Abstract: Studies were conducted to investigate the composition of essential oil of wormwood (Artemisia absinthium L.) growing wild in Iran. The wormwood aerial parts were harvested in full blooming time from an area between Deylaman and Asiabar villages, at Alborz altitudes in Guilan province in September 2005. Aerial parts were dried at shade (room temperature) for several days and their essential oil was extracted by hydrodistillation method in a Clevenger apparatus and analyzed by GC/MS. Results showed that essential oil yield was 1.3%. Twenty eight components representing 93.3% of the oil were identified, which were mostly monoterpenes. β- pinene and β- thujone were the main components, which their contents were 23.8 and 18.6% respectively. The largest part of the essential oil was formed by hydrocarbon monoterpenes (47.8%). The results proved that chemotype of the studied wormwood essential oil was specific and different from other wormwood essential oil chemotypes, which have been reported so far.
INTRODUCTION
Artemisia absinthium L. is an aromatic plant of the family Asteraceae, subfamily Asteroideae, tribe Anthemideae and is known by the common names wormwood (UK), absinthe (France), wermut (Germany) and afsantine (Iran) (Zargari, 1989; Wright, 2002). The plant grows in North and East of Iran (Zargari, 1989) and also is very common in Turkey (Aslan et al., 2005; Kordali et al., 2006). The herb is native to warm Mediterranean countries, usually found growing in dry waste places such as roadsides, preferring a nitrogen-rich stony and hence loose soil. Wormwood has been naturalized in northeastern North America, North and West Asia and Africa. The plant`s essential oil and bitter principles underlie its medicinal and commercial significance (Wright, 2002).
A. absinthium extracts and essential oils are used for healing various diseases (Lawless, 1999; Balz, 1996; Wright, 2002). Oil of A. absinthium has been found to repel fleas and flies (Duke, 1995) and mosquitoes (Morton, 1981) and to kill houseflies (Kaul et al., 1978). Anthelmintic, antibacterial, antifungal, insect repellent, narcotic, digestive, tonic and other bioactivities are characteristic of preparations from wormwood plants. Their stimulant property is dependent on bitter substances as artabsin (sesquiterpene lactone) and absinthin (dimmer of sesquiterpene lactones) present in plant extracts (Wright, 2002). Wormwood essential oil components 1, 8-cineole, cis (a) and trans (ß)-thujones help people to withstand cold and other hardships of the Himalayan region (Wright, 2002; Aslan et al., 2005; Kordali et al., 2006). Large amounts of the above compounds are toxic (Woolf, 1999). Seizures may be caused by 1, 8- cineole and thujones for chronic users. In addition, thujones may evoke dementia (Wright, 2002).
The major constituent of wormwood oil is thujone, present at levels of approximately 40-70% of the oil (Tucker et al., 1993). Other constituents present at significant levels include myrcene (<35%), a- pinene (6%) and nerol (3%) in wormwood oil of Russian origin (Goraev et al., 1962); camphor (6%), ?-cymene (4%), limonene (4%) and a- pinene (4%) in Spanish wormwood oil (Mugica and Ochoa, 1974); ß-phellandrene (10%), a-humachalene (7%) and ß- caryophyllene (5%) (Kaul et al., 1979; Aslan et al., 2005; Kordali et al., 2006). High levels of thujanol and thujyl acetate (60-70%), myrcene (35%), camphor and 1, 8-cineole were also determined in wormwood essential oils (Wright, 2002). Thujones, trans-sabinyl acetate, cis-chrysanthenyl acetate and cis-epoxyocimene are the most common constituents in wormwood essential oils (Arino et al., 1999; Wright, 2002; Juteau et al., 2003).
This study was conducted to obtain information about essential oil of wormwood growing wild in Iran. It presents the chemical composition of essential oil from aerial parts of A. absinthium L. collected in a habitat from North of Iran.
MATERIALS AND METHODS
Plant material: The aerial parts of A. absinthium were collected in full blooming time from an area between Deylaman and Asiabar villages, at Alborz altitudes in Guilan province in September 2005. The aerial parts were dried at room temperature (20-25°C) for several days until measurement of weight. Voucher specimens were deposited in the Herbarium of the faculty of agricultural sciences, Shahed University, Tehran, Iran.
Isolation procedure: The essential oil was prepared by hydrodistillation for 2.5 h using a Clevenger-type apparatus. The oil was dried over anhydrous calcium chloride and stored in sealed vials at low temperature (2°C) before analysis.
Gas chromatography: GC analysis was performed using a Shimadzu GC-9A gas chromatograph equipped with a DB-5 fused silica column (30 mx0.25 mm i.d., film thickness 0.25 µm). Oven temperature was held at 40°C for 5 min and then programmed to 250°C at a rate of 4°C min-1. Injector and detector (FID) temperature were 260°C; helium was used as carrier gas with a linear velocity of 32 cm sec-1.
Gas chromatography-mass spectrometry: GC-MS analyses were carried out on a Varian 3400 GC-MS system equipped with a DB-5 fused silica column (60 mx0.25 mm i.d.). Oven temperature was 40-250°C at a rate of 4°C min-1, transfer line temperature 260°C, carrier gas helium with a linear velocity of 31.5 cm sec-1, split ratio 1/60, ionization energy 70 eV, scan time 1 sec, mass range 40-300 amu.
