Eucalyptus species belong to the order myrtales and myrtaceae. It is a large genus of aromatic trees indigenous to Australia, Tasmania and the neighbouring islands but today can be found growing in subtropical regions of the world. The genus consists of about 700 species of evergreen trees and shrubs (Adeniyi et al., 2006).
The oils of the eucalyptus plant are frequently used as a remedy for cold and cough. They are used in pharmaceuticals such as cough syrups, lozenges, nasal drops and mouthwash. The leaves main medicinal ingredient, cineole, has demonstrated expectorant and nasal decongestant activities (Schulz et al., 1998). It may also provide potent antiseptic properties (Chao and Young, 1998). Eucalyptus is an ingredient in over-the-counter pharmaceuticals as temporary relief of minor aches and pains of muscles and for temporary relief of nasal congestion and coughs associated with cold.
The three main sources of eucalyptus supplements are the common eucalyptus (Eucalyptus globulus), Eucalyptus polybractea and Eucalyptus smithii (Gruenwald et al., 1998). The leaf extracts of E. globulus, E. maculata and E. viminalis have been reported to significantly inhibit six Gram-positive bacteria (Takahashi et al., 2004).
The fungicidal effect of E. camaldulensis against dermatophytic fungal isolates has also been reported (Essien and Akpan, 2004; Mehraban, 2005).
The increasing resistance of most synthetically derived antimicrobial agents is of utmost concern. The search for suitable medicinal plants with potent active principles against microorganisms becomes imperative. Since the antimicrobial activity of eucalyptus has been established from previous studies, the main objective of this study is to examine the antimicrobial activities of the methanolic and dichloromethane leaf extracts of E. camaldulensis and E. torelliana against pathogenic enteric isolates.
MATERIALS AND METHODS
This study was conducted in the Department of Pharmaceutical Microbiology,
Faculty of Pharmacy, University of Ibadan, Nigeria in August 2003.
Leaves of E. camaldulensis and E. torelliana were collected from the Department of Forestry, University of Ibadan and authenticated at the Herbarium of the Department of Botany and Microbiology, University of Ibadan. A voucher specimen was deposited at the Herbarium for reference purposes.
Dried powdered leaves of E. camaldulensis and E. torelliana were
extracted with methanol by soxhlet extraction. A part of the methanol extract
was then partitioned into dichloromethane. The extracts were allowed to evaporate
to dryness and stored in airtight bottles until ready for use.
Qualitative phytochemical screening of both plants was carried out using
the methods of Harbone (1984) and Trease and Evans (1989) to test for the presence
of these secondary metabolites: alkaloids, saponins, anthraquinones, tannins
and cardiac glycosides.
The organisms used in this study were Klebsiella sp., UCH 2101, Proteus
mirabilis UCH 2102, Proteus mirabilis UCH 2204, Salmonella typhi
UCH 2201, Escherichia coli CHO 3101, Escherichia coli UCH
2103, Pseudomonas aeruginosa CHO 3102 and Pseudomonas aeruginosa
UCH 2203. The organisms were clinical isolates obtained from the University
College Hospital, Ibadan, Nigeria and the Catholic Hospital, Oluyoro, Ibadan,
Determination of Antibacterial Activity
The antibacterial activity of the extracts was determined using the agar
well diffusion technique (Adeniyi et al., 1996). Sensitivity test agar
plates were seeded with 0.1 mL of an overnight culture of each bacterial isolate
(equivalent to 107-108 cfu mL-1). The seeded
plates were allowed to set and a standard cork borer of 8 mm diameter was used
to cut uniform wells on the surface of the agar. The wells were then filled
with 0.1 mL of each extract at a concentration of 10 mg mL-1. The
antibiotic gentamycin at 5 μg mL-1 was used as positive control.
The plates were incubated at 37°C for 24 h after which the diameter of zones
of inhibition were measured.
Determination of Minimum Inhibitory Concentration (MIC)
The determination of the minimum inhibitory concentration of the extract
was carried out using the agar well dilution method (Rusell and Furr, 1972).
Different concentrations of the extracts were prepared to give a final concentration in the range of 5.0, 2.5, 1.25, 0.625, 0.313, 0.157, 0.079 and 0.04 mg mL-1. Two milliliter of each dilution of the extract was mixed with 18 mL of Mueller Hinton agar, poured into Petri dishes and allowed to set. The agar was streaked with an overnight broth culture of the bacterial isolates and incubated overnight. The plates were then examined for the presence or absence of growth. The concentration that completely inhibited macroscopic growth was regarded as the minimum inhibitory concentration of the extracts.
