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Cytotoxic and Antimycotic Activities of Essential Oil ofArtemisia turanica Krasch from IranJavad Behravan a b , Mohammad Ramezani a b , M K Hassanzadeh c , Nasim Eliaspour b &Zahra Sabeti ba Biotechnology Laboratory, Biotechnology and Pharmaceutical Research Centersb Department of Pharmacognosy and Biotechnologyc Department of Medicinal Chemistry and Pharmaceutical Research Center , School ofPharmacy, Mashhad University of Medical Sciences , Mashhad , IranPublished online: 12 Mar 2013.
To cite this article: Javad Behravan , Mohammad Ramezani , M K Hassanzadeh , Nasim Eliaspour & Zahra Sabeti (2006)Cytotoxic and Antimycotic Activities of Essential Oil of Artemisia turanica Krasch from Iran, Journal of Essential Oil BearingPlants, 9:2, 196-203, DOI: 10.1080/0972060X.2006.10643492
To link to this article: http://dx.doi.org/10.1080/0972060X.2006.10643492
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Cytotoxic and Antimycotic Activities of Essential Oil of Artemisia turanicaKrasch from Iran
Javad Behravan1,2*, Mohammad Ramezani1,2, M K Hassanzadeh3,Nasim Eliaspour2 and Zahra Sabeti2
1Biotechnology Laboratory, Biotechnology and Pharmaceutical Research Centers,2Department of Pharmacognosy and Biotechnology,
3Department of Medicinal Chemistry and Pharmaceutical Research Center,School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
Abstract: The cytotoxic and anti-fungal activities of Artemisia turanica essential oil wereinvestigated. Also the composition of the essential oil of the plant was determined. For cytotoxicactivity tests, a potato disc tumor inhibition method was exploited. For antifungal activity tests, apoisoned food technique against standard strains of Trichophyton rubrum, Trichoderma reesei,Microsporum gypseum and Aspergillus niger was adopted. The essential oils of A. turanica exhib-ited tumor growth inhibition (100 % ) inhibition at concentrations equal to 0.02 % (w/v) and higher.Thirteen components were identified in the essential oil of Artemisia turania. The main compoundsconsisted of 1,8-cineol (40.9 %), cis-verbenyl actate (19.0 %) and camphor (11.0 %). A dose activityrelationship between the essential oil concentrations with inhibition of tumor growth on potato discs,and between the essential oil concentrations with antifungal activities was observed.
Keywords: Artemisia turanica, Chemical composition, Essential oil, Cytotoxic andantimycotic activities
Introduction: Plants have long been used as a very viable source of clinically rel-evant antimicrobial and anticancer compounds. Despite the wide availability of clinicallyuseful cytotoxic and antifungal agents, discovery and characterization of new substancesthat exhibit activity against tumors and infectious fungi are highly called for.
The genus Artemisia (Asreaceae), locally known as ‘Dermaneh’ is represented inIran by 34 species, of which two are endemic1. Most of Artemisia species are aromatic andyield essential oils which are used in perfumery and medicine. According to Napralert, somespecies have been used traditionally as carminative, stomachic and expectorant, and fortreatment of dysentery. Numerous constituents from Artemisia species have proven to
ISSN 0972-060X
Received 15 April 2006; accepted in revised form 27 July 2006
*Corresponding author: (Javad Behravan)E-mail: <[email protected]>
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be cytotoxic2, anti-oxidant3, antimicrobial4, and cell cycle arrestant5. Artemisinin, the mainactive principle isolated from Artemisia spp. has shown remarkable activities against multidrug resistant Plasmodium falciparum and P. vivax. It also has phytotoxic activity, even onmembers of Artemisa spp., and is a candidate as a natural herbicide6,7. Recently, significantanticancer activity of artesunate (a semi-synthetic derivative of artemisinin) has been re-ported by Efferth et al.8. Importantly it was demonstrated that otherwise multi drug resistantcell lines (resistant to doxorubicin, vincristine, methotrexate, or hydroxyurea) did not showany cross resistance to artesunate8. In a recent study by Ramezani et al.9, it was shown thatA. turanica essential oil was active against three standard bacterial strains including Sta-phylococcus aureus, Bacillus subtilis and Pseudomanas aeruginosa9.
