Chemical Composition and in-Vitro Evaluation of the Antimicrobial and Antioxidant Activities of Essential Oils Extracted from Seven Eucalyptus Species

Eucalyptus is well reputed for its use as medicinal plant around the globe. The present study was planned to evaluate chemical composition, antimicrobial and antioxidant activity of the essential oils (EOs) extracted from seven Eucalyptus species frequently found in South East Asia (Pakistan). EOs from Eucalyptus citriodora, Eucalyptus melanophloia, Eucalyptus crebra, Eucalyptus tereticornis, Eucalyptus globulus, Eucalyptus camaldulensis and Eucalyptus microtheca were extracted from leaves through hydrodistillation. The chemical composition of the EOs was determined through GC-MS-FID analysis. The study revealed presence of 31 compounds in E. citriodora and E. melanophloia, 27 compounds in E. crebra, 24 compounds in E. tereticornis, 10 compounds in E. globulus, 13 compounds in E. camaldulensis and 12 compounds in E. microtheca. 1,8-Cineole (56.5%), α-pinene (31.4%), citrinyl acetate (13.3%), eugenol (11.8%) and terpenene-4-ol (10.2%) were the highest principal components in these EOs. E. citriodora exhibited the highest antimicrobial activity against the five microbial species tested (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Aspergillus niger and Rhizopus solani). Gram positive bacteria were found more sensitive than Gram negative bacteria to all EOs. The diphenyl-1-picrylhydazyl (DPPH) radical scavenging activity and percentage inhibition of linoleic acid oxidation were highest in E. citriodora (82.1% and 83.8%, respectively) followed by E. camaldulensis (81.9% and 83.3%, respectively). The great variation in chemical composition of EOs from Eucalyptus, highlight its potential for medicinal and nutraceutical applications.


Introduction
The interest in use of natural medicines from plants has been increasing in the past few years in industrialized societies, particularly against microbial agents because of the ever growing problem compounds in each oil and (iii) determination of antioxidant and antimicrobial activities against some selected strains of bacteria and fungi.

Percentage Yield of EOs by Steam Distillation
The average yield of EOs from the different species of Eucalyptus was 1.84% (w/w) after 7 h of distillation. E. globulus had highest oil content (1.91%), while E. melanophloia contained least amount (1.73%). These findings are similar to results reporting a 2.4% yield of oil from E. gillii cultivated in Iran and 2.3% in Tunisia [21,22]. The small differences may be due to different climatic, geographic and ecological parameters.

Physiochemical Analysis of Essential Oils
The physiochemical properties of seven different Eucalyptus EOs revealed that initially Eucalyptus EOs were colourless, but after about 2 weeks they showed yellow colours ( Table 1). All the EOs were soluble in 2 mL of 80% ethanol, but insoluble in water. The specific gravity of the EOs extracted from the seven Eucalyptus species ranged from 0.84 to 0.94. The boiling point of E. melanophloia EO was the highest at 178˝C, followed by E. citriodora (177˝C) and the lowest was for E. globulus (161˝C).

Chemical Nature of Essential Oils
The chemical composition of the seven selected Eucalyptus EOs revealed great variation in the concentration and types of constituents ( Table 2). Identification of the components of EOs was based on their GC retention times and mass spectra, which were compared to the published data and spectra of respective standards to those constituents. The GC-MS analysis thus revealed the presence of 31 components in E. citriodora and E. melanophloia, 27 components in E. crebra, 24 components in E. tereticornis, 14 components in E. globulus, 13 components in E. camaldulensis and 12 components in E. microtheca. The results are comparable to the 25 major compounds at an average concentration greater than 0.9%˘0.2% reported earlier in eight Eucalyptus species EOs. E. gillii EO contained 34 compounds as reported by another study [23].

Antimicrobial Activity
The EOs extracted from the seven Eucalyptus species showed great potential for their application as antimicrobial agents. The activities were determined by using three strains of bacteria and two strains of fungi.

