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Contribution to the antiseptic effect study of two Eucalyptus Species.


Medicinal plants have been used as a source of remedies since ancient times (Abu-Shanab et al, 2004). The ancient Egyptians were familiar with many medicinal herbs and were aware of their usefulness in treatment of various diseases (Abu-Shanab et al, 2004). Phytomedicines derived from plants have shown great promise in the treatment of intractable infectious diseases including viral infections (Cowan, 1999). Single and Poly herbal preparations have been used numerously through out history for the treatment of various diseases. Many studies have been carried out to extract various natural products for screening antimicrobial activity but attention has not been focused intensively on studying the combinations of these products for their antimicrobial activity (Belaiche, 1979).

One of these medicinal plants, many species of the genus Eucalyptus from the Myrtaceae family are used in many parts of the world for the treatment of a wide variety of diseases including microbial infections (Ben Arfa et al, 2007).

The development of bacterial resistance to presently available antibiotics has necessitated the search for new antibacterial agents. The gram positive bacterium such as Staphylococcus aureus is mainly responsible for post operative wound infections, toxic shock syndrome, endocarditis, osteomyelitis and food poisoning (Benayache et al, 2001).

The gram negative bacterium such as Escherichia coli is present in human intestine and causes lower urinary tract infection, coleocystis or septicaemia (Benhassaini et al, 2003; Benjilali et al, 1986).

The aim of the present study was to examine the effects of essential oil of two eucalyptus species (E. globulus and E. camaldulensis) on S. aureus and E. coli.

Materials and methods

* Plant material and isolation of essential oils

Eucalyptus camaldulensis was collected from the "Nador-Mennaour" forest in the El bordj region located in Mascara willaya (city North West of Algeria).

Eucalyptus globulus was collected at the Mustapha Stambouli university campus of Mascara city. The essential oils of fresh plant leaves collected during the flowering stage were isolated by hydrodistillation method using a Clevenger-type apparatus. The oils were separated from the water by decantation and were dried by filtration over anhydrous sodium sulfate.

* Microorganisms

The microorganisms were obtained from microbiology department, Laboratory of medical analysis, Dr Yessaad Khaled hospital at Mascara city. They are two bacterial strains Escherichia coli and Staphylococcus aureus isolated and identified by classical methods (Menghini et al, 1987).

Staphylococcus. aureus was isolated in the urine of a patient, and Escherichia coli from a patient's coproculture.

The identification of organisms is based on cellular, cultural and biochemical characteristics. All species of Staphylococcus are Gram positive cocci. On nutrient agar they tend to be white, circular, entire, convex colonies. On blood agar Staphylococcus aureus may show hemolysis of the agar in the area around the colony.

Additional biochemical tests those are useful in separating the Staphylococcus species include catalase, coagulase, growth and fermentation of mannitol salt, andresistance or susceptibility to the antibiotic novobiocin. They were chosen for their high frequencies of food contamination and for their pathogenicites.

* Determination of the essential oil antiseptic activity

* The Aromatogram method.

This technique, which is performed in a clinical biology laboratory, is an in-vitro method of measuring the antibacterial power of chemotyped essential oils. To seed one ml of the bacterial inoculum in the petri dishes previously melted with Mueller Hinton agar.

Six disks of blotting paper (0.7 cm diameter) aseptically impregnated with the following essential oils quantities: 1 [micro]l, 2 [micro]l, 5 [micro]l, 7.5 [micro]l, 10 [micro]l, 20 [micro]l are deposited on the agar surface. After a latency period at 37.5[degrees]C, the inhibition halo surrounding the disks is then measured (Boland et al, 1991; Cimanga et al, 2002; Euzeby; Farah et al 2001; Franchomme, 1999).

* The microatmosphere method

This technique allows evidence to describe the diffusion of volatile essential oils components in petri dishes. A disc (with 2.5 cm diameter) impregnated with 50 [micro]l essential oils is deposited in the center of the lid and the petri dish is enclosed on it which the agar is previously dried under a hood for 15-20 minutes then incubated to 37 [degrees] C for 16-18 hours (Gamal and Sabrin, 2007; Gocho, 1991; Harkental et al, 1999; Iserin, 1997).

