In vitro kontrola patogenih bakterija poreklom iz humanog materijala delovanjem etarskih ulja i ekstrakata odabranih biljnih vrsta

Abstract

Poslednjih decenija jedan od vodećih problema u medicini je stvaranje rezistentnosti patogenih mikroorganizama na delovanje antibiotika. Bakterije koje pokazuju značajnu rezistentnost na postojeće antibiotike su: meticilin rezistentni Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Acinetobacter spp., itd. Prilikom delovanja na patogene bakterije, antibiotici neselektivno utiču i na nepatogene bakterije, izazivajući pri tom nepredvidive genetske promene. Pored korisnih efekata u delovanju na bakterije, raspoloživi antibiotici, takođe mogu prouzrokovati neželjene efekte kao što su hipersenzitivnost i imunosupresija. To je razlog zbog koga se traga za novim agensima sa antibiotskim delovanjem. Jedan od prirodnih izvora takvih agenasa su etarska ulja i ekstrakti aromatičnih biljaka koji se koriste u tradicionalnoj medicini za lečenje mnogih infektivnih bolesti i bolesti koje nastaju kao posledica oksidativnog stresa. Zbog toga je cilj istraživanja ove disertacije bio da se izvrši komparativna analiza hemijskog sastava, antibakterijske i antioksidativne aktivnosti odabranih biljnih vrstaAngelica pancicii, Angelica sylvestris, Laserpitium latifolium, Achillea grandifolia, Achillea crithmifolia, Artemisia absinthium, Tanacetum parthenium i Hyssopus officinalis koje su veoma zastupljene u tradicionalnoj medicini. Materijal i metode. Biljni materijal koji je korišćen u ovom istraživanju je prikupljan tokom 2012. i 2013. godine na teritoriji jugoistočne Srbije. Nakon sušenja biljnog materijala, pristupilo se procesu izolovanja etarskih ulja metodom hidrodestilacije u aparaturi po Klevindžeru, kao i pripremanju metanolnih ekstrakata alkoholnom ekstrakcijom. Hemijski sastav ulja je analiziran pomoću GH (gasna hromatografija) i GH/MS (gasna hromatografija sa spektrometrijom masa) metoda. Ukupni fenoli su određivani metodom po Folin-Sjoklto-u, sa malim modifikacijama, a ukupni flavonoidi su utvrđivani korišćenjem aluminijum hlorid (AlCl3) kolorimetrijskog metoda. Antioksidativna aktivnost etarskih ulja i ekstrakata je određivana pomoću ABTS (2,2'-azino-bis (3-etilbenzotiazolin-6- sulfonska kiselina), DPPH (2,2-difenil, 1-pikril hidrazil), TRP (total reducing power) i FRAP (Ferric reducing antioxidant power) metoda. Antibakterijska aktivnost izolovanih etarskih ulja,metanolnih ekstrakata i četiri antibiotika (ciprofloksacin, doksiciklin, gentamicin i eritromicin) je ispitivana mikrodilucionom metodom protiv 16 bakterijskih sojeva poreklom iz briseva rana, grla i nosa, sputuma i aspirata pacijenata (Escherichia coli, Pseudomonas aeruginosa, Klebsiella sp., Proteus mirabilis, Acinetobacter sp., Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae i Enterococcus faecalis). Sinergističko delovanje najaktivnijih ulja i antibiotika sa najslabijim delovanjem, eritromicinom, u cilju snižavanja njegovih aktivnih koncentracija, je određivno uz pomoć metode šahovske table tj. „checkerboard“ metodom.

During the last several decades one of the leading challenges in medicine is appearance of resistance of pathogenic microorganisms to antibiotic activity. Bacteria showing significant resistance on existing antibiotics include: methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Acinetobacter spp., etc. During the activity on pathogenic bacteria, the antibiotics simultaneously show non-selective impact on nonpathogenic bacteria, causing unpredictable genetic changes. In addition to beneficial effects on bacteria, the available antibiotics may also cause adverse effects such as hypersensitivity and immunosuppression. Therefore studies of new agents with antibiotic activity are constantly ongoing. One of the natural sources of such agents are essential oils and extracts of aromatic plants used in traditional medicine as a cure for many infectious diseases and ailments caused by oxidative stress. Therefore the goal of study presented in this dissertation was comparative analysis of chemical composition, antibacterial and antioxidant activity of eight chosen plant species regularly represented in traditional medicine: Angelica pancicii, Angelica sylvestris, Laserpitium latifolium, Achillea grandifolia, Achillea crithmifolia, Artemisia absinthium, Tanacetum parthenium and Hyssopus officinalis. The plant material used in this study was collected in 2012 and 2013 in southeastern Serbia. After the plant material was dried, essential oils were isolated by hydro-distillation method using the Clevenger apparatus, while methanol extracts were prepared by alcoholic extraction. The chemical composition of oil was analyzed by GC (gas chromatography) and GC/MS (gas chromatography with mass spectrometry) methods. The total amount of phenols was determined by Folin-Ciocalteu method with slight modifications, while total flavonoids were determined by aluminum chloride (AlCl3) colorimetric method. The antioxidant activity of essential oils and extracts was determined by ABTS (2,2'-azinobis (3-ethylbenzothiazoline-6-sulphonic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl), TRP (total reducing power) and FRAP (Ferric reducing antioxidant power) methods. The antibacterial activity of isolated essential oils, methanol extracts and four antibiotics (ciprofloxacin, doxycycline, gentamicin and erythromycin) was studied by micro-dilution method against 16 bacterial strains collected from swabs of wounds, throat, nose, sputum and aspirate of patients (Escherichia coli, Pseudomonas aeruginosa, Klebsiella sp., Proteus mirabilis, Acinetobacter sp., Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae and Enterococcus faecalis). The synergistic activity of most active oils and the lowest-activity antibiotic (erythromycin), in order to decrease its active concentrations, was determined by the so-called “checkerboard” method.