Antibacterial Activity
The EOs extracted from all seven Eucalyptus spp. showed antibacterial activity against S. aureus, B. subtilis and E. coli (Table 3). Maximum inhibition zones were observed for the E. citriodora EO against Gram positive bacteria (31 mm against S. aureus and 28 mm against B. subtilis). E. melanophloia and E. citriodora showed significant activity against the Gram negative bacterium E. coli. However, all the species showed higher activity for the Gram positive bacteria as compared to the Gram negative bacteria. Gram positive bacteria were found to be more sensitive to E. gillii EO and extracts than Gram negative ones [22]. E. oleosa EO exhibited an interesting antibacterial activity against all microorganisms tested (L. monocytogenes, S. aureus, E. coli, K. pneumoniae, S. cerevisiae, C. albicans, M. ramamnianus and A. ochraceus). The activity in this study was better against Gram-positive bacteria except for S. aureus and E. coli [23]. Results have proved that E. citriodora EO is more potent in antibacterial activity than the other six species. The EOs of Eucalyptus have previously shown antimicrobial and antiplasmid activities [32]. Previous studies also report that EOs against food spoilage organisms and food-borne pathogens are slightly more active against Gram-positive than Gram-negative bacteria [33].

Antifungal Activity
E. citriodora EO was found most effective against A. niger (29 mm) and R. solani (26 mm). Significant antimicrobial activity was shown by EO of E. microtheca against A. niger (21 mm) while EO of E. melanophloia was found least effective (12 mm) against R. solani ( Table 3). The results showed that all the tested Eucalyptus EOs presented significant antifungal activity against A. niger and R. solani and had equal or more antifungal effect than amoxil and nizoral. These properties could be correlated to the chemical composition of the oils with good phenolic, alcoholic or aldehydic contents that may be correlated to the geographical distribution and environmental effect on production of phytochemicals in plants.

Antioxidant Activity
The DPPH scavenging activity was highest in E. citriodora (82.1%), followed by E. camaldulensis (81.9%) and E. microtheca (81.8%) as compared to positive control BHT. The results are compared to the DPPH assay results for E. oleosa EO activity in the range of 12.0-52.8 mg/mL, whereas in the 2,2 1 -azinobis-3-ethylbenzothiazoline-6-sulfonate assay (176.5˘3.1 mg/L) it showed the best inhibition result [18]. All the EOs strongly suppressed the peroxide formation in linoleic acid system during incubation. The inhibition of oxidation of linoleic acid system was higher for the EOs of E. citriodora (83.8%) and E. camaldulensis (83.2%) than the other five Eucalyptus species (Figure 1). These findings are also supported by another study reporting 86.07% inhibition of linoleic acid for non-polar methanol extract of E. sargentii [34].  Table 3). The results showed that all the tested Eucalyptus EOs presented significant antifungal activity against A. niger and R. solani and had equal or more antifungal effect than amoxil and nizoral. These properties could be correlated to the chemical composition of the oils with good phenolic, alcoholic or aldehydic contents that may be correlated to the geographical distribution and environmental effect on production of phytochemicals in plants.

Antioxidant Activity
The DPPH scavenging activity was highest in E. citriodora (82.1%), followed by E. camaldulensis (81.9%) and E. microtheca (81.8%) as compared to positive control BHT. The results are compared to the DPPH assay results for E. oleosa EO activity in the range of 12.0-52.8 mg/mL, whereas in the 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate assay (176.5 ± 3.1 mg/L) it showed the best inhibition result [18]. All the EOs strongly suppressed the peroxide formation in linoleic acid system during incubation. The inhibition of oxidation of linoleic acid system was higher for the EOs of E. citriodora (83.8%) and E. camaldulensis (83.2%) than the other five Eucalyptus species (Figure 1). These findings are also supported by another study reporting 86.07% inhibition of linoleic acid for non-polar methanol extract of E. sargentii [34].

