INTRODUCTION:

Medicinal plants represent an economically important and healthy component of biodiversity components. It is necessary to do a full inventory of medicinal plants in any country flora in order to protect and ensure the continuity of use and therefore protect endangered medicinal species in the wild. The use of medicinal plants is considered to be more common in traditional medicine and pharmaceutical tool supplementary worldwide. Many communities support natural products collected from ecosystems for medicinal purposes as well as for food purposes^1,2.

Wild thyme Thymus vulgaris L. is a Herbaceous Perennial to dendritic Plant that belong to Lamiaceae Family, and grow in the wild and is cultivated for agricultural, medical, ornamental and edible purposes.

Received on 26.02.2015 Modified on 16.03.2015

Research J. Pharm. and Tech. 8(4): April, 2015; Page 437-442

DOI: 10.5958/0974-360X.2015.00073.6

Stem is square section list, coarse texture. Leaves are simple narrow gray, inflorescences are Rasimah found on the terminal pregnant, flowers are small purple or white in color. Height ranges 10-35 cm, native to the Mediterranean region. It is generally used as fresh, bruised, dried product or as Aromatic oil³.Economic and medical importance is increasing in North America, Europe and North Africa ⁴. It grows at different altitudes starting from sea level to a height of 2,000 meters above sea level ⁵. Thyme was used in medicine by the Greeks, Romans and ancient Egyptians, whose name is derived from the Greek word (thymos) means fragrance. Thyme oil is a volatile oil that has a strong aromatic smell, reddish brown color, drawn from the flowering tops and fresh and dried leaves by distillation with water or steam and yield 0.7-1.0%. The main chemical components are: “thymol, Carvacrol, α-pinene, β-pinene, Borneol, Linalool, β-Simine and Camphene”. Thyme oil is very effective and should not be used during pregnancy or in cases of high blood pressure due to the phenols (Thymol and Carvacrol), which can cause mucous membranes and skin irritation. Therapeutic properties: antibacterial, antispasmodic, antiseptic, anti-rheumatic, repelling gas, diuretic, expectorant, hypertensor, insecticide, stimulant and tonic⁶.

This oil is classified among the top ten essential oils and found that the best amount of oil and the highest content of Thymol was obtained at the beginning of flowering⁷. As some studies suggest that the thyme plant widely used for respiratory diseases (Cough) especially its aqueous extract, which is used in some countries as a raw material to preparate many pharmaceutical plant⁸. Survey of more than 60 component of thyme was done, including Thymol (72.900 - 482.600 ppm), Carvacrol (10.000- 63.800 ppm) ⁹.

Thymol or 2-Isopropyl-5-methylphenol (C₁₀H₁₄O) gives the thyme used as food eaten a strong distinct flavor ¹⁰, and is a prominent part of the natural compounds known as Biocides, and has the properties of an anti-bacterial when used alone or with other compounds such as Carvacrol, it also has an impact that can reduce bacterial resistance to some common drugs, penicillin ¹¹.

Carvacrol and Thymol have shown a strong antidote genetic mutations influence¹², In addition, there is evidence that Thymol has counter tumor properties¹³. Naturall , There is Carvacrol or 5-isopropyl-2-methylphenol (C₁₀H₁₄O) in the essential oil of thyme and each type contains the percentage of 5-75% of it. It is used as an additive to foods to prevent bacterial contamination 14. It inhibits several types of bacteria growth, such as Escherichia coli¹⁵. It also causes damage to the cell membrane of Pseudomonas aeruginosa bacterium¹⁶. One study suggests that Carvacrol stimulates programmed death of prostate cancer cells, Carvacrol was tested with different concentrations and stages against human prostate cancer cells, Its use has led to positive inhibition effect of cancer cells, and preliminary results of this research indicates to the existence of great potential for using Carvacrol as an anti-cancer¹⁷. Pinene or (1S,5S)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane (C₁₀H₁₆): is an organic compound of terpenes which has in nature two isomers α-pinene and β-pinene. This two figures are important components in pine resin and other conifers as well as in other plants such as Sage, thyme and others. Pinene is using in chemical industries where oxidation selectivity of Pinene with some stimuli gives many aromatic compounds, such as synthetic fragrances¹⁸. Extraction of essential oils from Sage cultivated and wild in southern Lebanon was done by steam distillation, oil content of Thymol and Carvacrol was determined by GC-MS. Carvacrol was the highest component in all samples (49.8%) compared to Thymol (31.5%)¹⁹. Thyme oil extracted from plant Coridothymuscapitatus leaves has been analysed with the same previous technique, and essential oil mainly contained the following compounds Carvacrol (35.6%), µ-Simine (21%), Thymol (18.6%) 20. The importance of this research lies in the economic, environmental and medical use of wild thyme and the continuous degradation which affects it, in addition to the current trend towards herbal remedies and the absence of a similar study on wild thyme at AL-Qadmous area. So the aim of this research is to study the variations in the chemical composition (statistically) of wild thyme plant which is wide spreaded in the Syrian Coast in terms of aromatic oil content “Thymol, Carvacrol and Pinene” and which is evaluated by gas chromatography - mass spectrometry GC-MS method.

