Available online at www.pelagiaresearchlibrary.com Pelagia Research Library Asian Journal of Plant Science and Research, 2014, 4(4):69-73
ISSN : 2249-7412 CODEN (USA): AJPSKY
Antimicrobial activity of essential oils of Eucalyptus camaldulensis from Mali Nah Traoréa*, Sékou Bouaréa, Lassine. Sidibea, Aimé A. Somboroa, Baba Fofanaa, Ousmane Tangaraa, Drissa Diallob, Jean C. Chalchatc and Gilles Figuiredoc a
Laboratoire de Chimie Organique et Substances Naturelles, Faculté des Sciences et Techniques de Bamako, Mali b Laboratoire de l’Institut National de Recherche en Santé Publique (INRSP), Département de Médecine Traditionnelle, Bamako, Mali c Laboratoire des Huiles Essentielles, Université Blaise Pascal de Clermont. Campus des Cézaux, Aubière Cédex, France _____________________________________________________________________________________________ ABSTRACT The essential oils of Eucalyptus camaldulensis collected at four sites in Mali were tested on Escherichia coli, Staphylococcus aureus and the fungus Candida albicans bacteria. We used the agar diffusion method for the determination of antibacterial activities. The greatest activity was noted with the sample from N'Débougou on Staphylococcus aureus. The antifungal activity was determined by thin layer chromatography (TLC). The four essential oils were active in anything on Candida albicans. Key words: Essential Oils, Escherichia coli, Staphylococcus aureus, Candida albicans. _____________________________________________________________________________________________ INTRODUCTION Eucalyptus camaldulensis is a large evergreen tree up to 20 m in the Sahel and neighboring Sudanese regions. Calcifuge this species gets its Latin name of the city Camaldule Australia. It is located in the east of the continent, especially near rivers. We are called River Gun. This tree easily reaches 40m high and its diverging branches are often spread [1]. Among the tests, Eucalyptus camaldulensis is best featured in Mali. It is part of the species that thrive on poor soils and withstand several months of drought (about 8 months) but also short floods. The main purpose of reforestation of Eucalyptus camaldulensis in Mali is timber production [2] The essential oil of Eucalyptus is endowed with antiseptic properties established in vitro and unequivocal on many germs [3]. Very few studies have been conducted on the antibacterial and antifungal activities of the essential oil of E. camaldulensis. However most of the work done has focused on other species. Traoré et al. [4] tested the antimicrobial activity of essential oils of Eucalyptus citriodora and Eucalyptus houseana by agar diffusion method. They found that they have some activity against Staphylococcus aureus and Escherichia coli; in addition they are active against Candida albicans. This is consistent with the result of the work of Adames et al. [5] on Essential Oil of Eucalyptus citriodora. Elaissi et al. [6] tested the antibacterial activity of essential oils of 20 varieties of Eucalyptus by the diffusion method. They found that E. odorata is most active against Staphylococcus aureus. In vitro, Bashir and Benali [7] tested the antibacterial activity of the essential oil of Eucalyptus globulus. They noted