RESULTS AND DISCUSSION
Phytochemical screening of the extracts of E. camaldulensis and E.
torelliana indicated that both plants had tannins, saponnins and cardiac
glycosides (Table 1). However E. torelliana had anthraquinones
which were absent in E. camaldulensis.
Antibacterial Activities of the Extracts of E. camaldulensis and
The results of the antimicrobial activity of the extracts against the
test organisms revealed that the methanolic extracts of the leaves of the two
plants inhibited the growth of all the test organisms. The dichloromethane fractions
also showed good activity on the isolates. However the dichloromethane fractions
of both plants as well as gentamycin used as positive control were inactive
against E. coli 2103 (Table 2).
The Minimum Inhibitory Concentration (MIC) of the Methanol and Dichloromethane
The MICs of the extracts of E. camaldulensis and E. torelliana
ranged between 0.04 and 10 mg mL-1. The MICs for Klebsiella
sp., Proteus mirabilis and Salmonella typhi were however lower
than that for Escherichia coli and Pseudomonas aeruginosa (Table
|| Phytochemical components of the leaf extracts of E. camaldulensis
and E. torelliana
|Note: + = Present; = Absent
|| Antibacterial activities of the leaf extracts of E. camaldulensis
and E. torelliana at 10 mg mL-1
|Resistance = No zone of inhibition, Positive control-Gentamycin
(5 μg mL-1)
||Minimum Inhibitory Concentration (MIC) of the leaf extracts
of E. camaldulensis and E. torelliana
|ME: Methanol Extract; DE: Dichloromethane Extract
Previous research into the phytochemistry of the leaves of E. camaldulensis and E. torelliana revealed the presence of tannins, saponins and cardiac glycosides and in addition to these, E. torelliana contained anthraquinones. Ahmad et al. (1998) and Shariff (2001) have independently reported the presence of these components in members of the family Myrtaceae to which the plants used in this study belong. Also, Babayi et al. (2004) had reported that the phytochemical analysis of the crude extracts of Eucalyptus species revealed the presence of saponin, saponin glycosides, steroid, cardiac glycosides, tannins, volatile oils, phenols and balsam gum. Thus the antimicrobial activity of the extracts on the test organisms may be due to the presence of the above phytochemical components.
One of the molecular actions of tannins is to form complex with proteins through so-called nonspecific forces such as hydrogen bonding and hydrophobic effects, as well as by covalent bond formation resulting in the inhibition of cell protein synthesis (Stern et al., 1996). The presence of tannins therefore plays a significant role in the antimicrobial activity of the extracts. The antimicrobial activity of E. torelliana could partly be explained by the presence of anthraquinones. The bacteriostatic and bactericidal activities of anthraquinone from Cassia italica, have been established (Kazmi et al., 1994).
This study showed that E. camaldulensis and E. torelliana were effective inhibitors of microbial growth as they showed varying degrees of activity against the test organisms. There were no significant differences in the activity of the methanol extracts and dichloromethane fractions and this shows that both extracts can be exploited as antimicrobial agents. The leaf extracts of both plants also showed higher activities against Salmonella typhi compared with all other test bacteria. This is in support of the antimicrobial activity of E. citriodora against S. typhi as reported by Akin-Osanaiye et al., 2007. The Minimum Inhibitory Concentration (MIC) for Klebsiella, Proteus mirabilis and Salmonella typhi (between 0.04 and 1.25 mg mL-1) were lower than that of Escherichia coli and Pseudomonas aeruginosa (between 0.625 and 10 mg mL-1). This means that the extracts of the two plants are very potent against Klebsiella, Proteus mirabilis and Salmonella typhi but higher doses of the antimicrobial agents will be required in infections caused by Escherichia coli and Pseudomonas aeruginosa.
In conclusion, all the crude extracts and dichloromethane fractions of both plants have antimicrobial activities and thus confirmed the historical use of eucalyptus oil as an antibacterial agent (Kumar, 1988). The results of this study therefore form a good basis for selection of E. camaldulensis and E. torelliana for further phytochemical and pharmacological investigation for their use as possible antimicrobial agents in the treatment of gastrointestinal infections.