The antibacterial activity of the leaf extracts of Eucalyptus camaldulensis was studied against Klebsiella spp, Salmonella typhi, Yersinia enterocolitica, Pseudomonas aeruginosa (Gram-negative), Staphylococcus aureus and Bacillus subtilis by the agar diffusion method. The methanol extract, dichloromethane fraction and methanol residue at 10mg mL-1 displayed broad spectrum activity against all the test organisms but the petroleum ether fraction showed no activity. The antibacterial activity of the extracts was compared to the drug gentamycin. The minimum inhibitory concentrations of the methanol extract and dichloromethane fraction determined by the agar dilution method ranged between 0.04 and 10mg mL-1 with that of Bacillus subtilis being the least. Phytochemical screening of the plant revealed the presence of tannins, saponins and cardiac glycosides. The results of this study support the traditional use of Eucalyptus camaldulensis leaves as an antibacterial agent.







Key words: Eucalyptus camaldulensis, antibacterial activity, minimum inhibitory concentration, methanol extract, dichloromethane fractio

Adeniyi, B.A

Ayepola, O.O

English

Covenant University Repository

Journal of Applied Sciences Research, 4(11): 1410-1413, 2008© 2008, INSInet PublicationCorresponding Author: Olayemi O. AYEPOLA, Department of Biological Sciences College of Science and Technology,Covenant University, PMB 1023, Ota, Ogun State, Nigeria.Tel. No.: +234 803 478 5269E-mail Address: hindsfeetus@yahoo.com,1410The Antibacterial Activity of Leaf Extracts of Eucalyptus camaldulensis (Myrtaceae)AYEPOLA O.O AND B.A. ADENIYI1 2 Department of Biological Sciences, College of Science and  Technology, Covenant University,1PMB 1023, Ota, Ogun State, Nigeria.Department of Pharmaceutical Microbiology, Faculty of Pharmacy, P.O. Box 22346, University2of Ibadan, Nigeria. Abstract: The antibacterial activity of the leaf extracts of Eucalyptus camaldulensis was studied againstKlebsiella spp, Salmonella typhi, Yersinia enterocolitica, Pseudomonas aeruginosa (Gram-negative),Staphylococcus aureus and Bacillus subtilis by the agar diffusion method. The methanol extract,dichloromethane fraction and methanol residue at 10mg mL  displayed broad spectrum activity against-1all the test organisms but the petroleum ether fraction showed no activity. The antibacterial activity of theextracts was compared to the drug gentamycin. The minimum inhibitory concentrations of the methanolextract and dichloromethane fraction determined by the agar dilution method ranged between 0.04 and10mg mL  with that of Bacillus subtilis being the least. Phytochemical screening of the plant revealed-1the presence of tannins, saponins and cardiac glycosides. The results of this study support the traditionaluse of Eucalyptus camaldulensis leaves as an antibacterial agent.Key words: Eucalyptus camaldulensis, antibacterial activity, minimum inhibitory concentration, methanolextract, dichloromethane fraction.INTRODUCTIONPlants have been used for the treatment of diseasesall over the world before the advent of modern clinicaldrugs and are known to contain substances that can beused for therapeutic purposes or as precursors for thesynthesis of useful drugs . Thus over 50% of these[14]modern drugs are of natural products origin and assuch these natural products play an important role indrug development in the pharmaceutical industry . [7]Eucalyptus camaldulensis is an importantethnomedicinal plant belonging to the family,Myrtaceae. It is used as a remedy for sore throat andother bacterial infection of the respiratory and urinarytracts. Essential oils of the leaves are used in thetreatment of lung diseases while the volatile oils areused as expectorants . Topical ointments containing[2]eucalyptus oil have also been used in traditionalAboriginal medicines to heal wounds and fungalinfections. Eucalyptus oil obtained by steam distillationand rectification of the fresh leaves has Eucalyptol(1,8-cineole) as its active ingredient and this isresponsible for its various pharmacological actions .[16]The antimicrobial activities of the methanolic extractsof E. camaldulensis have also been reported .