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Ethnopharmacological investigations of essential oils isolated from five Ethiopian medicinal plants against eleven pathogenic bacterial strains
Getachew Belay1,*, Yinebeb Tariku2, Taddesse Kebede3, Ariaya Hymete4, Yalemtsehay Mekonnen5
1 2

Ethiopian Health and Nutrition Research Institute (EHNRI), Addis Ababa, Ethiopia, 14842. Department of Chemistry, College of Natural Science, Jimma University, Jimma, Ethiopia 378. 3 Department of Microbiology, Immunology & Parasitology, Faculty of Medicine, Addis Ababa University, Addis Ababa, Ethiopia, 21656/1000. 4 Department of Pharmaceutical Chemistry, School of Pharmacy, Addis Ababa University, Addis Ababa, Ethiopia, 1176. 5 klilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia, 1176. *Corresponding Author: Email: gechbel@gmail.com
Received: 20 September 2011, Revised: 30 September 2011, Accepted: 1st October 2011

Abstract The aim of this study is to evaluate antibacterial activity of volatile fractions from Artemesia abyssinthium, Croton macrostachyus, Echnops kebericho and Satureja puncata against selected Gram-positive and Gram-negative bacterial strains. All the essential oils were active against all tested bacterial species. L. monocytogenes, B. cereus, and S. aureus were the three most sensitive bacteria with a mean concentration of 0.14, 0.8 and 0.62l/ml, respectively. Antibacterial activities were highest against S. paratyphi, P. mirabilis and E. coli with a mean concentration of 20.8, 18.8 and 15.7 l/ml, respectively. P. aeruginosa found to be the most sensitive bacteria to all essential oils among Gram-negative bacteria, showing MIC of 0.1 to 1.6 with overall mean values of 0.62l/ml while K. pneumonia ranked next with MIC mean values of 1.36l/ml. The mean of MIC values were significantly higher against Gram-positive bacteria than Gram-negative bacteria Key words: Essential oils, Ethiopian plants, Bacteria, Antibacterial activity, MIC, MBC, GC-MS
; Artemesia abyssinthium; Croton macrostachyus; Echnops kebericho; Satureja puncata; Gram-positive; Gram-negative; Artemesia abyssinica Sch. Bip. ex A. Rich; Origanum vulgare; Thymus vulgaris; thyme; Ocimun basilicum; Lippia sidoides; Plectranthus amboinicus; Eucalypthus citriodora; Syzygium cumini; Allium sativum; Melaleuca alternifolia; R. officinalis; Z. officinalis; C. citrates; M. piperita; Cinnamomum zeilanicum Blume; volatile oil; phenylpropanoids; Staphyloco-ccus aureus; Streptococcus pyogenes; Listeria monocytogenes; Bacillus cereus; Escherichia coli; Pseudomonas aeruginosa; Salmonella paratyphi; Shigella dysenteriae; Citrobacter; Klebsiella pneumonia; Proteus mirabilis; Mueller-Hinton broth; CFU/ml; Minimum Bactericidal concentration; MBC; artemisyl acetate; artemisia alcohol; santolina triene; 1,8-cineole; camphor; davanone; ethyl (E)-cinnamate; (E)-nerolidol; chamazulene; geranial; neral; abisabolol; (E)-nerolidol; dehydrocostus lactone; -phellandrene; germacrene B; -selinene; -pinene; -pinene; benzyl benzoate; -muurolene; linalool; -farnesene

Introduction Despite the huge budget spent on development of antibiotics, successful treatment of bacterial infections are still a great challenge in medicine due to the indiscriminate use these conventional drugs which led to emergence of multi drug resistant strains (Kenneth and George, 2004). The diversity of plant species and compounds they contained evoked researchers to screening wide variety of plants with the intention of getting clinically

