Volatolomics of Sardinian and Spanish Bituminaria: Characterization of Different Accessions Using Chemometrics

The present study aims to determine the volatile compositions of 15 different accessions of native Sardinian populations of Bituminaria morisiana (Pignatti & Metlesics) Greuter, Bituminaria bituminosa (L.) C. H. Stirt. (B. b.), and Spanish native accessions of B. bituminosa. Furthermore, we particularly focused on the essential oil characterization of these accessions and discriminated within populations with low furocoumarin content useful for fodder production in Mediterranean environments or furocoumarin extraction for pharmaceutical utilization. The plant extracts were analyzed by GC/MS, showing great variability in the content and composition. No differences were found in Bituminaria bituminosa (L.) C.H. Stirt. var. bituminosa essential oils, while the varieties Bituminaria bituminosa (L.) C.H. Stirt. var. crassiuscula P. Méndez, Fern. Galván & A. Santos and Bituminaria bituminosa (L.) C.H. Stirt. var. albomarginata P. Méndez, Fern. Galván & A. Santos are characterized by the presence of a high concentration of long-chain alcohols and of salicylic acid benzylic ester. In B. bituminosa var. albomarginata, we observed a different profile with predominance of a large concentration of alcohols as dodecanol and tetradecanol. The endemic B. morisiana can be identified for the predominant presence of farnesene. In methanolic fractions, we detected the presence of maltol, methyl citrate, methyl cumarate, santonin, and methyl linoleate. B. morisiana showed a low content of psoralens, and the accession of B. morisiana, from Siliqua indicated the presence of apocynin.


Introduction
Bituminaria genus (Fabaceae) was first established by Linnaeus in the second edition of the Linnaeus Gen. Plant., 358 (I742) and credited to Royen. In the first edition of the Sp. Plant., 762 (1753), eight species are described, only one of which is credited to America, and it is probably an introduced plant from the Island of Madeira [1].
The morphology of the genus Bituminaria Heist. ex Fabricius (Psoraleeae, Fabaceae) is very heterogeneous and it is widespread across the Mediterranean region and Macaronesian Islands, where it is estimated to have diverged from other Psoraleoid genera approximately 6.78 million years ago. The causes of diversification can be attributed to various factors, such as habitat modifications and reproductive biology. These speciation processes on the Bituminaria genus led to eight distinct species: B. bituminosa Stirt. [2].
In Europe, only Bituminaria bituminosa (L.) C.H. Stirt. var. bituminosa is present, which is a perennial legume from the Mediterranean Region and is, therefore, also able to resist drought and a hot climate. Bituminaria grows in the Mediterranean basin and Macaronesia, and it has a potential as pasture fodder for ruminants in semiarid environments, but only few selected ecotypes are cultivated in Canary Islands and Morocco for this purpose [3].
On Sardinia Island, two species of Bituminaria can be found in natural stands: B. bituminosa var. bituminosa and the endemic Bituminaria morisiana (Pignatti & Metlesics) Greuter. They can be easily distinguished on the basis of some morphological traits and the presence of a typical bitumen smell in B. bituminosa [4]. In Spain and the Canary Islands, B. bituminosa is spontaneously grown, which exhibits a large diversity with three botanical varieties Bituminaria bituminosa var. albomarginata (albo tedera), Bituminaria bituminosa var. crassiuscula (teide tedera), and Bituminaria bituminosa var. bituminosa (tedera). This genus also grows across the Mediterranean basin, and it is known as Arabian pea or pitch trefoil [3,5,6]. Both species have an appreciable content of furocoumarins, compounds that are related to a low palatability of fresh fodder [7], but that can be used in therapy combining the oral drug psoralen and high-intensity longwave ultraviolet light (PUVA) [8].
As far as the knowledge on the composition of the essential oil of Bituminaria genus is concerned, the paper of Bandeira et al. described the chemical composition of essential oil of Bituminaria basaltica Miniss., C. Brullo, Brullo, Giusso & Sciandr. [17], whereas other authors have investigated essential oils in Psoralea drupacea Bunge and P. corylifolia L. In these species, the presence of bakuchiol, angelicin, α-pinene, and limonene [18] and their antimicrobial activities [19,20] were documented. Volatile composition was also assessed in different organs of B. bituminosa collected in Tuscany [21]. Another study on a B. bituminosa germplasm collected in Italy was performed on the volatile composition of fresh leaves and flowers collected at the experimental field of the University of Tuscia, Viterbo [22]. El-Seedi et al. investigated the essential oil of Psoralea pubescence (Miq.) Standl in plants collected at the Reserva Forestal ENDESA (Ecuador) [23]. A study on B. bituminosa var. bituminosa essential oil growing wild in Elba Island was published in 2016 [14].
Bituminaria genus is widely present in Sardinia, along with the endemic B. morisiana, which is very interesting for its metabolites. Various articles on B. morisiana have revealed the presence of pterocarpans [24]; in particular, a paper on B. morisiana characterized two new pterocarpans and demonstrated a low cytotoxic activity of erybraedin C on all used cell lines, while erybraedin C induced necrosis in leukemia Jukart T cells [25].
In this view, we conducted a comparative analysis among native Sardinian populations of B. morisiana and B. bituminosa and Spanish native accessions of B. bituminosa and some of its varieties. The analyses of EOs of all studied plant species were examined using chemometric tools (PCA). The aim was to characterize the volatile compounds discriminating the population of these accessions to be used in the health or herbal field. In this way, it we had the possibility to divide accessions with low furocoumarin content to use for fodder production in Mediterranean environments and accessions with high furocoumarin content useful as pharmaceutical preparations.

