NATURAL COMPOUNDS OF PALESTINE FLORA. COMPARISON ANALYSIS BY STATIC HEADSPACE AND STEAM DISTILLATION GC-MS OF SEMIVOLATILE SECONDARY METABOLITES FROM LEAVES OF CULTIVATED PALESTINIAN

BACKGROUND
A comparative analysis by using static headspace (HS) and steam distillation (SD) GC-MS of the volatile and the semi-volatile secondary metabolites from leaves of cultivated Majorana syriaca.


METHODS
The essential oils endogenous to cultivated thyme were isolated and identified by HS-GC-MS technology and compared to those from SD-GC-MS.


RESULTS
The HS-GC-MS results showed that the Palestinian cultivated thyme is rich in monoterpene hydrocarbons and phenolic monoterpenes such as alpha-phellandrene, alpha-pinene, beta-myrcene, m-cymene, p-cymene, gamma-terpinene, thymol and carvacrol. In all the samples gamma-terpinene, p-cymene, thymol and carvacrol were the most abundant compounds.


CONCLUSIONS
HS and SD-GC-MS have proved that most of the cultivated thyme samples examined has thymol isomer as the major phenolic constituent.


INTRODUCTION
Given its small area, the West Bank is one of the richest places in the world in plant biodiversity 1 .The Palestine valleys and mountains are covered with a huge number of plant species.Horticulture was known at Jericho more than 10,000 years ago, crops included wheat, barley, lentils, and peas 2 .About 2600 species exist on this small Mediterranean area, out of which more than 700 species are cited in ethnobotanical data and therefore named as Palestinian medicinal plants 3 .Many of these plant species have been used in folk medicine for the treatment of diseases 1,3,4,[5][6][7][8] .One of the most popular herbs among Palestinians is thyme; its taxonomic name is Majorana syriaca L. Rafi n (equal to Origanum syriacum var.syriacum).
The green leaves of the herb are rich in essential oil, which is responsible for its characteristic fl avour and fragrance [9][10][11] .Flowering of Majorana syriaca occurs from May to October 12 .Leaves are normally collected from wild populations once a year, before fl owering.However, plants under cultivation are harvested three or four times a year.Cultivation of Majorana syriaca was initiated by transferring wild population growing in the central and northern parts of the West Bank to experimental fi elds particularly at the Ketf Al-wad area at the city of Jericho 13 .The production of cultivated thyme has increased dramatically in the recent years in response to the increasing local demands.
Oil of cultivated thyme is an important commercial product and is obtained mainly by steam distillation (SD) of the fresh leaves.The phenols, thymol and carvacrol are OH OH 1  2 Fig. 1.Major natural phenolic compounds thymol (1)  and its isomer carvacrol (2).
the principal constituents of thyme oil (Fig. 1).In medicine, thymol has been the most valuable compound for medicinal purposes, but carvacrol, its isomer, preponderate in oils that are extracted from wild origin 11,14,15 .The present work is a continuation to our previous work that explored the constituents in wild Palestinian thyme.The essential oils endogenous to cultivated thyme were isolated and identifi ed by HS-GC-MS technology and compared to those from SD-GC-MS.Qualitative correlation of some of the parameters including harvesting time, location, and fresh water irrigation to the isomeric distribution of the major isomers in the thyme were investigated.

Plant collection
Leaves of the Palestinian cultivated thyme samples were collected from six diff erent environmental sites in the West Bank territories.The chosen sites were distributed geographically as follows: the northern area: Jenin and Tulkarim, the southern area: Hebron (Tarqumia, Halhool, Dora and Bet O"la), the central area: Bethlehem, and Ramalla and the eastern area: Jericho (Tab.1).
The collection was carried out during the years 2003 and 2004.During this period, twenty-eight samples were collected.The cultivated thyme leaves were air dried in the absence of light at room temperature for a fi xed period of time (three months) for all of the samples, followed by storage in sealed paper bags.This is the most eff ective method to preserve the components of the essential oils from being damaged or altered 13 .Leaves were analyzed by HS-GC-MS without being grounded.The amounts of the dry leaves used for the analysis were 0.6000 g and 6.0000 g for the HS and SD experiments respectively.

