Comprehensive GC–FID, GC–MS and FT-IR spectroscopic analysis of the volatile aroma constituents of Artemisia indica and Artemisia vestita essential oils

1 Introduction

Artemisia is one of the largest genera in the family Asteraceae comprising of 200–500 species (depending on the author). 500 species of Artemisia are mainly found in Asia, Europe and North America. Asia seems to represent the greatest number of these species with 150 species found in China, 174 species in the ex USSR, about 50 species reported to occur in Japan, 35 species are found in Iran and 37 species are found in India mostly confined to the North Western Himalayas (Hu, 1965; Poljakov, 1961; Kitamura, 1939; Rechinger, 1986). The 500 species are characterized by common morphological characters: Herbs or small shrubs, highly aromatic, leaves alternate, capitate, racemose, paniculate or capitate inflorescences. Many Artemisia species have a high economic value in several fields as food plants and as anthelminthic and antimalarial in medicine (Shawl et al., 2009).

Artemisia indica Willd. (Asteraceae) commonly known as “Tite pati” is a perennial herb found in the western Himalayas. It has been traditionally employed by local people to alleviate chronic fever, dyspepsia and hepatobiliary ailments (Chopra et al., 1986; Manandhar, 2002; Said, 1984; Kunwar and Duwadee, 2003). Previous phytochemical investigation has led to the isolation of antimalarial compounds namely exiguaflavanone-A, exiguaflavanone-B, maacklain and 2-(2,4-dihydroxyphenyl)-5,6-methylenedioxy benzofuran from the crude methanol extract of the stem of A. indica (Chanphen et al., 1998). Our research group using state-of-the-art techniques has already reported the essential oil composition of various Artemisia species and they mostly contain 1,8-cineole, borneol, camphor, artemisia ketone, limonene, caryophyllene, germacrene-D, davanone as the major essential oil components (Shawl et al., 2009; Rather et al., 2011; Mir et al., 2012). A. indica essential oil has been the focus of some earlier studies from different parts of the world. In one study from Uttarakhand India, Shah et al. studied the steam-distilled essential oil from the aerial parts of A. indica Willd. and reported to have identified 29 constituents in the essential oil with β-caryophyllene (8.3%), germacrene D (6.3%), caryophyllene oxide (6.5%), cis-β-elemenone (10.4%) and selin-11-en-4-α-ol (8.5%) as the major constituents of the essential oil (Shah and Rawat, 2008). G.C. Shah and C.S. Mathela have also studied the essential oil obtained from the aerial parts of A. indica var. indica, a sub-variety of A. indica and have reported 35 chemical constituents with borneol (8.1%), β-cubebene (5.1%), β-caryophyllene (5.4%), germacrene D (5.2%), trans-guaiene (6.5%), δ-cadinene (6.9%), and vulgarone-B (18.9%) as the major components.

Artemisia vestita Wall. Ex DC. is a perennial herb widely used in Tibetan and Chinese traditional medicine for treating various inflammatory diseases. Phytochemical studies on this plant species have been reported and indicated that the plant contains a number of flavones, monoterpenes and sesquiterpenoids (Wang et al., 2005; Yin et al., 2008; Greger et al., 1982; Tan et al., 1999). The essential oil of A. vestita has been the focus of some earlier studies. Chu et al., have studied the essential oil composition of A. vestita aerial parts from China and reported grandisol (40.29%), 1,8-cineole (14.88%) and camphor (11.37%) as the principal oil constituents. In addition, they also reported the insecticidal activity of A. vestita essential oil which could be due to the presence of 1,8-cineole and camphor which are already reported in the literature as insecticidal compounds (Chu et al., 2010). Another study from a different geographical region revealed that the essential oil of A. vestita comprises α-, β- and γ-himachalene, caryophyllene, germacrene-D, himachalol, allohimachalol, α and γ-atlantone, 1,8-cineole, yomogi alcohol, artemisia and santolina alcohol, thujones and thujanols (Weyerstahl et al., 1987).

On scanning the literature reported on the essential oil composition of A. indica and A. vestita, it is evident that the essential oil composition of these two species exhibits a remarkable chemodiversity and is greatly influenced by the geographical region, our objective as part of our Institute’s program was to study the essential oil composition of these two species growing at high altitude locations of Kashmir Himalayas using a combination of capillary GC–FID, GC–MS and FT-IR analytical techniques.

2 Experimental

Plant material and Essential oil isolation: The leaves of A. indica and A. vestita were collected from Daksum, Kokerrnag (Kashmir) and the field station of Indian Institute of Integrative Medicine (IIIM) respectively during July-2011. Artemisia indica was identified by a well-known taxonomist Dr. A. R. Naqshi. A voucher specimen (Specimen number: KASH-769) was submitted to the Centre of Plant Taxonomy, Kashmir University. Two kg of each fresh plant material was subjected to hydrodistillation using a Clevenger type apparatus for 3 h according to the procedure described in the European Pharmacopoeia. Anhydrous Sodium sulfate was used to remove water after extraction. The essential oil was stored in an air tight glass container in a refrigerator at 4 °C. The oil yield was found to be 0.17% (V/W) for A. indica 0.18% (V/W) for A. vestita as calculated on fresh weight basis. The experiment was done in triplicate.