Chemical Composition of the Essential Oil Content
We analyzed 15 different accessions of plants belonging to the Bituminaria genus and growing in experimental fields of the Interdepartmental Center for the Conservation and Enhancement of Plant Biodiversity of Sassari University, coming from different areas of Sardinia and Spain. From the aerial part of fresh plants, we extracted and characterized the essential oils and the furocoumarins.
All the obtained essential oils were diluted at the same concentration and subjected to GC and GC/MS characterization; considering all the species, 96 constituents were identified.
In the six B. bituminosa var. bituminosa samples, three collected in Sardinia and three collected in Spain, the identified components were never lower than 91.45% (Table 1).
In the essential oil obtained from the accessions of B. bituminosa var. bituminosa coming from the Sardinian populations, we identified 63 different compounds, which accounted for 98.39%, 97.30%, and 94.34% of constituents.
In the three populations native to Sardinia, among all constituents, 22 had a concentration higher than 1%. In Loculi, the major constituent was caryophyllene oxide (19.56%). β-Caryophyllene was the major constituent of Sassari station (17.30%). Whereas germacrene D was the more important constituent in the accession coming from Siniscola (10.46%), it had a minimum level in Loculi (5.38%).
In samples from accessions of B. bituminosa var. albomarginata (Canary Islands) ( Table 2), we identified 47 constituents representing about 93.8% of the total and a different composition between the two stations was found.
In B. b. albomarginata accessions coming from Caleta de Famara, the main constituents were long-chain alcohols (cis,cis)-9,12-octadecadien-1-ol (14.96%) and (cis,cis,cis)-9,12,15octadecatrien-1-ol (9.86%). In B. b. albomarginata accessions coming from Arecife, an original profile was found. In fact, there was a predominance of lauryl alcohol (13.83%) (not present in Caleta de Famara) and tetradecanol (10.93%).          B. bituminosa var. crassiuscula belonging from Spanish native plants was characterized by the presence of 49 constituents, representing about 97% and 94% of the total. Among these constituents, 21 exceeded 1%. A common feature of oils at these two stations was represented by a high concentration of β-caryophyllene (22-25%) and caryophyllene oxide 11% in Vilaflor and about 7% in San Cristòbal de la laguna. A component present only in these two oils was benzyl salicylate, which is a compound frequently used in cosmetics as a fragrance or UV light absorber [42]. Another constituent present in high concentration in these varieties was benzyl benzoate (3.05% and 2.38%), which is also used in cosmetics as fragrance but is better known for the topical treatment of human scabies [43].
In Bituminaria morisiana Pign. et Metlesics (Sardinian endemism), it was possible to identify 54 constituents of the essence in toto, representing from 93.41% to 96.75% of the total (Table 3).
Twenty-six constituents reach a concentration higher than 1%. Five were present in a considerable amount. cis-β-Farnesene was the main constituent in all samples, from a minimum of 26.91% in Bitti accession to a maximum of 41.77% in Punta Giglio accession. The concentration in the other three stations varied from 34.36% to 37%. Other constituents present in very high concentration were two long-chain alcohols (cis,cis)-9,12-octadecadien-1-ol (linoleyl alcohol) from a minimum of 9.72% in Bitti to a maximum of 17.73% in Punta Giglio. Germacrene D was present in all samples with a high concentration in Monte Gonareddu and a minimum in Punta Giglio 2.84%. β-Caryophyllene was well represented in the two stations of Bitti and Siliqua with concentrations of 9.92% and 9%, respectively. In Bitti accession, 21.76% of alcohols deriving from fatty acids were present, as well as in Punta Giglio (22.32%), while, in the other stations, fatty alcohols reached a concentration range between 16% and 18%.

