Biological activity of essential oils of selected aromatic plants on species of the genera Staphylococcus, Streptococcus, Lactobacillus, Pseudomonas, Enterococcus and Candida isolated from the human oral cavity

Oralno zdravlje smatra se važnim delom opšteg zdravlja ljudi, ali je često zanemarena oblast, a razlozi tome su raznovrsni i kompleksni. Visoka učestalost oralnih oboljenja ima veliki socijalni i ekonomski uticaj. Najčešća oboljenja usne duplje su karijes, parodontalna oboljenja i oralne infekcije, dok su glavni uzročnici ovih oboljenja vrste rodova Streptococcus mutans-grupa, Staphylococcus, Lactobacillus, Pseudomonas, Enterococcus i Candida. Efikasan tretman i razvoj novih antiseptika koji poseduju antimikrobnu aktivnost je od velikog značaja za medicinu. Najčešće se koriste preparati koji sadrže hlorheksidin, povidon jod i cetilpiridinijum hlorid, zatim sintetička sredstva kao što su penicilin, cefalosporini, tetraciklini i njihovi derivati, nistatin, amfoterecin B, flukonazol itd. Ovi agensi poseduju antimikrobno dejstvo i kontrolišu upalu desni, ali imaju ograničenu efikasnost i veliki broj negativnih posledica kao što su bojenja zuba, iritacije sluzokože usne duplje, pojave alergije, nefrotoksičnost i endokrinu toksičnost, a povezuju se i sa kancerom i drugim sekundarnim oboljenjima. Problem rezistencije prema Svetskoj zdravstvenoj organizaciji je najveća pretnja globalnom zdravlju čoveka. Jedna od alternativa koja je poslednjih decenija u žiži naučnih interesovanja jesu etarska ulja kao izvor biološki aktivnih jedinjenja. U cilju dobijanja novih biološkli aktivnih jedinjenja, koja neće biti toksična za humane i animalne ćelije, u ovoj disertaciji analiziran je hemijski sastav i biološka aktivnost in vitro 60 komercijalnih uzoraka etarskih ulja ekstrahovano iz 47 biljnih vrsta: Cananga odorata, Foeniculum vulgare, Pimpinella anisum, Pastinaca sativa, Anethum graveolens, Matricaria recutita, Achillea millefolium, Helichrysum italicum, Boswellia carterii, Canarium luzonicum, Commiphora myrrha, Juniperus communis, J. virginiana, Gaultheria procumbens, G. fragrantissima, Pelargonium graveolens, Hyssopus officinalis, Mentha piperita, M. pulegium, Lavandula angustifolia, Thymus algeriensis, T. vulgaris, T. serpyllum, Rosmarinus officinalis, Salvia lavanudilifolia, S. officinalis, S. sclarea, S. triloba, Satureja montana, Laurus nobilis, Leptospermum petersonii, L. scoparium, Eucalyptus citriodora, E. globulus, Melaleuca alternifolia, M. quinquenervia, Syzygium aromaticum, Abies procera, A. grandis, A. alba, Pseudotsuga douglasii, Pinus silvestris, Piper nigrum, Cymbopogon martinii, Rosa centifolia, Citrus aurantium spp. aurantium i C.limon. Cilj rada je ispitivanje biološke aktivnosti i uticaja etarskih ulja i komponenti odabranih aromatičnih biljaka na mikroorganizme izolovane iz usne duplje čoveka i primena u prevenciji i lečenju oralnih bolesti i infekcija. Kvalitativna i kvantitativna analiza uzoraka etarskih ulja vršena je gasnohromatografski korišćenjem GC/MS i GC/FID procedura. Ukupno je identifikovano 342 komponente iz grupa jedinjenja oksidovanih monoterpena, monoterpenskih ugljovodonika, seskviterpenskih ugljovodonika, oksidovanih seskviterpena, fenilpropanoida, spiroetra, alifatičnih ketona, viših alkana, oksidovanih diterpena, alifatičnih ugljovodonika i oksidovana alifatična jedinjenja. U periodu 2010-2011. godine izvršena je izolacija i identifikacija mikroorganizama iz usne duplje pacijena sa Klinike za dečju i preventivnu stomatologiju Stomatološkog Fakulteta, Univerziteta u Beogradu. Antibakterijska aktivnost testirana je na 8 kliničkih izolata (Staphylococcus aureus, Streptococcus pyogenes, Streptococcus mutans, Lactobacillus acidophilus, Streptococcus salivarius, Streptococcus sanguinis, Pseudomonas aeruginosa i Enterococcus feacalis). Antifungalna aktivnost ispitana je na 58 kliničkih izolata (Candida albicans, C. glabrata i C. krusei) i dva referentna soja (C. albicans i C. tropicalis). Antimikrobna aktivnost etarskih ulja određivana je pomoću mikrodilucione i bioautografske metode na TLC pločama. Određivane su minimalne inhibitorne (MIK) i minimalne baktericidne i fungicidne (MBK/MFK) koncentracije. Kao pozitivne kontrole korišćeni su komercijalni antibiotici i mikotici (Ampicilin, Streptomicin i Flukonazol) i preparati za održavanje oralne higijene (Hexoral® i Curasept®), kao i koloidni rastvor srebrne vode i ozona. Rezultati mikrodilucione metode su pokazali da etarsko ulje T. serpyllum poseduje najbolji antibakterijski potencijal, dok su ulja M. recutita1 i J. virginiana