Biological effects of gentian root extracts (gentiana lutea)- enzyme inhibition, antioxidative and antimicrobial activity.

Predmet istraživanja doktorske disertacije je bio usmeren na ispitivanje bioloških efekata ekstrakata korena lincure i određivanje njihovog kvalitativnog i kvantitativnog sastava. Ispitivan je uticaj ekstrakata i konstituenata lincure (G. lutea) na aktivnost enzima mijeloperoksidaze (MPO), acetilholinesteraze (AChE) i ekto-ATPaze (E-NTPDaze) sa membrane sinaptozoma. Takođe, određivana je antioksidativna, antimikrobna, radioprotektivna i antiproliferativna aktivnost ekstrakata lincure i njenih konstituenata. Najzad, ispitan je i uticaj pojedinih konstituenata lincure na proces apoptoze humanih leukocita. Za proučavanje navedenih bioloških efekata pripremljeni su vodeni, kiseli (4% sirćetna kiselina, v/v) i etanolno vodeni (25, 50, 75 i 96% etanola u vodi, v/v) ekstrakti. Genciopikrozid, loganska kiselina, sverozid, svertiamarin, izoviteksin, amarogentin i izogentizin su izabrani kao konstituenti lincure čija je biološka aktivnost ispitivana. Kvalitativni sastav ekstrakata određivan je primenom tečno-masene hromatografije: HPLC-MS/ESI-TOF, MALDI-TOF i UPLC-PDA. Kvantitativna analiza ekstrakata kojom su određivane koncentracije navedenih konstituenata izvšena je pomoću UPLCTQD- MS/MS. Kvantitativnom analizom je utvrđeno da su genciopikrozid i loganska kiselina najzastupljenija jedinjenja u svim ekstraktima (0,9 do 2,5% genciopikrozid i 0,3 do 0,9% loganska kiselina), izogentizin je prisutan samo u etanolno vodenim ekstraktima (50- 96% EtOH, v/v) a amarogentin i izoviteksin su prisutni u tragovima. Kao najpotentniji inhibitori enzima MPO, AChE i E-NTPDaze su se pokazali etanolno vodeni ekstrakti. Pri koncentraciji od 100 g/mL, 50% i 75% EtOH ekstrakti inhibiraju 100% aktivnosti MPO, a u grupi ispitivanih konstituenata najjači inhibitor je genciopikrozid sa IC50=0,80,1 g/mL (2,25 M). Najviši stepen inhibicije AChE (54%) postignut je sa 75% EtOH ekstraktom, pri koncentraciji od 50 g/mL a od konstituenata sa loganskom kiselinom (39%) pri C= 6,77 M. Sposobnost inhibicije ENTPDaze su pokazali svi ispitivani ekstrakti u opsegu od 45 do 53% (vodeni i 50% EtOH) pri koncentraciji od 200 g/mL...The objective of the study of this doctoral thesis was focused toward investigation of the biological effects of G. lutea extracts and determination of their qualitative and quantitative composition. The activity of enzymes, myeloperoxidase (MPO), acetylcholinesterase (AChE) and synaptosomal membrane ecto-ATPase (ENTPDase) were investigated. Also, antioxidative, antimicrobial, radioprotective and antiproliferative activity of Gentian extracts and their constituents were determined. Finally, the influence of the individual G. lutea constituents on apoptosis of human leukocytes was examined. For the study of the mentioned biological effects water, acidic (4 % acetic acid, v/v) and ethanol-water extracts (25, 50, 75 and 96% ethanol in water, v/v) were prepared. Gentiopicroside, loganic acid, sweroside, swertiamarin, isovitexin, amarogentin and isogentisine were selected as G. lutea constituents whose biological activity was investigated. The qualitative composition of the extracts was determined by liquid chromatography coupled with mass spectrometry: HPLC-MS/ESI-TOF, MALDITOF and UPLC-PDA. Quantitative analysis of the extracts, which was used for the determination of the constituent’s concentration, was performed using UPLC-TQDMS/ MS. Quantitative analysis showed that the gentiopicroside and loganic acid are the most abundant compounds in all examined extracts (0.9 to 2.5% and 0.3-0.9% of gentiopicroside and loganic acid, respectively), while isogentisin is only present in the ethanol water extracts (50-96%, v/v) and amarogentin and isovitexin are present only in traces. The ethanol water extracts proved to be the most potent inhibitors of MPO, AChE and E-NTPDase enzymes. At a concentration of 100 μg/ml, 50% and 75% EtOH extracts inhibited 100% of MPO activity, and in the group of examined constituents the strongest inhibition was achieved with gentiopicroside with an IC50 = 0.80.1 g/mL (2.25 M). The highest degree of inhibition of AChE (54%) was obtained with 75% EtOH extract at a concentration of 50 g/mL while in the group of constituents the highest degree of..