MATERIALS AND METHODS:

1. Plant material and samples collection:

Wild thyme samples were collected from three different locations at AL-Qadmous area after dividing to sectors depending on height and distance from the sea (including plains and mountain slopes and on different types of soil):

· A₁: (250 Meter) above sea level.

· A2: (560 Meter) above sea level.

· A₃: (1000 Meter) above sea level.

Collection process was held in May and June of 2012, when plants were at the beginning of the flowering stage. All samples were dried aerially without lightning at room temperature for two months.

2. Essential oil extraction:

The amount of 200 g of dried plant from each of the studying sites was taken where essential oil extracted by distillation in accordance with the European Pharmacopeia, 1996 for a period of three hours using a homemade distillation device. Oil was withdrawn and placed in a glass test sealed tubes and then tubes were wrapped blocked from light and kept at a temperature of 4C^ο until GC-MS analysis.

3.Analysis by Gas Chromatography-Mass Spectrometry (GC-MS):

Qualitative analysis of oil samples was carried out using GC-MS QP2010 Plus device manufactured by Shimadzu Japanese company and shown in Figure (4). GC device is equipped with column of silica (30 m x 0.25 mm, and a thickness of 0.25 micrometer). MS-GC device turning conditions were: Use helium as a carrier gas, Injector temperature is 250 C^ο. Detector temperature is 280 C^ο. Column temperature has been raised from 50 C^ο to 130 C^ο at a rate of 20 C^ο/min, then heating temperature was kept for one minute and then lifted from 130 C^ο to 280 C^ο at a rate of 9 C^ο/min, then was kept on 280 C^ο for 8.33 minutes so that the total duration of heating program is 30 minutes. Size of Injected sample in the device is (1) microliter of oil. Chromatographic methods are used in qualitative and quantitative analysis of extracts of thyme T. vulgaris L. essential oil after extraction by Steam distillation 22.Identification of oil compounds depended on Retention Time (RT) and comparing these components spectrom electronically with the library included in the GC-MS system 23. Quantitative composition of oil components (%) was determined by calculating the areas of peaks which represented it in the Chromatogram and then the percentage of each of these values relative to the total value.

4. Statistical Analysis:

Microsoft Office Excel 2007 program was used in the statistical analysis of data through a test of significant differences analysis using ANOVA: Single Factor and then less significant difference LSD values were calculated When significant level of 5%.

5 RESULTS AND DISCUSSION:

The main objective of this study is to compare the essential oils extracted from wild thyme plants widespread naturally in three locations at AL-Qadmous area in terms of their containment of compounds “Thymol, Carvacrol and Pinene” as analyzed by GC-MS which has been described. Figures (1,2, and 3) describe the Chromatogram (ICS) of oil samples from three sites A₁, A2, A₃, respectively.

The results in Table (1) showed that there are differences between plants chemical compounds content in these locations. Statistical analysis has improved the significant differences and the results were:

Table (1): Percentage (%) of each of Pinene, thymol and Carvacrol in the wild thyme essential oil samples in the study locations and the value of LSD (5%) .

Compound	Site			LSD (5%)
Compound	A1	A2	A3	LSD (5%)
Pinene	14.57	12.15	22.58	0.51086
Thymol	6.28	7.80	2.09	0.26938
Carvacrol	79.15	80.05	75.32	0.48621

a. Content of Thymol:

Figure (4) recorded that the highest percentage of Thymol (7.80%) appeared at the site A2 while the lowest rate (2.09%) appeared at the site A₃. Statistical analysis of the existence of significant differences between plants content of Thymol in the study locations, where plants in the site A2 outperformed with high significantly on other sites plants, followed by site A1, and the lowest percentage was found at A3 site.

