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ANTIBACTERIAL AND ANTIOXIDANT ACTIVITIES OF ESSENTIAL OILS FROM LEAVES OF SEVEN EUCALYPTUS SPECIES GROWN IN PAKISTAN.

Byline: S. Siddique, Z. Parveen, Firdaus-e-Bareen, S. Mazhar and M. N. Chaudhary

Abstract

The chemical composition of essential oils from leaves of seven Eucalyptus species, viz., Eucalyptus crebra; Eucalyptus kitsoniana; Eucalyptus melanophloia; Eucalyptus microtheca; Eucalyptus pruinosa; Eucalyptus rudis and Eucalyptus tereticornis (Pakistan) was analysed by GC-FID and GC-MS. The main component of E. crebra, E. microtheca and E. rudis essential oils was 1,8-cineole (31.6-49.7 %). Eucalyptus melanophloia and E. tereticornis contained p-cymene (41.8-58.1 %) as a major component while E. kitsoniana and E. pruinosa essential oils were dominated by [alpha]-pinene (25.8-31.4 %). In vitro antimicrobial activity of the essential oils was studied by agar well diffusion method at concentration range 8-250 ug/ml. The Eucalyptus oils inhibited the growth of Gram positive bacteria (Bacillus spizizenii, Staphylococcus aureus) significantly with zones of inhibition (IZ) ranging from 15.3-46.0 mm.

The studied oil demonstrated moderate inhibitory effect against Gram negative bacteria (Enterobacter aerogenes, Escherichia coli, Salmonella enterica, Klebsiella pneumoniae and Pseudomonas aeruginosa) with IZ (0.0-22.2 mm). The oils exhibited bactericidal effects at 8 ug/ml in agreement with minimum inhibitory concentration against all tested foodborne pathogens except Pseudomonas aeruginosa (8-250 ug/ml). Time kill assay based on four weeks studies authenticated the potency of oils as food preservatives. Antioxidant assays (free radical scavenging activity and ferric reducing power test) demonstrated good activity of the oils. The evaluated essential oils exhibited strong antioxidant activity by 45.3-75.1 % inhibition of 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) and ferric reducing power (1.04 +- 0.05-1.95 +- 0.04 %) at 100 ug/ml. The good antioxidant and antibacterial activities of Eucalyptus essential oils validate their potential use as food preservatives.

Key words: 1,8-cineole, E. melanophloia, foodborne pathogens, GC-MS, ferric reducing power, bactericidal, time kill assay.

INTRODUCTION

The safety of food products is an important concern due to their vulnerability towards the microbes (bacteria and fungi) and atmospheric oxidation. Microbial growth is the major cause of food-borne ailments whereas the enzymatic oxidation of lipids affects the quality of food (Kanner and Rosenthal, 1992). Synthetic preservatives are preferred in food industry due to their effectiveness and low price to impede discoloration, spoilage and microbial contamination (Bajpai et al., 2008). However, apprehending their adverse environmental impacts, potential health hazards, development of microorganisms' resistance, the conventional synthetic chemicals are being substituted by natural products. Among the emerging natural preservatives, the interest in essential oils has been boosted up owing to their diverse biological (antimicrobial, antioxidants, anticarcinogenic) properties (Gutierrez et al., 2009).

Eucalyptus is one of the important genera of Myrtaceae and comprises about 800 species and subspecies (Gil et al., 2010). Most of the species are native to Australia. However, they have been cultivated throughout the tropics and subtropics including Africa, America, China, Europe, India, Mediterranean Basin and Middle East, (Guenther, 1952). Eucalyptus species have also been widely planted in many parts of NWFP and Punjab (Pakistan). Eucalyptus species are a rich resource of essential oil of medicinal and commercial importance. Eucalyptus essential oils also exhibit antibacterial (Elaissi et al., 2012), antioxidant, anti-inflammatory (Silva et al., 2003), antifungal (Somda et al., 2007), antiviral (Schnitzler et al., 2001) and insecticidal activities (Jemaa et al., 2014).

Keeping in view the recent trend of use of natural preservatives, seven common Pakistani Eucalyptus species, namely E. crebra, E. kitsoniana, E. melanophloia, E. microtheca, E. pruinosa, E. rudis and E. tereticornis have been selected to evaluate their antioxidant potential and antibacterial activity against foodborne pathogens.

