American Journal of Agricultural and Biological Sciences 9 (1): 78-88, 2014 ISSN: 1557-4989 ©2014 Science Publication doi:10.3844/ajabssp.2014.78.88 Published Online 9 (1) 2014 (http://www.thescipub.com/ajabs.toc) CHEMICAL COMPOSITION, ANTIMICROBIAL, ANTIOXIDANT AND CYTOTOXIC ACTIVITIES OF EUCALYPTUS CHAPMANIANA GROWN IN IRAQ 1Ghassan Mohammad Sulaiman, 1Thorria Radam Marzoog, 1Wasnaa Hatif Mohammed and 2Renzo Bagnati 1 Division of Biotechnology, Department of Applied Science, University of Technology, Baghdad, Iraq 2Department of Environmental Health Sciences, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, 20156 Milano, Italy Received 2013-10-31; Revised 2013-11-13; Accepted 2013-12-27 ABSTRACT The chemical composition of the essential oils extracted from immature flowers, leaves and seeds of Eucalyptus chapmaniana grown in Iraq were analyzed for the first time by gas chromatography/mass spectrometry. Twenty-four different compounds were identified and the predominant compound is eucalyptol, which accounted for 59.9, 55.6 and 8.6% of total compounds, respectively. To asses the possible therapeutic uses of the extracts, their antioxidant properties were assessed via DPPH free radical scavenging. The extracts showed significant antioxidant and antimicrobial activities against Escherichia coli , Pseudomonas aeruginosa , Klebsiella pneumonia , Proteus volgaris , Staphylococcus aureus and Candida albicans . The cytoxicity of flower extract against the Human Leukemia (HL-60) cells was evaluated and the extracts significantly reduced the viability of HL-60 cells in a dose- and time- dependent response relationship. The results indicated that essential oils from immature flowers are highly cytotoxic to HL-60 cells and that their antitumor potential was confirmed. Keywords: Eucalyptus chapmaniana , Essential Oils, Antimicrobial, DPPH, HL-60 1. INTRODUCTION properties, including antibacterial and antifungal activities (Cimanga et al ., 2002; Ramezani et al ., 2002; The Myrtaceae family contains 133 genera and 3,800 Sartorelli et al ., 2007). In addition, the leaf and plant species of trees and shrubs. This family can be found in extracts of the Eucalyptus species themselves possess temperate, subtropical and tropical regions and it is antibacterial and antifungal activities (Takahashi et al ., endemic in Australia, tropical America, Africa and Asia 2004; Salari et al ., 2006). Extracts and components (Wilson et al ., 2001). One important genera of isolated from several Eucalyptus species have been Myrtaceae is Eucalyptus , which is a large genus of shown to possess cytotoxic and antitumor activities evergreen trees and shrubs containing approximately 700 (Benyahia et al ., 2005; Ashour, 2008). species (Batish et al ., 2008). Although most plants are Antioxidant agents are compounds that function as native to Australia and Tasmania (Oyedeji et al ., 1999), scavengers of reactive oxygen species or free radicals, they have been successfully introduced worldwide and which have important functions in energy production, are cultivated in many other countries, including Iraq. synthesis of several biomolecules, phagocytosis and cell The leaves of the Eucalyptus species accumulate a growth in living systems (Packer et al ., 2008). An very large number of secondary metabolites and yield imbalance in the rate of production of free radicals or hydro distilled essential oils that possess many biological their removal via antioxidant defense mechanisms leads Corresponding Author: Ghassan Mohammad Sulaiman, Division of Biotechnology, Department of Applied Science, University of Technology, Baghdad, Iraq Science Publications 78 AJABS Ghassan Mohammad Sulaiman et al. / American Journal of Agricultural and Biological Sciences 9 (1): 78-88, 2014 to a phenomenon referred to as oxidative stress. In µm). The injector and detector temperatures were set at diseases such as diabetes, cardiovascular diseases and 200 and 270°C, respectively. The oven temperature was cancer, an aggravated imbalance may occur during gradually raised from 60 to 260°C at a rate of 5°C/min. deleterious oxidation of biomolecules, which results in cell The temperature was held for 15 min and finally raised or tissue damage (Arts and Hollman, 2005; Jung et al ., to 340°C at a rate of 40°C/min. Helium (purity 2009; Wells et al ., 2009). 