Antibacterial Activity of Essential oils Isolated from Eucalyptus globulus Labill and Eugnia caryophyllata Thunbery (Family Myrtaceae): A comparative study.

: The essential oils of Eucalyptus globulus Labill and Eugnia caryophyllata Thunbery analyzed by Thin Layer Chromatography TLC. The results showed that TLC plates separated five compounds from E. globulus oils and four compounds from E. caryophyllata oils with different Rf values. The obtained essential oils from leaves E. globulus and flower buds E. caryophyllata were tested on Escherichia coli and Staphylococcus aureus , by using agar well diffusion method, to determine its antibacterial activities. The strongest effected was noted in E. globulus oils against S. aureus and E.coli with diameter of zone of inhibition (25 mm and 23 mm respectively). While the E. caryophllata oils showed the least effect on S. aureus and E. coli in compare with E. globulus oils with diameter of zone of inhibition (20 mm and 18 mm respectively). We concluded that the essential oils obtained from the leaves of E. globulus offer significant antibacterial activity toward E.coli and S. aureus .


Plant material:
The plant materials of E.globulus (fresh leaves) collected from a garden in Najaf, Iraq. While the plant materials of E.caryophllata (flower buds) obtained from local market in Najaf. The identification and authentication of the plants materials was done by Botany Taxonomy Department, Kufa University.

Extraction of the essential oils:
The plant materials were extracted by steam distillation method, using "Clevenger apparatus" to extract its essential oils (14). The distillation was done for 4 hours and the oil was collected separately in airtight containers that were dried over anhydrous sodium sulphate (3). The oil was stored in a freezer at -4 C0 for further use. The Dimethyl Sulfoxide (DMSO) was used to prepare different concentrations of the oils.

Thin Layer Chromatography (TLC):
The essential oils, components were separated by TLC, Silica gel F254. Aluminum plates (20x20 cm) were used and Silica plates were activated by heating in an oven for 5 min at 100 C0 . The sample was loaded at one end of the TLC plate by capillary tube then entered in TLC Jar that contained appropriate solvent system, Hexane: Chloroform (6:4). When the solvent phase reached the top, the plate air-dried and checked the separated compounds by UV visible (254 nm), rate of Retention factor (Rf) values were recorded by calculated from below equation: Rf = distance a compound moves / distance solvent front (14).

Antibacterial assay:
The essential oils obtained from the E.caryophyllata (flower bud oil) and E. globulus (leave oil) were used to study their antibacterial activities, using Agar Well Diffusion method (11). Two species of bacteria were selected as target, Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus). Mueller-Hinton Agar (MHA) was poured in Petri dishes (Three Replication), after solidification. Suspension of the tested bacteria (1x106 CFU/ml) was spread onto media plates by sterile cotton swabs after 15 min. Four wells were made in plates with sterile borer (8 mm). The concentrations of essential oils prepared by mixing a definite amount of the oils with different volumes of Dimethyl Sulphoxide DMSO (1:1, 1:2, 1:3) to get the concentrations (50%, 33.3%, 25% respectively). 100 µl from each concentration of oils were loaded in the wells and control DMSO was loaded in a fourth well. The plates were left at the room temperature for 10 minutes to allow spreading the essential oils into the agar. The plates incubated at 37 C0 for 24 hours. The bacterial growth was determined by measuring the diameters of inhibition zones, measured in millimeters (mm), around each well in plates.

Thin layer chromatography (TLC):
Analysis of essential oils components through TLC gives distinct bands with different compounds. TLC plates separated four compounds from E. caryophyllata essential oils. They have detected according to Rf value for four spots (0,08, 0.38, o.55, 0.58 respectively). On other side, TLC plates separated five compounds from E. globulus essential oils with different Rf value for spots (0.14, 0.27, 0.48, 0.50, 0.52 respectively), (Table 1).  In this study, agar well diffusion method was used to evolution the antibacterial activity of the E.globulus and E. caryophllata essential oils. The leaves essential oils of E.globulus showed good antibacterial activity against the Staphylococcus aureus and Escherichia coli with zone diameter of inhibition (25 mm and 23 mm respectively) in oil´s concentration 50%, while in concentration 25% was 18 mm against S.aureus and 19 mm against E.coli ( Table 2). The effectiveness of antibacterial activity E. globulus essential oils attributed to the presence mixture of monoterpenes and oxygenated monoterpenes (1). As well as, present 1,8-cineol, linalool and pinocarveol. The 1,8-cineol and linalool are well-known substance with pronounced antimicrobial properties (34). The flower buds essential oils of E. caryophllata permitted antibacterial activity against S.aureus with inhibition zone diameter of 20 mm while E.coli inhibition zone diameter of 18 mm in oil´s concentration 50%, but concentration 25% the inhibition zone is 16 mm and 15 mm against S.aureus and E.coli respectively ( Table 2). Eugenol is the main constituent for essential oils of E. caryophyllata responsible for antibacterial effect and other medicinal properties (17, 25 and 30). The compound Eugenal is capable of proteins denaturation and reacts with phospholipids of the cell membranes to alters its permeability (5). Both E.globulus and E.caryophyllata essential oils types showed good activity against both Gram-negative and Gram-positive bacteria. Hydrophobicity of essential oils components enable them to partition in the lipids of the bacterial cell membrane and mitochondria disturbing the structure and rendering them more permeable. This will result in ions leakage and other cell contents (29). Extensive cell contents loss or draining out of critical molecules and ions will eventually lead to death of the cell (9). The results showed that Gramnegative bacteria were the least sensitive to the action of E. globulus and E. caryophyllata essential oils than Gram-positive bacteria. The outer membrane that surrounding the cell wall in Gram-negative bacteria restricts the diffusion of hydrophobic compounds through its lipopolysaccharide layer (13).

Conclusion:
The essential oils from the leaves of E. globulus have good antibacterial activity compared to that of flower buds of E. caryophllata. Hence, it is of medicinal importance and can be used as antibacterial agent.