Aroma Profiles Analysis of Two Populations of Salvia palaestina Benth. Collected from Mediterranean and Irano-Turanian Zones in Jordan

Principle Analysis （ （ kNN ） investigate the possi-Abstract: The volatile principles emitted from different aerial organs of two S. palaestina Benth. populations (Mediterranean (Med) and Irano-Turanian (IrT)) growing wild in Jordan were extracted by Solid Phase Micro-Extraction (SPME) and analysed by GC/MS technique. Sesquiterpene hydrocarbons dominated stems (59.38%, 49.67%) and leaves (93.28%, 32.39%) emissions from MeD and IrT zones, respectively while monoterpene hydrocarbons had the major contribution to the aroma of pre-flowering buds (78.62%, 74.96%), opened flowers (76.12%, 59.99%) and petals (69.57%, 54.28%) mostly represented by sabinene (in MeD zone) and ociemene isomers ( Z & E ) in IrT collection. Multivariate analysis classified the two populations into two different clusters based on their origin and indicated the occurrence of two ecotypes of this species. Different organs from same collection site showed emission profiles of similar chemical composition.


Introduction
Salvia genus, belonging to the Lamiaceae family, continues to be a hot topic for extensive pharmacognostic and phytochemical investigations due to the wide spectrum of interesting bioactivity properties and importance in food and cosmetics industries. Salvia species are also used as ornamental plants due to their beautiful eye-catching flowers. Many species are well recognized for their culinary, cosmetic and pharmaceutical value.
Salvia palaestina Benth. is a perennial herb branching from the base with long soft hairs and it is well recognized for its lemon scent. The plant reaches up to 60 cm high, and is characterized by its white to whitish lilac coloured flowers. Flowering occurs in spring season during the period extending from April to July. In Jordan, the plant has been recorded to grow wild in the different locations belonging to the Mediterranean Med and Irano-Turanian IrT zones 1 . Previously, our group reported the evaluation of the chemical constituents of the hydro-distilled essential oil of S. palaestina in addition to the isolation and structural elucidation of new secondary metabolites 2 4 . The plant has also been investigated for its essential oil composition from many origins including Palestine 5 , Lebanon 6 , Syria 7 , Turkey 8,9 , Saudi Arabia 10 and Iran 11,12

Solid Phase
Micro-Extraction SPME of organs aroma Extraction of volatile principles from the emissions of the aerial organs of the S. palaestina two populations was performed according to the procedure described in the literature and using the same SPME-fiber described there DVB/CAR/PDMS, Stable/DVB; fiber length 2 cm, Supelco, Bellefonte, PA, USA and according to the procedure described in the literature 13,14 . Briefly, fresh organ approximately 0.5 g was introduced into an amber glass vial 15.0 mL tightly capped with PTFE-coated septa. Using the conditioned fiber assembly, extraction was allowed to occur for 15 min at room temperature. Analytes were desorbed at 240 for 60 s. Each sample was repeated twice.

GC-MS, GC-FID analysis and identification of com-
ponents Analysis of the SPME extracted aromatic VOCs was performed by both experiments according to the procedure described in the literature, using the same instruments and columns GC/MS: Varian Chrompack CP-3800 GC/MS/ MS-200-Saturn, Netherlands; Capillary column: 5 diphenyl, 95 dimethyl polysiloxane, 30 m 0.25 mm i.d., 0.25 μm film thickness; GC/FID: Hewlett-Packard HP-8590 gas chromatograph equipped with an optima-5 fused silica capillary column 5 diphenyl, 95 dimethyl polysiloxan, 30 m 0.25 mm, 0.25 μm film thickness split-splitless mode, split ratio 1:50 and using the same temperature program described in our previous work 13 . A standard nalkane hydrocarbons mixture C 8 -C 20 was analyzed separately under the same chromatographic conditions. Relative peak areas of the different aroma constituents were used to calculate the concentration of the detected compounds.