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Belay et al. significant drugs, with different biological activities and novel structures. In this regard, aromatic medicinal plants are of great potential Medicinal properties of aromatic plants and their extracts have been recognised since time immemorial. They are still used in medicine, food and cosmetic industry (Lahlou, 2004). Among these plant species Origanum vulgare (oregano), Thymus vulgaris (thyme), Ocimun basilicum (basil), Lippia sidoides (rosemary-pepper), Plectranthus amboinicus (mint), Eucalypthus citriodora (eucalyptus), Syzygium cumini (clove), Allium sativum (garlic), Melaleuca alternifolia (tea tree), R. officinalis (rosemary), Z. officinalis (ginger), C. citratus (lemongrass), M. piperita (peppermint) and Cinnamomum zeilanicum Blume (cinnamon) were described as broad-spectrum antimicrobial agents in different monographs and journals (Mueller and Mechler, 2005; Silva and Fernandes, 2010). Therapeutic effect of these plants can generally be attributed to their volatile fractions (essential oils) rather than their extracts (Lahlou, 2004). Essential oils are aromatic oily liquids generally isolated from flowers, twigs, leaves, seeds, fruits, stem and roots of non-woody plants from such families as: Asteraceae, Lamiaceae, Cupressaceae, Apiaceae, Lauraceae, Verbenaceae, Monimiaceae, Myrtaceae, Geraniaceae, etc by steam or hydrodistillation and various other techniques. Chemically they are complex mixtures of numerous compounds usually composed of terpenoids (specifically monoterpenes and sesquiterpenes, and diterpenes in some cases) and variety of low molecular weight aliphatic hydrocarbons and organic acids, alcohols, aldehydes, acyclic esters or lactones, coumarines and homologues of phenylpropanoids (Lahlou, 2004). The fact that majority (ca. 60%) of the essential oils have been reported to possess antibacterial properties (Gangrade et al., 1991), previous data reported on antileishmanial effect of these oils by authors in this article (Tariku, et al, 2010a; Tariku, et al., 2010b & Tariku, et al., 2011) and the non-toxic, non-pollutive and biodegradable nature oils prompted us to evaluate the antibacterial activity of essential oils obtained from five aromatic medicinal plant species: viz. Artemisia absinthium L., Artemesia abyssinica Sch. Bip. ex A. Rich (Asteraceae/Compositae), Croton macrostachys Hochest. ex A.Rich; (Euphorbiaceae), Echnops kebericho Mesfin (Asteraceae/Compositeae), and Satureja punctata (Benth.) Briq. (Lamiaceae/Labiatae) against four pathogenic Gram-positive and seven Gram-negative bacteria. The antimicrobial activity of these oils was expressed as Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). Artemisia absinthium L., and Artemesia abyssinica Sch. Bip. ex A. Rich are erect annual or short lived perennial herbs, commonly used in Ethiopia for traditional medicine and rituals. With nearly 400 species occurring mostly in northern temperate regions of the world, the genus Artemisia constitutes one of the 1535 genera in the family Asteraceae/Compositae. Four of these species including the endemic A. abyssinica grow in Ethiopia (Hedberg et al., 2004). Artemisia sp. are used in the traditional treatment of intestinal problems, bronchitis and other inflammatory disorders, cold and fever, anorexia, colic, infectious diseases (bacterial, protozoal), headache, amenorrhoea, and dysmenorrhoeab (Abegaz, 1996; Kayser et al., 1996; Getahun, 2004; Teixeira, 2004). Besides these essential oils of Artemisia spp. were reported to produces sesquiterpene lactones, coumarins and acetylenes as main bioactive metabolites (Teixeira, 2004).

