Identification of the Volatile Components of Galium verum L. and Cruciata leavipes Opiz from the Western Italian Alps

The chemical composition of the volatile fraction from Galium verum L. (leaves and flowers) and Cruciata laevipes Opiz (whole plant), Rubiaceae, was investigated. Samples from these two plant species were collected at full bloom in Val di Susa (Western Alps, Turin, Italy), distilled in a Clevenger-type apparatus, and analyzed by GC/FID and GC/MS. A total of more than 70 compounds were identified, making up 92%–98% of the total oil. Chemical investigation of their essential oils indicated a quite different composition between G. verum and C. laevipes, both in terms of the major constituents and the dominant chemical classes of the specialized metabolites. The most abundant compounds identified in the essential oils from G. verum were 2-methylbenzaldheyde (26.27%, corresponding to 11.59 μg/g of fresh plant material) in the leaves and germacrene D (27.70%; 61.63 μg/g) in the flowers. C. laevipes essential oils were instead characterized by two sesquiterpenes, namely β-caryophyllene (19.90%; 15.68 μg/g) and trans-muurola-4(15),5-diene (7.60%; 5.99 μg/g); two phenylpropanoids, benzyl alcohol (8.30%; 6.71 μg/g), and phenylacetaldehyde (7.74%; 6.26 μg/g); and the green-leaf alcohol cis-3-hexen-1-ol (9.69%; 7.84 μg/g). The ecological significance of the presence of such compounds is discussed.


Results
Volatiles identified in the aerial parts of G. verum and C. laevipes are reported in Table 1, listed in order of elution on a DB-5 column. Leaves and flowers from G. verum were analyzed separately, while, due to their small size, flowers from C. laevipes could not be isolated and for this species the whole plant was analyzed.
In total, more than 70 compounds were identified in both species, on average amounting to 92%-98% of the total. Chemical investigations of their essential oils indicated a quite different composition between G. verum and C. laevipes, both in terms of major constituents and the dominant chemical classes of the specialized metabolites (Table 1; Figure 1). The chemical structures of the most abundant detected compounds are reported in Figure 2.
The sesquiterpene germacrene D was the main metabolite (27.70 ± 1.67%, 61.63 ± 2.87 µg/g) identified in the flowers from essential oils of the same species.
Among other classes of compounds, acids accounted for 4.27 ± 0.04% of the total oil, corresponding to 3.37 ± 0.01 µg/g fresh weight, mainly represented by hexadecanoic acid (2.25 ± 0.36%, 1.98 ± 0.27 µg g −1 ). Phenolics were also present at detectable amounts, quantified as 3.97 ± 0.38% of the total oil (3.13 ± 0.28 µg/g fresh weight). The main component of this class was eugenol, which is well known as a natural antimicrobial agent [22], and is detected at 3.67 ± 0.39% of the total volatiles corresponding to 2.89 ± 0.29 µg/g fresh weight.
Among the miscellaneous components worth mentioning was a relatively small amount of indole (0.34 ± 0.04%, 0.27 ± 0.03 µg/g), a metabolite that is possibly derived from the degradation of tryptophan, which is quite rare in plant volatiles and is associated with the presence of parasites in some cases [23].

Discussion
To the best of our knowledge, this is the first detailed investigation of the chemical composition of the essential oils produced by the two Rubiaceae species, G. verum and C. leavipes. In addition, this was the first characterization of these two species growing wild in the Italian Alpine environment.
Previous studies on the two species mainly dealt with the characterization of methanolic extractives [4,17]. The composition of volatiles obtained from the wild plants from East Europe was also reported [18,20] and in G. verum, only the flowers were analyzed. Data from the literature indicate a different chemical composition compared to our study, i.e., G. verum flowers were described to contain cis-3-hexen-1-ol as the most abundant component, followed by squalene [20]. Essential oils from C. laevipes were instead reported to produce borneol and verbenone, as the major terpenes [18].
Qualitative and quantitative differences can be possibly attributed to the different habitats in which the plant material used in our study was growing, i.e., the Italian Alpine environment.
Essential oils from the leaves of G. verum were characterized by a high amount of 2-methylbenzaldehyde, which is a compound that also naturally occurs in other aromatic plants such as Taraxacum officinale and Morinda officinalis [25,26], and was also reported as a component of the essential oils from G. humifusum [21]. This phytochemical and some derived molecules showed a strong anti-mite effect [26,27], thus suggesting its ecological contribution, and possibly of 4-methylbenzaldehyde, to prevent insect attacks.
On the other hand, essential oils distilled from G. verum leaves are very rich in cis-3-hexen-1-ol, and is well-known as a semiochemical acting as a repellant/attractant for herbivores [28].
The presence of germacrene D as the major metabolite in the flowers of the same species was also consistent with an ecological role. This sesquiterpene was reported to act as a pheromone with anti-herbivore properties and it has been reported to be repellent against aphids [29]. The same compound, however, often contributes to the floral scent of some plant species because of its importance as an attractant of pollinators [30].
With regards to C. leavipes, it should be underlined that the two major aromatic aldehydes, phenylacetaldeyde and benzaldehyde, largely exceeded the modest contribution of short-chained saturated and unsaturated aliphatic aldehydes (from C 6 to C 10 ). It is to be noted that most linear-chained aliphatic alcohols and aldehydes, also known as green-leaf volatiles, are derived from the enzymatic cleavage of C 18 unsaturated acids, and play a major role in plant signaling and defense mechanisms [23,31]. Consequently, it is worth noting the presence of linolenic acid as one of the precursors of green-leaf volatiles, in both G. verum and C. laevipes essential oils [32,33]. On the other hand, aromatic alcohols and aldehydes are synthesized through the phenylpropanoid pathway, together with other benzenoids and phenolics, and they can be enzymatically converted into one another through specific dehydrogenases [34]. Since aromatic aldehydes and alcohols are common volatiles in flowers [35], the interconversion of alcohols into aldehydes and vice-versa might play a significant role in modulating flower scent and might contribute to attract pollinators. The linear-chained alkanes might also play a significant role in pollinator attraction, besides having a possible function in preventing moisture loss from plant tissues [36][37][38].
Finally, the presence of compounds such as cis-3-hexenyl acetate and methyl salicylate can be reasonably associated with the mechanisms of active plant defense [23,24] in the species C. laeveipes.
In conclusion, the chemical composition of the essential oils obtained from the two Rubiaceae species, G. verum and C. laevipes, indicate a complex balance of phytochemicals to protect the plants in their environment. In addition, as shown for other studied Alpine plants [39,40] and plants producing essential oils with similar composition [27,41,42], G. verum and C. laevipes produce volatiles with valuable biological properties.