Instrumentation
Essential oils from cultivated thyme were analysed using Shimadzu GC-17A connected to MS-QP5050A.The GCMS was operated in the electron impact ionisation mode (EI) at 70 eV.Shimadzu auto sampler AOC-20i was used with 2ml vials.An equilibrium headspace Shimadzu HSS-4A auto sampler was used with 27-ml HS vials.The HS vials were sealed with silicon rubber septa and aluminium caps after introduction of the thyme sample while the AOC vials were sealed with 8mm double-faced rubber septa and screw cap with 12 mm hole.The GC is equipped with a fused silica capillary column; DB-5 MS containing (5 % diphenyl polysiloxane, 95 % dimethyl polysiloxane) 30 m x 0.25 mm i.d., coating thickness is 0.25 μm, Supelco (Sigma-Aldrich Inc., USA).

Extraction of the essential oil by Steam Distillation
Method A: Air-dried leaves of Magorana syriaca (6.0000g) were mixed with 350 ml of double distilled water (DDW) and 0.0300g 4-isopropyl phenol as internal standard was added and subjected to steam distillation for 3 hrs at atmospheric pressure.The water distillate containing oil was distributed two times with hexane (50 ml each) by using a separatory funnel.
A 50-ml of 10 % sodium hydroxide solution was added to the hexane layer and stirred at room temperature for 5 minutes and the two phases were separated.The aqueous fraction was washed with 50 ml of hexane, and the hexane fractions were combined, dried with anhydrous sodium sulfate and were labelled as "non-phenolic" fraction.To the basic water layer, 1 M HCl was added with stirring until pH 3. The solution was then extracted three times with hexane (50 ml each).The hexane fractions were combined and dried with anhydrous sodium sulfate and labelled as "phenolic" fraction.Aliquots(1μL) from the above solutions were injected to the GCMS.Air-dried leaves of Majorana syriaca L. (6.0000g) were mixed with 350 ml of double distilled water and 0.0300g 4-isopropyl phenol as internal standard was added and subjected to steam distillation for 3 hrs at atmospheric pressure.The water distillate containing oil was distributed two times with hexane (50 ml each) by using a separatory funnel.The hexane fractions were combined and dried with anhydrous sodium sulfate.A 0.2-ml of this solution was diluted to 1 ml with hexane and 1μL sample of it was injected to the GCMS.

A. Steam distillation samples
The carrier gas fl ow rate was 1.6 ml He/min.Injector and detector temperatures were 230 o C and 250 o C respectively.Split ratio was 1:30.The column temperature was held at 60°C for 2 minutes, then raised from 60 °C to 100 °C at 3 °C/min and from 100° to 280 °C at 30 °C/min and held there for 2 min.Solvent cut time was 4 minutes and the starting time of the chromatogram was 5 minutes.Mass range was from 30 to 350 u, and scan interval was 0.5 seconds.Detector voltage was set to 1.50 kV.The operating conditions were similar to that described in section A for the steam distillation samples except for the following: vial temperature was set to 100°C and the syringe temperature was set to 110°C.The volume of the thyme headspace injected to the GCMS capillary column was 0.2 ml.

Procedure of the GC-MS analysis
A. Steam distillation samples 1 ml of the hexane solution samples was placed in 2 ml vial and 1μl was injected.Before each injection the syringe of the autosampler was rinsed twice, fi rst with the solvent (hexane), and then with the sample solution.

B. Headspace sampling
Before each HS-GCMS analysis, a blank consisting of an empty vial is carried out to check whether any consti-Natural compounds of palestine fl ora.Comparison analysis by static headspace and steam distillation GC-MS of semivolatile secondary metabolites from leaves of cultivated palestinian Majorana syriaca tuents of the materials of the septum or the vial are being emitted.0.6000 g samples of dried leaves of thyme were placed in HS vials and immediately sealed with silicone rubber septa and aluminium caps.The vials were then transferred to the headspace tray.

B.1 Equilibration temperature
To reach the optimal conditions for HS analysis, fi ve diff erent temperatures were chosen at 40, 60, 80, 100, and 120°C at fi xed equilibration time.

B.2 Equilibration time
To select an optimal equilibration time for the extraction of the essential oils in the thyme, vials were equilibrated at 10, 20 and 30 minutes at fi xed temperatures.Analyses were performed in triplicates.