MATERIALS AND METHODS

Collection of Materials: Fresh leaves of E. crebra, E. kitsoniana, E. melanophloia, E. microtheca, E. pruinosa, E. rudis and E. tereticornis were collected from botanical garden, Pakistan Forest Research Institute (PFI), Faisalabad, Pakistan in October, 2014. Plant herbaria were authenticated by Dr. Nasir (Professor, Department of Botany, University of Punjab, Lahore, Pakistan) and voucher specimens have been deposited at the Department of Botany, University of the Punjab, Lahore, Pakistan under the following references: BDSS # 4023; BDSS # 4024; BDSS # 4025; BDSS # 4026; BDSS # 4027; BDSS # 4028 and BDSS # 4029.

Isolation of Essential Oils: Fresh leaves of Eucalyptus species were subjected to hydrodistillation for 3h using Clavenger-type apparatus according to the method recommended in the European Pharmacopoeia (EDQM, 2005. Essential oils distillates were dried over anhydrous sodium sulfate, filtered and kept at -4AdegC till analysis. The oil yields are listed in Table 1.

Chemical Analysis of Essential Oils: Gas chromatography analysis of the essential oils was carried out on Shimadzu GC 2010 using DB-5 MS (30 m x 0.25 mm id, 0.25 um film thickness) capillary column. The column oven temperature was programmed initially at 40-90AdegC at the rate of 2AdegC/min and then 90-240AdegC at the rate of 3AdegC/min. The final temperature was held constant for 5 min. Injector and detector temperatures were maintained at 240 and 280AdegC respectively. A sample of pure essential oil (0.5ul) was injected in a split mode ratio of 1:5. Helium was used as a carrier gas at the flow rate of 1 ml/min. GC-MS analysis was carried out on GCMS-QP 2010 Plus, Shimadzu, Japan operating in electron ionization mode at 70 eV. Column conditions were same as in GC analysis. The mass spectrometer was capable of scanning from 35 to 500AMU every second or less.

The data acquisition system continuously acquires and stores all data analyses. The components were identified by comparing their mass spectra with those of NIST mass spectral library (Mass spectral library 2001) and Adams (2001) as well as by comparing their retention indices either with those of authentic compounds or with literature values.

Evaluation of Antimicrobial Activities of Essential Oil:

Test Microorganisms

Seven bacterial strains from American Type Culture Collection (ATCC, Rockville) were selected for in vitro antibacterial activity of the essential oil. Gram positive bacteria comprised of B. spizizenii (ATCC 6633) and S. aureus (ATCC 25923) while Gram negative strains were E. aerogenes (ATCC 13048), E. coli (ATCC 8739), S. enterica (ATCC 14028), K. pneumoniae (ATCC 13882) and P. aeruginosa (ATCC 27853). All the bacterial strains were sub-cultured at 35AdegC for 24 h on nutrient agar slants prior to being grown in nutrient broth overnight.

Antibacterial Activity: Antibacterial activity of Eucalyptus essential oils was checked by agar well diffusion method (Zaika, 1988). Molten agar medium (20 ml) was inoculated with bacterial suspension containing indicator strain at 106 cfu/ml .The inoculated medium was poured into a petri plate and allowed to solidify. Wells were made in triplicate on solidified agar and 90 ul of the tested oil was added to each. The plates with bacterial strains were incubated at 35AoC for 24 h. The diameters of inhibition zones were measured in millimeters and results were recorded in triplicate.

Minimum Inhibitory Concentration (MIC) Assay: Serial dilutions of 8 ug/ml, 15 ug/ml, 65 ug/ml and 250 ug/ml were used in triplicate to determine MIC levels by agar well method (Zaika, 1988). The lowest concentration of oil inhibiting visible growth of each microbe after incubation was taken as the MIC.

Minimal Bactericidal Concentration (MBC): Minimum Bactericidal Concentration (MBC) was determined by the method of Rabe, et al., (2002). Bacterial spore load (106 cfu/ml) was poured into the tubes containing respective culture broth and oil with concentration of MIC. Broth tubes with and without bacterial load were used as controls. The tubes were incubated for 24 h at 35AdegC. After incubation, 100 ul from tubes having no visible growth was removed and poured in plates along with agar to enumerate total viable counts. The lowest concentration with no visible growth after 24 h of incubation at 35AdegC was defined as the MBC, indicating 99.5 % killing of the original inoculum.