Identification of components: The components of the oil were identified by comparison of their mass spectra with those of a computer library or with authentic compounds and confirmed by comparison of their retention indices, either with those of authentic compounds, or with data published in the literature (Adams, 1995; Shibamoto, 1987).
RESULTS AND DISCUSSION
The chemical composition of the A. absinthium essential oil is shown in Table 1. The components are listed in order of their elution on the DB-5 column.
| Table 1: | Chemical composition of the
essential oil of Artemisia absinthium |
| RI: Retention index on DB-5 column | |
Twenty eight compounds were identified in the essential oil of A. absinthium, representing 93.3% of the total oil components. The major components of the oil were found to be ß- pinene and ß- thujone, their contents being 23.8 and 18.6%, respectively. The other major components were sabinene, Cubenol, linalool, myrcene, a-pinene, a- dehydro- ar-himachalene, a-phellandrene and germacrene D. Monoterpenoids comprised a large part (75.4%) of wormwood oil. The largest part of the essential oil was formed by hydrocarbon monoterpenes (47.8%) and the other parts included oxygen-containing monoterpenes (27.6%), hydrocarbon sesquiterpenes (9.1%) and oxygen-containing sesquiterpenes (8.8%). Compounds with a sabinene carbon skeleton (sabinene, a and ß- thujone, iso-3-thujanol) made up 29.3% of total oil.
The oil isolated by hydrodistillation from the aerial parts of A. absinthium was found to be dark green to brown liquid that can be attributed to the presence of chamazulene and its yield was 1.30% (v/w). Essential oil contents between 0.2 and 1.5% in the crude drug have been reported in the literature (Wichtl, 1989). The most abundant constituent in the essential oil was ß-pinene, which has not been reported for the species from other countries till now. It can be said that ß-pinene is a new component for wormwood oil and possibly restricted to Iran. It has been reported that A. absinthium plants produce several chemotypes of essential oils in different countries and the main component of wormwood essential oils is thujone (Juteau et al., 2003; Wright, 2002). A sample collected in Argentina, had the ß-thujone (60%) as main constituent (Sacco and Chialva, 1988). The plants and their parts from Croatia contained notable amounts of (Z)-6, 7-epoxyocimene besides the first major constituent trans-thujone (Juteau et al., 2003). Some volatile oils from North Italy (Wright, 2002), France (Juteau et al., 2003; Wright, 2002) and Spain (Arino et al., 1999) did not contain thujones. The predominant components of the above oils were (Z)-6, 7-epoxyocimene and/or (Z)-chrysanthenyl acetate. Two chemotypes were determined in Spain: one oil contained both the above compounds as the main ones, but another oil was characterized by (Z)-6, 7-epoxyocimene with minor amounts of other constituents (Arino et al., 1999). Carnat et al. (1992) found cis- chrysanthenol as the major component in the oil of plants grown in central France. Sabinyl acetate prevailed in some oils from different countries (Arino et al., 1999; Wright, 2002). Chiasson et al. (2001) reported ß-thujone as a main component (32.1%) in wormwood essential oil from Canada. Oils isolated from native and cultivated wormwood plants growing in Oregon contained ß-thujone (40%) and cis-epoxyocimene (25%) in the native plants and sabinene (30%) and myrcene (30%) in the cultivated plants (Tucker et al., 1993). Bornyl acetate was reported as the major constituent (23%) of a sample collected in Cuba (Pino et al., 1997), but it was not observed in the present study.
It is suggested that the studied wormwood in this work synthesizes a kind of essential oil component, which is different from other components reported so far. The quality and yield of essential oils from Anthemideae plants are influenced by the various factors such as harvesting season (Cornu et al., 2001), fertilizer and the pH (ideal in acidic, pH 4.5-5.4) of soils (Alvarez-Castellanos and Pascual-Villalobos, 2003), the choice and stage of drying conditions (Tateo and Riva, 1991), the geographic location (Maffei et al., 1994), subspecies (Goren et al., 2001), choice of plant part or genotype (Mishra et al., 1999; Nori-Shargh et al., 1999; Keskitalo et al., 2001) or extraction method (Scalia et al., 1999).
Monoterpenoids comprised a large part of wormwood oil in the study. This corroborates the finding of Judpentiene and Mockute (2004) which reported that monoterpenoids comprise a large part (55.7-80.2%) of wormwood oil in Lithuania. The largest part of the essential oil was formed by hydrocarbon monoterpenes (47.8%) and the other parts included oxygen-containing monoterpenes (27.6%), hydrocarbon sesquiterpenes (9.1%) and oxygen-containing sesquiterpenes (8.8%). Judpentiene and Mockute (2004) reported that the largest part of the essential oil in wormwood is formed by oxygenated monoterpenes (47.1-66.7%) and the hydrocarbon monoterpenes were 8.2-24%.
Compounds with a sabinene carbon skeleton made up 29.3% of total oil. These compounds were comprised (33.9-61.0%) of wormwood oil in Lithuania (Judpentiene and Mockute, 2004). Chamazulene (0.9%) was present in the oil which contributed to the characteristic colour of wormwood oil. A small percentage of chamazulene (0.2%) was found in the Cuban oil (Pino et al., 1997) and in two chemotypes studied of Spanish oil it was 3 and 0.3% (Arino et al., 1999).
ACKNOWLEDGMENTS
The authors gratefully acknowledge the financial support of the University of Shahed and scientific assistance of Dr. H. Abbasipour.