Figure (4): Percentages of Thymol compound in the wild thyme essential oil in three locations at AL-Qadmous area (%)

b. Content of Carvacrol:

Figure (5) showed that the highest percentage of Carvacrol (80.05%) appeared at the site A₂, while the lowest percentage (75.32%) appeared at the site A3.Statistical analysis has shown significant differences between plants content of Carvacrol in the study sites where the plants on the site A₂ outperformed by high significantly on the plants at all the other sites, followed by plants of the two sites A₁ and A3, which outperformed by all other sites with significant differences.

Figure (5): Percentages of Carvacrol compound in the wild thyme essential oil in three locations at AL-Qadmous area (%)

c. Content of Pinene:

Figure (6) indicated that the highest percentage of pinene (22.58%) appeared at the site A3, while the lowest percentage (12.15%) appeared at the site A₂.

Statistical analysis of proved the significant differences between plants content of Pinene in the study locations where outperformed plants in the site A3 sign by high.significant on all the other sites, then A1 site.

Figure (6):Percentages of Pinene compound in the wild thyme essential oil in three locations at AL-Qadmous area(%)

Depending on the percentages of the main components of Thymol and Carvacrol in this study, the greatest component in all samples was Carvacrol and rate values ranged between 75.32 and 80.05%, while Thymol values ranged between 2.09 and 7.80%. This is in agreement with the results of 19, where Carvacrol dominated the essential oil composition for all samples and obtained the highest peak of 49.8% versus 31.5% for Thymol. Also Thymol had low concentration (0.24%) in the results of Imelouane ²⁴. Thymol isa major compound in thyme oil.The results of studies conducted by Abu-Lafi ²⁵ on the Palestinian Thyme grown that the most compounds percentage in oil are Thymol, Carvacrol and α-pinene. GC-MS analysis has demonstrated that Thymol is the main phenolic compound.Pinene compounds percentage ranged between (11.86 - 22.58%). This is in agreement with the findings of 24 where the percentage was 13.25%.

CONCLUSIONS:

Chemical composition of thyme essential oil has changed by geographic location (altitude, soil and other ) where the Site A2 was distinguished by its plants which contained the highest percentage of Thymol and Carvacrol, whereas the site A3 was distinguished by its plants which contained the highest percentage of pinene. Significant differences were apparent between the studied samples according to their locations. Thyme essential oil in the study sites at AL-Qadmous area contains mainly Carvacrol compared with the other two components (Thymol and pinene compounds).

ACKNOWLEDGEMENT:

I would like to thank Dr. Oussama Mansour , Faculty of Pharmacy- Alandalus University-Syria, for his scientific contribution and guidance not to forget the language assistance provided by Mr. Ayham Aljghami, instructor at The Higher Institute of Languages-Tishreen University-Syria, during the writing process.

REFERENCES:

1. Al-SaidM Set al- Medicinal plant diversity in the flora of Saudi Arabia 1: a report on three plant families – Science Direct - Fitoterapia / Volume 75, Issue 2, March 2004:149-161.

2. Youssef A, Mansour O. A Histological – Chemical Comparative Study of Two Species of Inula (I. Viscosa& I. Graveolens) Distributed in The Syrian Coast, Tishreen University Journal for Research and Scientific Studies – Health Sciences Series., vol. 35, no. 1, 2013: 71- 92.

3. Anonymous. Thyme: Thymus spp. Labiatae. In: Fortin, F. (Ed.) , The Visual Food Encyclopedia: The Definitive Practical Guide to Food and Cooking. MacMillan, New York.1996: 477.

4. Letchamo, W. , Gosselin, A. Transpiration, essential oil glands, epicuticular wax and morphology of Thymus vulgaris are influenced by light intensity and water supply. J. Hort. Sci.71, 1996 :123–134.

5. Morales, R. Taxonom´ıa de los g´eneros Thymus (excluida la secci´ on Serpyllum) Thymbra en la Pen´ınsulaIb´erica. Ruizia, 3. Monograf´ıas Del Real Jard´ınBot´anico, Madrid.1986.