Ispitivanje dejstva persirćetne kiseline na lipidnu komponentu bakterijskih spora i doprinos standardizaciji testa za ocenu efikasnosti

The aim of this research was to contribute to a more detailed insight in the action of peroxygen disinfectants based on peroxoacetic acid (PAA), on lipid components of sporogenic forms of microorganisms. Sporogenic forms are recognized to be significantly more resistant in comparison to vegetative forms. Bacillus cereus ATCC 11778 and Bacillus subtilis NCTC 10480 where chosen as referent bacterial sporogenic strains used to investigate the acting mechanism of PAA. After treatment of the above mentioned bacterial strains with PAA, fatty acids have been isolated from untreated and treated spores and consequent changes in the lipid component were analyzed. The obtained methyl-esters of fatty acids (MEFA) were analyzed by gas chromatography/mass spectrometry (GC/MS), using a standard of bacterial MEFA. Results confirmed that after treatment, in Bacillus cereus certain quantitative changes occurred, which included the decrease in quantity of fatty acids with 16 and 17 carbon atoms, i.e. 16:0, 16:1, /so 17:0 and anteiso 17:0. At the same time, in B. Subtilis the significant decrease in quantity of fatty acids with 15 and 17 carbon atoms occurred, i.e. anteiso 15:0 and anteiso 17:0.Cilj ovog istraživanja je bio da se stekne potpuniji uvid u dejstvo peroksigenih dezinfektanata na bazi peroksosircetne kiseline (u daljem tekstu: PSK) na lipidnu komponentu sporogenih formi mikroorganizama, za koje je poznato da pokazuju veću otpornost od vegetativnih oblika. Bacillus cereus ATCC 11778 i Bacillus subtilis NCTC 10480 su odabrani kao referentni sojevi - bioindikatori, na kojima je ispitivano delovanje PSK (nakon formiranja spora). Nakon tretmana spora pomenutih bakterijskih sojeva sa PSK, izolovane su masne kiseline iz netretiranih i tretiranih spora i analizirane su promene u lipidnoj komponenti. Dobijeni metil-estri masnih kiselina (MEMK) analizirani su pomoću gasne hromatografije i masene spektrometrije (GC/MS) uz poređenje sa standardom bakterijskih MEMK. Dobijeni rezultati su ukazali da je kod B. cereus ATCC 11778 nakon tretmana došlo do kvantitativne promene odnosno smanjenja količine masnih kiselina sa 16 i 17 Catoma, odnosno 16:0,16:1, ante/so 17:0,/so 17:0. Kod B. subtilis NCTC 10480 došlo je do značajnog smanjenja količine masnih kiselina sa 15 i 17 C atoma, odnosno anteiso 15:0 i anteiso 17:0

Inhibition of Vascular Smooth Muscle Cell Proliferation by Gentiana lutea Root Extracts