Methanolic Extracts
The methanolic extracts were partitioned using H 2 O/CHCl 3 and analyzed by GC/MS (see Section 4) [44]. From the methanolic extracts, we obtained 15 samples that were analyzed by GC and GC/MS. The content of furocoumarins is expressed in mg/100 g of fresh plant (Figure 1). In the six analyzed samples of B. b. var. bituminosa, Monte Rosello showed a very high content of furocoumarins. In these samples, we found 750.2 mg/kg angelicin and 108.7 mg/kg psoralen calculated in fresh plant. In general, all samples from B. b. var. bituminosa showed a larger quantity of angelicin than psoralen. The analyzed extracts from B. b. var. bituminosa species from plants coming from Sardinia and Spain showed, in addition to the expected furocoumarin content, good amounts of maltol, trimethyl citrate, methyl coumarate, santonin, and methyl linoleate. In the crude extracts derived from our samples, we found a good concentration of maltol (e.g., 34.71% in Calnegre and 31.56% in San Cristòbal de la Laguna). In the Sardinian samples, the maximum content of maltol in the crude extract was found in the accession derived from Loculi (14.89%). In the Spanish varieties, salicylic acid was found in the crude methanol extract, varying from 2.78% in San Cristòbal de la laguna B. b. var. bituminosa to 0.35% in LIano del Beal B. b. var. bituminosa. The other varieties from Spain showed the highest concentration of maltol in B. b. var. crassiuscula from Vilaflor (39.32%), while a high concentration (33.96%) was also found in San Cristòbal de la laguna station.      339.4 mg/kg and angelicin 154.3 mg/kg). In terms of the other characterized compounds, in the endemic B. morisiana, maltol reached a maximum of 19.63% in the crude extract derived from the Siliqua station accession. Only in the extract derived from B. morisiana from Siliqua was a small quantity of apocynin found (1.54%).

Statistical Analysis
The obtained data were submitted to multivariate statistical evaluation to verify the biodiversity through secondary metabolites. The PCA analysis performed using only essential oil components (96 different compounds were identified) ( Figure 2 The endemic species of Sardinia, B. morisiana, generally showed a low content of furocoumarins, while the accessions native to Burcei did not contain more than 55 mg/kg of these substances. An exception is constituted by B. morisiana derived from the accession of Monte Gonareddu, which surprisingly had a high content of furocoumarins (psoralen 339.4 mg/kg and angelicin 154.3 mg/kg).
In terms of the other characterized compounds, in the endemic B. morisiana, maltol reached a maximum of 19.63% in the crude extract derived from the Siliqua station accession. Only in the extract derived from B. morisiana from Siliqua was a small quantity of apocynin found (1.54%).

