The essential oil and various extracts of plants have provoked interest as sources of natural products. They have been screened for their potential uses as alternative remedies for the treatment of many infectious diseases and preservation of foods from the toxic effects of oxidants. Particularly, the antimicrobial and antioxidant activities of plants oils and extracts have formed the basis of many applications, including low and processed food preservation, pharmaceuticals and alternative medicine and natural therapies (Lis-Balchin and Deans, 1997). Moreover, they offer an effective way to prevent the development of various off-flavours and undesirable compounds that result from lipid peroxidation in foods (Wang et al., 1998). Because of the possible toxicities of the synthetic antioxidants, butylated hydroxyanisol (BHA) and butylated hydroxytoluene (BHT), increasing attention has been directed toward natural antioxidants (Namiki, 1990).
Steam distillated oils obtained from the flowers and leaves of T. polycephalum were already investigated. The main components of the oil of flowers were camphor (59.1%), camphene (14.9%) and 1,8-cineol (10.1%), wherease the leaves oil comprised mainly camphor (53.5%), bornyl acetate (12.1%), camphene (10.9%), 1,8-cineol (7.8%) and borneol (6.1%) (Nori-Shargh, et al., 1999). Camphor (18.2%), 1,8-cineol (17.0%), carveol (9.1%), trans-isopulegone (8.0%) and α-thujone (6.1%) as major constituents also are reported of the aerial parts oil of this plant (Mojab and Nickavar, 2006).
The genus Tanacetum (Compositae) is represented by 26 species in the flora of Iran, 12 of them are endemic (Mozaffarian, 1996). Tanacetum polycephalum are used in folk medicine to treat many disorders (Zargari, 1996), therefore, it seem interesting to investigate its biological activity and chemical analysis.
Present study deals with the analysis, antimicrobial and antioxidant activity of the essential oil of Tanacetum polycephalum grown wild in West of Iran.
MATERIALS AND METHODS
The aerial parts of Tanacetum species were collected during flowering period (May 2005) from Aleshtar, Lorestan province, Iran. The specimens were then subjected to hydrodistillation using a Cleavenger-type apparatus for 2.5 h subsequent to decanting and drying over anhydrous sodium sulfate.
GC analysis: GC analysis was performed on a Shimadzu 15A gas chromatograph equipped with a split/splitless injector and a flame ionization detector at 250°C. N2 was used as a carrier gas (1 mL min1) and a DB-5 type was utilized as the capillary (50 mx0.2 mm, film thickness 0.32 μm). Temperature within the column for 3 min was retained at 60°C, after that the column was heated at a rate of 5°C min1 until it reached at 220°C and maintained in this condition for 5 min. The percentage of relative amounts were calculated from peak area using a shimadzu C-R4A chromatopac without applying correction factors.
GC/MS analysis: The analysis of the essential oil was performed using a Hewlett-packard 5973 with a HP 5MS column (30 mx0.25 mm, film thickness 0.25 μm). The column temperature was kept at 60°C or 3 min and programmed to reach 220°C at rate of 5°C min1 and stayed steady at 220°C for 3 min. The components of each oil were then identified by comparison of their mass spectra and Retention Indices (RI) with those given in literature and those authentic samples (Adams, 1995).
Antimicrobial activity : The antimicrobial tests were carried out by using the following microorganisms: Staphylococcus aureus PTCC 1113, Staphylococcus epidermidis PTCC 1349, Staphylococcus saprophyticus PTCC 1379 (Gram-positive bacteria ), Salmonella typhi PTCC 1185, Shigella flexneri PTCC 1234, Escherichia coli PTCC 1330, Pseudomonas aeroginosa PTCC 1310 and Klebsiella pneumoiae PTCC 1053 (Gram-negative bacteria) identified by Iranian Research organization for Science and Technology (IROST).
Microorganisms (obtained from enrichment culture of the microorganisms in 1 mL of Muller-Hinton broth, incubated at 37°C for 12 h) were cultured on Muller Hinton Agar medium. After drilling wells on medium oils dissolved in hexane and 40 μL from solutions in each well was poured. After incubation at 37°C during 24 h, incubation zone diameter was measured.
Inhibition of lipid peroxide formation: Free radical scavenging activity was evaluated by the 5-lipoxygenase test in sample and in positive controls. The activity of the enzyme was assayed spectrophotometrically according to Holman. This method was modified by Sudina et al. (1993).
The assay mixture (1 mL) contained: 10 mM linoleic acid, the sample (or the same quantity of solvent as reference) and 50 mM sodium phosphate, pH 6.8. This mixture was maintained at 20°C for 20 min.
Subsequently, 0.18 μg mL1 commercial 5-lipoxygenase was added to mixture and formation of hydroperoxides from linoleic acid was observed spectrophotometrically at 235 nm at 20°C.
DPPH assay: Radical scavaging activity was determined by spectrophotometric method based on the reduction of an ehanol solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Mellors and Tappel, 1966). Tests were carried out in triplicate.