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The genus Satureja consists of ca. 450 species that grow in Europe, Africa, Asia, and America. In Ethiopia, the genus is represented by eight species (Hedberg et al., 2006). One of these, Satureja punctata (Benth.) Briq. ssp. punctata (Benth.) Briq. (Lamiaceae/ Labiatae) is a small shrub that grows to a height of 10-100 cm, with distinctive purple/violet flowers and leaves with pleasant lemon-scented fragrance. Preparations made from the aerial parts of several of these species are used in Ethiopian traditional medicine to treat headache, stop menstruation, relieve stomach pains (Abate, 1989), and improve the quality of milk (Hedberg et al., 2006). Essential oils obtained from the leaves and flowers of different Satureja sp. are commonly used in various industrial applications as flavoring material, medicine, and in perfumers (Teklu et al., 1998). Echinops kebericho Mesfin, locally called kebericho, is an erect massive root stockbearing perennial herb or shrub that grows up to a height of 1.2 m. The plant belongs to the genus Echinops that comprises 125-130 species in the family Asteraceae/Compositae distributed in semi-humid zones of tropical and North Africa, Mediterranean basin, and temperate regions up to Central Asia (Hedberg et al., 2004; Teresa et al., 2005). Twelve species occur in Ethiopia confined to the highlands of the country (Tadesse and Abegaz, 1990). Echinops species were reported to contain a wide range of chemical structures comprising alkaloids, saponins, phytosterols, polyphenols, carotenoids, sesquiterpene lactones/alcohols, lignans, acetylenic and thiophene compounds, and essential oil (Tadesse and Abegaz, 1990; Hymete et al., 2005). Reports and ethnobotanical surveys also evidence long traditional use of the plant for preparation of medicines against migraine, mental illness, heart pain, lung TB, leprosy, kidney disease, malaria, billharzia, syphilis and amoebic dysentery (Abebe and Ahadu, 1993). Extracts and essential oils of the roots of E. kebericho were also assessed for their antimicrobial, antihelminthic and molluscicidal activities (Ashebir and Ashenafi, 1999; Hymete et al., 2005). Distributed mostly in the New World, the genus Croton includes 800 species of about 8,100 species in the family Euphorbiaceae,. It belongs to family. Croton macrostachyus Delil is shrub or deciduous tree that grows up to 30 m high at an altitude of 200-2000m and widely distributed in most parts of Ethiopia. The plant is native to Eritrea, Ethiopia, Kenya, Nigeria, Tanzania and Uganda (Hedberg et al., 1995; Schmelzer and Gurib-Fakim, 2008). The genus Croton is rich in terpenoids (diterpenoids and triterpenoids), alkaloids, flavonoids, lignoids, proanthocyanidins and volatile oils containing monoterpenoids, sesquiterpenoids, and some shikimate-derived compounds (Salatino et al., 2007). Previous studies revealed exinstence of crotin (a chalcone), crotepoxide (a cyclohexane diepoxide), lupeol (a triperpene), proteins, fatty acids, saponins, resins and alkaloids (Schmelzer and Gurib-Fakim, 2008; Salatino et al., 2007) in C. macrostachyus. In Ethiopia, Croton macrostachyus has folk medicinal uses as purgative and vermifuge, various skin problems, management of helminths and venereal diseases induce abortion (Getahun, 2004; Abate, 1989; Schmelzer and Gurib-Fakim, 2008). Materials and Methods Plant Collection and Identification The study was conducted in the Blacklion Teaching Hospital, Addis Ababa University, Ethiopia. Fresh plant parts were collected from Inchini (86km W of Addis

