Biološka aktivnost ekstrakta krušine (Frangula alnus) i njegove dominantne komponente, emodina, u prokariotskim i eukariotskim test sistemima

Abstract

Frangula alnus (krušina) ima dugu istoriju korišćenja u tradicionalnoj medicini, a u ovom radu su ispitana njena određena nova svojstva, naročito antibiofilm aktivnost. Pripremljen je etil-acetatni ekstrakt kore krušine koji je hemijski okarakterisan te su ispitane njegova biološke aktivnosti. GC×GC/MS analiza je pokazala da je u ekstraktu dosta zastupljena 4-etoksi-benzoeva kiselina, , dok je LC-MS/MS analiza pokazala da je bogat fenolnim i flavonoidnim jedinjenjima među kojima je najdominantniji emodin koji je odabran za dalje testiranje uz ekstrakt. Pokazano je da ekstrakt ima snažnu antioksidantnu aktivnost za razliku od emodina. MIK vrednosti su pokazale da su svi testirani sojevi Staphylococcus aureus izuzetno osetljivi na delovanje ekstrakta i emodina, posebno klinički izolati. Rezultati ispitivanja antibiofilm aktivnosti su pokazali da ekstrakt i emodin imaju izuzetno snažan efekat na inhibiciju formiranja biofilmova sojeva S. aureus, ali da je efekat na već formirane biofilmove slabiji. Pokazano je da obe test supstance imaju inhibitorni efekat na koncentraciju ekstracelularnih polisaharida, da menjaju količinu eDNK u biofilmu kao i broj ćelija perzistera, ali i deluju na metabolizam ćelija. SEM i AFM mikroskopija su potvrdile antibiofilm efekat ukazujući na promene u morfologiji, strukturi i hrapavosti biofilmova. Dodatno, obe supstance su modulisale ekspresiju icaA, icaD, srrA, srrB gena i RNA III. Rezultati MTT testa su ukazali na jaku citotoksičnu aktivnost ekstrakta i emodina prema HepG2 i HCT 116 ćelijskim linijama, dok na imortalizovanoj MRC-5 liniji nije bilo efekta. Međutim, pokazano je da supstance remete ćelijski ciklus svih linija, kao i da indukuju apoptozu i nekrozu i remete njihov mitohondrijski membranski potencijal. Obe test supstance su ispoljile snažan genotoksični efekat na svim odabranim ćelijskim linijama.

Frangula alnus (alder buckthorn) has a long history of use in traditional medicine, thus in this study its certain new properties, especially antibiofilm, were examined. Ethyl-acetate extract of buckthorn bark was prepared, chemically characterized and its biological activities were examined. GC×GC/MS analysis showed that 4-ethoxy-benzoic acid, is abundant in the extract, while LC-MS/MS analysis showed that extract is rich in phenolic and flavonoid compounds, with emodin as dominant one, which was selected for further testing along with extract. It was shown that extract has strong antioxidant activity unlike emodin. MIC values showed that all tested strains of Staphylococcus aureus were extremely sensitive to the action of the extract and emodin, especially clinical isolates. The results of the study of antibiofilm activity showed that the extract and emodin have an extraordinary inhibitory effect on biofilm formation of S. aureus strains, but that the effect on already formed biofilms is less pronounced. It was demonstrated that both test substances have inhibitory effect on the concentration of extracellular polysaccharides, alter the amount of eDNA in the biofilms as well as the number of persister cells, but also affect cell metabolism. SEM and AFM microscopy confirmed the antibiofilm effect, indicating changes in the morphology, structure and roughness of the biofilms. Additionally, both substances modulated the expression of icaA, icaD, srrA, srrB genes and RNA III. The results of the MTT test indicated strong cytotoxic activity of the extract and emodin against HepG2 and HCT 116 cell lines, while there was no effect on the healthy MRC-5 line. However, the substances disturbed the cell cycle of all cell lines, as well as induced apoptosis and necrosis and modulated mitochondrial membrane potential. Both test substances showed a strong genotoxic effect on all selected cell lines.