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxlic acid), BHT (butylated hydroxytoluene) and ascorbic acid were used as positive controls and purchased from Merk company.
Hydroxyl radical scavenging: Hydroxyl radical scavenging was carried
out by measuring the competition between deoxyribose and the extract for hydroxyl
radicals generated from the Fe3+/ascorbate/EDTA/H2O2
system. The attack of the hydroxyl radical on deoxyribose leads to TBARS formation
(Kunchardy and Rao, 1990). Various concentration of the extracts were add to
reaction mixture containing 3.0 mM deoxyribose, 0.1 mm FeCl3, 0.1
mM EDTA, 0.1 mM ascorbic acid, 1 mM H2O2 and 20 mM phosphate
buffer (pH 7.4), making up a final volume of 3.0 mL. The reaction mixture was
incubated at 37°C for 1 h. The formed TBARS were measured by the methods
given elsewhere (Ohkawa et al., 1979). On mililitre of thiobarbituric
acid, TBA (1%) and 1.0 mL trichloroacetic acid, TCA (2.8%) were added to test
tubes and incubated at 100°C for 20 min. After cooling, absorbance was measured
at 532 nm against a blank containing deoxyribose and buffer. Reactions were
carried out in triplicate. Inhibition (I) of deoxyribose degradation in per
cent was calculated in following way:
Where A0 is absorbance of control reaction (containing all reagents
except the test compound) and A1 is the absorbance of the test compound.
RESULTS AND DISCUSSION
The yield of essential oil obtained by hydrodistillation from dried plant material was 0.45%. The composition of the volatile oil of Tanacetum polycephalum is listed in Table 1. Thirty-nine constituents, representing 94% of the total components in the oil, have been identified in the essential oil extracted from the aerial parts of this plant. There was a high content of monoterpene compounds including borneol (28.30%), β-pinene (10.10%), α-pinene (6.5%), camphene (6.04%), α-terpineol (5.16%) and 1,8-cineol (5.10%) as major components. Among the identified sesquiterpenes in the oil spathuenol (4.17%) and caryophyllene oxide (2.33%) were most abundant. The studied by us is different from the other Iranian samples (Nori-Shargh, et al., 1999; Mojab and Nickavar, 2006).
According to Nori-Shargh et al. (1999) camphor (59.1%), camphene (14.9%)
and 1,8-cineol (10.1%) in flowers oil and camphor (53.5%), bornyl acetate (12.1%),
camphene (10.9%), 1,8-cineol (7.8%) and borneol (6.1%) in the leaves oil were
the main components of essential oil of this plant, wherease Mojab and Nickavar
(2006) are reported camphor (18.2%), 1,8-cineol (17.0%), carveol (9.1%), trans-isopulegone
(8.0%) and α-thujone (6.1%) as major constituents of the aerial parts oil
of T. polycephalum.
||Composition of the aerial parts oil of Tanacetum polycephalum
On the other hand, in the present study camphor was detected only in low amount
wherease is a abundant compound. These differences might have been derived from
harvest time, extraction method, locality, climatic and seasonal factors.
The essential oil of T. polycephalum remarkably inhibited the growth of the tested bacteria. The oil was shown to possess the strongest antibacterial activity particulary against Salemonella typhi (52 mm), Staphylococcus aureus (45 mm) and Klebsiella pneumoniae (41 mm) corresponding to the largest inhibition zone diameters (Table 2). The antibacterial activities of T. polycephalum can be attributed to the presence of high concentration monoterpene compounds. The antimicrobial activities of borneol (an oxygenated monoterpene) as major compound have been previously reported (Dorman and Deans, 2000). Other than the main compounds, β-pinene, α-pinene, camphene and 1,8-cineol as well as other minor constituents of the essential oil of T. polycephalum have antibacterial activity (Sivropoulou et al., 1997; Sur et al., 1991).
The antioxidant activity of T. polycephalum essential oil was determined using three in vitro assays: Scavenging effect on DPPH and hydroxyl radicals and inhibition of lipid peroxide radicals formation. The antioxidant activity of the test sample, expressed as IC50 (μg mL1), was compared with activity of know antioxidants such as ascorbic acid, BHT and Trolox. The data collected in Table 3 show that, in reduction of the stable radical DPPH, the highest activity was obtained with the essential oil and with Trolox (IC50 = 12.4 and 8.9 μg mL1, respectively).
||Antibacterial activity of Tanacetum polycephalum oil
|-: Not determined
Hydroxyl radical scavenging assay was not performed with ascorbic acid since this compound was already present in the test medium. Hydroxyl radical scavenging of the essential oil was better than BHT (IC50 = 1.7 and 33.1 μg mL1, respectively) but lesser than Trolox (IC50 = 0.1 μg mL1). In the inhibition of lipid peroxidation the IC50 values obtained showed that the essential oil antioxidant activity were higher than ascorbic acid and BHT (IC50 = 31.2 and 23.6 μg mL1, respectively) and less than that of Trolox (IC50 = 6.7 μg mL1).