* The suspension of germs method

The microorganisms (S.aureus, E. coli) present in suspension are dissolved in broth, then diluting a series of 10' to 10-4. The essential oils are added to each ml from dilution to a certain volume (10, 50, and 100) w 1 which is then added an aqueous solution of 1% (v / v) of tween 80. Microbial growth was checked by spectrophotomtric determination at 600nm after 10 minutes, 20 minutes and 24 hours of incubation. The surviving bacteria are then counted and transferred to a agar culture medium Mueller Hinton for their development (Kouokam et al, 2002; Larrondo et al, 1995; Le Minor and Richard, 1993; Levine, 1987).

Standard antibiotic discs such as ampicillin, Amoxicillin, Chloramphenicol, Erythromycin, Nitroxoline and Pristinamycine were used for antibiogram test.

Results and discussion

* The antibiotic sensitivity testing

According to the results of antibiogram, we noticed that the S. aureus strain was sensitive to Chloramphenicol and Pristinamycine, has withstood Ampicillin and Doxycycline, as she presented an intermediate resistance to the Amoxicillin, Erythromycin and Nitroxoline. The E. coli was sensitive to Chloramphenicol, resistant to ampicillin, Doxycycline and Pristinamycine and intermediary resistance to Erythromycin and Nitroxoline.

The data show that, E. coli and S. aureus were resistant to the low doses of Eucalyptus oil like 1 [micro]l and 2 [micro]l; but from 5 [micro]l until 20 [micro]l the inhibition zone Diameter are measurable [Table 02]. With an increasing dose of oil of eucalyptus, the resulting diameter of the zone of inhibition increased for the two bacterial strains. Escherichia coli is more resistant than Staphylococcus aureus. These results are similar to those found by (Trivedi and Hotchandani, 2004; Farah et al, 2001; Gamal and Sabrin, 2007; Nair et al, 2008).

According to the results presented in the photo (01 and 02), we see the action of Eucalyptus essential oils vapor against Staphylococcus aureus and Escherichia coli, this action is reflected by translucent inhibition zone. The vapor of essential oil of Eucalyptus inhibited the growth of Staphylococcus aureus and Escherichia coli in culture medium. The essential oil antimicrobial activity is almost similar to those of Eucalyptus globulus and Eucalyptus camaldulensis.

Iserin (1997), Inouye et al (2001), Cimanga et al (2002) and many other researchers reports that the antimicrobial activity of an essential oil is to be linked to its chemical composition, especially the functional groups of compounds majority (alcohol, phenols, terpene compounds and ketones).



Pibiri (2006) has demonstrated in his doctoral thesis that the tested essential oils in gaseous phase do have a lethal effect on two strains Staphylococcus aureus and Pseudomonas aeruginosa, even in small doses.

The essential oil constituents of the genus Eucalyptus (Myrtaceae) have been well characterized (Batista-Pereira et al, 2006) Eucalyptus species produce numerous volatile compounds in large amounts, especially isoprenoids (here referred to as terpenes), which are accumulated in glands abundantly distributed throughout the leaf parenchyma and bark. (Rakotonirainy and Lavedrine, 2005; Moleyar, and Narasimham, 1986).

The chemical compositions of the leaf oils of Eucalyptus from various parts of the world have been reported. And even in Algeria (Singh et al, 2000). A major component of eucalyptus oil in percentage weight was 1,8-cineole, but a major contributor for the bioactivity was assumed to be [alpha] -terpineol, which showed eight fold higher activity than 1,8-cineole against S. aureus. 1,8-Cineole has been reported not to be an active principle in other eucalyptus oils (Inouye et al, 2001).

It has already been shown that the antimicrobial activity of volatile compounds results from the combined effect of direct vapour absorption on microorganisms and indirect effect through the medium that absorbed the vapour (Trivedi and Hotchandani, 2004). A significant contribution of the volatile compounds through agar absorption was reported for E. coli (Younis Haggag, 1996).

In referring to figures 01,02,03 and 04, there has been a remarkable decrease in the bacterial growth rate which is defined by the optical density during all the incubation time from 0.5 to 0.2 with 10 [micro]l for S. aureus, and 0.5 to 0.4 for E. coli then it reaches zero (total inhibition) after 24 hours at 50 [micro]l and after 2h at 100 [micro]l for S. aureus, in the time where it is reported some resistance to E. coli, which would not disappear completely under the influence of a high concentration of E. globulus essential oil and in a long incubation period as is is the case for a dose of 50 [micro]l and after 24 hours or 100 [micro]l after two hours.