In the present study, we evaluated the cytotoxic and anti-fungal activities of essentialoil of the herb A. turanica by examining its capability to inhibit tumor growth on a potato discmodel. This paper also reports the results of composition and antifungal perspectives of theessential oil from A. turanica.
ExperimentalPlant material: Aerial parts of A. turanica K. were collected from Torbat-Jam near
Mashhad (a city in northeast of Iran) in July 2002. A voucher specimen was deposited inherbarium of School of Pharmacy, Mashhad University of Medical Sciences (MUMS), Iran(Herbarium No. 763).
Microorganisms and culture media: The fungal species used in this study con-sisted of four standard strains of filamentous fungi, Trichophyton rubrum, Trichodermareesei, Microsporum gypseum and Aspergillus niger. All the strains were obtaind fromPersian Type Culture Collection. The fungi were maintained on Sabouraud Dextrose Agar(SDA) slants. The inoculated agar slants were incubated at 250C for 48 hours to 10 days.Agrobacterium tumefaciens strain B6 was maintained on slopes of nutrient agar (NA,Oxoid). Cultures for antitumor tests were grown in Nurient Broth (NB, Oxoid) at 280C on arotrary shaker at 200 rpm.
Preparation of essential oil: The essential oil was obtained from dried aerial parts(moisture content 6 %) of A. turanica K. (100 g) by a hydrodistillation method using aClevenger type apparatus10. The essential oil was separated from aqueous layer and driedover anhydrous sodium sulfate and stored in a refrigerator until use. The yield was 0.3%(v/w).
Cytotoxic activity assay: Cytotoxic activities of A. truranica K essential oil wasassessed with modification of a method of McLaughlin’s group. The intact white potatotubers of moderate size were disinfected by immersion in 1% (w/v) sodium hypochlorite for20 minutes. This was followed by two washes in sterile distilled water. The two ends ofpotato were removed and a tissue cylinder of 1.5 cm diameter was taken with a sterile corkborer under a laminar air cabinet. A one centimeter length from each end of the cylinder wasremoved and the reminder of potato cylinder was cut into discs of 0.5 cm thickness. Fivediscs were transferred onto surface of sterile agar (3 %) in a 12-cm Petri dish. Plant essen-
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tial oil was dissolved in 2 mL dimethyl sulfoxide and filtered through a sterile 0.45 mM filter.An aliquot of 0.5 mL was then added to 1.5 mL sterile distilled water (for essential oil distilledwater with 1% Tween 80 was used). This was inoculated with 2 mL of a culture of A.tumefaciens (1x 109 CFU/mL) and, 50 µL of the final mixture was added onto the surfaceof potato discs in triplicate plates. For controls, 0.5 mL of DMSO was used instead of theextract or essential oil. The Petri dishes were then transferred to an incubator at 250C for20-30 days. To determine the number of tumors, the potato discs were stained with a solu-tion of I
2 (5% w/v), KI (10% w/v) in distilled water (Figure 1). Significant antitumor activity
was indicated when a test concentration on average gave 20% or more tumor growth inhi-bition compared to blank controls.
Figure 1. Representation of tumor growth on potato disc tubers byAgrobaterium tumefaciens strain B6.
The tumors were developed and pictured after 20-30 day of incubation. A: beforestaining B: after staining with the dye.
Antifungal activity assay: The minimum inhibitory concentrations (MICs) of theessential oil against the test filamentous fungi were determined by poisoned food techniqueas reported elsewhere11. Briefly, the requisite quantity of the oil samples were mixed inacetone and then added in pre-sterilized SDA medium, pH 5.6, to give a final concentrationof oil and acetone mix of 2% by volume. In control sets, sterilized water (in place of the oil),and acetone were included in the medium in appropriate amounts. Mycelial discs of 5 mmdiameter cut out from the periphery of seven day old cultures were aseptically inoculatedupside down on the agar surface of the medium. Inoculated Petri plates were incubated at27 ± 10C and the observations were recorded on the seventh day. Percentage of MycelialGrowth Inhibition (MGI) was calculated according to the formula:
where dc = fungal colony diameter in control sets, dt = fungal colony diameter intreatment sets.