Statistical Analysis
The obtained data were submitted to multivariate statistical evaluation to verify the biodiversity through secondary metabolites. The PCA analysis performed using only essential oil components (96 different compounds were identified) ( Using all identified volatile metabolites coming from water distillation and methanolic extraction for PCA statistical analysis, it was possible to distinguish B. b. var. morisiana from B. bituminosa and all its varieties. Figure 3 reports the plot score of this analysis.

Discussion
The plants we analyzed showed great variability with regard to the content and the composition of essential oils. It is not easy to identify the accessions of B. b. var. bituminosa because there are no decisively discriminating constituents. Comparing the samples coming from Sardinian accessions and that from native plants from Spain, we can note that the most represented constituents were always the same, albeit in different  Using all identified volatile metabolites coming from water distillation and methanolic extraction for PCA statistical analysis, it was possible to distinguish B. b. var. morisiana from B. bituminosa and all its varieties. Figure 3 reports the plot score of this analysis.

Discussion
The plants we analyzed showed great variability with regard to the content and the composition of essential oils. It is not easy to identify the accessions of B. b. var. bituminosa because there are no decisively discriminating constituents. Comparing the samples coming from Sardinian accessions and that from native plants from Spain, we can note that the most represented constituents were always the same, albeit in different

Discussion
The plants we analyzed showed great variability with regard to the content and the composition of essential oils. It is not easy to identify the accessions of B. b. var. bituminosa because there are no decisively discriminating constituents. Comparing the samples coming from Sardinian accessions and that from native plants from Spain, we can note that the most represented constituents were always the same, albeit in different concentrations. A twofold higher concentration of β-caryophyllene was found in LIano del Beal compared with Sardinian samples. In other compounds, e.g., caryophyllene oxide, there were no significant differences in concentration.
Comparing B. b. var. albomarginata and B. b. var. crassiuscula, it was possible to note some differences; in B. b. var. albomarginata with respect to B. b. var. crassiuscula, we found a higher concentration of α-copaene, germacrene D, and palmitic acid. Moreover, in B. b. var. albomarginata, some compounds were present in contrast to B. b. var. crassiuscula, such as lauryl alcohol, β-sesquiphellandrene, n-tetradecanol, and viridiflorene. Benzyl benzoate, also present in some other varieties, reached its maximum in B. b. var. crassiuscula. B. b. var. crassiuscula also contained long-chain alcohols and benzyl salicylate.
In B. morisiana, we observed the preponderant presence of farnesene, unique to this essential oil; moreover, germacrene D was present in all samples with a high concentration in Monte Gonareddu and a minimum concentration in Punta Giglio. This volatile sesquiterpene belonging to the germacrene family is typically produced in plants as an antimicrobial and insecticidal or as an insect pheromone.
The concentration of essential oils distilled from Bituminaria genus was to predict their use in pharmaceutical applications, especially in terms of furanocoumarins such as psoralen and angelicin. Psoralens are widely used to treat human skin diseases and for their antimicrobial activity and anti-HIV effects. Angelicin has calmative, sedative, and anticonvulsant activities and is used for the treatment of thalassemia (US Patent No.: US2006/0111433(A1)). On this basis, we proceeded to quantify the concentration of furocoumarins and other volatile products, which are easily extracted by methanol, such as maltol, trimethyl citrate, methyl coumarate, santonin, and methyl linoleate, which might have medicinal interest. All these substances give to these plants an interest that goes beyond the simple furocoumarin content and enhance their effect. For instance, maltol itself is known for its odor of cotton candy and caramel and is used to impart a sweet aroma to fragrances. Some synthetic derivatives of maltol showed limited in vitro antiproliferative activity toward cancer cells lines [45].
Analyzing all the varieties, we found many other compounds having pharmaceutic interest. For example, in all Spanish varieties, we found salicylic acid, which is an antiinflammatory that might increase the effect of furocoumarins. In the extract derived from B. morisiana from Siliqua, we found a small quantity of apocynin, which is a very interesting compound, since it is a selective inhibitor of the phagocyte NADPH oxidase Nox2, which can be applied orally and is remarkably effective at low dose [46].
The quantified furocoumarins in our samples ( Figure 1) were very significant, indicating a strong correlation between the unpleasant smell of the plant and the content of psoralens. In particular, the accessions native to Burcei did not contain more than 55 mg/kg of these substances, and it is very interesting to note that these species did not exhibit the characteristic odor of bitumen. On the contrary, other accessions contained a relatively large amount of furocoumarins; these plants could be really exploited in phytotherapy as a furocoumarin source, e.g., in the treatment of psoriasis according to the BALNEO-PUVA methodology (BATH-PUVA) [47]. In this view, the fraction extracted with the methanol/acidic water solution partitioned in H 2 O/CHCl 3 (see Section 4) proved to be a valuable source of anti-inflammatory compounds and may be well suited for the treatment of psoriasis using the BALNEO-PUVA methodology. In particular, the accession of B. b. var. bituminosa from Monte Rosello would be able to easily provide the concentration of furocoumarin necessary for this health treatment.
B. b. var. albomarginata and B. b. var. crassiuscula derived from Spain accessions showed a low content of psoralens.
The obtained data coming from essential oil analyses or methanol extract analyses were submitted to multivariate statistical evaluation to verify the biodiversity through secondary metabolites and furnished a very interesting indication, enabling the distinction of B. morisiana from B. bituminosa and its varieties (Figure 3). Moreover, using only the essential oil components, it was possible to divide the accessions into different groups of plants.

