© Borgis - Postępy Fitoterapii 2/2016, s. 80-86
*Małgorzata Nabrdalik, Katarzyna Grata
Antibacterial activity of Ocimum basilicum L. essential oil against Gram-negative bacteria
Aktywność przeciwbakteryjna olejku eterycznego z bazylii pospolitej (Ocimum basilicum L.) wobec bakterii gram-ujemnych
Independent Chair of Biotechnology and Molecular Biology, University of Opole
Head of the Chair: Professor Adam Latała, DVM, PhD
Wstęp. Bazylia pospolita (Ocimum basilicum L.) jest powszechnie stosowaną w medycynie ludowej rośliną zielną. Ziele bazylii za-wiera do 2,5% olejku eterycznego, a składnikami dominującymi są: linalol, 1,8-cyneol, metylochawikol oraz eugenol. Prozdrowotne właściwości olejku bazyliowego związane są między innymi z jego działaniem: przeciwbakteryjnym, przeciwgrzybiczym, przeciwwirusowym oraz przeciwutleniającym.
Cel pracy. Celem pracy było określenie aktywności przeciwbakteryjnej olejku bazyliowego wobec bakterii Gram-ujemnych.
Materiał i metody. W pracy oceniono wpływ olejku bazyliowego oraz czas jego działania na przeżywalność Gram-ujemnych bakterii. Badanie wrażliwości bakterii na olejek bazyliowy, w stężeniach 0,25-4,0% (v/v), przeprowadzono metodą rozcieńczeń w podłożu bulionowym. Inkubację prowadzono przez okres 4-168 godz., określając liczbę żywych bakterii w 1 ml hodowli. Za MIC uznano najniższe stężenie olejku, które całkowicie hamowało wzrost bakterii.
Wyniki. Badania wykazały, że olejek bazyliowy wykazywał wysoką aktywność przeciwbakteryjną, w szczególności wobec Aeromonas hydrophila, Citrobacter freundii, Escherichia coli, Hafnia alvei i Klebsiella pneumoniae. Wartość MIC dla tych szczepów mieściła się w granicach 0,25-1,0% (v/v). Żadne z zastosowanych stężeń olejku bazyliowego nie hamowało całkowicie wzrostu Pseudomonas aeruginosa oraz Salmonella enteritidis.
Wnioski. Znacząca aktywność olejku bazyliowego wobec bakterii Gram-ujemnych pozwala uznać go za skuteczny preparat naturalny, który może być stosowany jako składnik preparatów farmaceutycznych i kosmetycznych.
Medicinal plants have been for ages a valuable and an indispensable source of natural products used in the treatment of human diseases and ailments. Their applicability initiated the production of today’s pharmaceutical and para-pharmaceutical products. It has been assumed that plant extracts possess biocidal properties against bacteria, fungi and viruses (1-3). The growing interest in the antimicrobial activity of plant extracts results from the necessity of looking for the new products as an alternative to antibiotics, which have limited time of acting effectively. Other reason is the society, which has become more aware of the consequences it may have if antibiotics are applied too often. Moreover, the growing autonomy in the treatment process and an accessibility to herbal products contribute to an annual growth of interest in the plant extracts as an alternative way of therapy. Additional advantage of herbal products application is their lower toxicity to people and higher environmental safety due to the fact that their production process causes less pollution.
Many species of plants, are not only the alternative to synthetic medicines but they are also spices, flavoring agents and an addition to cosmetics. Ocimum basilicum L. is an example of such plant. The raw material is a herb containing 0.5-2.5% of essential oils of variable chemical constituents (4). Essential oils are very complex natural mixtures which can contain about 20-60 components at quite different concentrations. They are characterized by two or three major components at fairly high concentrations (20-80%) compared to other components present in a trace amount. Generally, these major components determine the biological properties of essential oils. The components include two groups of distinct biosynthetical origin. The main group is composed of terpenes or terpenoids and the other of aromatic and aliphatic constituents, all characterized by a low molecular weight (1, 5-6).The main components of the essential oils from O. basilicum depend on the geographical origin of the plant and include the following: methyl eugenol and eugenol, methyl chavicol, methyl cinnamate, linalool, α-cubebene, 1,8-cineole, nerol, geranial, estragole, epi-α-cadinol, α-bergamotene, α-muurolene, 3,7-dimethyloct-1,5-dien-3,7-diol, β-cubebene and β-elemene (7-16).
