Introduction

Rosmarinus officinalis L. is a perennial herb with an evergreenneedle-like leaves that belong to the Lamiaceae (mint) family^1-3. 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, whichcan be extracted from the harvested plants when flowers are in buds⁵.

Essential oils arenatural, concentrated, hydrophobic liquids containing volatile aroma compounds from plants¹. 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 distillation⁶.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 chemotype⁸. 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.

Materials and Methods

Sample Collection

Fresh leaves of RosmarinusofficinalisL.were collected 15minutes 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, 250g of freshly collected leaves weretaken 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 sampleswas collected and dried over anhydrous sodium sulphate^3,11. Oil was then stored at 4^oC 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 (30m x 0.32 mm; 0.25 μm film thickness). The oven temperature was programmedfrom 60^oC to 250^oCat 5^oC per minute.The injector and detector temperatures were set at 250^oC and 270^oC, 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 samplesand authentic compounds were determined.The relative amounts of the identified compounds were calculated based on GC peak areas without using correction factors.

Results and Discussion

Yield of Essential Oil

The yield of essential oil from each of the foursamples ranged from 0.88% to 1.2% (Figure 1). It is clear from Figure 1that the highest essential oil content was found from plant – P₅₅/B₂(1.2%)and lowest yield from plant P₅₇/B₂(0.88%).

Figure 1: Percentage yield of essential oil from different Rosemary (Rosmarinus officinalis L.) plants   Click here to View figure

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 P₅₅/B2; 19 components representing 97.6266% of the essential oil of P₅₇/B₂; 17 components representing about 76.5109% of the essential oil of P₆₇/B₂ and 22 components representing about 86.0686% of the essential oil of P₁₇₈/B₄.All of the identified compounds and their percentagepresent 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 Vermaet al.¹⁰followed 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.

Table 1: Composition of essential oil of Rosemary (Rosmarinusofficinalis L.) (P₅₅/B₂)

Figure 2: Gas chromatogram of essential oil Rosmarinusofficinalis L. (P55/B2)   Click here to View figure

Table 2: Composition of essential oil of Rosemary (Rosmarinusofficinalis L.) (P₅₇/B₂)

RT: Retention time; KI: Kovat index (relative to n-alkane)

Figure 3: Gas chromatogram of essential oil Rosmarinusofficinalis L. (P57/B2)   Click here to View figure

Table 3: Composition of essential oil of Rosemary (Rosmarinusofficinalis L.) (P₆₇/B₂)

RT: Retention time; KI: Kovat index (relative to n-alkane)

Figure 4: Gas chromatogram of essential oil Rosmarinusofficinalis L. (P67/B2)   Click here to View figure

Table 4: Composition of essential oil of Rosemary (Rosmarinusofficinalis L.) (P₁₇/B₄)

RT: Retention time; KI: Kovat index (relative to n-alkane)

Figure 5: Gas chromatogram of essential oil Rosmarinusofficinalis L. (P17/B4)   Click here to View figure

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 byVermaet al.¹⁰ and Shawl¹², 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 calledchemotypes. Three principal chemotypes of R. officinalis L. have been reported which include camphor/borneol, cineole and verbenon¹³.Typical components of rosemary are 1,8-cineole, α- pinene and camphor¹⁴and 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, α-pinene and camphor¹⁵. These findings are in concomitance with our present results.

Acknowledgment

Author is highly grateful to Indian Institute of Integrative Medicine, Sanatnagar, Srinagar, Jammu& Kashmirfor providing an opportunity to work in their lab.

Conflict of Interest

The author reports no conflict of interest.

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