Chemicals and Instrumentation
The fertilization of plant material was carried out using P 2 O 5 (NIT Greenhouse grade 25 kg) from Haifa-Italia srl (Bologna, Italy). The distilled water used for the essential oil isolation was produced in the laboratory, whereas anhydrous Na 2 SO 4 was obtained from Merck S.p.A. (Sigma-Aldrich) (Milano, Italy). The quantitative GC analyses were performed using a Hewlett-Packard Model 5890A GC equipped with an automatic injector HP 7673 (now Agilent Technologies Italia S.p.A, Milano, Italy) and GC/MS analyses were conducted using an Agilent Technologies model 7820A Milano, Italy connected with a MS detector 5977E MSD (Agilent Technologies Italia S.p.A, Milano, Italy) equipped with an automatic injector HP 7673 for qualitative analyses. The carrier gas helium was obtained from SAPIO, and the columns (Phenomenex ZB-5, Torrance, CA, USA) for GC and GC/MS analyses were from ThermoFisher scientific Italia (Monza, Italy). The 400 MHz NMR spectra were recorded using a VARIAN (Mercury plus) spectrometer (now Agilent Technologies Italia S.p.A, Milano, Italy).

Plant Material
An experimental field was established in CBV (Interdepartmental Center for the Conservation and Enhancement of Plant Biodiversity Center) of Sassari University located in Surigheddu district (40 • 35 49" N; 8 • 22 47" E) close to Alghero in northwest Sardinia. The site has a Mediterranean climate with an average annual rainfall of 540 mm and an alluvial soil with a high limestone content and neutral pH (6.9). The cultivation of different species and varieties of B. bituminosa was carried out by researchers of ISPAAM Institute (Institute for the Animal Production System in the Mediterranean Environment) of CNR (Research National Council), who handled the germplasm. They oversaw the bio-morphological screening concerning the accessions collected from different stations of Sardinia, and they took care of all agronomic factors, seed production, and its components. They also planted the seeds from the Spanish accessions. The samples of plant material derived from certified seeds or plantlets were treated after collection to avoid the degradation of biomass. The collecting stations for Sardinian B. b. var. bituminosa plants are reported in Table 4 and shown in Figure 4.    The species located in Canary Islands showed a large diversity, with three botanical varieties found in habitats ranging from the coastal semiarid areas on Lanzarote Island with an annual rainfall of 150-300 mm (B. b. var. albomarginata) to the high elevation subhumid area (1700-2200 m, 500 mm) of Tenerife (B. b. var. crassiuscula). The third (B. b. var. bituminosa) displayed a wide adaptation across the Canary Islands (300-1000 m) and is the only one present in the Mediterranean basin. In the Iberian Peninsula, it has been found in environments ranging from 250-1000 mm of rainfall and up to 1250-1500 m of altitude [48]. Germplasms of Bituminaria species and varieties were collected in different Spanish areas (Table 4). The experiments were carried out in plots with 12 plants per accession in a completely randomized design with three replicates. Plants were grown from scarified seeds sown in jiffy pots in a greenhouse and then transplanted to the field in February. Fertilization was done before planting with 46 kg·ha −1 of P 2 O 5 . Occasional irrigation was supplied to plants, when necessary, from late spring to early summer in the first year.