[3 ,10]The emergence of bacterial resistance to thecurrently available antimicrobial drugs necessitatesfurther research in the discovery of new safe andeffective antibacterial agents . The investigation of[9]certain indigenous plants for their antimicrobial activityis therefore of utmost importance. This study is aimedat investigating the antimicrobial activity of Eucalyptuscamaldulensis against Gram-positive and Gram-negativebacteria thereby establishing it as a potentialantimicrobial agent.MATERIALS AND METHODSPlant M aterial: Eucalyptus camaldulensis leaves werecollected around the Department of Forestry, Universityof Ibadan and authenticated at the Department ofBotany and Microbiology, University of Ibadan. Avoucher specimen was deposited at the Herbarium forreference purposes. The leaves were air-dried and thenground before use for this study.Extraction Procedure: The dried and powdered leaves(400g) were subjected to soxhlet extraction withmethanol as the extraction solvent. A part of themethanol extract was then partitioned into petroleumether and dichloromethane. The extracts were filteredand allowed to evaporate to dryness. Each extract wastransferred into clean and dried airtight vials until readyfor use.J. Appl. Sci. Res., 4(11): 1410-1413, 20081411Phytochemical Screening: Samples of Eucalyptuscamaldulensis leaves were screened phytochemicallyfor the presence of secondary metabolites using thestandard methods of Harbone,  and Trease and Evans[6]. The secondary metabolites screened for are[15]saponins, alkaloids, anthraquinones, tannins and cardiacglycosides.Bacterial Strains: The organisms used in this studywere four Gram-negative bacteria and two Grampositive bacteria namely, klebsiella spp, Salmonellatyphi, Yersinia enterocolitica, Pseudomonas aeruginosa(Gram-negative), Staphylococcus aureus and Bacillussubtilis (Gram-positive). The organisms were obtainedfrom the Department of Pharmaceutical Microbiology,Faculty of Pharmacy, University of Ibadan.Determination of antibacterial activity: Theantibacterial activity of the extracts was determinedusing the agar cup diffusion as described by Adeniyi etal . A 1 mL of an overnight culture of each bacterial[1]isolate (equivalent to 10 -10 cfu mL ) was used to seed7 8 -1sensitivity test agar plates maintained at 45 C. Theoseeded plates were allowed to set and a sterile corkborer of 8mm diameter was used to cut equidistantwells on the surface of the agar. The wells were filledwith 0.1mL solution of each extract reconstituted withmethanol at a concentration of 10mg mL . Gentamycin-1at 5µg mL  was included as positive control. The-1plates were incubated at 37 C for 24h after which theodiameter of zones of inhibition were measured.Determination of M inimum Inhibitory Concentration(MIC): The determination of the minimum inhibitoryconcentration of the methanol extract and thedichloromethane fraction was carried out using the agardilution method described by Lajubutu et al .[8]Different concentrations of the extracts were preparedto give a final concentration in the range of 10 to0.039 mg mL . 2ml of each dilution was mixed with-118ml of Mueller-Hinton agar, poured into Petri dishesand allowed to set. The agar was streaked with anovernight broth culture of the test organisms andincubated overnight. The lowest concentration inhibitinggrowth was regarded as the minimum inhibitoryconcentration of the extracts.RESULTS AND DISCUSSIONQualitative phytochemical screening of the extractsof E. camaldulensis (Table 1) demonstrated thepresence of tannins, saponins and cardiac glycosideswhile anthraquinones and alkaloids were absent.The results of the antibacterial screening of theplant leaves (Table 2) revealed that the methanolextract, dichloromethane fraction and the methanolresidue represent a broad spectrum of activity. Allextracts showed varying degrees of inhibition on thetested  microorganisms  at a concentration of 10 mgmL . The methanol extracts showed greater activity-1against Salmonella typhi, Staphylococcus aureus andBacillus subtilis (15 -16mm) than Klebsiella spp,Yersinia enterocolitica and Pseudomonas aeruginosa(14mm). The dichloromethane fraction exhibited higheractivity against Klebsiella spp, Salmonella typhi,Yersinia enterocolitica and Bacillus subtilis (15–16mm)than Staphylococcus aureus and Pseudomonasaeruginosa (13-14mm). The methanol residue had alower activity against all the test organisms exceptKlebsiella spp and Salmonella typhi. The result alsoshowed that Klebsiella spp and Yersinia enterocoliticawhich were not inhibited by gentamycin used as apositive control in this study were inhibited by theextracts.  