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Belay et al. Ababa) and Addis Ababa areas in their natural habitats. All plant materials were identified and voucher specimens of each species were deposited at the National Herbarium, Department of Biology, Addis Ababa University under voucher numbers YT-004/06, YT005/06, YT-006/06, YT-007/06 and YT-008/06. Isolation of volatile oil The fresh parts (50-400g) of the plants were subjected to hydrodistillation in a glass type Clevenger-type apparatus for 3 h after the mixture started boiling. The oils were carefully separated; yields were calculated, and samples were finally kept in a refrigerator at 200C in a sealed vial before further analysis was carried on the oils. Test organisms Microorganisms used in this study included four strains of gram-positive (Staphylococcus aureus (ATCC 25923), Streptococcus pyogenes, Listeria monocytogenes, and Bacillus cereus) and seven gram-negative (Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Salmonella paratyphi, Shigella dysenteriae, Citrobacter spp, Klebsiella pneumonia and Proteus mirabilis). These samples were obtained from the Ethiopian Health and Nutrition Research Institute (EHNRI) and Blacklion Teaching Hospital. The bacteria were grown in nutrient broth at 37C and maintained on nutrient agar slants at 4C. Preparation of inoculum Active cultures for experiments were prepared by transferring a loop-full of bacterial cells from nutrient agar slants in to test tubes containing Mueller-Hinton broth (MHB). Then the test tubes were incubated without agitation for 24 hrs at 37C. The suspension turbidity was adjusted to a 0.5 McFarland turbidity standard (1x108CFU/ml). Antimicrobial activity test Minimum Inhibitory Concentration (MIC) MIC was carried following reported method (CLSI, 2009). Mueller Hinton Broth (MHB) (100l) was dispensed in each well and stock solution of plant extracts were added in the first wells and a two-fold serial dilution was carried. The standard bacterial suspension was diluted 1:20 to yield 5x 105 CFU/ml (CLSI, 2009) and 100l of bacterial suspension were inoculated in to each dilution to the wells. The final concentration of extract dilutions ranged from 50.0 to 0.1 l/ml. The plates were coated with plastic and incubated at 37C for 24hr and examined for bacterial growth. Negative control (equal volume of DMSO and Tween-80) wells without the test extracts and positive control wells using gentamicin were assayed simultaneously. All extracts were tested in triplicates. Afterwards, the MIC of each sample was confirmed by measuring the optical density in spectrophotometer (620 nm), comparing the sample read out with the non-inoculated Mueller Hinton Broth (CLSI, 2009).

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Table 1. Yield for extraction of essential oil from claimed medicinal plants.
Scientific name (Part used)/Voucher No. A. abyssinica (L)-/YT-004/06/ A. abyssinithum (L)- /YT-005/06/ C. macrostachys (B)- /YT-006/06/ E. kebericho(T)- /YT-007/06/ S. puncata(L)- /YT-008/06/ Site of location Inchini* Inchini Addis Ababa Inchini Inchini* Amount used for extractions (g) 175 400 400 400 150 Oil colour Pale yellow Blue Pale yellow Yellow Yellow Yield (w/v) 0.60 1.39 0.038 0.13 0.67

(L)= Leaf; (B) = Berries; (T) = Tuber; *: 85Km W of Addis Ababa

Minimum Bactericidal concentration (MBC) To determine the MBC, all the micro-wells used in the MIC, which did not show any growth of the bacteria after the incubation period were first diluted (1:4) in fresh MHB and then subcultured on to the surface of the freshly prepared Mueller Hinton Agar plates and incubated at 37C for 24 h. The MBC were recorded as the lowest concentration of the extract that did not permit any visible bacteria colony growth on the agar plate after the period of incubation (Mueller and Mechler, 2005). All assays were carried out in triplicate using fresh inoculums . Data entry and analysis The means of MIC and MBC value were analyzed by one way analysis of variance (ANOVA) followed by multiple comparison test using SPSS software package version 13.0 for windows. The results were expressed as mean value and P<0.05 was considered as significant. Results Yield of volatile oil Volatile oil yields were expressed based on dry weight of plant material is shown in Table 1. The highest (1.39 % v/w) and lowest (0.038% v/w) yields were obtained from A. absinthium and C. macrostachyus respectively. Except for A. abissinthium oil which is blue the rest oils were yellowish in color. Chemical constituents of the oils isolated were analyzed by GC and GC-MS, and the data published by the Authors in this paper (Tariku, et al, 2010a; Tariku, et al., 2010b & Tariku, etal., 2011). The GC chromatograms of tested essential oils were also indicated in the previous papers. The major constituents were yomogi alcohol (38.47%), artemisyl acetate (24.88%), artemisia alcohol (6.70%), santolina triene (1.78%), and 1, 8-cineole (1.56%) (in A. abyssinica); camphor (27.40%), davanone (16.43%), ethyl (E)-cinnamate (5.81%), (E)-nerolidol (4.63%) and chamazulene (4.00%) (in A. abssinthium); geranial (27.62%), neral (21.72%), abisabolol (13.62%),and (E)-nerolidol (4.82%) (in S. Puncata); dehydrocostus lactone (41.83%), -phellandrene (10.84%), germac-rene B (5.38%), -selinene (4.13%), -pinene (3.63%) and -pinene (3.62%) (in E. keberic-ho) and