Time Kill Assay: A time kill study was carried out with the MIC values found previously agar well method to discern whether the Eucalyptus oils have bacteriostatic or bactericidal effect over a period of time to be used as food preservatives (White, 1996). Bacterial suspension with 106 cfu/ml and oil having concentration equal to MIC were added respectively in the tube of corresponding culture medium. Broth tubes with and without microbial suspension were used as controls. The cultures were incubated for one month at 35AdegC. An inoculant of 100 ul, removed after 2, 5, 8, 11, 14 and 30 d was poured in agar plates in triplicate to determine the total reduction in viable counts. The mean number of the colonies (cfu/ml) was counted and compared with that found in the control culture at the end of the incubation period.

The test tubes with turbidity after a certain time period of incubation depict bacteriostatic effect of the evaluated essential oils at the applied concentration. To determine the bactericidal concentration of Eucalyptus essential oils against that particular strain, the higher concentrations (15 ug/ml, 65 ug/ml and 250ug/ml) were applied and lethal effect of essential oils were observed as mentioned above.

ANTIOXIDANT ACTIVITY

DPPH Assay: The antioxidant activity of the essential oils from Eucalyptus species was assessed by measuring its ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) stable radical. The assay was carried out spectrophotometrically as described by Shimada et al., (1992) with some modifications.

Various concentrations of oils samples were prepared in methanol. To the 0.1ml of each test concentration, 3ml of methanolic solution of DPPH (0.004 %) were added. Resulting mixtures were incubated in dark for 30 min at 25AdegC. The decrease in absorbance was measured at 517 nm using a spectrophotometer (Cecil CE7200). Scavenging (%) exhibited by essential oils was calculated as follows:

Scavenging (%) = (Ablank - Asample/Ablank) x 100

Where Ablank is the absorbance of the control reaction (containing all reagents except the test compound) and Asample is the absorbance of the test oil. All determinations were performed in tripilcate.

Total Reduction Ability by Fe3+-Fe2+ Transformation: The capacity of essential oils to reduce the ferric ion (Fe3+) to the ferrous ion (Fe2+) was determined by measuring absorbance at 700 nm (Oyaizu et al., 1986.). Different concentrations of the essential oils were added to 2.5ml of phosphate buffer (0.2M, pH 6.6) and 2.5ml of potassium ferricyanide (1 %). The mixture was incubated at 50C for 20 min and 2.5 ml of trichloroacetic acid (10 %) were added thereafter. The mixtures were revolved at 3000 rpm for 10 minutes. The supernatant (2.5 ml) was mixed with distilled water (2.5 ml) and ferric chloride 0.5 ml. The resultant mixture was allowed to stand for 30min and absorbance was measured on UV spectrophotometer at 700 nm. Higher absorbance of reaction mixture indicated greater reducing power. The comparison of ferrous reducing antioxidant potential was made with butylated hydroxytoluene (BHT).

Statistical Analysis: The mean values, +- standard deviations were calculated using MS Excel 2007.

RESULTS AND DISCUSSION

The yield of essential oils ranged from 0.1-1.9 % for different Eucalyptus species [Table 1]. The highest yield was obtained from E. rudis (1.8 %) while E. tereticornis gave the lowest yield (0.1 %). The oil yields of E. rudis and E. crebra were in accordance to the previous reports (Iqbal et al., 2003; Ahmad et al., 2005; Haq et al., 2007; Elaissi et al., 2011; Jemaa et al., 2012; Sliti et al., 2015). Eucalyptus microtheca, E. melanophloia, E. kitsoniana and E. tereticornis oil yields were found lower than the former reports (Bachheti et al., 2011; Elaissi et al., 2011; Iqbal et al., 2003; Ghaffar et al., 2015). No data was available to compare oil yield of E. pruinosa in literature.

Essential oils of seven Eucalyptus species were found rich in monoterpene hydrocarbons and oxygenated monoterpenes. [alpha]-Phellandrene (0.0-2.0 %), [alpha]-pinene (0.7-39.4 %), p-cymene (0.7-58.1 %), 1,8-cineole (1.0-49.7 %) and terpinolene (1.3-7.2 %) were found as common components in volatile oils of all Eucalyptus species in varying concentrations.