In referring to the figures 05, 06, 07 and 08, illustrating two microbial strains growth tested under the action of the E. camaldulensis essentials oils in their culture medium, where they found the inhibitory effect of these herbal extracts on these germs.

A decrease in the colonies number of two species was marked with increasing concentration of E. camaldulensis essential oil, for example, a concentration of 10 [micro]l oil added, he spends more than 300. [10.sup.-1] CFU / ml to an incubation period of 10 minutes over 200. [10.sup.-1] CFU / ml germs of S. Aureus for 24 hours. In parallel, it signals the optical density dimunition from 0.5 for 10 minutes to 0.2 for 24 hours.

An important simultide of microbial growth of E. coli was reported with that of S. Aureus after adding the E. camaldulensis essential oil to the culture medium in which evolves, noting a slight resistance of the first (of E. coli) than the second to high concentrations of this plant extract, and to the long incubation periods (2 and 24 hours).

Like that of E. Globulus, the inhibitory action of the E. camaldulensis essential oil is much remarkable in large doses as 50 [micro]l and 100 [micro]l and at long incubation time of about two or 24 hours, during which, we are witnessing a total disappearance of two germs in their culture medium.

The results of the study revealed that oil of eucalyptus has antibacterial activity against Gram-positive as well as Gram-negative bacteria resistant to commonly used antimicrobial agents.





The present study confirmed antimicrobial properties of essential oils from E.globulus and E. camaldulensis. The two Eucalyptus species oil presented an important growth inhibition for S.aureus and E.coli tested whose the problem relates to the emergence of strains that possess multiple resistance to a range of antibiotics, thereby making them difficult to treat. The encouraging results indicate the E. globulus and E. Camaldulensis might be exploited as naturel antibiotic for the traitemnt of several infectious diseases caused by these two germs, and could be useful in understanding the relations between traditional cures and current medicines.


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Bachir Raho ghalem and Benali Mohamed

Biotoxicology laboratory. biology institute-science faculty-Djillali Liabes university of Sidi Bel Abbes--Algeria Phone: 00213771063841

Mr. Bachir Raho ghalem and Mr. Benali Mohamed Contribution to the Antiseptic Effect Study of Two Eucalyptus Species: Adv. in Nat. Appl. Sci., 2(3): 170-177, 2008

Corresponding Author: Mr. Bachir Raho ghalem, Biotoxicology laboratory. biology institute-science faculty-Djillali Liabes university of Sidi Bel Abbes--Algeria Phone: 00213771063841
Table 1. Antibiogram results of S.aureus and Exoli R: resists 1:
intermediarv resistance S: sensitive

 S.aureus Exoli

 inhibition inhibition
 zone zone
Antibiotic Diameter (cm) Results Diameter (cm) Results

Ampicillin 0 R 0 R
Amoxicillin 1.5 I 2.8 S
Chloramphenicol 2.7 S 0 R
Doxycycline 0 R 1.4 I
Erythromycin 1.4 I 2.4 I
Nitroxoline 2.1 I 1.2 R
Pristinamycine 2.6 S 0 R

Table 2. Effects of essential oils on a microbial mat of
Staphylococcus aureus and Escherichia coli.

microbial Species Staphylococcus aureus

The dose 1 [micro]l 2 [micro]l

inhibition zone Eucalyptus. 0 0
Diameter (cm) camaldulensis

 Eucalyptus globulus 0 0

microbial Species Escherichia coli
inhibition zone
 Eucalyptus. 0 0
Diameter (cm)
 Eucalyptus globulus 0 0

microbial Species Staphylococcus aureus

The dose 5 [micro]l 7.5 [micro]l

inhibition zone Eucalyptus. 1.40 1.9
Diameter (cm) camaldulensis

 Eucalyptus globulus 1.60 2.5

microbial Species Escherichia coli
inhibition zone
 Eucalyptus. 1 1.2
Diameter (cm)
 Eucalyptus globulus 1.30 1.5

microbial Species Staphylococcus aureus

The dose 10 [micro]l 20 [micro]l

inhibition zone Eucalyptus. 2 3
Diameter (cm) camaldulensis

 Eucalyptus globulus 2.9 4

microbial Species Escherichia coli
inhibition zone
 Eucalyptus. 1.5 1.8
Diameter (cm)
 Eucalyptus globulus 2 2.4
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Title Annotation:Original Article
Author:Raho ghalem, Bachir; Mohamed, Benali
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Geographic Code:6ALGE
Date:Sep 1, 2008
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