49 research outputs found
The effects of reference monoterpene compounds and Gentiana lutea root extract on apoptosis and necroptosis in human peripheral blood mononuclear cells
Gentiana lutea je lekovita biljka koja se vekovima koristi u narodnoj medicini, a njena primena je u mnogim aspektima potkrepljena nauÄnim podacima. S obzirom da je zapaženo da u zavisnosti od eksperimentalnih uslova osim pozitivnih može ispoljavati i citotoksiÄni i genotoksiÄni uticaj, cilj ove doktorske disertacije je bila procena odgovora neizmenjenih mononuklearnih Äelija periferne krvi Äoveka (PBMC) i stimulisanih na deobu fitohemaglutininom (PHA-PBMC) na tretman referentnim monoterpenskim komponentama (MRK) i ekstraktom korena Gentiana lutea (EKG), identifikacija Äelijske smrti tipa apoptoze i nekroptoze izazvane uticajem tretmana i procena selektivne aktivacije ispitivanih tipova Äelijske smrti.
Rezultati ove doktorske disertacije pokazali su da citotoksiÄni i genotoksiÄni potencijal EKG zavise od koncentracije i dužine izlaganja, kao i od karakteristika Äelijske kulture. EKG sadrži znaÄajne koliÄine MRK, a posebno genciopikrozid (Gp), koje pojedinaÄno imaju veÄi genotoksiÄni potencijal od samog EKG, stoga je uoÄeni citotoksiÄni i genotoksiÄni uticaj EKG verovatno uslovljen odnosima MRK i drugih sekundarnih metabolita dobijenih ekstrakcijom. CitotoksiÄne koncentracije MRK aktiviraju kaspazno-zavisnu apoptozu usled indukovanog oÅ”teÄenja molekula DNK, tako da je aktivacija apoptoze neselektivna i može biti praÄena drugim tipovima Äelijske smrti. Dodatno, Gp i svertiamarin (Sm) tretmani u Äelijama mogu aktivirati i Äelijsku smrt tipa nekroptoze.
Ovim saznanjima se ne osporava blagotvorno dejstvo EKG primenjenog u manjim dozama i u kraÄem vremenskom periodu, veÄ se ukazuje da neadekvatna priprema i primena lako dostupnog biljnog materijala može imati i Å”tetne posledice.Gentiana lutea is a medicinal plant that has been used in folk medicine for centuries, while its application is, in many aspects, scientifically proven. Since its effects vary from positive to cytotoxic and genotoxic depending on the experimental conditions, the aim of this doctoral dissertation was to evaluate the response of primary human peripheral blood mononuclear cells (PBMC) and those stimulated with phytohemagglutinin (PHA-PBMC) to reference monoterpene components (MRK) and Gentiana lutea root extract (EKG) treatment; to identify the type of treatment-provoked cell death (apoptosis or necroptosis) and to assess the selectivity of investigated cell death typesā activation.
The obtained results indicate that the cytotoxic and genotoxic potential of EKG depends on concentration and time of exposure to EKG, as well as on the characteristics of cell culture. EKG contains significant amounts of MRK, especially gentiopicroside (Gp), which individually have a higher genotoxic potential than the EKG, so the observed cytotoxic and genotoxic effects of the EKG are probably due to the ratios of MRK and other secondary metabolites acquired after extraction. Cytotoxic concentrations of MRK activate caspase-dependent apoptosis due to treatment-induced DNA damage, indicating that activation of apoptosis is nonselective and may be accompanied by the other types of cell death. Additionally, Gp and swertiamarin (Sm) treatments can activate necroptosis-type cell death.