Essential Oil Extraction
Plant material (200 g of aerial part) of 15 accessions of Bituminaria genus was submitted to hydrodistillation for 4 h using a Clevenger-type apparatus. The oils were collected by separation from the aqueous phase, dried over anhydrous Na 2 SO 4 , and stored at −20 • C before being analyzed. The reached yields (w/w) are reported in Figure 5. seeds sown in jiffy pots in a greenhouse and then transplanted to the field in February. Fertilization was done before planting with 46 kg·ha −1 of P2O5. Occasional irrigation was supplied to plants, when necessary, from late spring to early summer in the first year.

Essential Oil Extraction
Plant material (200 g of aerial part) of 15 accessions of Bituminaria genus was submitted to hydrodistillation for 4 h using a Clevenger-type apparatus. The oils were collected by separation from the aqueous phase, dried over anhydrous Na2SO4, and stored at −20 °C before being analyzed. The reached yields (w/w) are reported in Figure 5.

Gas Chromatography and Mass Spectrometry (GC/MS) Analysis
Three replicates of the essential oils were separately analyzed using a GC (Hewlett-Packard Model 5890A, Agilent Technologies Italia S.p.A, Milano, Italy) equipped with a flame ionization detector and fitted with a 60 m × 0.25 mm, thickness 0.25 μm ZB-5 fused silica capillary column (Phenomenex). The injection port and detector temperatures were 280 °C. The column temperature was programmed from 50 °C to 135 °C at 5 °C/min (1 min), 5 °C/min up 225 °C (5 min), 5 °C/min up 260 °C, and held for 10 min. Oil samples of 0.2 μL injection volume were analyzed, diluted in hexane using 2,6-dimethylphenol as an internal standard. Injection was performed using a split/splitless (used in split mode, ratio 50:1) automatic injector HP 7673 and helium as a carrier gas. Several measurements of peak areas were performed through an HP workstation with a threshold set to 0.00 and peak width set to 0.02. Compound quantification was expressed as absolute weight percentage using internal standard (n-tetradecane) response factors (RFs). Since oxygenated

Gas Chromatography and Mass Spectrometry (GC/MS) Analysis
Three replicates of the essential oils were separately analyzed using a GC (Hewlett-Packard Model 5890A, Agilent Technologies Italia S.p.A, Milano, Italy) equipped with a flame ionization detector and fitted with a 60 m × 0.25 mm, thickness 0.25 µm ZB-5 fused silica capillary column (Phenomenex). The injection port and detector temperatures were 280 • C. The column temperature was programmed from 50 • C to 135 • C at 5 • C/min (1 min), 5 • C/min up 225 • C (5 min), 5 • C/min up 260 • C, and held for 10 min. Oil samples of 0.2 µL injection volume were analyzed, diluted in hexane using 2,6-dimethylphenol as an internal standard. Injection was performed using a split/splitless (used in split mode, ratio 50:1) automatic injector HP 7673 and helium as a carrier gas. Several measurements of peak areas were performed through an HP workstation with a threshold set to 0.00 and peak width set to 0.02. Compound quantification was expressed as absolute weight percentage using internal standard (n-tetradecane) response factors (RFs). Since oxygenated compounds have lower detectability than hydrocarbons by FID, detector RFs were determined for key components relative to 2,6-dimethylphenol and assigned to other components on the basis of functional group and/or structural similarity. MS analyses were carried out with an Agilent Technologies model 7820A connected to an MS detector 5977E MSD (Agilent), using the same conditions and column described above. The column was related to the ion source of the mass spectrometer. Mass units were monitored from 10 to 900 at 70 eV. The identification of compounds was based on a comparison of their retention times with those of authentic samples and/or by comparison of their mass spectra with those of published data [22,49,50].