pokazala najslabije dejstvo na ispitivane bakterije. Najbolji antifungalni potencijal pokazalo je etarsko ulje S. montana, dok je najslabiju aktivnost imalo etarsko ulje H. italicum2. Prema rezultatima korišćenja bioautografske metode na TLC pločama sva etarska ulja i izolovana jedinjenja pokazala su antimikrobnu aktivnost. Za potvrđivanje antimikrobne aktivnosti i ispitivanje mehanizma dejstva etarskih ulja i komponenti korišćena je skening elektronska mikroskopija (SEM), kojom je potvrđena njihova aktivnost na destabilizacije ćelijske membrane ćelija C. albicans. Korišćenjem kriterijuma jačine antimikrobne aktivnosti etarska ulja odabrana su za ispitivanje međusobne interakcije, antiquorum sensing, antibiofilm i citotoksičnog efekta/potencijala su bila C. limon, P. nigrumZ, M. alternifolia2, T. algeriensis, T. serpyllum, T. vulgaris, G. fragrantissima, P. graveolens, C. limon, R. centifolia, L. petersonii, S. montana, C. martinii i C. myrrha. Međusobne interakcije etarskih ulja i njihovih komponenti testirane su na reprezentativne kliničke izolate S. mutans i C. albicans. Rezultati ispitivanja pokazuju sinergističko, aditivno i antagonističko dejstvo. U okviru antiqourum-sensing aktivnosti, ispitan je uticaj ulja na proces kretanja ćelija P. aeruginosa PAO1 (twiching i flagella test), produkciju piocijanina kao i sposobnost formiranja biofilma. Odabrana etarska ulja su se pokazala vrlo efikasnim u inhibiciji quorum-sensing zavisnih procesa i pokazala jak antiqourum sensing potencijal. Testirane minimalne inhibitorne i sub-inhibitorne koncentracije etarskih ulja inhibirale su proces formiranja biofilma kod S. mutans i C. albicans, u odnosu na kontrolnu grupu. Citotoksičnost etarskih ulja ispitivana je pomoću sulforodamin B testa (Sr-B test) i test redukcije tetrazola (MTT test) na ćelijske linije MCF-7, NCI-H 46, HCT15, HeLa, HepG2, PLP2, LS-174, K562, A549, Fem-X i MRC-5. Kvantifikacijom stepena citotoksičnosti odabranih etarskih ulja, utvrđeno je da pokazuju dejstvo na tumorske ćelijske linije. Prikazana biološka aktivnost odabranih etarskih ulja na mikroorganizme iz usne duplje čoveka ukazuje na mogućnost izrade preparata na bazi etarskih ulja i njegove upotrebe u terapijske i profilaktičke svrhe.Oral health is considered an important part of general health, but is often neglected area, and the reasons for this are diverse and complex. The high incidence of oral disease has a huge social and economic impact. The most common diseases of the oral cavity are dental caries, periodontal disease, and oral infections, while the main causal agents of these diseases are species of genus Streptococcus mutans-group, Staphylococcus, Lactobacillus, Pseudomonas, Enterococcus and Candida. Effective treatment and developing new drugs that have antimicrobial activity is of great significance for medicine. The most commonly used preparations containing chlorhexidine, povidone iodine and cetylpyridinium chloride, followed by synthetic drugs such as penicillin, cephalosporins, tetracyclines and their derivatives, nystatin, amphotericin B, fluconazole, etc. These agents possess antimicrobial activity and control gum inflammation, but have limited efficacy and a large number of negative effects such as tooth staining, mucosal irritation, allergy, nephrotoxicity and endocrine toxicity, and are also associated with cancer and other secondary illnesses. The problem of resistance to the World Health Organization is the biggest threat to global human health. One of the alternatives that in recent decades are in focus of scientific interest are essential oils as a source of biologically active compounds. In order to obtain new biologically active compounds, which will not be toxic to human and animal cells, in this dissertation analysis of the chemical composition and biological activity in vitro of 60 commercial samples of essential oils extracted from 47 plant species were used: Cananga odorata, Foeniculum vulgare, Pimpinella anisum, Pastinaca sativa, Anethum graveolens, Matricaria recutita, Achillea millefolium, Helichrysum italicum, Boswellia carterii, Canarium luzonicum, Commiphora myrrha, Juniperus communis, J. virginiana, G . fragrantissima, Pelargonium graveolens, Hyssopus officinalis, Mentha piperita, M. pulegium, Lavandula angustifolia, Thymus algeriensis, T. vulgaris, T. serpyllum, Rosmarinus officinalis, Salvia lavanudilifolia, S. officinalis, S. sclarea, S. triloba, Satureja montana, Laurus nobilis, Leptospermum petersonii, L. scoparium, Eucalyptus citriodora, E. globulus, Melaleuca alternifolia, M. quinquenervia, Syzygium