The findings of this study do not dispute the beneficial effects of EKG applied in smaller doses and over a shorter period of time, but they do emphasize the necessity of proper preparation and application of easily available herbal material that otherwise may have an adverse impact
Lomljivost telomera u bolniÄkih radnika profesionalno izloženih niskim dozama ionizirajuÄega zraÄenja
AbstractBioloÅ”ki uÄinci ionizirajuÄega zraÄenja (IZ) pripisuju se oÅ”teÄenjima DNA i indirektnim uÄincima kroz poveÄanu proizvodnju reaktivnih vrsta kisika. Iako se telomere rabe kao pokazatelji radioosjetljivosti, o njihovu ponaÅ”anju kao odgovoru na ionizirajuÄe zraÄenje u uvjetima profesionalne izloženosti i dalje se raspravlja. U ovom radu željeli smo istražiti duljinu i strukturu telomera u bolniÄkih radnika koji su profesionalno izloženi ionizirajuÄem zraÄenju te povezati te nalaze s oksidacijskim biomolekulama i kromosomskim aberacijama. Uzorci krvi izloženih ispitanika i zdravih kontrola uzeti su za analizu tijekom rutinskoga godiÅ”njeg zdravstvenog pregleda. Osim kromosomskih aberacija, u uzorcima plazme izmjereni su i parametri oksidacijskoga stresa [prooksidacijska/antioksidacijska ravnoteža (PAB), lipidna peroksidacija i 8-okso-dG], a procjena duljine i strukture telomera provedena je metodom Q-FISH na metafaznim kromosomima. Analiza kromosomskih aberacija pokazala je da od 34 ispitanika njih 14 ima kromosomske aberacije (skupina 1), a 20 nije imalo aberacije (skupina 2). Nije bilo znaÄajne razlike u spolu ili dobi ni u duljini telomera izmeÄu skupina. MeÄutim, incidencija lomljivih telomera bila je znaÄajno veÄa u objema skupinama ispitanika izloženih IZ-u u usporedbi s kontrolnim ispitanicima. Produkti peroksidacije lipida i 8-okso-dG takoÄer su bili znaÄajno viÅ”i u objema skupinama. UÄestalost lomljivih telomera u pozitivnoj je korelaciji (statistiÄki znaÄajna) s razinama 8-okso-dG
Optimizacija metode za izolaciju epitelnih stanica iz nežljezdanog dijela želuca Å”takora za protoÄnu citometriju
Traditional methods in cell proliferation studies are based on immunohistochemical detection of proliferating cells in the target tissue. Since they are time consuming, optimization of novel, more efficient methods is important for large scale proliferation studies. In this study, we aimed to optimize the isolation of single epithelial rat forestomach cells for flow cytometry. As a marker of cellular proliferation we used the Ki-67 antibody to detect this nuclear protein expressed in proliferating cells. We also performed immunohistochemical detection of Ki-67 positive cells and propidium iodide staining to validate the results. 3-tert- butyl -4-hydroxyanisole was used as the positive control to ensure cellular proliferation. The results showed that isolation of epithelial cells with collagenase, trypsin and cell strainer ensures great cell viability (>95%) and the purity of the samples. Flow cytometry and immunostaining with the Ki-67 antibody indicated that 3-tert- butyl-4-hydroxyanisole treatment leads to a significant increase in proliferation. A significant positive correlation was observed between the results obtained by immunohistochemistry and flow cytometry, but the flow cytometric data had a smaller measurement error, suggesting the equal sensitivity and greater accuracy of this method. Propidium iodide staining showed that the percentage of cells in the G2+S phase of the cell cycle correlated positively with the percentage of Ki-67 positive cells assessed by flow cytometry, indicating that Ki-67 positive cells reflect an active dividing cell pool. We conclude that the isolation of forestomach epithelial cells described is a simple and reliable method for obtaining viable cells for use in flow cytometry. Compared to immunohistochemistry, flow cytometric detection of the Ki-67 antigen is equally sensitive, but much faster and provides more accurate results.Tradicionalne metode u ispitivanju staniÄne proliferacije temelje se na imunohistokemijskom otkrivanju proliferacijskih stanica u ciljanom tkivu. Kako su dugotrajne, optimizacija novih i uÄinkovitijih metoda važna je za velika istraživanja o proliferaciji. U ovom smo radu željeli optimizirati izolaciju epitelnih stanica prednjeg želuca Å”takora za protoÄnu citometriju. Kao marker staniÄne proliferacije koristili smo Ki-67 protutijelo za otkrivanje ovoga nuklearnog proteina izraženog u proliferacijskim stanicama. TakoÄer smo uÄinili imunohistokemijsku detekciju Ki- 67 pozitivnih stanica i bojenje propidij-jodidom kako bismo potvrdili rezultate. Butil-hidroksianizol koriÅ”ten je kao pozitivna kontrola da se osigura staniÄna proliferacija. Rezultati su pokazali da izolacija epitelnih stanica s kolagenazom, tripsinom i