Peaks identifi cation
The identifi cation of the compounds was based mainly on their retention times in comparison with those from authentic standards.The standards were injected separately in addition to adding them to the thyme matrix (spiking) to enhance the relevant peaks of interest.Identifi cation of some peaks was based on matching of their MS spectra with NIST/EPA/NIH Mass spectral library (NIST 98).

RESULTS AND DISCUSSION
Yield of cultivated thyme oils The essential oils of cultivated Majorana syriaca leaves were isolated by normal steam distillation.The thyme samples were collected from twenty-seven individual plants cultivated in six populations from Palestine.The collections were made between the years 2003 and 2004.The harvesting time, location and origin of thyme (i.e., cultivated) eff ects on the yield of essential oils are summarised in Table 2.
From Table 2 it is obvious that there are distinct differences in oil yield when comparison is made between the following:

Wild vs. cultivated thymes
The yield of the essential oils from cultivated thyme ranged from 1.328 % (Bethlehem, Menia, February 2004) to 5.52 % (Tulkarim, Rameen, June 2003).The diff erences in the yield of the cultivated samples are as much as 74.7 %.Almost the same observation was noticed when diff erent wild thyme samples from diff erent locations were examined 11 .
This result simply indicates that harvesting the thyme in May-June months would produce more essential oils than earlier in the year, namely, January-March months no matter what type of thyme was isolated (Fig. 2-4).A possibility of variation in the oil yield may be attributed to the vegetation stages of the natural plant growth.Usually fl owering of thyme occurs from May to June (Fig. 2-4).According to Dudai et.al. 12 leaves are normally collected from wild thyme populations once a year between March and April, just before fl owering.This fi nding gives oil industries some degree of control over the amount of essential oil of thyme produced upon distillation.It is worthwhile mentioning that drying the leaves of thyme causes a dramatic decrease in the amount of water especially of the irrigated thyme, hence increasing oil percentage.Therefore, there is no signifi cant contribution apparently to the type of thyme (i.e., wild vs. cultivated) on the yield of the total oil.

Sampling sites
Our study reveals that there are no dramatic diff erences in the oil yields as it related to diff erent geographical sites (Tab.2).

Steam distillation (SD) and GC-MS analysis
Upon SD of the cultivated thyme samples that were collected as shown previously (Tab 2) a relatively acceptable percentage of the oil was obtained (1.3-5.5 %) based on dry weight.GCMS analysis reveals that the oil contained mainly the semi-volatile phenolic isomers, namely, carvacrol and thymol.None of the volatiles, particularly, p-cymene and γ-terpinene, appeared in the TIC analysed samples.This is most probably related to the method of sample preparation, which involved concentration of hex-Natural compounds of palestine fl ora.Comparison analysis by static headspace and steam distillation GC-MS of semivolatile secondary metabolites from leaves of cultivated palestinian Majorana syriaca ane solvent volume on high vacuum.Nevertheless, the ratio of phenolic isomers refl ected almost exactly the ratio we have seen when analysing the thyme leaves using the HS technique after peak normalization.An interesting variation appeared when the comparison is made between the wild thyme and the cultivated one.Almost exclusively the wild samples contained carvacrol as the major constituent 11 , while the cultivated samples contained thymol as the major isomer.
This variation appears to be related to the water stress and is not aff ected signifi cantly by the geographical location or the harvesting time (Fig. 5-7).This is consistent with previous work that appeared in the literature lately 16 .Thus the ratio of carvacrol to thymol in the wild sample obtained from northern areas (Nablus/Beta) in June 2003, for example, is 85.8%:14.2%,while the ratio for the cultivated sample obtained from southern areas (Hebron/ Tarqumia) in June 2003 is 7.10%:92.9%(Tab.3) 11 .On the other hand, the ratio of carvacrol to thymol in the wild sample obtained from Ramallah (Kafer Malek) in June 2003 is 86.1%:13.9%while the ratio for the cultivated sample obtained from the same location in March 2004 is 22.1%:77.9%respectively.This observation is almost consistent in most of the samples studied (Fig. 5-7).The dominance of thymol over carvacrol isomer in cultivated thyme by using SD-GCMS is depicted in Fig. 8.