Plant Material
Cruciata laevipes Opiz and Galium verum L. were identified according to Pignatti [3]. Aerial parts were collected at full bloom in the vicinity of Dravugna, Val di Susa, Western Alps (1250 m. asl; N 45 • 08 47", E 7 • 16 46") in the province of Turin, Italy. Plants were cut at about 1 cm height above ground to avoid soil impurities, samples were weighted and then placed in sealed bottles, half-filled with CH 2 Cl 2 , as a preservative. The G. verum flowers were separately collected and stored. Samples were taken to the laboratory within the day and stored at 4 • C, until distillation. Specimens of C. laevipez (CL1908) and G. verum (GV1935) are deposited at CREA, Lodi, Italy.

Isolation of the Oil
The plant material (about 50 g of the C. laevipes whole plant and the G. verum leaves and about 35 g of the G. verum flowers), to which 0.352 mg of 3-methylcyclohexanone (Sigma-Aldrich (St. Louis, MO, USA), 99% purity) and 0.511 mg of octadecane (Sigma-Aldrich, 99% purity) were added as internal standards, was steam-distilled with odor-free water in a Clevenger-type apparatus, for 1 h. The distillate was saturated with NaCl, extracted with freshly distilled Et 2 O (3 × 100 mL), dried over anhydrous Na 2 SO 4 , and concentrated with a rotary evaporator to give a pale-yellow oil with a yield of 0.01%-0.02%, (weight/fresh weight basis). The resulting oil was then diluted with Et 2 O and analyzed by GC/FID and GC/MS.

Analysis of the Essential Oil
GC/FID analysis was carried out using a Perkin Elmer model 8500 GC (Perkin Elmer Italia Spa, Milano, Italy) equipped with a 30 m × 0.32 mm i.d., Elite-5MS capillary column (0.32 µm film thickness). The sample (0.5 µL) was injected in the "split" mode (1:30), with a column temperature program of 40 • C for 5 min, then increased to 260 • C at 4 • C/min and finally held at that temperature for 10 min. Injector and detector were set at 230 • C and 280 • C, respectively; the carrier gas was He with a head pressure of 12.0 psi. GC/MS analysis was carried out using a Perkin Elmer Clarus 500 GC equipped with a Clarus 500 mass spectrometer, using the same capillary column and chromatographic conditions as for the GC/FID analysis. Mass spectra were acquired over the 40-500 amu range at 1 scan/sec with ionizing electron energy 70 eV, ion source 230 • C. The transfer line was set at 270 • C, while the carrier gas was He at 1.0 mL/min.

Identification and Quantitation of the Oil Components
The identification of the volatile oil components was performed by their retention indices (AI), their mass spectra, by comparison with the NIST database mass spectral library [43], as well as with literature data [44,45]. Authentic reference compounds purchased from Sigma-Aldrich were also used. Retention indices were calculated using an n-alkane series (C 6 -C 32 ) under the same GC conditions as that for the samples. The relative amount of individual components of the oil were expressed as percent peak area relative to total peak area from the GC/FID analysis of the whole extracts. The quantitative data were obtained with GC/FID analysis by the internal standard method, using 3-methylcyclohexanone as the internal reference for compounds with an AI < 1350 (Rt < 25.0 min.; compounds 1-35 in Table 1), and octadecane for compounds with an AI > 1350 (Rt > 25.0 min.; compounds 36-75 in Table 1). A linear proportion between the areas was used, assuming an equal response factor for all detected compounds. Supervision, A.T. and P.A.; Funding Acquisition, A.T. and P.A. All authors have read and agreed to the published version of the manuscript.
Funding: This research was partially funded by the project: "Composti naturali e microorganismi per la difesa ed il priming di colture biologiche mediterranee" of the Italian Ministry of Agriculture, Alimentation, and Forestry Policies.