Table 1. Chemical Composition of Essential Oils from Eucalyptus Species.

###Content (%)

###Compounds###RI

###E. cre###E.kit###E. mel###E. mic###E. pru###E.rud###E. ter

[alpha]-Pinene###930###16.9###25.8###24.1###31.0###29.5###32.5###0.7

Camphene###944###0.2###0.2###0.3###0.6###0.4###0.1###-

[beta]-Pinene###974###-###-###-###-###0.3###-###-

4-Carene###1001###0.1###0.2###0.3###-###-###tr###0.2

[alpha]- Phellandrene###1002###0.2###2.0###1.0###tr###0.1###tr###0.3

Limonene###1011###4.1###4.0###6.2###5.7###4.6###8.4###-

p-Cymene###1026###16.1###24.8###41.8###2.4###8.2###1.6###58.1

1,8-Cineole###1029###49.7###3.3###3.3###31.6###24.2###48.5###6.5

[beta]-cis-Ocimene###1043###Tr###tr###tr###-###-###0.1###-

I3-Terpinene###1055###0.2###1.2###0.8###0.1###-###0.2###0.5

Terpinolene###1083###0.1###0.6###0.6###0.1###tr###0.1###0.2

Terpin-4-ol###1089###4.1###7.2###2.9###4.8###3.9###2.4###2.1

[beta]-Linalool###1095###-###tr###0.8###-###0.1###-###0.5

[alpha]-Campholenal###1127###0.1###-###0.1###0.1###0.1###-###-

trans-Pinocarveol###1135###2.0###0.2###-###2.2###1.3###0.9###0.1

Camphene Hydrate###1145###0.1###0.1###0.1###0.2###0.2###tr###-

p-Cymen-3-ol###1183###0.1###0.1###0.1###tr###0.1###0.4###0.4

p-Cymen-8-ol###1196###0.5###0.3###0.8###0.3###0.4###0.2###3.6

Compounds###RI###Contents %

###E. cre###E.kit###E. mel###E. mic###E. pru###E.rud###E. ter

Nerol acetate###1365###-###-###-###-###-###0.1###0.2

[alpha]-Copaene###1374###-###-###-###tr###-###-###0.1

Fenchol###-###-###-###-###-###-###tr###-

Eugenol methyl ether###1402###0.1###0.2###0.5###-###0.1###0.8###0.7

[alpha]-Caryophyllene###1444###Tr###0.1###0.4###0.2###0.2###0.1###-

Humulen- (IV)###1452###-###-###-###0.1###-###-###-

Germacrene D###1484###Tr###-###0.1###tr###tr###0.5###0.2

Monoterpenes###37.9###58.8###75.1###39.9###43.1###43.0###60.0

Oxygenated monoterpenes###56.6###11.2###8.1###39.2###30.3###52.5###13.4

Sesquiterpenes###Tr###0.1###3.3###0.3###0.2###0.6###0.5

Oxygenated sesquiterpenes###2.5###0.5###6.9###2.5###9.7###1.0###-

Phenolic compounds###0.1###0.2###0.5###-###0.1###0.8###0.7

Others###-###tr###-###-###-###-###-

Unidentified###2.9###29.2###6.1###18.1###16.6###2.1###25.4

Oil Yield (%)###0.5###2###0.4###0.4###0.9###1.9###0.1

The richness of E. crebra, E. rudis, E. microtheca and E. pruinosa essential oils in 1,8-cineole and [alpha]-pinene was in conformity with the previous reports (Isiaka et al. 2003; Sefidkon et al., 2007; Joseph et al.,2008). Eucalyptus rudis oil contained higher concentration of 1,8-cineole (48.5 %) and [alpha]-pinene (32.5 %) as compared to Tunisian variety of E. rudis (19.9 % and 3.9-14.5 %, respectively). Limonene which has been identified in E. rudis oil was absent in the Tunisian variety of E. rudis (Haouel et al 2010; Elaissa et al.2012; Sliti et al., 2015). p-Cymene was the major component in the studied species of E. tereticornis contrary to 1,8-cineole and [alpha]-pinene as reported previously (Pino et al., 2001; Kaur et al., 2010). [alpha]-Pinene was found as a major component in E. kitsoniana oil whereas, 1,8-cineole was reported as principal constituent in Tunisian variety of E. kitsoniana (Elaissi et al., 2011).