99.99999%) was used as the carrier gas at a flow rate of − Numerous investigations were performed on volatile 1 mL min 1. The total analysis time was 57 min. About components of essential oils from different species of 1.0 µL of the diluted sample (1/100 in petroleum ether, Eucalyptus . Previous studies on the compositions of v/v) was injected in the split mode (ratio 1:10). several Eucalyptus spp. leaves, flowers (Giamakis et al ., Quantitative data were obtained electronically from the 2001; Tsiri et al ., 2003) and recently, stem and fruit FID area percent data without the correction factors. essential oils have been reported (Marzoug et al ., 2011). Peak integration and quantification were performed However, no previous studies on the chemistry and the automatically by using Saturn 2100 Workstation antioxidant, antimicrobial and cytotoxic activities of software. The integration of each peak was manually essential oils from leaves, flowers and seeds of E. chapmaniana have been presented. In the current study checked and corrected when needed. the composition of essential oils obtained from adult 2.4. Gas Chromatography/Mass Spectrometry leaves, immature flowers and seeds of E. chapmaniana (GC-MS) collected in Iraq has been studied. The antioxidant, antimicrobial and cytotoxic activities of E. Chapmaniana The essential oils were analyzed in the same were also investigated. conditions as those of GC (column, oven temperature, flow rate of the carrier gas) by using a Varian Star 2. MATERIALS AND METHODS 3400 (Les Ulis, France) gas chromatograph equipped with a Varian Saturn GC/MS/MS 4D mass selective 2.1. Chemicals detector in the electron impact mode (70 eV). The injector and MS transfer line temperatures were set at All chemicals used were of analytical reagent grade. 200 and 300°C, respectively. MS was adjusted with an Penicillin and streptomycin were purchased from Bio emission current of 10 µA and an electron multiplier Source International (Belgium). RPMI 1640, Fetal Bovine voltage of 1,500 V. The trap temperature was set at Serum (FBS), 3-(4,5-dimethyl-thiazol-2-yl)-2 (MTT) and 250°C and mass scanning was performed from 40 amu S-diphenyl tetrazolium bromide were purchased from to 650 amu. The components were identified by Sigma Chemical Co. (St. Louis, MO, USA). 2, 2- comparing their Kovats Indices (KI), by co-injection Diphenyl-1- Picrylhydrazyl (DPPH) (Sigma-Aldrich) was of standards and by MS experimental data of used for the spectrophotometrical determination of the free commercial or literature libraries (NIST 02 version radical scavenging activities of the extracts. 2.62). Alkanes (C5-C24) were used as reference points in KI calculation. The GC and GC-MS analysis 2.2. Plant Materials and Extraction of Oils results are given in Table 1 . All determinations were Plant materials, such as the leaves, flowers and seeds performed in duplicate and the average was obtained. from the E. chapmaniana , were collected from several 2.5. Antioxidant Assay specimens located in the At-Tarmyia (a region 60 km north-east of Baghdad, Iraq) in 2012. The collected leaf, The DPPH assay was measured by bleaching a purple seed and flower materials were kept at room temperature methanol solution of DPPH (Milliauskas et al ., 2004). and left to dry. After air-drying, the plant materials were About 0.5 mL of 1 mM DPPH solution was added to 3 hydrodistilled for 3 h by using a Clevenger type mL of various concentrations of sample extracts of E. apparatus. The resulting oil was collected, preserved in a chapmaniana . After 30 min of incubation at room sealed sample tube and stored until analysis. temperature, the absorbance was obtained against a blank sample at 517 nm. The decrease in the actual absorption 2.3. Gas Chromatography was measured against that of the control sample. All The essential oils were analyzed using a Varian Star experiments were carried out in triplicates and percentage 3400 (Les Ulis, France) Cx gas chromatograph equipped inhibition values were calculated based on the equation: with a Flame Ionization Detector (FID) and DB-5MS =( −) × capillary column (30m×0.25 mm, film thickness 0.25 % Inhibition A0 AT /A0 100 Science Publications 79 AJABS Ghassan Mohammad Sulaiman et al. / American Journal of Agricultural and Biological Sciences 9 (1): 78-88, 2014 Table 1. Chemical composition of E. chapmaniana extracts identified by GC-MS Total ion current (%) Molecular Molecular ------------------------------------------ Compound RT min formula weight Leaves Flowers Seeds 2,5-Furandione, dihydro-3-methylene- 3.781 C5H4O3 112.083 0.26 - 1.320 Propanal, 2-methyl- 11.698 C4H8O 72.105 0.70 0.33 - 5,9-dodecadien-2-one, 6,10 11.811 C14H 24 O 208.339 - - 1.870 -dimethyl-dodecadien-2-one Nonanal dimethyl acetal 17.508 C11 H24 O2 188.307 - 0.51 - α-Pinene 20.206 C10 H16 136.230 - 0.34 - α- phellandrene 21.920 C10 H16 136.240 - 4.16 - β-Cymene 22.508 C10 H14 134.218 2.14 3.57 - Eucalyptol 22.817 C10 H18 O 154.136 55.62 59.97 8.610 γ-Terpinen 23.042 C10 H16 136.125 - 2.88 - α- Terpinolen 23.752 C10 H16 136.234 - 0.17 - Undecane 23.273 C11 H24 156.188 0.79 0.47 2.770 trans-Pinocarveol 26.215 C10 H16 O 152.230 1.30 - - Terpinen-4-ol 26.627 C10