the germacrene type hydrocarbons, aromadendrene, allo-aromadendrene, and bicyclogermacrene to tricyclic sesquiterpenes [50].

Conclusions
Compounds biosynthesized from the plant can be identified with the shuttle analysis that is with the withdrawal of the secretion directly from the head of the secretor trichomes by means of microneedles followed by GC/MS analysis. As reported by numerous works, during hydrodistillation new compounds are formed (artifacts) and in the case of hydrodistillation of the leaves of O. basilicum have formed 26 new compounds. Some of these artifacts were not reported in the literature. Some compounds reported as artifacts may also be present as primary compounds. Further studies are needed to explain the kinetics of artifact formation from compounds really biosynthesized by the plant.