Gentiana lutea belonging to the Gentianaceae family of flowering plants are routinely used in traditional Serbian medicine for their beneficial gastro-intestinal and anti-inflammatory properties. The aim of the study was to determine whether aqueous root extracts of Gentiana lutea consisting of gentiopicroside, gentisin, bellidifolin-8-O-glucoside, demethylbellidifolin-8-O-glucoside, isovitexin, swertiamarin and amarogentin prevents proliferation of aortic smooth muscle cells in response to PDGF-BB. Cell proliferation and cell cycle analysis were performed based on alamar blue assay and propidium iodide labeling respectively. In primary cultures of rat aortic smooth muscle cells (RASMCs), PDGF-BB (20 ng/ml) induced a two-fold increase in cell proliferation which was significantly blocked by the root extract (1 mg/ml). The root extract also prevented the S-phase entry of synchronized cells in response to PDGF. Furthermore, PDGF-BB induced ERK1/2 activation and consequent increase in cellular nitric oxide (NO) levels were also blocked by the extract. These effects of extract were due to blockade of PDGF-BB induced expression of iNOS, cyclin D1 and proliferating cell nuclear antigen (PCNA). Docking analysis of the extract components on MEK1, the upstream ERK1/2 activating kinase using AutoDock4, indicated a likely binding of isovitexin to the inhibitor binding site of MEK1. Experiments performed with purified isovitexin demonstrated that it successfully blocks PDGF-induced ERK1/2 activation and proliferation of RASMCs in cell culture. Thus, Gentiana lutea can provide novel candidates for prevention and treatment of atherosclerosis

Protective effect of Gentiana lutea root and leaf extracts against heterocyclic aromatic amines IQ and PhIP produced in thermally processed meat

During high-temperature cooking of protein rich foods, especially meat and fish, heterocyclic aromatic amines can be formed. These amines are a class of potent mutagens that can cause alterations in the structure of DNA and chromosomes. In recent decades, research has been focused on investigating plants and their phytochemicals as potential antimutagens. The aim of this study was to examine the anti-genotoxic effect of methanolic root and leaf extracts of Gentiana lutea against the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) produced in thermally processed meat. To determine the protective potential of extracts, the alkaline comet assay was applied. The results obtained indicated strong anti-genotoxic effect of both extracts against the tested mutagens. The highest inhibition of IQ-induced genotoxicity was recorded for leaf extract (72%). Regarding PhiP, root extract achieved inhibition of 80% of DNA damage, so was more successful than leaf extract. The data obtained in this study stimulates further research of G. lutea extracts and its constituents as potential dietary supplements in improving human health. © Published under licence by IOP Publishing Ltd.Conference of 60th International Meat Industry Conference, MEATCON 2019 ; Conference Date: 22-25 September 2019; Conference Code:15329

Antioxidative properties and antigenotoxic potential of Gentiana lutea extracts against the heterocyclic aromatic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine, PhIP

Lipid oxidation that occurs in different types of food can cause alterations in nutritional qualities, flavour, texture and shelf life of foods. Furthermore, high temperature cooking of protein-rich food can lead to formation of heterocyclic aromatic amines capable of compromising the integrity of DNA molecules. To reduce these harmful effects, research has been focused on investigating plants as a source of potential natural food additives and preservatives. Thus, the aim of this study was to estimate antioxidant and antigenotoxic activities of 50% ethanolic-aqueous root and leaf extracts of the medicinal plant, Gentiana lutea . Antioxidative effect was investigated using the DPPH assay, while antigenotoxicity against the mutagen 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP) was determined using Salmonella Typhimurium TA 1535 in the SOS/ umuC assay. Leaf extract showed high antioxidative effect with the ability to neutralize up to 87% of free radicals at 400 µg mL -1 . Antigenotoxicity testing revealed that both extracts exhibited remarkable genoprotective activity against PhIP-induced DNA damage, with the highest inhibition levels being 70% and 85% for root and leaf extracts, respectively. Results obtained are encouraging and suggest further research of G. lutea extracts as potential food preservatives and additives in improving food quality and human health

Нови ПГК/TiO2 нанокомпозит добијен од поли(γ -глутаминске киселине) из новоизолованог соја bacillus subtilis 17B