Static Headspace (HS) and GC-MS analysis
In Palestine, little information is available dealing with local thyme.Nonetheless, there is an investigation conducted by using GC-FID analysis on very limited number of thyme samples 17 .We have investigated wild thyme by using HS-GCMS for the fi rst time 11 .The optimal HS parameters chosen were 10 min of equilibration time and 100 o C of vial temperature 18 .Figure 9 shows a typical HS-GCMS chromatogram of a cultivated thyme at the optimised conditions of temperature and time.Baseline separation with good resolution was obtained for almost all the compounds separated (Fig. 9).The results of the HS-GCMS of all the samples appear in Table 2 show obvious diff erences in the percentages of essential oil constituents as follows: Thymol vs. Carvacrol percentages Thymol and carvacrol are isomers that have boiling points of 232 o C, 237 o C respectively.Hence thymol gets eluted earlier than carvacrol in the GCMS (Fig. 8,9).We noticed that the wild growing Palestinian thyme is characterised by the dominant presence of carvacrol irrespective of the harvesting time or the location.Conversely, fresh water irrigation (cultivated) has revealed predominance of the thymol isomer concentration (Fig. 8,9 and Tab.3).This consistent result explains why wild thyme, which is rich in carvacrol, has a distinct worm pungent taste a distinctive property of carvacrol 19 .

Comparison between HS and SD-GCMS analysis of cultivated thyme
We have developed a sensitive and rapid SD,HS-GCMS methods for analysis of volatile, and semivolatile compounds in Palestinian cultivated thyme.Although both the HS and SD complement each other, the combined HS with GCMS has certain advantages over SD technique that have been used in cultivated thyme extraction.It senses directly all volatile and semivolatile terpenoids present in thyme examined whereas using SD large amounts of the volatiles escape, leaving only the semivolatiles, namely thymol and carvacrol isomers.Natural compounds of palestine fl ora.Comparison analysis by static headspace and steam distillation GC-MS of semivolatile secondary metabolites from leaves of cultivated palestinian Majorana syriaca Upon careful comparison between the isomeric distribution of thymol and carvacrol in SD and HS, we found that the isomeric ratios using both methods are very close to each other.Therefore, HS technology would off er faster, reliable, and simultaneous method for the determination of essential oils present in Palestinian thyme.

Fig. 2 .
Fig. 2. Left part, Thyme oil % (W/W) at diff erent harvesting time isolated from cultivated samples collected from Hebron (Tarqumia); Right part, Thyme oil % (W/W) at diff erent harvesting time isolated from cultivated Samples collected from Bethlehem (Menea).

Fig. 3 .
Fig. 3. Left part: Thyme oil % (W/W) at diff erent harvesting time isolated from cultivated samples collected from Ramallah (Kafer Malek); Right part, Thyme oil % (W/W) at diff erent harvesting time isolated from cultivated samples collected from Jenin (Ya'bad).

Fig. 6 .
Fig. 6.Left part, Histograms of the isomeric distribution of thymol and carvacrol from Ramallah (Kafer Malek) at diff erent harvesting time by SD-GCMS; Right part, Histograms of the isomeric distribution of thymol and carvacrol from Jenin (Ya"bad) at diff erent harvesting time SD-GCMS.

Fig. 7 .
Fig. 7. Left part, Histograms of the isomeric distribution of thymol and carvacrol from Tulkarim (Rameen) at diff erent harvesting time by SD-GCMS; Right part, Histograms of the isomeric distribution of thymol and carvacrol from Jaricho at diff erent harvesting time by SD-GCMS.

Fig. 8 .
Fig. 8.Typical TIC of SD-GCMS of cultivated thyme sample harvested from Hebron (Tarqumia) by using DB-5 capillary column.Chromatographic conditions are stated in the experimental sections.I.S stands for the internal standard (4-isopropylphenol) that has been used for quantifi cation of thymol and carvacrol.

Table 1 .
Cultivated thyme samples collection from diff erent areas in the West Bank.Natural compounds of palestine fl ora.Comparison analysis by static headspace and steam distillation GC-MS of semivolatile secondary metabolites from leaves of cultivated palestinian Majorana syriaca

Table 2 .
Variation in yield of the essential oils in cultivated thyme leaves.

Table 3 .
Composition of the major essential oils components in cultivated Majorana syriaca in a diff erent districts at diff erent harvesting time by using HS and SD-GCMS.