The antimicrobial activity of Eucalyptus essential oils was evaluated by measuring the inhibition zones against the common food borne pathogens (B. spizizenii, S. aureus, E. aerogenes, E. coli, S. enterica, K. pneumoniae, P. aeruginosa).. Preliminary screening of antibacterial activity of Eucalyptus essential oils was done through agar well diffusion assay. The degree of antibacterial activity was evaluated by determining the minimum inhibitory concentration (MIC) of the oils while the bactericidal activity was assessed by broth dilution method. Eucalyptus species manifested good antibacterial properties against all the tested microbes. However, the level of bacterial growth inhibition was dependent on the oils concentration and the bacterial strain.

The results of antibacterial activity and MIC and MBC values have been summarized in Table 3 and 4. The selected gram positive strains (B. spizizenii and S. aureus) showed high sensitivity towards Eucalyptus oils at concentrations of 8-250 ug/ml (Table 2). Bacillus spizizenii was most sensitive towards E. microtheca essential oil with zones of inhibition (ZI = 14.5-46.0 mm) followed by E. rudis (13.8-37.0mm), E. microtheca (ZI = 12.0-33.0 mm), E. melanophloia (ZI = 15.7-22.0 mm), E. pruinosa (ZI = 13.2-20.0 mm), E. kitsoniana (ZI= 12.0-17.0 mm) and E. tereticornis (ZI = 15.0 mm). Eucalyptus rudis and E. kitsoniana (ZI = 11.3-21.2 mm) showed good activity against S. aureus followed by E. microtheca (11.2-19.2 mm), E. crebra (12.3-18.7 mm), E. tereticornis (13.0-17.0 mm) and E. melanophloia (13.2-16.8 mm).

Among gram negative strains, the Eucalyptus oils demonstrated significant inhibitory effect against K. pneumoniae and E. coli (11.5-22.2 mm) followed by E. aerogenes, S. enterica (11.0-16.8 mm) and P. aeruginosa (0.0-15.7 mm). Elaissa et al., 2011 reported smaller zones of inhibition (7.7-10.3 mm) against S. aureus, E. coli and P. aeruginosa. Our results on antibacterial activity are in accordance with those described by Fawad et al., 2011 and Ghaffar et al., 2015.The larger inhibitory zones induced by studied Eucalyptus oils when compared to Elaissa et al., 2011 showed their effectiveness as antibacterial agents.

Table 2. Antibacterial activity of Eucalyptus Essential Oils by Agar well diffusion method.

Essential###Conc.###Tested Microbial Strains

Oil###ug/ml###B. spizi###S. aure###E. aerog###E. coli###K. pneum###P. aerug###S. enter

E. cre###250###46.0 +-0.0###18.7 +-0.4###13.7 +-0.4###15.7 +-0.6###18.7 +-0.4###15.7 +-0.4###13.3 +-0.2

###65###17.7 +-0.5###16.0 +-0.0###12.3 +-0.4###13.0 +-0.0###15.3 +-0.4###14.7 +-0.4###12.3 +-0.4

###15###15.7 +-0.4###13.0 +-0.0###11.7 +-0.4###11.8 +-0.5###14.0 +-0.0###12.0 +-0.0###11.7 +-0.4

###8###14.5 +-0.0###12.3 +-0.4###11.0 +-0.0###11.7 +-0.4###12.0 +-0.0###10.3 +-0.4###11.3 +-0.4

E. kit###250###17.0 +-0.0###20.5 +-0.3###15.8 +-0.2###22.2 +-0.5###22.2 +-0.5###15.7 +-0.2###16.2 +-0.5

###65###13.8 +-0.2###13.2 +-0.2###13.5 +-0.3###15.7 +-0.2###15.7 +-0.2###12.3 +-0.2###13.5 +-0.0

###15###13.0 +-0.0###12.0 +-0.0###12.5 +-0.0###14.2 +-0.2###13.5 +-0.3###12.0 +-0.0###12.3 +-0.2

###8###12.0 +-0.2###11.3 +-0.2###12.0 +-0.0###13.3 +-0.2###13.0 +-0.0###11.3 +-0.2###11.3 +-0.2