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A B
MGI = (dc-dt) x 100 /dc
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Assay for antimicrobial activity against A. tumefaciens: A mixture of Tween 80/essential oil at a ratio of 6:4 was diluted with Muller-Hinton Broth (MHB) medium to obtaina final test dilution of 1:10 to 1:10000 (v/v). The test solution (1mL) was serially diluted with1 ml of a 3-hour old culture of A. tumefaciens (to a final concentration of 106 CFU/mL) andwas added to each well. The covered 24 well microplates were incubated at 280C overnight.
To indicate bacterial growth, 500 µL of 2,3,5-triphenyltetrazolium chloride (TTC, tet-razolium red, Sigma) dissolved in water (50 mg/mL) was added to the microplate wells andincubated at 370C for 10-30 minutes. Three replicates were maintained for the assay. 2 mLof MHB for sterility control and 1 mL of MHB diluted with 1 mL of microbial solution forpositive control were used each in two wells12,13.
Gas chromatography-mass spectrometric analysis: The GC-MS analysis of theessential oil was performed on a Varian Saturn 3 GC-MS spectrometer. The essential oilwas analyzed under the following conditions: column DB-5, 0.32 mm × 30 m (J & W Scien-tific); carrier gas, He; flow rate 2 mL/min; oven temperature: 60-2400C with rate of 30C/min; injector mode: split injection; ionization mode: electron impact (EI) at 70 eV; interfacetemperature: 2700C; scan range 40-300 u. The essential oil components were identified bycomparison of their relative retention time (RRT) and their mass spectra with those ofauthentic samples, literature data14, and computerized MS-data bank (Saturn, version 4).The peak area method was followed for quantitative determination of different constituents;the percentage was calculated relatively14.
ResultsCytotoxic activity of essential oil of A. turanica: The essential oil was tested for
cytotoxic activity against tumor galls on potato discs induced by A. tumefaciens. TumorGrowth Inhibition activity (TGI) was considered significant with TGIs of more than 20 % ascompared to those of the controls. The essential oil exhibited tumor inhibition activity atvalues higher 1% (v/v) showing 100% inhibition at concentrations of 2-10 % (v/v) (Fig. 2).
Figure 2. Crown gall tumor growth inhibition of essential oil of A. turanica
Essential Oil dilution (v/v)
Percent inhibition
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0
20
40
60
80
100100 100
1.00E-04 0.001 0.002 0.01 0.02 0.12.00E-04
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Antifungal activity of essential oil of A. turanica: The highest antimycotic activi-ties were observed against M. gypseum with 100% Mycelial Growth Inhibition (MGI) ofthe oil at 1 µL/mL (0.1 % v/v) (Table 2). Among the filamentous strains tested M. gypseumwas the most sensitive (MGI, 48.75 % at essential oil dilutions of 0.0625 µL/mL), while A.niger was the most resistant strain (MGI, 68.6 % at essential oil dilutions of 1 µL/mL,Table 1).
Activity against A. tumefaciens: The anti Agrobacteium test results indicated thatthe essential oil of A. turanica did not exhibit any antimicrobial effects on the test strain atthe concentrations used in anti-tumor assay.
The essential oil components: GC-MS analysis of the volatile oil of A. turanicaresulted in identification of 13 compounds corresponding to 86.4% of the total composition ofthe essential oil. The percentage of each component is presented in Table 2.
Discussion: The genus Artemisia contains a class of medicinal plants used to treatvarious maladies. The activities include anti infectious2, tonic, febrifuge, anthelmintic, anti-inflammatory and anti-tumor activities15. The importance of cytotoxicity properties ofartesunate (a semi-synthetic derivative of artemisinin) against multi drug resistant tumor celllines has just recently been appreciated8.