Poly(γ-glutamic acid) (PGA), naturally produced by Bacillus species, is a biodegradable, non-toxic, biocompatible, and non-immunogenic negatively charged polymer. Due to its properties, it has found various applications in the food, cosmetic and pharmaceutical industries. In this work, Bacillus subtilis 17B was selected as the best PGA producer among fifty wild-types Bacillus strains tested and characterized as a glutamate-independent producer. The production of PGA by the newly identified strain was optimized and increased tenfold using the Box-Behnken experimental design. The purity of PGA after recovery and purification from the fermentation broth was confirmed by SDS-PAGE followed by Methylene Blue staining. PGA was characterized by ESI MS and used for the preparation of a new nanocomposite with TiO2. The synthesis of PGA/TiO2 nanocomposite, its structural analysis, and cytotoxic effect on the cervical cancer cell line (HeLa cell) was investigated to determine the potential anti-cancer usage of this newly prepared material. Encouraging, PGA/TiO2 nanocomposite showed an increased cytotoxic effect compared to TiO2 alone.Поли(γ-глутаминска киселина) (ПГK), коју производе бактерије рода Bacillus, је биоразградив, нетоксичан, биокомпатибилан и неимуноген негативно наелектрисани полимер. Због својих својстава нашао је разноврсну примену у прехрамбеној, козметичкој и фармацеутској индустрији. У овом раду, Bacillus ѕubtilis 17Б је изабран као најбољи ПГК продуцер међу педесетак тестираних природних изолата бактерија из овог рода и окарактерисан као глутамат независтан продуцер. Производња ПГК овим новоидентификованим сојем је оптимизована и десетоструко увећана коришћењем Box-Behnken експерименталног дизајна. Чистоћа ПГК након изоловања и пречишћавања из ферметационе течности је потврђена електрофорезом (SDS-PAGE) након бојења метиленским плавим. ПГК је окарактерисана масеном спекроскопијом (ESI MS) и коришћена за добијање новог нанокомпозита са ТiО2. Синтеза ПГК/ТiО2 нанокомпозита, његова структурна анализа и цитотоксични ефекат на ћелијску линију рака грлића материце (HeLa ћелије) је испитан да би се утврдила потенцијална употреба овог новодобијеног материјала у борби против ћелија рака. Нанокомпозит ПГК/ТiО2 показао је повећан цитотоксични ефекат на поменуте ћелије рака у поређењу са самим ТiО2

Analysis of 4,4′-bis(2,2′diphenyl vinyl)-1,1′- biphenyl using the atmospheric-pressure solids analysis probe for ionization

An Atmospheric pressure Solids Analysis Probe (ASAP) mass spectrometer are used for investigation the ionization mechanism and fragmentation pathways of 4,4′-bis(2,2′diphenyl vinyl)-1,1′-biphenyl (DPVBi). DPVBi is material used in OLEDs (organic light-emitting diode). Results obtained indicate that by controlling ion source conditions it is possible to optimize forming of desired precursor ion, primarily radical cation and in less content protonated ion of DPVBi. The results presented illustrate the usefulness of ASAP MS in the characterization of DPVBi compounds

