Volatile Compounds of the Leaves and Flowers of Lavandula dhofarensis A

The leaves and flowers of Lavandula dhofarensis were collected from the Dhofar region of Oman and hydro-distilled to give low boiling volatiles, which did not condense at 10 C. The dichloromethane extract of the hydrosol was analyzed by GC/FID and GC/MS. Sixty four compounds were identified in the volatiles of the leaves, accounting for 78.7% of the total. The major components were caryophyllene oxide (8.0%), germacrene (7.9%), spathulenol (7.8%), and caryophyllene (6.6%). Eighty six compounds were also identified in the volatiles of the leaves plus flowers, comprising 94.5% of the total. The major compounds were camphor (12.9%), viridiflorol (10.5%), -terpinyl acetate (7.5%), valerenal (7.2%), -gurjunene (5.6%), and spathulenol (5.5%). Compounds such as linalool, linalyl acetate, 1,8-cineole, and -ocimene, which are usually found as the major components of lavender oils, were either absent or detected at low levels (<0.1%) in the hydrosol of L. dhofarensis. This investigation showed that the fragrance essence of L. dhofarensis is different from the other Lavandula species. L. dhofarensisis is regionally endemic to wetter areas of Oman.


Introduction
is a leafy wild-growing perennial herb, producing an aromatic smell of lavender or faintly of lemons (Miller and Morris, 1988).It grows in clumps; the stems are hairy L and much-branched and the leaves are ovate with up to 5 pairs of segments.The leaves are 7.5 to 50 mm long and 3 to 20 mm wide.The flowers are lilac in color and 15 to 70 mm long.It is endemic to the Dhofar region of Oman where it is commonly known as 'heryen ekúlún' and 'hilbēn' in Jabbali vernacular (Miller and Morris, 1988).L. dhofarensis is not found in drier areas of Oman.
Based on botanical features, the lavenders fall into four categories: L. latifolia, L. angustifolia, L. stoechas, sometimes known as French lavender, and Lavandula x intermedia, a sterile breed between L. latifolia and L. angustifolia.From a literature review, the most frequently identified volatile components of the aerial parts of the lavenders irrespective of group or analysis method are linalool, linalyl acetate 1,8-cineole, camphor, terpinen-4-ol, and -ocimene (Porto et al., 2009).Broadly, the lavenders may be grouped as linalool or camphor chemotypes, based on the most abundant component of the floral oil.The percentage composition of each of the major components varies from one species to the other and the relative levels of each component determine the market value, smell and medicinal application of their oils.However, no references to the components of the oil or volatiles produced by L. dhofarensis were found in the literature.
As part of our investigation of the composition and bioactivity of the essential oil from fragrant endemic plants in Oman, we report here, for the first time, the chemical composition of steam-distilled volatiles of L. dhofarensis subspecies dhofarensis.

Plant material
The fresh leaves and flowers of L. dhofarensis subspecies dhofarensis were collected from the Dhofar region of Oman, 4.4 km from the Ma'amura roundabout on the Salalah-Marbat road at an altitude of 40 m in September 2002 and identified by Dr. Shahina Ghazanfar.A voucher specimen was deposited in the Herbarium of the Botanical Garden at Sultan Qaboos University under the code name NP020.
Approximately 600 g of fresh plant material (either leaves only or combined leaves and flowers) were subjected to hydro-distillation using a 10-Liter Stove Still apparatus (Essential Oil University, New Albany, IN, USA) for 3 h.No condensed oil was visible in the arm of the modified Clevenger-type apparatus used but the condensed steam had a distinct and pleasant odor.The hydrosol was extracted twice with 2.0 ml of dichloromethane and dried over anhydrous sodium sulfate.The organic solution was transferred to GC-MS auto-sampler vials, ready for analysis.