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Nah Traoré et al Asian J. Plant Sci. Res., 2014, 4(4):69-73 _____________________________________________________________________________ that it has some activity against Staphylococcus aureus and Escherichia coli. The same essential oils of Eucalyptus olida, E. staigeriana and E. dives were tested by Martin et al. [8]. They got the greatest activity with the essential oil of Eucalyptus staigeriana Staphylococcus. aureus. To enhance its natural resources, we considered important to study the antibacterial and antifungal activities of essential oils of Eucalyptus camaldulensis from Mali. MATERIALS AND METHODS Vegetable material Essential oils are extracted from the dried leaves of Eucalyptus camaldulensis harvested from Koulikoro (HE 1) Magnambougou (HE 2) Niono (HE 3) and N'Débougou (HE 4) (Parcel N° P -79- 2 of the Institute of Rural Economy Niono) The extraction was made by steam distillation of water on a device Kaiser Land modified in the laboratory of Organic Chemistry and Natural Products, Faculty of Science and Technology Bamako (Mali). Biological tests Antibacterial and antifungal tests were carried out in the service of Serology and Bacteriology Sterilization of the National Institute of Public Health Research (INRSP) in Bamako (Mali). Pathogens tested Bacteria : clinical strain Escherichia coli, Staphylococcus aureus Reference strain: Escherichia coli ATCC922. Champion Candida albicans (clinical isolate) Solutions test and control Essential oils having undergone biological test were used at the concentration of ½ dissolved in DMSO and methanol. Different families of antibiotics were used as control ( Pefloxacin , Gentamicin , Colistin, Chloramphenicol , Tetracyclin, Erythromycin, pristinamycin, oxacillin , lincomycin). The antifungal test on Candida albicans recommends Nystatin as control. Antibacterial activities We used the agar diffusion method for determining the activity. Discs impregnated with the test solutions are deposited on the Petri dishes. After spending 24 hours in an oven at 37 ° C, the activities are assessed by measuring the inhibition zones around the discs The bacterial suspension prepared was cast on Mueller Hinton medium (MH) for bacilli and staphylococci. After flooding the entire surface of the medium with the bacterial suspension, the supernatant was discarded by suction with a transfer pipette. Each box has received five discs placed on an identification number stamped on the bottom of the box. The media were then incubated in an oven for 24 hours at 37 ° C. Media Reisolation is stored in the refrigerator. We performed the measurement of the diameter of the inhibition zones around each disc. The standard antibiotics were tested in the same conditions Antifungal activities To test the antifungal activity of our essential oils, we used bio autographic method [9]. It involves rapid dilution and to the isolation of active components through a target. Chromatograms are covered with a culture medium incorporated microorganism. After incubation for 24 hours at 37 ° C a suitable developer can observe the activity [10]. RESULTS AND DISCUSSION Table I gives the results of the antibacterial activity. It is readily noticeable that the dissolution solvent does not
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Nah Traoré et al Asian J. Plant Sci. Res., 2014, 4(4):69-73 _____________________________________________________________________________ affect the observed activity. The essential oil HE 4 dissolved in DMSO was more active against Staphylococcus aureus with 4 µl. The diameter of the inhibition zones is 16 mm. The essential oil HE 3 dissolved in the same solvent was more active on Escherichia coli with 4 µl. and 11 mm as a diameter of inhibition. The four essential oils tested showed an activity against Candida albicans, as shown in table II. Table I. Results of the antibacterial activity of essential oils of Eucalyptus camaldulensis solvents and antibiotics references Quantities
2 µl
4 µl
2 µl
4 µl
Germs Inhibition diameter in mm Products