E. mel###250###21.8 +-0.2###16.8 +-0.2###11.8 +-0.2###13.5 +-0.3###16.2 +-0.5###0.0 +-0.0###12.7 +-0.2

###65###19.5 +-0.5###14.7 +-0.4###11.3 +-0.2###12.5 +-0.3###14.2 +-0.2###0.0 +-0.0###12.3 +-0.4

###15###17.0 +-0.3###13.5 +-0.0###11.2 +-0.2###12.2 +-0.2###13.0 +-0.0###0.0 +-0.0###11.7 +-0.2

###8###15.7 +-0.2###13.2 +-0.2###11.0 +-0.0###11.5 +-0.0###12.2 +-0.2###0.0 +-0.0###11.3 +-0.4

E. mic###250###33.3 +-0.5###19.2+-0.5###16.7 +-0.4###15.8 +-0.5###16.7 +-0.2###15.5 +-0.5###15.7 +-0.6

###65###19.3 +-0.4###13.0 +-0.3###15.8 +-0.5###15.2 +-0.5###14.8 +-0.4###13.8 +-0.2###14.8 +-0.2

###15###14.5 +-0.5###12.0 +-0.3###14.8 +-0.2###13.5 +-0.3###14.5 +-0.0###11.8 +-0.2###13.2 +-0.2

###8###12.0 +-0.0###11.2 +-0.4###14.0 +-0.0###12.8 +-0.2###13.8 +-0.2###11.3 +-0.4###12.5 +-0.0

E. pru###250###20.0 +-0.0###19.0 +-0.6###16.8 +-0.5###19.2 +-0.2###21.8 +-0.5###13.7 +-0.4###16.3 +-0.4

###65###16.5 +-0.3###15.0 +-0.0###13.3 +-0.5###17.3 +-0.5###16.8 +-0.5###12.2 +-0.2###14.5 +-0.3

###15###13.5 +-0.0###13.0 +-0.3###12.5 +-0.0###15.3 +-0.4###14.2 +-0.2###11.5 +-0.5###13.8 +-0.2

###8###13.2 +-0.2###12.3 +-0.2###11.5 +-0.3###13.7 +-0.4###13.8 +-0.2###10.3 +-0.4###12.8 +-0.2

E. rud###250###36.8 +-0.2###21.2 +-0.2###16.2 +-0.2###17.8 +-0.2###18.0 +-0.0###14.8 +-0.5###16.7 +-0.3

###65###17.8 +-0.7###18.2 +-0.2###15.0 +-0.0###14.5 +-0.3###16.8 +-0.2###12.2 +-0.2###14.0 +-0.0

###15###14.7 +-0.5###13.5 +-0.3###13.8 +-0.2###13.0 +-0.3###14.7 +-0.2###11.8 +-0.6###13.0 +-0.0

###8###13.8 +-0.2###12.2 +-0.2###13.0 +-0.0###12.3 +-0.4###13.7 +-0.4###10.0 +-0.0###11.8 +-0.3

E. ter###250###15.3 +-0.5###17.0 +-0.5###15.5 +-0.5###13.2 +-0.2###18.3 +-0.5###14.5 +-0.3###13.0 +-0.0

###65###14.3 +-0.4###15.3 +-0.4###14.0 +-0.5###12.5 +-0.3###15.0 +-0.0###12.7 +-0.4###12.5 +-0.3

###15###13.5 +-0.2###13.7 +-0.5###12.8 +-0.5###12.3 +-0.4###13.5 +-0.3###12.3 +-0.4###12.0 +-0.0

###8###13.0 +-0.2###13.0 +-0.0###12.0 +-0.0###11.8 +-0.2###13.3 +-0.2###11.3 +-0.4###11.7 +-0.4

The high degree of antibacterial activity was further confirmed by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). All Eucalyptus oils except E. tereticornis and E. melanophloia exhibited bacteriostic and bactericidal effect at concentration of 8ul/ml against tested strains. The MIC and MBC values of E. tereticornis and E. melanophloia were found 15 ug/ml and 250 ug/ml respectively (Table 3). Sliti et al., 2015 reported MIC (0.075-0.5 %) and MBC values (0.5-3.5 %) of E. rudis against different pathogens. They have been found higher than Pakistan grown variety of E. rudis. Based on the MIC results, the oil or plant extract is considered strong inhibitor with MIC below 0.5 mg/ml;moderate inhibitor with MIC 0.6-1.5 mg/ml and weak inhibitor with MIC above 1.6 mg/ml (Duarte et al. 2005).