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Belay et al. benzyl benzoate (51.46 %), -muurolene (11.98 %), linalool (10.06 %) and -farnesene (3.21 %) (in Croton macrostachyus). Antimicrobial activity The essential oils were evaluated for antimicrobial activity against eleven Grampositive and Gram-negative bacteria strains. As shown in Table 2 all the essential oils were active against all tested bacterial spp. The results show a variable effect of the oils on the microorganisms with MIC values of the oils ranged from 25 to 0.1l/ml, while that of gentamicin varied from 2.5 to 0.04 g/ml. Among all the essential oils included in this study, A. abyssinica and C. macrostychyus have exhibited effect against all bacterial spp. But E. kebericho and A. abyssinithium had no effect against E. coli, S. paratyphi and Citrobacter species at a concentration range of this study. Among the bacteria, E. coli was affected by only two plant spp. (A. abyssinica and C. macrostychyus) followed by S. paratyphi and Citrobacter species which were affected by three of the study plants. Based on the mean MIC values of all essential oils against each bacterium, L. monocytogenes, B. cereus, and S. aureus were the three most sensitive bacteria with a mean concentration of 0.14, 0.8 and 0.62l/ml, respectively. Almost all the essential oils had a minimum MIC and MBC values against L. monocytogenes. In contrast, higher MIC values were shown against S. paratyphi, P. mirabilis and E. coli with a mean concentration of 20.8, 18.8 and 15.7 l/ml, respectively. The mean MIC values of the essential oils against B. cereus were significantly higher than the control (P = 0.013) whereas the essential oils against L. monocytogenes and S. aureus had comparable antibacterial activity with the control drug (P>0.05). The mean value of MIC and MBC of the essential oils had no significant difference (P = 0.90) whereas the difference is significant in case of gentamicin (P<0.005). The overall mean of MIC and MBC of the essential oils had significant difference with the positive control (P<0.001). S. punctata and A. abyssinica have shown the lowest and highest mean of MICs against all the bacteria with a mean concentration of 3.7 and 8.6l/ml, respectively. The MIC and MBC of S. punctata and the gentamicin drug have shown similar MIC (0.1l/ml) and MBC (0.1l/ml) values against P. aeruginosa, and A. abyssinithium and E. kebericho had similar MIC (0.2 l/ml) against S. aureus. The mean of MIC values have shown significantly higher antibacterial effect against the Gram positive than Gram-negative bacteria (P<0.001) and all essential oils were active against all the four Gram-positive bacteria. Among the Gram-negative bacteria, P. aeruginosa found to be the most sensitive bacteria to all essential oils, showing MIC of 0.1 to 1.6 with overall mean values of 0.62l/ml while K. pneumonia ranked next with mean MIC value of 1.36l/ml. In addition, the lowest MIC (0.1l/ml) and MBC (0.2l/ml) values were observed on both K. pneumonia and P. auroginosa using essential oils of E. kebericho and S. punctata, respectively. Discussion The MIC and MBC of the five essential oils were determined against seven Gramnegative and four Gram-positive bacterial strains. This study results demonstrated that all essential oils had antimicrobial activity against eight or more of the eleven bacterial strains