E. coli
S. aureus
HE 1
9
9
0
7
HE 2
10
8
8
8
HE 3
8
11
8
7
HE 4
10
8
12
16
DMSO
0
0
0
0
Methanol
0
0
0
0
Pefloxacin (PEF) (5 µg)
0 (R)
-
Gentamicin (GM) (15 µg)
17 (S)
22 (S)
Colistin (CL) (50 µg)
15 (S)
-
Chloramphenicol (C) (30 µg)
22 (I)
-
Tetracyclin (Te) (30 UI)
0 (R)
-
Erythromycin (E) (15 UI)
-
23 (S)
Pristinamycin (PR) (15 µg)
-
26 (S)
Oxacillin (OX) (5 µg)
-
15 (R)
Lincomycin (L) (15 µg)
-
28 (S)
HE 1: essential oils of Eucalyptus. camaldulensis from Koulikoro, HE 2: essential oils of Eucalyptus camaldulensis from Magnambougou, HE 3: essential oils of Eucalyptus camaldulensis from Niono, HE 4: essential oils of Eucalyptus camaldulensis from N’Débougou Table II. Results of the antifungal activity of essential oils of Eucalyptus camaldulensis and nystatin on Candida albicans Products
Spots
Rf
HE 1
1
0,73
HE 2
1 2
0,76 0,83
HE 3
1 2
0,72 0,79
HE 4
1
0,64
Nystatin
1
0
The antibacterial activity was investigated on Escherichia coli and Staphylococcus aureus. It is easily noticeable that the solvents do not influence dissolution observed activity. Essential oils tested showed a definite activity against the used bacteria. The greatest activity was observed with HE
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Nah Traoré et al Asian J. Plant Sci. Res., 2014, 4(4):69-73 _____________________________________________________________________________ 4 on Staphylococcus aureus. We noted with some essential oils that the activity does not always increase with the increase in amount of the deposit. This could be explained on the one hand by the dissolution more or less good, on the other hand by the dissemination or load of petri dishes or the composition of essential oils (case HE 2 and HE 4 on Escherichia coli, and HE 3 on Staphylococcus aureus). The antibacterial activity of essential oils tested could be explained by the presence of the following compounds: 1.8- cineol (eucalyptol), p -cymene and α -pinene (table 3). Indeed, the main component of 1.8- cineole for most of the essential oils of Eucalyptus component is active on Staphylococcus aureus and Escherichia coli by Prakash and al. [11]. With regard to the oils tested, three are rich in a 1.8- cineole of which one is active against Escherichia coli (HE 3). Table III: Composition of essential oils of Eucalyptus camaldulensis Yield (%) 1.9 2.7 2.5 2.9 Compounds HE 1 HE 2 HE 3 HE 4 2,9 0.1 0.1 α-thujene 3.7 2.7 10.0 5.7 α-pinene Camphene 0.2 0.1 0.2 0.2 β-pinene Myrcene 0.1 0.1 0.1 2.8 α-phellandrene 0.1 α-terpinene 1.6 0.7 P-cymene 15.4 39.4 Limonene 15.4 1,8-cineole 90.4 78.8 57.6 19.9 1.0 3.5 19.3 γ-terpinene Terpinolene 0.1 0.5 0.3 P-cyménene 0.2 0.2 0.1 Linalool Tr Fenchool Tr Trans Pinocarveol Tr 4.1 1(7),5 Mentha-diene-2-ol 0.3 0.3 Pinocarvone 0.2 0.3 Borneol 1.2 Tr 0.3 0.7 0.2 2.2 0.3 Terpinene-4-ol Cis Mentha-1(7).8 Diene-2-ol 0.9 0.5 0.7 0.5 1.1 α-terpineol 0.2 Cryptone Myrtenol 0.1 Trans Carveol 0.1 0.1 Trans Menta- 1(7).8-Diene-2-ol 0.1 0.1 Cuminaldehyde 0.7 Carvotanacetone 0.3 Pipéritone 0.3 0.1 Carvacrol 0.5 0.1 0.3 Methyl eugénol 0.1 0.1 α-Gurjunene 0.1 β-caryophyllene g-Maaliene 0.2 0.1 0.1 0.3 β-gurjunene Aromadendrene 0.2 0.5 Selina-5,11-diene 0.1 1.2 Allo-aromadendrene 0.2 0.2 0.1 0.3 Spathulenol 0.6 0.3 0.3 2.6 Globulol Epiglobulol Tr Tr 0.7 Isospathulenol Tr 0.4 0.1 0.4 α-elemol 0.1 0.2 γ-eudemol Total 97 99 99 98.3 Tr : trace (< 0.5%)
However, it is surprising to notice that some essential oils which are very rich in 1.8-cineole were found weakly active. It is possible in these cases that the presence of one or more components of the oil inhibits the antimicrobial [12] activity; this could be the case of α -pinene in HE 1 and HE 2.