Hereby, considering Duarte et al. 2005 plants classifications for potential antimicrobial activity, the Eucalyptus oils with powerful antibacterial activity at 8-250 ug/ml concentrations against the evaluated bacterial strains fall within the group of strong inhibitors.

The time kill assay was carried out to check the potency of Eucalyptus essential oils to be used as a food preservative. The study was carried out to evaluate the bacteriostatic or bactericidal effects of tested oils for four weeks. All the tested oils showed bactericidal effect against B. spizizenii at 15-250 ug/ml while it ranged from 8-250 ug/ml for S. aureus. K. pneumoniae was found sensitive towards all tested oil with lowest MBC i-e. 8 ul/g among gram negative strains followed by E. coli with MBC values (8-15 ug/ml), E. aerogenes and S. enterica (15-250ug/ml). P. aeruginosa was found most resistant among the tested strains with MBC values ranging from 65-250 ug/ml (Table 4).

Table 3. Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) ug/ml of Eucalyptus essential oils.

Tested###E. cre###E. kit###E. mel###E. mic###E. pru###E. rud###E. ter

Microbial###MIC###MBC###MIC###MBC###MIC###MBC###MIC###MBC###MIC###MBC###MIC###MBC###MIC###MBC

Strains

B. spizizenii###8###8###8###8###8###8###8###8###8###8###8###8###8###8

S. aureus###8###8###8###8###8###8###8###8###8###8###8###8###8###8

E.###8###8###8###8###8###8###8###8###8###8###8###8###8###8

aerogenes

E. coli###8###8###8###8###8###8###8###8###8###8###8###8###8###8

K.###8###8###8###8###8###8###8###8###8###8###8###8###8###8

pneumoniae

P.###8###8###8###8###250###250###8###8###8###8###15###15###15###15

aeruginosa

S. enterica###8###8###8###8###8###8###8###8###8###8###8###8###8###8

Table 4. Time kill Assay; bactericidal Effects (MBC) ug/ml of Eucalyptus essential oils for 30 days.

Tested Microbial###E. cre###E. kit###E. mel###E. mic###E. pru###E. rud###E. ter

Strains

B. spizizenii###65###65###250###65###15###15###250

S. aureus###8###8###250###8###15###15###8

E. aerogenes###65###250###250###250###15###15###65

E. coli###8###15###15###8###15###8###8

K. pneumoniae###8###8###8###8###8###8###8

P. aeruginosa###250###250###250###65###65###65###250

S. enteric###15###15###250###15###15###15###15

Table 5. DPPH Redical Scavenging Activity of Eucalyptus Essential Oils.

###DPPH Redical Scavenging (%)

