Chemotyping the Essential Oil in Different Rosemary ( Rosmarinus officinalis L . ) Plants grown in Kashmir Valley

The aim of the present study was to evaluate the yield, chemical constituents and determine the chemotype of the essential oil obtained from different rosemary plants growing in different accessions of rosemary fields. About four plant samples were analyzed for essential oil yield and the essential oil yield varied from 0.88% to 1.2%. The essential oil samples were further analyzed by Gas Chromatography (GC) for the purpose of identification of chemical constituents present in them. It was contended from the results that the selected plants differed from each other in terms of chemical constituents. Camphor content was found in higher amount in all the four samples, thus it could be inferred that the plants are camphor chemotype.

Rosmarinus officinalis L. is a perennial herb with an evergreen needle-like leaves that belong to the Lamiaceae (mint) family [1][2][3] .Rosemary is a widely used aromatic and medicinal plant 4,5 .The leaf of rosemary is an indispensable spice of the French, Italian and Spanish cuisine.Rosemary is cultivated for the valuable oil, which can be extracted from the harvested plants when flowers are in buds 5 .
Essential oils are natural, concentrated, hydrophobic liquids containing volatile aroma compounds from plants 1 .They are also known as volatile or ethereal oils.The volatile or essential oils correspond to a mixture of hemiterpenoids, monoterpenoids and some sesquiterpenoids that are in conjunction with oil.These mixtures are highly volatile when exposed to air at room temperature, thus the name ethereal oils.They are almost insoluble in water and soluble in alcohol and usually lighter than water.They have high refractive index and many of them are optically active.Essential oils are generally extracted by distillation.The chemical composition of an essential is quite different from one plant to another and the main chemical constituents present in essential oil determine its aroma, taste and biological activity.
There is a wide range of techniques which have been used for extraction and concentration of essential oils as well as chromatographic separation and identification of chemical constituents present in essential oils.Extraction of volatile terpenoids from plant materials and from a wide variety of other matrices is often carried out using hydrodistillation or steam distillation 6 .Rosemary oil was notified for Generally Recognized as Safe (GRAS) status by the Fragrance and Essence Manufacturers Association of the USA (FEMA) in 1965 and has been listed by the U.S. Food and Drug Administration (FDA) for food use (GRAS) 2 .In 1970, the Council of Europe included rosemary oil in the list of substances, spices and seasonings deemed admissible for use, with a possible limitation of the active principles in the final product 2,7 .
It is generally known that the components of essential oils from aromatic plants of the same scientific name could be different according to the plant's habitats, or parts and methods for extraction.This variation in composition is called chemotype 8 .Chemotype occurs when aromatic plants grow under different climatic and soil conditions 8,9 .
The present study was therefore, carried out to evaluate the yield, chemical composition and determine the chemotype of different rosemary plants grown in Kashmir valley.

Sample collection
Fresh leaves of Rosmarinus officinalis L. were collected 15 minutes prior to distillation of essential oil, from the fields of Indian Institute of Integrative Medicine (IIIM) Sanatnagar, Srinagar, Jammu & Kashmir, India.

Distillation of essential oil
The essential oil was obtained by hydrodistillation in a Clevenger (PERFIT, INDIA) for 3 hours 3,10 .Briefly, 250 g of freshly collected leaves were taken in 500 ml round-bottom flask (PERFIT, INDIA) followed by addition of water in the ratio of 1:6 (w/v) and was distilled for about 3 hours.Essential oil from each of the samples was collected and dried over anhydrous sodium sulphate 3,11 .Oil was then stored at 4 o C until analysis with Gas chromatography (GC) 3 .

Chromatography
The analysis of the oil was carried out by following the method of Amin et al. ( 2013) on a gas chromatograph Perkin Elmer -Auto XL equipped with head space analyzer and FID, using a fused-silica capillary column (30 m x 0.32 mm; 0.25 ¼m film thickness).The oven temperature was programmed from 60 o C to 250 o C at 5 o C per minute.The injector and detector temperatures were set at 250 o C and 270 o C, respectively.Nitrogen at a pressure of 8 psi was used as the carrier gas.The identification was done on the basis of retention time, Kovats index, MS Library search (NIST & WILEY).Retention indices (RI) of the chemical components of samples and authentic compounds were determined.The relative amounts of the identified compounds were calculated based on GC peak areas without using correction factors.

Yield of essential oil
The yield of essential oil from each of the four samples ranged from 0.88% to 1.2% (Figure 1).It is clear from Figure 1 that the highest essential   oil content was found from plant -P 55 /B 2 (1.2%) and lowest yield from plant P 57 /B 2 (0.88%).

Identification of compounds
Gas chromatographic analysis of the essential oil resulted in the identification of 18 different components representing about 97.2217% of the essential oil of P 55 /B2; 19 components representing 97.6266% of the essential oil of P 57 /B 2 ; 17 components representing about 76.5109% of the essential oil of P 67 /B 2 and 22 components representing about 86.0686% of the essential oil of P 178 /B 4 .All of the identified compounds and their percentage present in each of the plant are summarized in Tables 1, 2, 3 & 4.
Camphor was found to be present in the highest concentration (53.3871%) which is much higher than that reported by Verma et al. 10 followed by 1, 8-cineole (11.6516%), alpha-pinene (5.6862%) and camphene (5.4258%).It is thus clear from the results that all the plants are camphor chemotype.
It is clear from the results that the yield of essential oil from samples of different plants varied considerably.The composition of the essential oil varies from plant to plant as can be seen in the tables 2, 3, 4 and 5.The camphor content of the essential oil for all the 4 samples is higher than the value of 15.64% and 22.01% reported by Verma et al. 10 and Shawl 12 , respectively.The camphor content also varies within the samples.Since the camphor content of essential oil is higher, therefore, all the samples are camphor chemotype.The chemical composition of oil depends on how and where the plant was grown, harvested and distilled.When the conditions cause permanent variation in the chemical composition of essential oil of rosemary plants, such plants are called chemotypes.Three principal chemotypes of R. officinalis L. have been reported which include camphor/borneol, cineole and verbenon 13 .Typical components of rosemary are 1,8-cineole, ±-pinene and camphor 14 and the relatively stable ratio of these components defines each chemotype.It is known that the rosemary oils are widely divided into two chemotypes by the ratio of major components; one with more than 40% of 1,8-cineole and the other with almost the same percentage of 1,8-cineole, ±-pinene and camphor 15 .These findings are in concomitance with our present results.