Identi cation of VOC constituents
Identification of VOCs was done by comparing their ex-perimentally obtained arithmetic index relative to the n-alkane hydrocarbons standard mixture C 8 -C 20 to those listed in the literature 15 and through computer matching against commercially available libraries NIST and Wiley, USA . Moreover, the identity of several compounds including α -pinene, β -pinene, p-cymene, 1,8-cineol, α -terpineneol, limonene, ocimene, linalool, sabinene, caryophyllene, aromadendrene and alloaromadendrene Sigma-Aldrich, Buchs, Switzerland were further confirmed by the analysing authentic standards under the same GC/MS conditions.

Multivariate data analysis
Multivariate statistical analysis for data processing and chemometric analysis was performed on MAT LAB 7.0.4 Math Works, MA, USA with PLS Toolbox 4.0 Eigenvector Research, Inc, WA and USA .

Results and Discussion
A total of 69 compounds have been detected in the aerial organs aroma emissions of S. palaestina two populations MeD and IrT zones . The chemical compositions of components of the aroma are listed in Table 1. The structures of the main compounds identified in the emission profiles of the two populations are shown in Fig. 1. Figure 2 shows the relative percentage of the main classes of VOCs detected in the investigated aromas.  Among the different classes detected in the emission profiles of stems from IrT origin, sesquiterpene hydrocar-bons was the main class that had the highest contribution and was mainly represented by δ -elemene 13.91 ; α -gurjunene 11.21 and β -selinene 8.64 . However, β -ocimene monoterpene isomers were the main components that had the highest concentrations in the emission profile of this emission Z-:14.09 , E-: 12.77 . The main VOCs emitted by fresh leaves included α -gurjunene 10

HS-SPME-GC/MS Statistical analysis
Multivariate analysis has been applied to the SPME-GC/ MS data shown in Table 1 to investigate the possible differences or similarities in chemical composition between the studied two populations of S. palaestina according to both, origin and organ. The first eight PCs were calculated and best PCA score model obtained involved using the second and fifth PCs that accounts about 35 of the total variation in the data set. Figure 3 represents the resulted score PCA plot. Statistical analysis showed that the emissions of all organs belonging to one site were located in an opposite location to those extracted from the other site, those from the MeD zone were located to the positive part while those corresponding to IrT zone were located in the negative side of the same PC. Accordingly, PCA classified the two populations into two clusters based on site of col-lection. To confirm this, Cluster analysis CA was applied to the same data matrix used for the PCA. The resulted dendrogram is shown in Fig. 4 using k nearest neighbour kNN distances between the samples organs . The obtained results were very consistent to those obtained from the PCA application. Samples of same composition were grouped in an independent cluster Fig. 4 . The obtained PCA and kNN results suggest that all organs of S. palaestina Benth. exhibited almost similar chemical composition. However, this chemical composition was quite different based of sampling location.
Previously, the emission profiles of two other Salvia species, namely S. verbenaca 16 and S. dominica 17 , both collected from the same two geographical zones from Jordan were reported. Despite the variation in the chemical composition observed among the three different Salvia species, statistical analysis revealed that the emission profiles of the different organs from each collection had similar composition but this chemical composition was different   Table  1 .
based of sampling location. It was noticed that each of trans-sabinene hydrate, trans-sabinene hydrate acetate in S. verbenaca ; trans-sabinine hydrate acetate S. dominica ; β -ociemene isomers Z & E in S. palaestina are among the stable components that were detected in the emission profiles of the three species, and hence can be considered as stable chemotypes of these species.

Conclusions
The current study confirms the effect of environmental and climatic zones on the emission profiles of Salvia species and the occurrence of different ecotypes. The results obtained in this investigation are in total agreement with those obtained on S. verbenaca 16 and S. dominica 17 from Jordan collected from the Mediterranean and Irano-Turanian zones, in which two ecotypes for each species were recognized based on bio-geographical zone.