###20 ug/ml###40 ug/ml###60 ug/ml###80 ug/ml###100 ug/ml###IC50ug/ml

BHT###30.8 +- 0.6###52.4 +- 1.0###66.5 +- 0.7###76.8 +- 0.6###84.9 +- 0.3###44.5 +- 0.4

E. crebra###25.9 +- 0.5###30.8 +- 0.7###38.0 +- 0.4###45.2 +- 0.6###50.7 +- 0.7###97.3 +- 2.3

E. kitsoniana###28.7 +- 0.4###35.5 +- 0.7###42.8 +- 0.6###51.4 +- 1.1###59.3 +- 1.0###76.7 +- 1.0

E. melanophloia###29.8 +- 1.0###37.3 +- 0.7###46.6 +- 1.4###58.3 +- 1.1###64.9 +- 1.7###66.8 +- 1.8

E. microtheca###29.3 +- 0.7###38.6 +- 0.8###52.6 +- 1.7###64.3 +- 0.7###74.4 +- 0.7###56.8 +- 0.9

E. pruinosa###21.5 +- 1.3###25.7 +- 0.8###35.2 +- 0.8###39.9 +- 0.8###45.5 +- 0.7###113.0 +- 2.2

E. rudis###25.0 +- 0.8###28.6 +- 1.3###37.3 +- 0.9###49.4 +- 0.7###55.2 +- 0.6###86.9 +- 1.1

E. tereticornis###27.5 +- 0.7###35.3 +- 0.9###45.6 +- 0.8###55.2 +- 1.2###63.6 +- 0.9###69.9 +- 1.0

The bioactivity of the essential oils depends upon their major volatile component (Cimanga et al., 2002). [alpha]-Pinene, p-cymene and 1,8-cineole; the principal constituents in the Eucalyptus oils have been reported to exhibit antibacterial activities (Khamis et al., 2005; Sonbolia et al., 2006; Jiang et al., 2011). The minor components (linalool, [alpha]-terpineol; cis-ocimene, limonene, terpinolene and [alpha]-phellandrene) in the Eucalyptus oils also possess antibacterial properties (Khamis et al., 2005; Magwa et al., 2006; Donsi et al., 2011). Therefore, the antimicrobial activity of tested essential oils could be due to synergistic effects of major and minor components.

DPPH assay and reducing power assay were used to assess antioxidant potential of essential oils. The synthetic antioxidant BHT was used as an equivalence parameter for the antioxidant activity of the essential oils. The Eucalyptus essential oils showed DPPH scavenging activity (45.34-75.11 %). The DPPH scavenging activity of tested oils was found lower than synthetic antioxidant; butylated hydroxytoluene (BHT) (Table 5). Highest antioxidant activity was displayed by E. microtheca essential oil with IC50 value of 56.8 +- 0.9 ug/ml while E. pruinosa demonstrated lowest activity with IC50 value 113.0 +- 2.2 ug/ml among the Eucalyptus oils.

In the reducing power assay, the Eucalyptus essential oils showed lower ferric reducing power than BHT. Eucalyptus microtheca essential oil showed highest absorbance at tested concentrations (20-100 ug/ml) corresponding to good antioxidant activity (1.77 +- 0.02 ug/ml) while E. pruinosa demonstrated lowest activity with percentage absorbance of 1.04 +- 0.05 ug/ml among the Eucalyptus oils at 100 ug/ml (Table 6).

Table 6. Total Ferric Reducing Abilityof Butylatedhydroxytoluene and Eucalyptus Essential oils.

###Absorbance (%) at 700nm

###20 ug/ml###40ug/ml###60ug/ml###80ug/ml###100ug/ml

BHT###1.16 +- 0.18###1.36 +- 0.03###1.57 +- 0.05###1.82 +- 0.07###1.95 +- 0.04

E.crebra###0.51 +- 0.02###0.68 +- 0.03###0.81 +- 0.02###0.98 +- 0.02###1.17 +- 0.03

E.kitsoniana###0.60 +- 0.01###0.80 +- 0.02###0.95 +- 0.02###1.17 +- 0.01###1.31 +- 0.01

E. melanophloia###0.66 +- 0.02###0.84 +- 0.04###1.06 +- 0.04###1.25 +- 0.06###1.50 +- 0.10

E.microtheca###0.75 +- 0.02###1.01 +- 0.02###1.25 +- 0.02###1.46 +- 0.02###1.77 +- 0.02

E.pruinosa###0.45 +- 0.01###0.61 +- 0.02###0.75 +- 0.02###0.90 +- 0.03###1.04 +- 0.05

E.rudis###0.54 +- 0.02###0.70 +- 0.01###0.87 +- 0.02###1.02 +- 0.03###1.23 +- 0.06

E. tereticornis###0.61 +- 0.02###0.82 +- 0.03###1.05 +- 0.03###1.25 +- 0.05###1.44 +- 0.00

Antioxidant activity of monoterpene and monoterpenoids present in studied oils has been reported by many researchers. Wei and Shibamoto 2007 related antioxidant activity to the [alpha]-pinene in Citharexylum caudatum L. Limonene, terpinolene, and I3-terpinene have also been reported to show considerable activity (Ruberto and Baratta, 2000; Song et al., 2001; Elmastas et al. 2006). Among other monoterpenoids, 1,8-cineole and terpen-4-ol has demonstrated antioxidant and anti-inflammatory activities (Kim et al., 2004; Porres et al., 2014). Thus, antioxidant activity of Eucalyptus essential oils can be attributed to the synergy among the different oil constituents.

Conclusion: The Eucalyptus species have remarkable antibacterial activity against common food-borne pathogens and antioxidant potential so these may be suggested as new potential source of natural antimicrobial and antioxidant agents.

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