Chemotaxonomic Study of Four Subspecies of Pinus nigra Arn. Grown in Common Garden Based on Essential Oil Composition

The aim of this study was to investigate the chemical diversity of Pinus nigra Arn. essential oils. The research was carried out on the needles collected from eighteen provenances of black pine grown in common garden located in West-Northern Tunisia and belonging to four different subspecies (Pinus nigra subsp. nigra, Pinus nigra subsp. salzmannii, Pinus nigra subsp. pallasiana, and Pinus nigra subsp. laricio). Essential oil yields ranged from 0.19% to 0.68%. The obtained essential oils have been analyzed by GC-FID and GC-MS apparatus. Twenty-three constituents accounting about 98% of total essential oil composition were identified. The essential oil compositions appeared to be very different according to their origin. Thus, five main essential oil chemotypes were identified in Pinus nigra plants: caryophyllene oxide, camphene, ß-caryophyllene, α-amorphene, and germacrene D. The chemotaxonomic value of the essential oil compositions was discussed in relation to the results of the multivariate statistical test, including a detailed survey of the available literature data.


Introduction
Pinus nigra Arnold (black pine) is a circum-Mediterranean species belonging to the Pinaceae family. e species is divided into six subspecies: Pinus nigra subsp. nigra (Arn.), Pinus nigra subsp. salzmannii (Dun.), Pinus nigra subsp. dalmatica (Vis.), Pinus nigra subsp. pallasiana (Lamb.), Pinus nigra subsp. mauretanica (Mair. & Pey.), and Pinus nigra subsp. laricio (Poir.) [1]. is species is discontinuously distributed from Southwest Europe to Asia Minor, extending to the Crimea, and is also found in North Africa (Morocco and Algeria). Pine essential oils were widely used as fragrances, flavoring, intermediates in the synthesis, and beverages. Traditional therapeutic and pharmaceutical uses of its oils have been recorded all around the world. Pine needle essential oils were mainly used in folk medicine for the treatment of cardiovascular and cholesterol-lowering benefits and respiratory infection, and they have powerful antioxidant and anti-inflammatory effects and the ability to enhance microcirculation by increasing capillary permeability [2]. Black pine was characterized by a genetic, morphological, phenotypic, and biochemical diversity. e chemical composition and the intraspecific variation of Pinus nigra essential oil have been the subject of numerous studies [3][4][5][6][7][8][9][10][11]. According to analysis of variance, these investigations indicated a significant variability in chemical composition of black pine essential oils between provenances and subspecies. In Tunisia, four subspecies from eighteen provenances of Pinus nigra have been introduced in a common garden in the northwest of the country since 1966. erefore, and based on the chemotaxonomic classification tool which was known as one of the most important guides for researchers in their studies for new industrial and medicinal plants, this work was conducted to study, for industrial and medicinal plants. is work was conducted to study, for the first time, the variability in essential oils obtained from the needles of 18 provenances of Pinus nigra growing under humid bioclimatic conditions in the northwest of Tunisia. is study could be helpful to highlight the variability of intraspecific terpene profile of Pinus nigra species growing in the southern limit of its range.

Essential Oil Isolation and Analysis.
Needles from the eighteen Pinus nigra provenances were subjected to hydrodistillation in a Clevenger-type apparatus for 3 h in order to isolate essential oils. e essential oils were measured directly in the extraction burette, and the amount of oil obtained (%) was calculated as volume (ml) of essential oils per 100 g of dry plant material. e essential oils were dehydrated over anhydrous Na 2 SO 4 and kept in a cool and dark place prior to analysis. Analysis of the essential oils were carried out by combination of gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS) according to a standard analytical procedure using a DB-5MS column (30 m, 0.25 mm, and 0.25 µm film thickness). e flow rate of the carrier gas (helium) was 1.0 ml/min. e GC oven temperature started at 100°C and then held for 1 min at 260°C and then for 10 min with a program rate of 4°C min −1 . Besides, the injector and detector temperatures were set at 250 and 230°C, respectively. e mass range was scanned from 50 to 550 amu. A sample of 1.0 µl was injected, using split mode (1 : 100). Compounds of the essential oils were identified by both their Kovats indices and mass spectra. Kovats indices were calculated by linear interpolation relative to retention times of nalkanes (C 8 -C 24 ). Mass spectra were matched with the reference spectra from the Wiley/NIST database, published data, and the spectra of authentic compounds. Relative amounts of individual components were calculated based on GC peak areas without FID response factor correction [2].