Synthesis of lutetium(III) complex with a polyazamacrocyclic ligand

Radijacionо zračenje predstavlja specifičan način onkološkog lečenja, kod kojeg se antitumorski efekat postiže dejstvom jonizujućeg zračenja.Visokoenergetsko jonizujuće zračenje oštećuje genetski materijal ćelija tumorskog tkiva i tako ograničava ili onemogućuje njihovu sposobnost daljnjeg deljenja. Radioizotopi koji se koriste u lečenju u većini slučajeva grenerišu beta zračenje koje izaziva uništenje obolelih ćelija. Interesovanje za razvoj 177Lu kao radiofarmaceutika može se pripisati podobnosti njegovih nuklearnih svojstava za terapeutsku upotrebu, emisija β(-) i γ zračenja i njegovo dobro kompleksiranje sa raznim ligandima. U ovom radu je prikazana preliminarna sinteza kompleksa Lu(III) sa poliazamakrocikličnim ligandom.Radiation is a specific method of oncological treatment, in which the action of ionizing radiation achieves the antitumor effect. High-energy ionizing radiation damages the genetic material of tumor tissue cells and thus limits or disables their ability to divide further. Radioisotopes used in treatment in most cases generate beta radiation that causes the destruction of diseased cells. The interest in developing 177Lu as a radiopharmaceutical can be attributed to the suitability of its nuclear properties for therapeutic use, the emission ofβ(-) and γ radiation, and its good complexation with various ligands. This paper presents the preliminary synthesis of Lu(III) complex with a polyazamacrocyclic ligan

Synthesis of Lutetium(III) Complex With a Polyazamacrocyclic Ligand

Radijacionо zračenje predstavlja specifičan način onkološkog lečenja, kod kojeg se antitumorski efekat postiže dejstvom jonizujućeg zračenja.Visokoenergetsko jonizujuće zračenje oštećuje genetski materijal ćelija tumorskog tkiva i tako ograničava ili onemogućuje njihovu sposobnost daljnjeg deljenja. Radioizotopi koji se koriste u lečenju u većini slučajeva grenerišu beta zračenje koje izaziva uništenje obolelih ćelija. Interesovanje za razvoj 177Lu kao radiofarmaceutika može se pripisati podobnosti njegovih nuklearnih svojstava za terapeutsku upotrebu, emisija β(-) i γ zračenja i njegovo dobro kompleksiranje sa raznim ligandima. U ovom radu je prikazana preliminarna sinteza kompleksa Lu(III) sa poliazamakrocikličnim ligandom.Radiation is a specific method of oncological treatment, in which the action of ionizing radiation achieves the antitumor effect. High-energy ionizing radiation damages the genetic material of tumor tissue cells and thus limits or disables their ability to divide further. Radioisotopes used in treatment in most cases generate beta radiation that causes the destruction of diseased cells. The interest in developing 177Lu as a radiopharmaceutical can be attributed to the suitability of its nuclear properties for therapeutic use, the emission ofβ(-) and γ radiation, and its good complexation with various ligands. This paper presents the preliminary synthesis of Lu(III) complex with a polyazamacrocyclic ligand.XXXII Simpozijum Društva za zaštitu od zračenja Srbije i Crne Gore, 4-6. oktobar 2023; Budva, Crna GoraProceedings: [https://vinar.vin.bg.ac.rs/handle/123456789/11602

Influence of Gamma Radiation on the Stability of Aflatoxin in Milk

Aflatoksini su značajan problem u oblasti bezbednosti hrane i rizika po zdravlje ljudi. Gama zračenje uništava opasne zagađivače u hrani kao što su bakterije, virusi, gljivice, pesticidi i toksini. U ovoj studiji je korišćeno gama zračenje da bi se smanjila količina aflatoksina u mleku. Rezultati su pokazali smanjenje aflatoksina u mleku za 9 % - 35,26 % u poređenju sa kontrolnim uzorkom. Smanjenje koncentracija aflatoksina u mleku nije proporcionalno primenjenoj dozi grama zračenja usled zgrušavanja mleka.Aflatoxins are a significant problem in the field of food safety and risk to human health. Gamma irradiation destroys dangerous contaminants in foods such as bacteria, viruses, fungi, pesticides, and toxins. In this study, we used gamma radiation to reduce the amount of aflatoxins in milk. The results showed a 9 % - 35.26 % reduction of aflatoxin in milk compared to the control sample. Reductions in aflatoxin concentrations in milk are not proportional to the applied dose of grams of radiation due to milk coagulation.XXXI Симпозијум Друштва за заштиту од зрачења Србије и Црне Горе, 06-08. октобар 2021.Proceedings: [https://vinar.vin.bg.ac.rs/handle/123456789/9668