Analysis of the volatile extract
GC/FID analyses were performed on a Focus GC gas chromatograph (Thermo Electron Corporation, Italy) equipped with FID detector, and a DB-1 column, 30 m x 0.25 mm, 0.25 µm film thickness (J and W Scientific, Folsom, CA, USA).Analyses were conducted under the following conditions: the carrier gas was He; flow rate 2.7 ml/min; injection port temperature, 250 o C; oven temperature, programmed from 35 -250 C at 5 C /min up to 200 C and 20 C/min and then held at the upper limit of 250 C.Split/split less injection: solutions of oil in dichloromethane were injected in split mode at a ratio of 1:20.
GC/MS analyses were performed on a Shimadzu (Kyoto, Japan) GCMS-QP5050A using a 30 m × 0.250 mm × 0.25 µm DB-1 column from J and W Scientific (Folsom, CA, USA).The carrier gas was helium at a flow rate of 2.7 ml/min and the split mode had a ratio 1:20.The injector and detector temperatures were 275 C.After injection, the oven temperature was kept at 35 C for 2 minutes, and then programmed at a rate of 2 C/min to a temperature of 200 C for 5 minutes followed by an increase to the final temperature of 240 C at 5 C/min.For the mass spectra, the electron impact ionization was at 70 eV, and the acquisition scan was from m/z 40 to 500 (1000 amu/sec at 0.5 sec intervals).
Qualitative data were obtained electronically from area percent data.Some compounds were identified by comparison of GC retention times with those of standards on a GC-FID instrument, by computer matching of mass spectral fragmentation patterns using digital library (Wiley spectral library of 229,000 spectra) of the GC-MS instrument or by comparison of their calculated retention indices relative to C 8 -C 20 standard n-alkane with literature values (Shellie et al., 2002;Da Porto and Decorti 2010;Migel et al., 2004;Baratta et al., 1998;Gancel et al., 2003).
Qualitative studies of lavender have also shown variable composition of the major components: linalool (35-37%), linalyl acetate (21-34%), 1,8-cineole (4-11%) and camphor (5-12%) (Da Porto and Decorti, 2010).Surprisingly, linalool, linalyl acetate, the two major components of several lavender flower oils, and 1,8-cineole, a major component of lavender herb oil, were found only at low levels (≤ 0.6%) in the volatiles of L. dhofarensis (Table 1).One possible reason for the difference observed is that other workers used mostly flower heads whereas in this study, the flowers were steam-distilled with the leaves and the amount of flowers compared to leaves was low.The flowers were not collected, extracted and analyzed separately because L. dhofarensis grows naturally as a small crop in Oman; the ecosystem could be harmed if, for example, 600 g of flower heads were taken from the wild.From Table 3, camphor, viridiflorol, -terpinyl acetate, valerenal, and -gurjunene were the major components of the flowers.Surprisingly, germacrene, torreyol, humulene oxide, 2-undecanone, and -elemene were present in the leaf volatiles at higher levels (Table 3) suggesting their absence or presence at trace levels in the flower essence.L. stoechas and L. lanata have high camphor levels in flowers while L. augustifolia, L. dentate and L. pinnata are low in camphor (< 2%).L. dhofarensis flowers have low levels of linalool and linalyl acetate (< 1%), and high levels of camphor (12.9%) and valerenal (7.2%).There is thus some similarity between L. dhofarensis, L. lanata and L. stoechas.The low levels of linalool and linalyl acetate, (Table 1) and the high level of camphor (12.9%) in the floral oil volatiles of L. dhofarensis (Table 1 and Table 3) distinctly support the grouping L. dhofarensis as a camphor chemotype.

Conclusion
The results taken together, this investigation showed that caryophyllene oxide, germacrene, spathulenol, viridiflorol, valerenal, camphor, and -terpinyl acetate are the major components of the hydrosol of L. dhofarensis.L. dhofarensis is thus different from other lavenders and the presence of camphor could lower the market value and applications of the fragrance essence of L. dhofarensis in aromatherapy.

Table 1 .
Composition of L. dhofarensis subspecies dhofarensis volatiles for leaves only and combined leaves and flowers.
a List of compounds in elution order from DB-1 column.b RI relative to C8-C20 n alkanes on DB-1 column.c GC Peak area % d Identification by standard e Identification by MS/RI

Table 2 .
Compound distribution in the analyzed volatiles of L. dhofarensis subspecies dhofarensis.

Table 3 .
Major components of the volatiles of L. dhofarensis subspecies dhofarensis.