Chemicals and Microbial Strains
All the chemicals used in this study were of analytical grade and purchased from Sigma Aldrich (Taufkirchen, Germany). S. aureus, B. subtilis, E. coli, A. niger

Chemicals and Microbial Strains
All the chemicals used in this study were of analytical grade and purchased from Sigma Aldrich (Taufkirchen, Germany). S. aureus, B. subtilis, E. coli, A. niger and R. solani were obtained from and identified by the Department of Microbiology, University of Agriculture (Faisalabad, Pakistan). The bacterial strains were preserved and cultured separately in Lauria and Bertani (LB) media and fungal strains in Eggins and Pugh (E and P) medium broth by following the standard microbiological protocols. All microorganisms were stocked at´4˝C in standard conditions and were revived twice before use in the manipulations.

Collection of Samples
The fresh leaves from E. citriodora, E. melanophloia, E. crebra, E. tereticornis, E. globulus, E. camaldulensis and E. microtheca were collected from the Gutwala Forest Research Institute, (Faisalabad, Pakistan) in May 2014. All the leaves were washed with water to remove dirt particles and shade dried to reduce the moisture contents.

Procedure to Extract EOs
The EO was extracted from each plant material through a hydrodistillation method as described elsewhere [35]. In summary, the samples (leaves) were shade dried and a weighed amount (50 g) of crushed plant material was immediately charged to the distillation flask. Pressurized steam was circulated through the plant material. The vapors of the pure EO along with steam were condensed while passing through a water condenser and collected in a receiver flask kept in ice water in order to prevent the evaporation of the low boiling point constituents. The upper oily layer (2-3 mL) of condensed material was dissolved in diethyl ether (40 mL) and then separated from the distilled water component with the help of a separating funnel. Total EO was obtained after careful removal of the solvent by evaporation. The whole process of extraction was repeated till about 14-17 mL of oil was collected. The oils were dehydrated with Na 2 SO 4 and the average percent yield was calculated. The EO was stored in a cool place away from heat and light.

Physiochemical Properties
The physiochemical properties of the oils like colour, odour, appearance, solubility in aqueous ethyl alcohol, boiling point, specific gravity at 20˝C and refractive index were determined by the methods described earlier [36][37][38][39].

Chemical Composition through GC-Mass Spectroscopy
Qualitative and quantitative determination of chemical composition for each EO was carried out through gas chromatography mass spectroscopy by using a GC-17A (Shimadzu, Kyoto, Japan) fitted with a DB-wax (30 mmˆ0.25 mm) column (Agilent Technologies, Waldbornn, Germany) and a flame ionization detector (FID). Injector and detector temperatures were set at 250˝C and 260˝C, respectively. Column temperature was programmed from 90˝C for 2 min to 180˝C with a gradient of 2˝C/min. A second gradient was applied to 240˝C at 3˝C/min. Helium was used as a carrier gas at a flow rate of 30 mL/min at 150 psi. One µL sample of each EO was injected through the injector port. The chemical composition was determined by identifying the peaks with available data as standard and reported as a relative percentage of the total peak area. The quantitative measurements were made on chromatography station CSW 32 software of Data Apex (Prauge, Czech Republic, version 5.0).