Results and Discussion
Statistical analysis showed a high variability between the four studied subspecies (p < 0.0001).
e highest yield was recorded by needles of nigra subspecies (Table 2), while the lowest values were reached by both calabrica and salzmannii subspecies. Furthermore, a significant variability was recorded between the eighteen provenances. e most important oil yield (0.68%) was reached by needles from P6 (Pinus nigra austriaca; Puget-éniers, France) while P2 (Pinus nigra calabrica; Trenta, Italy) showed the lowest oil yield with 0.19%. e results of the identified compounds by GC-MS are shown in Table 3. Twenty-three constituents accounting about 98% of total essential oil composition were identified. e essential oil compositions appeared to be very different among provenances. e major essential oil components were especially variable in occurrence and concentration among the different provenances, ranging from almost absent in some samples to more than 90% of the total essential oil composition in others.
ere appear to be five basic essential oil chemotypes in Pinus nigra plants investigated ( e results of the principal component analysis showed that β-caryophyllene, caryophyllene oxide, linalool, myrcenol, and c-muurolene were the most significant variables for the classification of the Pinus nigra essential oils. ese parameters were considerably loaded into the two major principal components (Dim 1 and Dim 2) explaining more than 50% of the variance. According to the analysis, five different groups were revealed (Figure 2). e first group contained P6, P10, and P19 samples, which had the main concentrations of β-caryophyllene (15.65, 23.28, and 51.10%, respectively) and the lowest rate of caryophyllene oxide (1.23, 8.33, and 0.99%, respectively). On the other hand, the second group enclosed only P12 and P13 samples, which were characterized by its high amount of both β-caryophyllene (30.29-32.16%) and caryophyllene oxide (38.71-41.85%). e third group regrouped P1, P5, P11, and P14, which showed the most important amount of c-muurolene (2.23-4.02%). e fourth group contained P2 and P3 oils characterized by the highest amount of linalool (5.83 and 2.97%, respectively). e fifth group regrouped all the other samples studied, which showed the highest rate of caryophyllene oxide.
When considering the variability between the four studied subspecies, statistical results showed that oils from nigra subsp. were the richest in α-pinene. is richness is related to the high amount found in P6 (Puget-éniers, France) oil (19.34%). e results of the principal component analysis revealed the presence of three groups ( Figure 3)-the first group regrouped laricio and salzmannii subspecies, which showed the highest rate of camphene and limonene; the second group contained pallasiana subsp., which demonstrated the most important amount of caryophyllene oxide; and the third one enclosed only nigra subsp. representing the highest amount of α-pinene.
Various terpenoid compounds, which are characteristic constituents of conifers, have been reported in Pinus nigra needles. Several studies that noted the chemical composition of the essential oils extracted from needles of Pinus species growing in Tunisia (Pinus halepensis, Pinus pinea, and Pinus pinaster) found that β-caryophyllene, amorphene, limonene, and germacrene D were the major components in all essential oils of pine needles [2,[12][13][14]. ese results were supported by our study.
In our study, caryophyllene oxide was the major compound in oils of pallasiana subsp. from Turkey. ese results were not similar to those found by Dogan and Bagci [15] that demonstrated that the main compounds of this subspecies were α-pinene, limonene, and β-caryophyllene. In the same context, Sezik et al. [11] proved that α-pinene and β-pinene were the main constituents of Pinus nigra essential oils from Turkey.
Several studies investigated the chemical composition of Italian essential oils. Macchioni et al. [10] indicated that α-pinene was the principal constituent. In the same context, Bader et al. [16] claimed that the essential oils from laricio subsp. were rich in α-pinene, β-caryophyllene, and germacrene-D. According to our results, the main compounds in Italian oils were β-caryophyllene, camphene, and caryophyllene oxide.
Pinus nigra subsp. laricio essential oils from Corsica were investigated by Rezzi and co-workers [9] who demonstrated that α-pinene, manoyl oxide, and germacrene-D were found     ese findings were not in accordance to that found by our research team. In our case, laricio subsp. essential oils showed three main compounds, which were β-caryophyllene, α-amorphene, and caryophyllene oxide.
According to Jurc et al. [17], the terpene profile of Pinus nigra subsp. salzmannii from France was characterized by a high amount in c-cadinene and δ-cadinene. Our results differ considerably from those reported previously. In this study, oils from salzmannii subsp., France, were characterized by a significant rate of camphene and caryophyllene oxide.
In most previous literature that focused on Pinus nigra essential oils composition, α-pinene was mentioned as the major compound, while in our study, this compound was absent in almost all the studied provenances excepting nigra  subsp. austriaca var. that was mainly composed of germacrene and α-pinene. is finding was supported by Jurc et al. [17]. ese comparisons with earlier studies indicated that, when planted in a common garden in Tunisia, where the pedoclimatic conditions were constant, Pinus nigra from four subspecies and deriving from eighteen provenances has undergone a significant change in its chemotypes. Plants of black pine seem to be adapted to local climate and soil conditions. is finding was supported by Amri et al. [12] who mentioned that there was a significant difference in the chemical composition of the essential oils from Pinus nigra subsp. laricio grown in Tunisia and those from other countries.
It has long been known that pedoclimatic conditions affect the volatile oil content as well as its chemical composition. Ormeño and Fernandez [18] mentioned that both biotic and abiotic conditions influence terpenoid production in plants, especially light and temperature. On the other hand, Staudt and Lhoutellier [19] determined the effect of these two environmental factors on monoterpene and sesquiterpene leaf emissions. In addition, water availability was known to be one of the most important environmental factors controlling volatile organic compounds from plants [20].
Under the same pedoclimatic conditions of the studied provenance trials, Pinus nigra plants showed a significant chemical variability. is could be explained by an eventual genetic variability among the eighteen provenances.

Conclusion
From the present study, we can conclude that the analyzed essential oils belonged to five different chemotypes. In addition, the obtained results showed differences in the quantitative and qualitative composition. Caryophyllene oxide was the main component of most of the studied oils.
erefore, this type of study will be useful for pharmacologists to promote pharmaceutical products and resolve several economic problems.

Data Availability
No data were used to support this study.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this article.

Authors' Contributions
Sondes Fkiri and Faten Mezni performed some practical experiments, wrote and coordinated all the analysis, and calculated the results, statistics etc. Ghayth Rigane followed and checked the obtained results. Hanene Ghazghazi participated in some practical experiments. Ridha Ben Salem, M. Larbi Khouja, Zouheir Nasr, and Abdelhamid Khaldi supervised the scientific paper.