staniÄnim cjedilom osigurava veliku vijabilnost stanica (> 95 %) i ÄistoÄu uzoraka. ProtoÄna citometrija i Ki-67 bojenje pokazali su da tretman butil-hidroksianizolom dovodi do znakovitog porasta proliferacije. PrimijeÄena je znakovita pozitivna korelacija izmeÄu rezultata dobivenih imunohistokemijom i protoÄnom citometrijom, dok su protoÄni citometrijski podaci imali manju pogreÅ”ku mjerenja, Å”to upuÄuje na jednaku osjetljivost i veÄu toÄnost ove metode. Bojenje propidij-jodidom pokazalo je da postotak stanica u G2+S fazi staniÄnog ciklusa pozitivno korelira s postotkom Ki-67 pozitivnih stanica procijenjenih protoÄnom citometrijom, Å”to upuÄuje na to da Ki-67 oslikava stanice u aktivnoj diobi. ZakljuÄujemo da je opisana izolacija epitelnih stanica prednjeg želuca Å”takora jednostavna i pouzdana metoda za dobivanje održivih stanica za upotrebu u protoÄnoj citometriji. U usporedbi s imunohistokemijom, protoÄna citometrijska detekcija antigena Ki-67 jednako je osjetljiva, ali mnogo brža i daje toÄnije rezultate
Prolonged exposure to mesoporous silica decrease cell viability in vitro
āContemporary Materials 2021ā : XIV International Scientific Conference : programme and book of abstracts : September 9-10, 2021; Banja Luka, Bosnia and Herzegovin
Synthesis and characterisation of SBA-15 with spherical particles
āContemporary Materials 2021ā : XIV International Scientific Conference : programme and book of abstracts : September 9-10, 2021; Banja Luka, Bosnia and Herzegovin
Vodeni ekstrakt kaduljina liÅ”Äa umanjuje upalu i oksidacijsku genotoksiÄnost u ljudskim mononuklearnim stanicama periferne krvi
Traditional medicine has used sage (Salvia officinalis L.) preparations for centuries to prevent and treat various inflammatory and oxidative stress-induced conditions. The aim of this in vitro study was to determine the bioactive properties of a sage leave extract obtained with environmentally friendly aqueous extraction and lyophilisation in primary human peripheral blood cells. To that end we measured the total phenolic and flavonoid content (TPC and TFC, respectively) with gas chromatography-mass spectrometry (GC-MS). Non-cytotoxic concentrations determined with the trypan blue assay were used to assess the antioxidant (DPPH, ABTS, and PAB assay), antigenotoxic (CBMN assay), immunomodulatory (IL-1Ī² and TNF-Ī±), and neuroprotective effects (AChE inhibition). The extract contained high TPC (162 mg GAE/g of dry extract) and TFC (39.47 mg QE/g of dry extract) concentrations, while Ī²-thujone content was unexpectedly low (below 0.9 %). Strong radical-scavenging activity combined with glutathione reductase activation led to a decrease in basal and H2O2-induced oxidative stress and DNA damage. A decrease in TNF-Ī± and increase in IL-1Ī² levels suggest complex immunomodulatory response that could contribute to antioxidant and, together with mild AChE inhibition, neuroprotective effects. Overall, this study has demonstrated that aqueous sage leave extract reduces the levels of thujone, 1,8-cineole, pinene, and terpene ketones that could be toxic in high concentrations, while maintaining high concentrations of biologically active protective compounds which have a potential to prevent and/or treat inflammatory and oxidative stress-related conditions.Salvia officinalis L. stoljeÄima se koristi u tradicionalnoj medicini za prevenciju i lijeÄenje raznih upalnih i oksidacijskim stresom izazvanih poremeÄaja. U ovoj studiji željeli smo ekstrahirati kaduljino liÅ”Äe koriÅ”tenjem ekoloÅ”ki prihvatljivog, āzelenogā pristupa vodenom ekstrakcijom i liofilizacijom te odrediti njegova bioaktivna svojstva u primarnim ljudskim perifernim krvnim stanicama. Ukupni sadržaj fenola i flavonoida i GC-MS koriÅ”teni su za karakterizaciju ekstrakta. NecitotoksiÄne koncentracije, odreÄene metodom bojenja s bojom tripan plavo, analizirane su za procjenu antioksidacijskih (DPPH, ABTS i PAB test), antigenotoksiÄnih (CBMN test), imunomodulacijskih (IL-1Ī² i TNF-Ī±) i neuroprotektivnih uÄinaka (AChE inhibicija). Ekstrakt je sadržavao visoku koncentraciju ukupnih fenola (162 mg GAE/g liofilizata) i flavonoida (39,47 mg QE/g liofilizata), dok je sadržaj Ī²-tujona bio neoÄekivano nizak (niži od 0,9 %). Snažna aktivnost hvatanja radikala u kombinaciji s aktivacijom glutation reduktaze dovela je do smanjenja bazalnog i H2O2 induciranog oksidacijskog stresa i oÅ”teÄenja DNA. Smanjenje TNF-Ī± i poviÅ”enje razine IL-1Ī² sugeriraju kompleksan imunomodulatorni odgovor koji bi mogao pridonijeti antioksidacijskim i, zajedno s blagom inhibicijom AChE, neuroprotektivnim uÄincima. Sveukupno, ova je studija pokazala da vodena ekstrakcija kaduljina liÅ”Äa smanjuje toksiÄne spojeve kao Å”to su tujon, 1,8-cineol, pinen i terpenski ketoni, a održava visoku koncentraciju bioloÅ”ki aktivnih zaÅ”titnih spojeva u ekstraktu, Å”to bi moglo imati potencijal za prevenciju i/ili lijeÄenje oksidacijskih i upalnih poremeÄaja