6. Esoteric Oils. Thyme oil (Thymus vulgaris) - information on the origin, source, extraction method, chemical composition, therapeutic properties and uses, Esoteric Oils CC and Sallamander. 2011.

7. Badi, N.H. et al. Effects of spacing and harvesting time on herbage yield and quality/quantity of oil in thyme, Thymus vulgaris L. Elsevier B.V Industrial Crops and Products . 19; 2004 : 231–236 .

8. Al-Sheibany , et al.Isolation and Identification of Volatile oils from Iraqi Thyme ( ThymbraSpicata ) and study the antimicrobial activity - Babylon University.Journal of Chemistry. volume 18 , 2005 : 289-298.

9. Nijssen, L.M., et al. Volatile Compounds in Foods and Beverages. TNO Nutrition and Food Research Institute, Zeist, The Netherlands.1999.

10. Fenaroli, Fenaroli's Handbook of Flavor Ingredients, Volume I, 3rd Edition. CRC Press. London.2005.

11. Holley, R.A. and Palaniappan, K. Use of natural antimicrobials to increase antibiotic susceptibility of drug resistant bacteria , International Journal of Food Microbiology - Volume 140, Issues 2–3, 2010: 164–168 .

12. Mezzouga N. et al. Investigation of the mutagenic and antimutagenic effects of Origanum compactum essential oil and some of its constituents Mutation Research, Genetic Toxicology and Environmental Mutagenesis- Vol. 629, Issue 2, 2007 : 100–110 .

13. Andersen A. Final report on the safety assessment of sodium p-chloro-m cresol, p-chloro-m-cresol, chlorothymol, mixed cresols, mcresol, o-cresol, p-cresol, isopropyl cresols, thymol, o-cymen-5-ol, and Carvacrol. Int J Toxicol. 25 Suppl 12006:29-127.

14. Ultee A, Smid EJ. "Influence of carvacrol on growth and toxin production by Bacillus cereus". Int. J. Food Microbiol. 64 (3);2001 : 373–8.

15. Olsen CE, et al."Storage Stability and Antibacterial Activity against Escherichia coli O157:H7 of Carvacrol in Edible Apple Films Made by Two Different Casting Methods". J. Agric. Food Chem. 56 (9); 2008 :3082

16. Cox SD, Markham JL."Susceptibility and intrinsic tolerance of Pseudomonas aeruginosa to selected plant volatile compounds". J. Appl. Microbiol. 103 (4); 2007: 930–6.

17. Bavadekar S.www.liu.edu / Brooklyn / Press – Releases / 2012 /April / BK-PR-Apr25-2012.aspx.

18. Dorman, J.D., Deans, S.G. "Antimicrobial agents from plants: antibacterial activity of plant volatile oils". J. Appl. Microbiol , Vol 88; 2000 : 308–316.

19. Zein S. , et al .Variation of thymol, carvacrol and thymoquinone production from wild and cultivated Origanum syriacum of South Lebanon / Journal of Medicinal Plants Research, Vol. 6(9), 2012 :1692-1696.

20. Goren A. C. , et al. Analysis of Essential Oil of Corido thymus capitatus (L.) and Its Antibacterial and Antifungal Activity / Naturforsch. 58c; 2003 : 687-690 .

21. European Pharmacopoeia. Pennsylvania 1996 .

22. Zeković Z. , Lepojeviíc Z. and Vujić DJ. Supercritical extraction of thyme (Thymus vulgaris L.) .Springer-Chromatographia. Volume 51, Issue 3-4,2000 : 175-179 .

23. Adams R.P. Identification of Essential Oil Components by Gas Choromatography/ Mass Spectroscopy (GC-MS). Allured Publication Corp. Carlo Stream, Il -USA; 1995 :456 .

24. Imelouane, B., et al. Chemical composition of the essential oil of thyme (Thymus vulgaris) from Eastern Morocco. Int. J. Agr& Bio ,V. 11- 2009: 205–208.

25. Abu-Lafi S et al. Natural Compounds of Palestine Flora. Comparison Analysis by Static Headspace and Steam Distillation GC-MS of Semivolatile Secondary Metabolites from Leaves of Cultivated Palestinian Majorana syriaca Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. V. 151 I. (1): 2007:21–29.