Essential oils seem to have no specific cellular targets because of the great number of their constituents. The antibacterial properties of these constituents are, inter alia, associated with their lipophilic character (17). They pass through the cell wall and cytoplasmic membrane, disrupt the structure of their polysaccharides, fatty acids and phospholipids and permeabilize them. Cytotoxicity appears to include such membrane damage. In bacteria, the permeabilization of the membranes is associated with loss of ions and reduction of membrane potential and depletion of the ATP. Essential oils can also damage lipids and proteins. Damage to the cell wall and membrane can lead to the leakage of macromolecules and to lysis (1).
Plant-derived essential oils are known to be active against a wide variety of microorganisms. Cytotoxic effects were observed in vitro in most of pathogenic Gram-positive and Gram-negative bacteria generally by a disc diffusion method or a dilution method. In own research, the biocidal activity of O. basilicum against selected pathogenic Gram-negative strains was assessed with an application of a dilution method with some modifications. Applied method enabled an assessment of the survival rate of the Gram-negative bacteria under study in reference to the time length of basilium oil activity and its concentration.
The aim of conducted research was to determine antibacterial activity of basil essential oil against Gram-negative bacteria.
Material and methods
Extraction of the essential oil
For the experiment, the natural basil oil was obtained by a steam distillation of the herb O. basilicum L., containing linalool (amounting 62%) as its dominant component. Specimen sample was kept under refrigerated condition for future references.
Bacterial strains and growth conditions
Bacterial strains applied in the experiment had been isolated from infected skin. The identification of selected Gram-negative bacteria was based on their morphological and biochemical features with the use of ID32GN (Biomerieux) tests. In the experiment seven different Gram-negative bacterial strains were applied, as follows: Aeromonas hydrophila, Citrobacter freundii, Escherichia coli, Hafnia alvei, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella enteritidis.
All the bacteria used in this study were grown at 37°C in nutrient broth for 24 h. Throughout the experiments, the strains were subcultured every week on TSA (tryptone soya agar) containing (g/l): pepton SP 5.0, pepton K 15.0, sodium chloride 5.0, agar 15.0. The cultures were stored at 4oC. Before their application, liquid cultures prepared from a single colony were transferred twice into fresh nutrient broth and incubated at 37°C for 24 h. to adapt the inoculum’s density to ca. 108 cfu/ml (turbidity = McFarland barium sulfate standard 0.5).
Antimicrobial activity of the essential oil from O. basilicum (EOOB) against bacteria was determined by the modified dilution method. The experiment was run in five replicates with the following concentrations of the essential oil from O. basilicum EOOB: 0.25, 0.5, 1.0, 2.0 and 4.0 % (v/v) prepared in the nutrient broth with an addition of 0.05% (v/v) of Tween 80. The solutions were inoculated with bacterial strains under study of 108 cfu/ml density obtained after one day of culturing. The control trial contained nutrient broth only, without tested strain, with the same concentration of Tween 80, in order to eliminate its potential inhibition of the growth of the microorganisms. The culturing media were incubated at 37°C for 4, 24, 48 and 168 h. After the incubation time the number of bacteria per 1 ml of the culturing medium (cfu/ml) was determined for both test trials and the control. The number of active bacteria was assessed with the culturing-plate method on TSA medium.
Minimum inhibitory concentration (MIC) was defined as the lowest concentration of EOOB expressed in % (v/v), which inhibits the colonial growth of tested bacteria on TSA medium.
Evaluation of antibacterial effects
The antibacterial activity of EOOB was also expressed as a percentage of the growth reduction. The obtained results underwent statistical analysis of variance (ANOVA) with Duncan’s test. Values were considered significantly different at p < 0.05.
Results and discussion
Powyżej zamieściliśmy fragment artykułu, do którego możesz uzyskać pełny dostęp.
