© 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
Streszczenie
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.
Introduction
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.
Aim
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).
Antibacterial activity
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
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