Antimicrobial Activity
The antimicrobial activity of EOs was determined by using the disk agar diffusion method. The growing stock cultures were stabilized through various cycles for uniform growth. Sterilized Muller Hinton agar (Sigma-Aldrich, Taufkirchen, Germany) was cooled to 50˝C and inoculated with 100 µL fresh culture of each one of the above mentioned bacteria (10 5 -10 6 bacteria/mL), separately. The inoculated medium (15 mL) was poured into sterilized petri dish of 9 cm diameter and swirled to distribute homogenously. Disks (9 mm diameter, Whatman filter paper no. 3) injected with 20 µL either oil or standard antibiotics (see below) were applied on solid agar medium. The plates were placed at 4˝C for 1-2 h and then incubated at 37˝C for 24 h. The zones of inhibition (including the size of the disk as well = 0.9 mm) on the media were measured with ruler [40]. Antifungal activity was determined on Sabouraud dextrose agar medium as described earlier [41]. The microorganisms were cultured in test tubes on the agar for 24 h. Fresh Sabouraud medium was inoculated with these spores to the desired concentration of cells (10 5 spores/mL) and plates were prepared. Disks containing 20 µL EO were applied on the medium. The plates were incubated at 30˝C for three days and zones of inhibition were measured. The standard antibiotic control discs (containing 15 µg of the medicine on each disc) of Amoxil for bacteria and nizoral for fungi were used as controls, respectively.
The sensitivity of microorganism to each individual EO was determined by the diameter of the zones of inhibition with a small modification as described somewhere else [42]. Thus, the sensitivity was characterized as follows: not sensitive for total diameters smaller than 10 mm; sensitive for total diameters of 10-15 mm; very sensitive for total diameters of 16-20 mm; extremely sensitive for total diameters larger than 20 mm.

DPPH Scavenging Activity
The free radical scavenging activities of the seven Eucalyptus EOs were assessed by measuring their scavenging abilities for stable 2,2 1 -diphenyl-1-picrylhydazyl DPPH radicals [43]. To do this, the samples (0.5 µg/mL) were mixed with 1 mL of 90 µM DPPH solution and made up with 95% methanol to a final volume of 4 mL. The mixture was incubated at 25˝C for 1 h and absorbance was measured at 515 nm using a spectrophotometer (U-2001, Hitachi, Tokyo, Japan). Butylated hydroxytoluene (BHT) was used as a positive control. All the samples were analyzed in triplicate. The free radical scavenging activity was determined as percent inhibition by using the following equation: ( 1) The antioxidant activities of EOs were expressed as IC 50 values, which represented the concentrations of EOs that caused 50% neutralization of DPPH radicals and were calculated from the plot of inhibition percentage against concentration.

Antioxidant Activity in Linoleic Acid System
The antioxidant activity of EOs were also determined using inhibition of linoleic acid oxidation [44]. To do this, the test samples (50 µL) were dissolved in ethanol (1 mL) then mixed with linoleic acid (52 µL), ethanol (4 mL) and 0.05 M sodium phosphate buffer (pH 7, 4 mL). The solution was incubated at 40˝C for 175 h. The colorimetric method was used to measure the extent of oxidation by peroxide value as described earlier [45]. For 0.2 mL sample solution, 10 mL of ethanol (75%), 0.2 mL of aqueous solution of ammonium thiocynate (30%) and 0.2 mL of ferrous chloride solution (20 mM in 3.5% HCl) were added sequentially. The contents were stirred for 3 min and the absorbance was measured at 500 nm. BHT was used as a positive control. The percentage of the inhibition of linoleic acid oxidation was calculated as follows: % inhibition of linolic acid oxidation " 100´" Abs.increase of sample at 175 h Abs.increase of control at 175 h ˆ1 00 (2)

Statistical Analysis
Statistical analysis was performed with the IBM Statistical Package of Social Sciences (SPSS, version 19, SPSS Inc., Chicago, IL, USA). Statistically significant differences were found by using two factorial ANOVA and significant results were represented at p-values < 0.001.

Conclusions
Applications of natural extracts are growing rapidly in the food, cosmetic and pharmaceutical industry. The present study systematically explored the potential of some local Eucalyptus species. The chemical composition of EOs from selected Eucalyptus species showed great variation. The chemical compounds of essentials oils reported in these species were also different from other reported Eucalyptus species. The antimicrobial potential of the EOs extracted from seven Eucalyptus species was higher against Gram positive bacteria than Gram negative ones and two types of fungi. All the results proved that these EOs were very effective and could be used in medicines, cosmetics, food and flavors industries. This report is so far first systematic study and comparison of EOs of local prevalent Eucalyptus species and their potential for uses in health and industries.