Influence of synthesis conditions on adsorption capacity of SBA-15/alginate adsorbent for removal ions of nickel(II) and lead(II) from aqueous solution
āContemporary Materials 2021ā : XIV International Scientific Conference : programme and book of abstracts : September 9-10, 2021; Banja Luka, Bosnia and Herzegovin
Nanotechnology approach for diminishing quercetin toxicity toward peripheral blood mononuclear cells
Quercetin (Q) is one of the most common and well researched antioxidant flavonoids, which usually occurs in plant-based foods, and medicinal plants. It was shown that quercetin exerts many beneficial effects on human health, including prevention of cancer and heart diseases. Quercetin was found to be toxic toward various types of cancer cell, still, at higher concentrations it was also shown toxic toward normal human cells [1,2]. One of the approaches to overcome this shortcoming offers nanotechnology which enables the novel perspective of phytochemical usage in contemporary medicine [3]. The strategy of binding quercetin to the gold nanoparticles during their synthesis was used, which resulted in quercetin capped gold nanoparticles (NPQ) [4]. Trypan blue exclusion test [5] was used to evaluate peripheral blood mononuclear cells (PBMC) viability after their exposure to either NPQ or free Q during 24, 48 and 72 h, at 37 Ā°C, in the range of quercetin concentrations from 5 to 50 Ī¼g/mL. A significant reduction in the cell count was observed in PBMC cultures treated with 10, 20, and 50 Ī¼g/mL of free Q, for all exposure times. The treatments of increasing concentrations and exposure times lowered the cells viability, resulting in 63% of the viable cells, following 72 h of the treatment with 50 Ī¼g/mL of free Q. Although NPQ treatments affected the cells viability in a concentration- and time-dependent manner the treatment with 50 Ī¼g/mL of NPQ for 72 h, had a milder effect on PBMC cultures than free Q, resulting in 81% of the viable cells (Figure 1). According to the obtained results, NPQ were shown less toxic toward PBMC than free Q.1-EuSPMF - 1st European Symposium on Phytochemicals in Medicine and Food; 7-9 September, 2022; Belgrad
Photocatalytic Degradation of Carbofuran in Water Using Laser-Treated TiO2: Parameters Influence Study, Cyto- and Phytotoxicity Assessment
This study investigates the impact of changing parameters on the photocatalytic degradation of carbofuran (CBF) using laser-treated TiO2 nanotube arrays on a Ti mesh under simulated sunlight irradiation and assessing toxicity during photocatalytic degradation. Various parameters, including the stirring effect, light intensity, initial CBF concentration, and variation in the active surface area of laser-treated TiO2 photocatalysts, were examined to determine their impact on degradation efficiency. The photodegradation kinetics were monitored using ultra-performance liquid chromatography with a PDA detector (UPLC-PDA) and UV-Vis spectrophotometry, while mineralization was assessed by a total organic carbon (TOC) analyzer. The photocatalytic degradation of CBF is enhanced by an increase in the active surface area of the TiO2 photocatalyst, light intensity, and the introduction of stirring, but it decreases with an increase in the initial concentration of CBF. The toxicity assessments revealed that the cytotoxicity of CBF initially increased during the degradation process but decreased after further treatment, indicating the formation and subsequent breakdown of toxic intermediates. The phytotoxicity test showed that longer degradation times resulted in higher toxicity to plant growth. This study provides new insights into the photocatalytic degradation of CBF with TiO2, the importance of parameter optimization for more efficient treatment, and the use of toxicity tests to confirm the success of the photocatalytic proces
The influence of hydrochloric acid on the features of SBA-15 particles
The template method synthesis of both SBA-15 materials with elongated and spherical particles was performed using a surfactant Pluronic P123. The HCl (p.a.) was used to synthesize material with elongated particles connected in chain structures grouped into shapes resembling sheaves of wheat. In the synthesis of spherical SBA-15 with diameters ranging from 0.5 to 2 Ī¼m, a spent HCl solution which was obtained after chemical treatment of clay was used where the dominant presence of the spheres was confirmed by the SEM method. In addition to the methods mentioned above, XRD, EDS and FTIR methods characterize SBA-15 materials