However the petroleum ether fraction showedno activity on all test organisms.The minimum inhibitory concentration of themethanol extract and dichloromethane fraction of theleaves ranged between 0.04 and 10mg mL  (Table 3).-1The lowest MICs for both extracts were those forBacillus subtilis (0.04 - 0.079 mg mL ).-1Table 1: Phytochem ical com ponents of the leaf extracts of E.camaldulensis Phytochemical components E. camaldulensisAlkaloids -Tannins +Saponins +Anthraquinones -Cardiac Glycosides +Note: + = Present- = AbsentDiscussion: The antibacterial activity of the leafextracts of Eucalyptus camaldulensis can be attributedto the action of the phytochemical compounds itcontains. Babayi et al . reported the presence of[4]saponins, saponin glycosides, stero id, cardiacglycosides, tannins, volatile oils, phenols and balsam(gum) in the plant. The presence of these compoundsin the family Myrtaceae to which E. camaldulensisbelong has been reported by Pamplona-Roger .[11]Polyphenolic compounds and /or volatile oils areknown to inhibit a wide range of organisms . The[5]antimicrobial activity of the extracts could be explainedby the presence of tannins. The mechanism of actionof tannins is based on their ability to bind proteinsthereby inhibiting cell protein synthesis . There was[13]no significant difference in the antimicrobial activity ofthe extracts on Gram-negative and Gram positivebacteria despite the differences in their cell wallcomponents. Akin-Osanaiye et al . reported the[3]antibacterial activity of E. camaldulensis extractsagainst Staphylococcus aureus while Sherry et al  [12]J. Appl. Sci. Res., 4(11): 1410-1413, 20081412Table 2: Antibacterial activities of the leaf extracts of E. camaldulensis at 10mg mL-1Organisms M eOH Pet. Ether Dichl. M eOH-residue GentamycinKlebsiella spp 14 - 15 15 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Salmonella typhi 16 - 15 15 17---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Yersinia enterocolitica 14 - 16 13 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Pseudomonas aeruginosa 15 - 14 13 26---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Staphylococcus aureus 15 - 13 14 28---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bacillus subtilis 16 - 16 14 28Resistance= no zone of inhibition, Positive control-Gentamycin (5µg mL ), M eOH- M ethanol, Pet. Ether- Petroleum ether fraction, Dichl--1Dichloromethane fraction.Table 3: M inim um  Inhibitory Concentration (M IC) of the leafextracts of E. camaldulensisOrganisms ME (mg mL ) DF (mg mL )-1 -1Klebsiella spp 10 10Salmonella typhi 10 10Yersinia enterocolitica 0.157 0.625Pseudomonas aeruginosa 10 10Staphylococcus aureus 1.25 0.625  Bacillus subtilis 0.04 0.079M E= M ethanol Extract, DF= Dichloromethane Fractionrevealed that topical applications of eucalyptus oilclears methicillin resistant Staphylococcus aureusinfections. The methanol extract of E. camaldulensishas been found to be effective against staphylococcusaureus and Bacillus subtilis . The results of the tests[4]for minimum inhibitory concentration (MIC) revealedtha t  the  M IC  fo r  Y ers in ia  e n te r o c o l i t ic a ,Staphylococcus aureus and Bacillus subtilis (between0.04mg mL  and 0.625mg mL ) were lower than that-1 -1of Klebsiella spp, Salmonella typhi and Pseudomonasaeruginosa (10mg mL ). This means that higher doses-1of the antimicrobial agent will be needed in thetreatment of infections caused by Klebsiella spp,Salmonella typhi and Pseudomonas aeruginosaprovided they are not toxic to the tissues.Conclusion: The results of this study have shown thatthe leaf extracts of E. camaldulensis have