3 GC–FID analysis

GC/FID was carried out on Perkin Elmer auto system XL Gas Chromatograph 8500 series with flame ionization detector (FID) and head space analyser using a fused silica capillary RTX-5 column (30 m × 0.32 mm, film thickness 0.25 μm) coated with dimethyl polysiloxane. Oven temperature was programmed from 60 to 280 °C at 3 °C/min, with injector temperature at 230 °C and detector temperature at 250 °C. Injection volume 1 μl, nitrogen was used as a carrier gas (1.0 ml/min).

4 GC–MS analysis

GC–MS analysis was carried on a Varian Gas Chromatograph series 3800 fitted with a VF-5 ms fused silica capillary column (60 m × 0.25 mm, film thickness 0.25 μm) coupled with a 4000 series mass detector under the following conditions: injection volume 0.5 μl with split ratio 1:60, helium as carrier gas at 1.0 ml/min constant flow mode, injector temperature 230 °C at 3 °C, oven temperature was programmed from 60 to 280 0C at 3 °C/min. Mass spectra: electron impact (EI+) mode, 70 eV and ion source temperature 250 °C. Mass spectra were recorded over 50–500 a.m.u. range.

5 FT-IR spectroscopy measurement

The Fourier Transform-Infrared spectroscopy was carried out on a Perkin Elmer FT-IR using Spectrum software version 10.3.2 using a liquid sampler.

6 Results and discussion

The various chemical constituents identified in the essential oils of A. indica and A. vestita are shown in Table 1, in order of their elution from an RTX-5 column. The corresponding chromatograms are shown in Figs. 1 and 2, respectively. A total of 45 constituents were identified in the essential oil of A. indica constituting 97.6% of the total oil composition. The oil composition is dominated by the presence of oxygenated monoterpenes constituting 65.2% of the total oil composition followed by sesquiterpene hydrocarbons (16.5%), and monoterpene hydrocarbons (10.7%). The principal chemical constituents were found to be artemisia ketone (42.1%), germacrene D (8.6%), borneol (6.1%), chrysanthenyl acetate (4.8%), p-cymene (2.7%), α-thujone (2.7%) and β-pinene (2.4%). The essential oil composition of A. vestita was found to contain a total of 18 components representing 94.2% of the total oil composition. The principal components were found to be 1,8-cineole, (E)-citral, limonene, α-phellandrene, camphor, (Z) and (E)-thujones. Oxygenated monoterpenes were the dominant group of terpenes in the essential oil constituting 73.1% of the total oil composition followed by monoterpene hydrocarbons (17.3%). The results of our study on the essential oil of A. indica and A. vestita reveal that the essential oil composition of these two species growing in Kashmir Himalayas exhibit further chemodiversity. The current results show some similarity with the already published literature, but more importantly exhibit a greater difference as far as the major essential oil components of these two species are concerned. Artemisia ketone prevails in the essential oil of A. indica and has not been reported so far from this plant species in the previously studied reports. Previously reported grandisol was not present in the essential oil of A. vestita growing in Kashmir Himalayas, but the 1,8-cineole content in it goes as high as 46.8% in the essential oil of this plant growing in high altitudes of Kashmir Himalayas. The essential oils of both the species were characterized by the high content of oxygenated monoterpenes imparting a strong characteristic aroma to these two Artemisia species. Literature survey reveals that the essential oil composition of the Artemisia genus has been thoroughly investigated and the chemodiversity in the oil composition has led to many oil-dependent chemotypes for the genus, among which 1,8-cineole/camphor chemotype appears to be predominant in most Artemisia species (Lutz et al., 2008; Govindraj et al., 2008; Lawrence, 1982; Lamberg, 1982). In some other well-known Artemisia species such as A. annua, A. vulgaris, A. diffusa, A. santonicum, A. spicigera, A. afra, A. abiatica, A. austriaca and, A. pedemontana, bornane derivatives (camphor, borneol and bornyl acetate) and 1,8-cineole are the major biochemical marker compounds (Perez-Alonso et al., 2003) Figs. 3 and 4.

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Figure 1 GC–MS Total Ion Chromatogram (TIC) of the leaf essential oil of Artemisia indica.

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Figure 2 GC–MS Total Ion Chromatogram (TIC) of the leaf essential oil of Artemisia vestita.

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Figure 3 FT-IR spectrum of the essential oil of Artemisia indica.

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Figure 4 FT-IR spectrum of the essential oil of Artemisia vestita.

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