The results of our in vitro study have demonstrated a dose dependence activity of A.turanica essential oil against tumor galls induced by A. tumefaciens on potato discs. It hasbeen shown by different groups that the potato disc tumor inhibition assay correlates wellwith other antitumor activity assays16,17.
The essential oil of A. turanica was analyzed by GC-MS contained 13 compoundsmaking up 86.4% of the total composition. The major components identified in the essen-tial oil of this plant were 1.8-cineol (40.9 %), cis-verbenyl acetate (19.0 %), and camphor(11.0 %). The essential oil was shown to be very rich in oxygen containing monotrepenoids(76.3 %), while it is quite poor in monoterpene hydrocarbons (3.0%).
Inhibition of crown gall tumors could be caused by two effects: directly by antitumoractivity of essential oil, or indirectly by decreasing the viability of the A. tumefaciens inocu-lum. Therefore, anti A. tumefaciens tests were carried out to distinguish between thesepossibilities. The results indicated that the essential oil at the concentrations used did nothave antibacterial activities against A. tumefaciens. This suggests that the observed inhibi-tion of tumor formation is likely to be due to anti-tumor activity and not to the inhibition of A.tumefaciens itself.
Although, once the anti-tumor activity is established for a certain plant, it is necessaryto conduct further anti proliferation studies, it is also advantageous that crown gall tumorbioassay be used as an initial step in the screening of potential antitumor compounds. Par-ticularly, the advantages of such a system with respect to costs, animal friendly approachand time are obvious.
This study also showed that the essential oil of A. turanica is active against certainstrains of filamentous fungi. Antifungals are in great demand in medicine, food production
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and agriculture. Species of Microsporum and Trichophyton are amongst the most commonfungal pathogens in skin infection. Although at a low incidence, Trichophyton and Mi-crosporum spp. are also reported to cause fungal nail infections (oncomycoses). Otherfilamentous fungi such as Aspergillus spp. can cause white superficial oncomycosis18.
In conclusion, the results of this study show that A. turanica has beneficial effects byinhibition of tumor growth on potato disc and growth of test filamentous fungal strains.However, further studies are needed to further elucidate the activity against other tumormodels as well as other pathogenic fungi, to establish mechanism of these activities and, toidentify active compounds present in extracts of A. turanica.
Acknowledgements: The authors are indebted to the Budget and Planning Organi-zation of Iran (BPOI) and the Research Committee, Mashhad University of Medical Sci-ences of Iran for approval and financial support of this project.
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12. Ellof, J.N. (1998). A sensitive and quick microplate method to determine the minimalinhibitory concentration of plant extracts for bacteria. Planta Medica, 64: 711-713.
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Table 1. Minimum inhibitory concentrations of the oil of A. turanica K.against test fungi
Concentrations Mycellial growth inhibition (MGI) (%) (µµµµL/mL) A. niger T. rubrum T. reesei M. gypseum
1.00 68.6 83.33 76.7 1000.50 25.34 70.33 31.3 82.50.25 8.95 54.16 16.04 77.5
0.125 0 53.33 0 54.250.0625 0 31 0 48.75
Table 2. Chemical composition (%) of the volatile oil of Artemisa turanica
Components Kovats index Percentage
Camphene 956 1.20p-Cymene 1024 1.841,8-Cineole 1036 40.94Artemisia alcohol 1083 1.36trans-Pinocarveol 1140 1.10Camphor 1144 11.03Terpineol 1180 1.52cis-Verbenyl acetate 1279 19.03Isobornyl acetate 1282 0.86β-Patchoulene 1376 0.75Methyl Eugenol 1397 0.48Spathulenole 1579 4.86Caryophyllene oxide 1584 1.46Total 86.43Grouped compounds:Monoterpene hydrocarbons 3.04Oxygen-containing monoterpenes 76.32Sesquiterpens hydrocarbons 0.75Oxygen-containing sesquiterpens 6.32
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