greatpotential as antimicrobial agents in the treatment ofinfectious organisms. Further detailed investigation ofthe active components of the plant for the exactmechanism of action will contribute greatly to thedevelopment new pharmaceuticals.REFERENCES1. Adeniyi, B.A., H.A. Odelola and B.A. Oso, 1996.Antimicrobial potentials of Diospyros mespiliformis(Ebenaceae). Afr. J. Med. Sci., 25: 179-184.2. Adeniyi, B.A, T.O. Lawal and S.B. Olaleye, 2006.Antimicrobial and gastroprotective activities ofEucalyptus camaldulensis (Myrtaceae) crudeextracts. J. Bio. Sci., 6(6): 1141-1145.3. Akin-Osanaiye B.C, A.S. Agbaji and M.A. Dakare,2007. Antimicrobial activity of oils and extracts ofCymbopogon citrates, Eucalyptus citriodora andEucalyptus camaldulensis. J. Med. Sci., 7(4): 694-697.4. Babayi H, I. Kolo, J.I. Okogun and U.J.J. Ijah,2004. The antimicrobial activities of methanolicextracts of Eucalyptus camaldulensis andTerminalia catappa against some pathogenicmicroorganisms. Biokemistri, 16: 106-111.5. Cheesbrough, M., 1984. Tropical HealthTechnology. Cambridge University Press, U.K.6. Harbone, J.B., 1984. Phytochemical Methods: AGuide to Modern Technique of Plant Analysis. (2ndedn). Chapman and Hall, London, 1-168.7. Jeyachandran, R. and A. Mahesh, 2007.Antimicrobial evaluation of Kigelia Africana(Lam). Res. J. Microbiol., 2(8): 645-649.8. Lajubutu, B.A., R.J. Pinney, M.F. Robert, H.A.Odelola and B.A. Oso, 1995. Antimicrobial activityof diosquinone and plumbagin from Diospyrosmespiliformis (Hostch) (Ebenaceae). Phytother.Res., 9: 346-350.9. Lokhande, P.D., K.R. Gawai, K.M. Kodam, B.S.Kuchekar, A.R. Chabukswar and S.C. Jagdale,2007. Antibacterial activity of isolated constituentsand extract of roots of Inula racemosa. Res. J.Med. Plant, 1(1): 7-12.10. Mehraban, F., N.O. Tabrizib and F. Jahaniani,2005 . Antidermatophyte a c tiv ities o f E .camaldulensis in comparison with griseofulvin.Iranian J. Pharm & Ther., 4(2): 80-83.11. Pamplona-Roger, G.D., 1999. Encyclopedia ofMedicinal Plants. 1 and 2, (2  Edn). Educationndand Health Library, The European Union, U.K.,128-150.12. Sherry, E., H. Boeck, P.H. W arnke, 2001. Topicalapplication of a new formulation of eucalyptus oilphytochemical c lears  methicillin  resis tantStaphylococcus aureus infection. Am. Infect.Control., 29(5): 346.J. Appl. Sci. Res., 4(11): 1410-1413, 2008141313. Stern, J.L., A.E. Hagerman, P.D. Steinberg andP .K .  M a so n ,  19 9 6 .  P h lo ro tann in-p ro te ininteractions. J. Chem. Ecol., 22: 1887-1899.14. Sofowora, A., 1982. Medicinal Plants andTraditional Medicine in Africa. 2  Edn., JohnndWilley and Sons ltd., Ibadan, 8-14.15. T rease , G .E . and W .C Evans, 1989 .Pharmacognosy (13  edn). English Language BookthSociety, Bailliere Tindall, Britain, 378-480.16. Trivedi, N.A and S.C. Hotchandani, 2004. A studyof the antimicrobial activity of the oil ofEucalyptus. Ind. J. Pharmacol., 36: 93-94.

The Antibacterial Activity of Leaf Extracts of Eucalyptus camaldulensis (Myrtaceae)

The antibacterial activity of the leaf extracts of Eucalyptus camaldulensis was studied against Klebsiella spp, Salmonella typhi, Yersinia enterocolitica, Pseudomonas aeruginosa (Gram-negative), Staphylococcus aureus and Bacillus subtilis by the agar diffusion method. The methanol extract, dichloromethane fraction and methanol residue at 10mg mL-1 displayed broad spectrum activity against all the test organisms but the petroleum ether fraction showed no activity. The antibacterial activity of the extracts was compared to the drug gentamycin. The minimum inhibitory concentrations of the methanol extract and dichloromethane fraction determined by the agar dilution method ranged between 0.04 and 10mg mL-1 with that of Bacillus subtilis being the least. Phytochemical screening of the plant revealed the presence of tannins, saponins and cardiac glycosides. The results of this study support the traditional use of Eucalyptus camaldulensis leaves as an antibacterial agent

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The Antibacterial Activity of Leaf Extracts of Eucalyptus camaldulensis (Myrtaceae)

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