Methanolic Extracts
The vegetable biomass deriving from the various accessions was cold extracted using methanol acidified with 0.1% HCl (37%). The fresh aerial plant parts (100 g) were treated with a grinder and then subjected to maceration three times with 300 mL of acidified CH 3 OH at room temperature in a flask with a magnetic stir bar at 400 rpm (30 h). At the end of this period, the resulting solution was dried by evaporating the solvent under vacuum, taking care that the temperature of the water bath did not exceed 50 • C. The residue was dissolved with 250 mL of distilled water and extracted three times with portions of 100 mL of CHCl 3 . The solvent was evaporated under vacuum at room temperature, and the residue was used for subsequent analyzes. The yields of extracts varied between 1.5% and 2.4% (Table 5). Table 5. Yield of methanolic crude extracts of Bituminaria varieties.

Bituminaria Varieties (Station)
Yield: g/100 g; (Std. Error) × 2.5 mm, Kieselgel 60, 0.015-0.040 mm), before eluting with n-hexane/Et 2 O at different ratios with increasing Et 2 O concentration. All collected fractions were analyzed by TLC, by elution with n-hexane/Et 2 O (1:1 v/v). Pure fractions containing angelicin (40 mg) and psoralen (5 mg) were obtained. The same procedure was applied to 1 g of crude extract of B. morisiana derived from the Monte Gonareddu accession; in this case, we obtained 8 mg of angelicin and 19 mg of psoralen.
The identity of these compounds was assessed by 1 H and 13 C nuclear magnetic resonance (NMR), using a VARIAN (Mercury plus) spectrometer operating at 399.93 MHz for 1 H and 100.57 MHz for 13 C, with the sample dissolved in CDCl 3 , using tetramethylsilane (TMS) as an internal reference. Angelicin

Statistical Analysis
Data analyses of three replicates were performed for every sample. ANOVA was applied with a factorial design (MSTAT-C, software developed by the Crop and Soil Sciences Department of Michigan State University of the United States). Mean separation was tested by application of Tukey's test. To investigate chemical variation in the 15 different accessions of Bituminaria based on GC and GC/MS, we submitted the data to multivariate statistical evaluation (PCA). PCA is an unsupervised pattern recognition technique that converts data consisting of many interrelated variables to a new coordinate system, thereby reducing dimensionality while maintaining the variance [52]. PCA reveals trends in a dataset such as groupings and clusters based on chemical similarities or differences, while outliers within the dataset are also identified. The results of PCA were observed in a score scatter plot, displaying the spatial distribution of observations. Prior to chemometric analysis, the total integral areas were set to 100 to normalize the data, and the generated ASCII file was imported into Microsoft Excel for labeling. The matrix was then imported into SIMCA-P software version 12.0 (Umetrics AB, Umea, Sweden) for statistical analysis.

Conclusions
The aim of the present research was to discriminate Bituminaria populations with low furocoumarin content, useful for fodder production in Mediterranean environments, or Bituminaria populations with high furocoumarin content, useful for pharmaceutical purposes. For this reason, we characterized the volatile compounds of 15 different accessions of native Sardinian populations of B. morisiana and B. bituminosa and Spanish native accessions of B. bituminosa and its varieties. We demonstrated that it is not easy recognize the various plants belonging to the genus Bituminaria using secondary metabolites, because most represented constituents are often the same (although in different concentrations); however, considering all characterized volatile compounds in our work and carrying out a principal component analysis, it was possible to clearly distinguish the species B. morisiana and B. bituminosa. Moreover, using only components of the essential oils, we also evidenced the differences between B. b. var. crassiuscula and the other B. bituminosa varieties, as well as B. morisiana, as reported in the score plot of PCA analysis performed using the essential oil components, where a clear differentiation of the varieties B. b. var. crassiuscula and B. morisiana from B. b. var. bituminosa and B. b. var. Albomarginata is shown.