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Nah Traoré et al Asian J. Plant Sci. Res., 2014, 4(4):69-73 _____________________________________________________________________________ In addition, other essential oils seem to have a much greater activity than might have been supposed, given their compositions: the case of oils HE 4 with p-cymene and 1.8 -cineole as majority compounds and HE 3 with 1.8cineole and γ-terpinene. Note that larger diameters were observed with HE 4 (16 mm and 12 mm on Staphylococcus aureus) and HE 3 (11 mm in Escherichia coli), we assume that there is a synergistic effect between the p- cymene and 1.8- cineole on the one hand and between the 1.8- cineole and γ -terpinene on the other hand. The antifungal activity has been sought on Candida albicans; HE1 and HE 4 showed a single zone of inhibition which are Rf 0.73 and 0.64. The only area for inhibition of HE 1 may be due to 1.8- cineole which is the main compound with a majority rate of 90.4 %. P-cymene (39.4%) and 1.8 -cineole (19.9%) major components of HE 4 seem to have a synergistic effect. The activity was remarkable with HE 2 and HE 3 with two zones of inhibition. This activity could be explained by the presence of 1.8-cineole (78.8 %) and α -pinene (10%) for HE 2 and 1.8- cineole (57.6 %) and γ- terpinene (19.3%) for HE 3 compounds which are the majority of these oils. The essential oils of Eucalyptus camaldulensis tested have significant activity against bacteria and fungi. In the literature similar results were obtained for the essential oil of other Eucalyptus species. Therefore essential oils of Eucalyptus crebra, Eucalyptus citriodora, Eucalyptus camaldulensis. Eucalyptus robusta and Eucalyptus rudis of Madagascar were tested by Razafindrakoto. She noted that the essential oils of Eucalyptus citriodora , Eucalyptus camaldulensis and Eucalyptus robusta are classified as active species [12] Similarly Oyedeji et al [13] showed significant antibacterial activity on gram-positive and gram-negative and antifungal activity on Candida albicans with essential oils of five varieties of Eucalyptus from Nigeria. Beylier [14] tested essential oils of Eucalyptus citriodora, Eucalyptus dives and Eucalyptus radiate by Simeon method [15].He noted that they showed some activity against Staphylococcus aureus, Escherichia coli and fungi Candida albicans and Aspergillus Niger [14]. The works of Gilles and al. [16] have demonstrated the antimicrobial activity of the essential oil of Eucalyptus staigeriana. Acknowledgment We are grateful to The Embassy of France in Mali for its financial support, The Institute of Rural Economy (IER) Niono, The laboratory of the National Institute of Public Health Research (INRSP) and the Department of Traditional Medicine Bamako research of antimicrobial activity, The laboratory of Essential Oils to Blaise Pascal University of Clermont for his collaboration in the analysis REFERENCES [1] .Maydell HJ, Arbres et arbustes du sahel: leurs caractérisations et leurs utilisations, Weikersheim : Marggraf, 1990, 9, 480-483. [2] Collection FAO Forets N°11, Les Eucalyptus dans les reboisements, Organisation des Nations Unies pour l’Alimentation et l’Agriculture, Rome, 1982, 1-339 [3] Bruneton J, Pharmacognosie, Phytochimie, Plantes médicinales, 4eme Edition, Tec & Doc Lavoisier, Paris, 2009. [4] Traoré N, Sidibé L, Bouaré S, Harama D, Somboro A, Fofana B, Diallo D, Figueredo G et Chalchat JC, Int. J. Biol. Chem. Sci., April 2013, 7(2) : 800-804. [5] Adames M, Mendoza E et De Nigrinis LSO, Revista Colombiana de Ciencias Quimico Farmaceuticas, 1983, Volume 4, Issue 1, Pages 95. [6] Elaissi A, Salah KH, Mabrouk S, Larbi KM, Chemli R and Chemli R, Food Chemistry, December 2011, Volume 129, Issue 4, Pages 1427–1434. [7] Bachir RG and Benali M, Asian Pacific Journal of Tropical Biomedicine, September 2012, Volume 2, Issue 9, Pages 739–742. [8] Gilles M J, Zhao M and Agboola S, Food Chemistry, 2010, Volume 119, Issue 42, Pages 731–737. [9] Diallo D, Thèse de doctorat d’université, (Lausanne, 2000). [10] Ouattara F, Thèse Pharmacie (Université de Bamako, 2005). [11] Prakash S, Sinha GK and Pathak RC IO & SJ, 1972, 230. [12] Razafindrakoto BS, Thèse de Doctorat (Université du Languedoc, 1988). [13] Oyedeji AO, Ekundayo O, Olawore ON, Adenivi BA, Koenig WA, Fitoterapia, 1999, Volume 70, Issue 5. [14] Beylier MF, Perfum & Flavor, 1979, pp 23 [15] Simeon de Buochberg M, Thèse Doct. Pharm (Montpellier, 1976). [16] Gilles MJ, Zhao M, And Agboola S, Food Chemistry, 2010,Volume 119, Issue 2, 15.
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