It has long been recognized that naturally occurring substances in higher plants have anti bacterial activity (Cha et al., 2005). The genus Artemisia is one of the largest in the Asteraceae family, consisting of more than 800 species which are wide spread all over the world (Judzentiene and Buzelyte, 2006).
Artemisia species has been known as a folk medicine resource, which used for its anti-inflammatory, diuretic agent and treatment of epidemic hepatitis (Qiu Guo et al., 2004). Essential oils make a major contribution in to the plants biological activity as well (Judzentiene and Buzelyte, 2006). Sesquiterpene lactones and acetylenes have been reported from the some species of Artemisia such as A. assoana, A. lantana and A. pedemontana (Preze-Alonso et al., 2003) and artemisia ketone, 1, 8-cineole, davanone, camphor, β-thujone, myrcene and germacrene-D have been also reportedin essential oil of A. absinthium, A. scoparis and A. vulgaris (Chericoni et al., 2004; Preze-Alonso et al., 2003; Morteza Semnani et al., 2004; Rana et al., 2003; Perazzo et al., 2003). Numerous studies in the literature have reported the antibacterial and antifungal activity of oils isolated from various species of Artemisia (Qiu Gua et al., 2004). Essential oil of aerial parts of Artemisia annua with camphor (44%) and germacrene-D (16%) inhibited the growth of Entrococcus faecalis (Juteau et al., 2002).
Artemisia siberi Besser subsp. Siberi has been known as endemic medicinal herbs with wide dispersal that used by the rural healers in traditional medicine in Chaharbagh region located in north of Iran.
In this research project study, the chemical constituents and antibacterial activity of volatile oil obtained from A. siberi in this region were assessed.
MATERIALS AND METHODS
Plant material: Aerial parts in blooming of Artemisia sieberi was collected in late August of 2005 from Chaharbagh region, 70 km east of Gorgan in Golestan province in the north of Iran. Subsequently, it was dried in the shade for one week and was powdered. Their botanical name identified in the Plant Systematic Laboratory, College of Science, Islamic Azad University of Gorgan Branch, Iran where voucher specimens were deposited.
Two hundred grams of the dried powders were separately subjected to hydro distillation for 2 h, in full glass apparatus. The oil was isolated using a Clevenger type apparatus and stored frozen in dark glass bottles until they were used (Orav et al., 2006).
Oil analysis: The oils were analyzed by GC (9-A-shimadzu) and GC-MS
(Varian-3400) column (DB-1, 60 mm 0.25 mm fused silica capillary column film
thickness 0.25 μm using a temperature program of 50-250°C at a rate
of 4°C min-1, injector temperature 260°C, carrier gas: Helium,
the constituents were identified by comparison of their mass spectra with those
in the computer library and with authentic compounds. The identifications were
confirmed by comparison of their retention indices with those of authentic compounds
or with literature data. The components of the oils were identified by matching
their mass spectra and retention indices with those of the Wiley 275 library
in the computer library and literature. The yield of each component was calculated
per kg of the plant material, while its percentage composition was determined
from the peak areas of the total oil composition.
Tested organisms: The test organisms used in the study were obtained from Persian Type Culture Collection, Tehran, Iran (PTCC), namely: E. coli (PTCC No. 1330), P. aeroginosa , Staphylococcus aureus (PTCC No. 1112). Suspension equal to 0.5 McFarland were prepared for each tested organism in Muller Hinton Broth.
Essential oil dilution and disc preparation: The essential oil was serial diluted for antibacterial study by Dimethylsulphoxide (DMSO) with final concentration: 100, 50, 25, 12.5 and mg mL-1. Blank disc were saturated by each dilution of essential oil and after drying used for study.
Disc that saturated by DMSO were prepared as negative control and disc contain Gentamycin (Pad Tan Teb, Tehran, Iran) were used as positive control.
Antimicrobial activity: The antibacterial effects were tested by the disc diffusion method, briefly, Muller Hinton Agar plates were cultured with a standardized inoculums (1.5x108 cfu mL-1 equal to 0.5 McFarland) of each bacterial strains, then the saturated discs with different concentration of crude essential oils were carefully placed on the plates, then were incubated aerobically at 37°C and inhibition zones were measured after 24 h. The inhibition zones were compared with the control disc containing Gentamycin as positive control. Each test was repeated 3 times and means inhibition zone were recorded. Inhibitory zone≥12 mm used as good inhibitory effect of extract.
Artemisia siberi is a perennial herb, 50-150 cm in height, known as locally name Dermaneh. Flowering time in August and setting seeds in September. This species grows in open fields, road sides and wast ground. Often forming dense colonies in arid and semi-arid stepic grasslands in mountainous region in Golestan province over the 2500 m above sea level.
Chemical composition: Water distillation of dried aerial parts in blooming
of A. siberi yielded 94% (v/w) and about 22 constituents were identified
by means of GC /MS analysis ( Table 1). Artemisia ketone (48.5%)
and 1,8-cineole (19.6%) were the major constituents of this essential oil.
|| Chemical composition of Artemisia siberi L.
|| Diameter of inhibitory zone (mm) of crude essential oil,
Artemisia sieberi against three tested bacteria
|R = Resistant
Anti bacterial activity: The best antibacterial effect were found against
P. aeroginosa with Inhibitory zone 18 mm ( Table 2).
Literature studies showed that there are not enough research about ethno pharmacology, the essential oil components and in vitro antibacterial activity of A. siberi.
Appearance of phonological state of plants is affected by the genetic and environmental factors, especially in medicinal plants, these factors affected on the rates of not only the yield but also the secondary metabolites.
Therefore for obtaining the best yield of plants the surveying on and determining the phonological stage of the plant is necessary (Fieldsend and Morison, 2000). The rural healers believed that the proper therapeutically effect of A. siberi is in flowering stage in the late of August.
In present study Artemisia ketone and 1, 8-cineole were the major constituent of this species, similar to some other species of Artemisia such as A. pedemontana (Perez-Alonso et al., 2003), A. annua (Jutteau et al., 2002), A. absanthium and A. parviflora (Orav et al., 2006).
Cha et al. (2005) and Zhang et al. (2005) in two separated studies were reported that the A. lavandulaefolia, A. scoparia and A. capillaris have a good anti microbial activity against different genera of bacteria with the presence of β-caryophyllene, 1,8-cineole, α-terpineole and β-pinene in their essential oil. All of these constituents were present in high or low concentration in present studied Artemisia species, it means that this species can show good antibacterial activity. We found that the Gram-negative bacteria especially P. aeroginosa is the most sensitive bacteria to essential oil of A. siberi and S. aureus was a Gram positive sensitive tested bacteria.
P. aeruginosa is a leading cause of nosocomial infections, ranking second among the Gram-negative pathogens reported to the NNIS and is important for immunosuppressed patients and in patients hospitalized with cancer, cystic fibrosis and burns (NNIS, 1998 ). The increasing frequency of multi-drug-resistant Pseudomonas aeroginosa stimulated the medical researcher to look for efficacious antimicrobial options which are severely limited (Orav et al., 2006). The conclusion drown from this study indicated that this herb, demonstrated a suitable anti Pseudomonas activity with good in vitro inhibitory effect. Further study are suggested to determine which of the chemical constituent of this essential oil carry the best anti Pseudomonal activity and also we recommend in vivo study of the same substance in an animal model.