Płatny dostęp do wszystkich zasobów Czytelni Medycznej
1. Bakkali F, Averbeck S, Averbeck D et al. Biological effects of essential oils – A review. Food ChemToxicol 2008; 46:446-75. 2. Bassolè IHN, Juliani HR. Essential oils in combination and their antimicrobial properties. Molecules 2012; 17:3989-4006. 3. Shafique M, Khan SJ, Khan NH. Comparative study for antibacterial potential of in vitro and in vivo grown Ocimum basilicum L. plant extracts. Pak J Biochem Mol Biol 2011; 44:113-7. 4. Nurzyńska-Wierdak R. Ocimum basilicum L. – a valuable spice, medicinal and oleiferous plant. A review. Ann UMCS Sectio EEE 2012; 22:20-30. 5. Tomar US, Daniel V, Shrivastava K et al. Comparative evaluation and antimicrobial activity of Ocimum basilicum L. (Labiatae). J Global Pharma Tech 2010; 2:49-53. 6. Verma S, Kothiyal P. Pharmacological activities of different species of Tulsi. Int J Biopharm Phytochem Res 2012; 1:21-39. 7. Govindarajan M, Sivakumar R, Rajeswary M et al. Chemical composition and larvicidal activity of essential oil from Ocimum basilicum (L.) against Culex tritaeniorhynchus, Aedes albopictus and Anopheles subpictus (Diptera: Culicidae). Exp Parasitol 2013; 134:7-11. 8. Hussain AI, Anwar F, Sherazi STH et al. Chemical composition, antioxidant and antimicrobial activities of basil (Ocimum basilicum) essential oils depends on seasonal variations. Food Chem 2008; 108:986-95. 9. Kathirvel R, Ravi S. Chemical composition of the essential oil from basil (Ocimum basilicum L.) and its in vitro cytotoxicity against HeLa and HEp-2 human cancer cell lines and NIH 3T3 mouse embryonic fibroblasts. Nat Prod Res 2012; 26:1112-8. 10. Özcan M, Chalchat JC. Essential oil composition of Ocimum basilicum L. and Ocimum minimum L. in Turkey. Czech J Food Sci 2002; 20:223-8. 11. Politeo O, Jukic M, Milos M. Chemical composition and antioxidant capacity of free volatile aglycones from basil (Ocimum basilicum L.) compared with its essential oil. Food Chem 2007; 101:379-85. 12. Saha S, Dhar TN, Sengupta C et al. Biological activities of essential oils and methanol extracts of five Ocimum species against pathogenic bacteria. Czech J Food Sci 2013; 31:194-202. 13. Sastry KP, Kumar RR, Kumar AN et al. Morpho-chemical description and antimicrobial activity of different Ocimum species. J Plant Develop 2012; 19:53-64. 14. Shirazi MT, Gholami H, Kavoosi G et al. Chemical composition, antioxidant, antimicrobial and cytotoxic activities of Tagetes minuta and Ocimum basilicum essential oils. Food Sci Nutr 2014; 2:146-55. 15. Verma RS, Bisht PS, Padalia RC et al. Chemical composition and antibacterial activity of essential oil from two Ocimum spp. grown in sub-tropical India during spring-summer cropping season. J Trad Med 2011; 6:211-7. 16. Beatovic D, Krstic-Miloševic D, Trifunovic S et al. Chemical composition, antioxidant and antimicrobial activities of the essential oils of twelve Ocimum basilicum L. cultivars grown in Serbia. Rec Nat Prod 2015; 9:62-75. 17. Helander IM, Alakomi HL, Latva-Kala K et al. Characterization of the action of selected essential oil components on Gram-negative bacteria. J Agric Food Chem 1998; 46:3590-5. 18. Hammer KA, Carson CF, Riley TV. Antimicrobial activity of essential oils and other plant extracts. J App Microbiol 1999; 86:985-90. 19. Gupta PC, Batra R, Chauhan A et al. Antibacterial activity and TLC bioautography of Ocimum basilicum L. against pathogenic bacteria. J Pharm Res 2009; 2:407-9. 