aromaticum, Abies procera, A. grandis, A. alba, Pseudotsuga douglasii, Pinus silvestris, Piper nigrum, Cymbopogon martinii, Rosa centifolia, Citrus aurantium spp. aurantium and C. limon. The aim was to test their biological activity and the impact of essential oils and components from selected aromatic plants to microorganisms isolated from human oral cavity for the application in the prevention and treatment of oral diseases and infections. Qualitative and quantitative analysis of essential oils Gas chromatographic was performed using GC / MS and GC / FID procedures. A total of 342 compounds were identified belonging to different groups such as oxidized monoterpenes, monoterpenskih hydrocarbons, sesquiterpene hydrocarbons, oxidized sesquiterpenes phenylpropanoids, spiro ethers, aliphatic ketones, higher alkanes, oxidized diterpenes, aliphatic hydrocarbons and oxidized aliphatic compounds. In the period 2010-2011 the isolation and identification of microorganisms from the oral cavity were collected of patients who attend the Clinic for Pediatric and Preventive Dentistry, Faculty of Dentistry, University of Belgrade. The antibacterial activity was tested on 7 clinical isolates of bacteria (Staphylococcus aureus, Streptococcus pyogenes, Streptococcus mutans, Lactobacillus acidophilus, Streptococcus salivarius, Streptococcus sanguinis, Pseudomonas aeruginosa and Enterococcus faecalis). Antifungal activity was investigated on 58 clinical isolates (Candida albicans, C. glabrata and C. krusei) and two reference strains of fungi (C. albicans and C. tropicalis). The antimicrobial activity of essential oils was determined by microdilution and bioautography method on TLC plates. Determination of minimal inhibitory concentrations (MIC) and minimum bactericidal and fungicidal (MBC / MFC) concentration were done. As a positive control we used a commercial antibiotics and antimycotics (Ampicillin, Streptomycin and Fluconazole), and preparations for oral hygiene (Hexoral® and Curasept®), as well as colloidal silver, and a ozone gas. Results from the microdilution method showed that the essential oil of T. serpyllum has the best antibacterial potential, while oil M. recutita1 and J. virginiana showed the lowest effect on the examined bacteria. Best antifungal potential was achived by the essential oil of S. montana, while the lowest activity had essential oil of H. italicum2. According to the results of bioautography method used on TLC plates, all essential oils and isolated compounds showed antimicrobial activity. To confirm the antimicrobial activity and the potential mechanism of action of essential oils and components scanning electron microscope was used, which confirmed their activity on the cell membrane destabilisation to C. albicans. The essential oils with the best antimicrobial activity, as main criteria, were selected for further study as testing interactions, antiquorum sensing, antibiofilm and cytotoxic effect / potential C. limon, P. nigrumZ M. alternifolia2, T.algeriensis, T. serpyllum, T. vulgaris, G. fragrantissima, P . graveolens, R. centifolia, L. petersonii, S. montana, C. martinii and C. myrrha. Mutual interactions of essential oils and their components are tested on a representative clinical isolates of S. mutans and C. albicans. The test results demonstrate the synergistic, additive and antagonistic effects. Within antiqourum-sensing activities, the effects of the oils on the process of moving of P. aeruginosa PAO1 (twiching and flagella test), piocyanin production, as well as the ability to form biofilms were investigated. Selected essential oils have proven very effective in the inhibition of quorum sensing dependent processes and showed strong antiqourum sensing potential. Tested minimum inhibitory and sub-inhibitory concentrations of essential oils were found to inhibit the process of biofilm formation of S. mutans and C. albicans in comparison to the control group. The cytotoxicity of the essential oils was tested using a sulforhodamine B assay (Sr-B test) and the test of the tetrazole salt reduction (MTT assay) in the cell lines MCF-7, NCI-H 46, HCT15, HeLa, HepG2, PLP2, LS-174, K562, A549, Fem-X and MRC-5. The results of the quantification of cytotoxicity of the essential oils selected, it has been found to exhibit the effect on all tumor cell lines. Displayed biological activity of selected essential oils on microorganisms from the oral cavity as man point show the possibility of making preparations based on essential oils and their use in therapeutic and prophylactic purposes