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Belay et al. used at test concentrations ranging from 0.2 to 25 l/ml (Table 2). The observed activities were significantly higher against Gram-negative bacteria (with mean MICs 0.25 to 1.6l/ml) than Gram-negative bacteria (with average MICs > 7.63l/ml).Among bacterial species tested, L. monocytogenes was found to be the most sensitive to all essential oils, with MIC ranged from 0.1 to 0.2l/ml and the MBC ranged from 0.2 to 1l/ml, while B. cereus and S. aureus ranked next. MBC values of the oils were generally higher than MIC values for each bacterial strains (Table 2). The high antimicrobial activities of the essential oils against L. monocytogenes and B. cereus indicated significance of these oils in combating food born pathogens. The observed difference in antibacterial activities between Gram-negative and Grampositive could be attributed to differences in composition and structure of bacterial outer membrane and cell wall which are among primary site of drug action in these organisms (Kenneth and George, 2004). Outer membrane of Gram-negative bacteria is rich in lipopolysaccharde molecules which can hinder penetration of different antibiotic molecules, and the proteins in the periplasmic space (Kenneth and George, 2004; Abdollah et al., 2010). Among tested oils that of S. punctata have shown the highest antibacterial activity (lowest mean MIC). In addition, this oil has shown similar MIC and MBC values compared to gentamicin against P. aeruginosa. Similar studies conducted on other Satureja species also shown that the antibacterial activity was comparable to positive controls and even better to certain bacteria like P. aeruginosa, S. aureus and Salmonella typhimurium (Ahmet et al., 2004). This indicates that the antibacterial activity of S. punctata in this study is in agreement with previous reports on other Satureja species which revealed that the genus has antimicrobial activity against human, food, and plant pathogens (Ahmet et al., 2004; Adiguzel et al., 2007). The MIC values of S. punctata has shown better antibacterial activity with very low MIC values ranging (0.1 to 12.5l/ml) as compared to the previous studies on different species of Satureja (>15.62 l/ml) (Azaz et al., 2002; Adiguzel et al., 2007). S. pyogenes was one of the Gram-positive bacteria affected by all essential oils with low MIC values. The essential oils might also be used as inhalation therapy against bacterial respiratory tract pathogens as S. pyogenes which can cause pharyngitis though it needs to determine the different factors such as the minimal exposure time for efficacy, applicability and the possibility of toxicity needs to be further evaluated. In this study the essential oils have shown strong antimicrobial effect on most of the bacteria, even the mean of cumulative MIC of all essential oils against B. cereus indicated there was highly significant antibacterial activity than the positive control drug which suggests that the essential oils were highly bactericidal. Almost all essential oils tested have shown strong antibacterial potential against pathogenic bacteria and both Gram-positive and Gram-negative bacteria were found susceptible to essential oils. Essential oils are complex systems containing many compounds. In these oils many of the potentially active constituents could function synergistically, while inactive components might influence resorption rate and bioavailability of the active compounds (Azaz et al., 2002). In this regard, several monoterpenoids such as linalool, eugenol, cinnamaldehyde, thymol, carvacrol and menthol (Gallucci et al., 2009; Neslihan et al., 2009), diterpenes (Batista et al., 1994), sesquiterpenes (Barrero et al., 2005) were reported to

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possess antibacterial activities. These classes of compounds were revealed in various quantities in volatile fraction of our study plants (Tariku, et al, 2010a; Tariku, et al., 2010b & Tariku, et al., 2011). Lipophilic constituents of essential oils, either inhaled or topically applied, have been suggested to successfully inhibit microbial growth by means of reacting with the lipid parts of the cell membranes, rendering more permeable microbial cell membranes and mitochondria membrane, which results in death of bacterial cells after massive ion leakage (Di Pasqua et al., 2007) and interrupting enzyme function in specific metabolic pathways (Neslihan et al., 2009). Essential oils are also able to inhibit the synthesis of DNA, RNA, proteins and polysaccharides in bacterial cells (Zani et al., 1991). In recent years due to an upsurge in antibiotic-resistant infections, the search for new prototype drugs to combat infections is an absolute necessity and in this regard plant essential oils may offer great potential and hope. The study results indicated that all the essential oils have antimicrobial activity and these plants are a potential source of natural antibacterial agents both for medical and for more commercial applications. Further steps should be performed in order to describe the antimicrobial activities in more detail and investigate the suitability of these antimicrobial properties in practical applications. Acknowledgement
We would like to acknowledge the Aklilu Lemma Institute of Pathobiology and the School of Pharmacy, Addis Ababa University for financial support and the laboratory technicians of Blacklion Hospital, Addis Ababa, Ethiopia.

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