20. Opalchenova G, Obreshkova D. Comparative studies on the activity of basilan essential oil from Ocimum basilicum L. against multidrug resistant clinical isolates of the genera Staphylococcus, Enterococcus and Pseudomonas by using different test methods. J Microbiol Meth 2003; 54:105-10. 21. Adeola SA, Folorunso OS, Amisu KO. Antimicrobial activity of Ocimum basilicum and its inhibition on the characterized and partially purified extracellular protease of Salmonella typhimurium. Res J Biol 2012; 2:138-44. 22. Ahonkhai I, Ayinde BA, Edogun O et al. Antimicrobial activities of the volatile oils of Ocimum bacilicum L. and Ocimum gratissimum L. (Lamiaceae) against some aerobic dental isolates. Pak J Pharm Sci 2009; 22:405-9. 23. Orhan IE, Özcelik B, Kartal M et al. Antimicrobial and antiviral effects of essential oils from selected Umbelliferae and Labiatae plants and individual essential oil components. Turk J Biol 2012; 36:239-46. 24. Shafique M, Khan SJ, Khan NH. Study of antioxidant and antimicrobial activity of sweet basil (Ocimum basilicum) essential oil. Pharmacology online 2011; 1:105-11. 25. Sharma U, Agnihotri RK, Ahmad S et al. Antibacterial activity of some medicinal plants of family Lamiaceae from Braj region. Glob J Med Plant Res 2013; 1:72-6. 26. Usman LA, Ismaeel RO, Zubair MF et al. Comparative studies of constituents and antibacterial activities of leaf and fruit essential oils of Ocimium basilicum grown in north central Nigeria. Int J Chem Biochem Sci 2013; 3:47-52. 27. Wan J, Wilcock A, Coventry MJ. The effect of essential oils of basil on the growth of Aeromonas hydrophila and Pseudomonas fluorescens. J Appl Microbiol 1998; 84:152-8. 28. Smith-Palmer A, Stewart J, Fyfe L. Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Lett Appl Microbiol 1998; 26:118-22. 29. Eriotou E, Anastasiadou K, Nikolopoulos D, Koulougliotis D. Antimicrobial and free radical scavenging activities of basil (Ocimum basilicum) essential oil isolated from five plant varieties growing in Greece. J Nutr Food Sci 2015; 5(3):1-9. 30. Veras HNH, Rodrigues FFG, Colares AV et al. Synergistic antibiotic activity of volatile compounds from the essential oil of Lippia sidoides and thyme. Fitoter 2012; 83:508-12. 31. Alakomi HL, Skytta E, Saarela M et al. Lactic acid permeabilizes Gram-negative bacteria by disrupting the outer membrane. Appl Environ Microb 2000; 66:2001-5. 32. Pagès JM, James CE, Winterhalter M. The porin and the permeating antibiotic: a selective diffusion barier in Gram-negative bacteria. Nat Rev Microbiol 2008; 6:893-903. 33. Paparella A, Taccogna L, Aguzzi I et al. Flow cytometric assessment of the antimicrobial activity of essential oils against Listeria monocytogenes. Food Control 2008; 19:1174-82. 34. Xu J, Zhou F, Ji BP et al. The antibacterial mechanism of carvacrol and thymol against Escherichia coli. Appl Microbiol 2008; 47:174-9. 35. Kuorwel KK, Cran MJ, Sonneveld K et al. Essential oils and their principal constituents as antimicrobial agents for synthetic packaging films. J Food Sci 2011; 76:164-77. 36. Bassolè IHN, Lamien-Meda A, Bayala B et al. Composition and antimicrobial activities of Lippia multiflora Moldenke, Mentha x piperita L. and Ocimum basilicum L. essential oils and their major monoterpene alcohols alone and in combination. Molecules 2010; 15:7825-39. 37. Rattanachaikunsopon P, Phumkhachorn P. Antimicrobial activity of basil (Ocimum basilicum) oil against Salmonella enteritidis in vitro and in food. Biosci Biotechnol Biochem 2010; 74:1200-4. 38. Kim J, Marshall MR, Wei CI. Antibacterial activity of some essential oil components against five foodborne pathogens. J Agric Food Chem 1995; 43:2839-45.