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..

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

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

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

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..

Oxidation of chlorpyrifos, azinphos-methyl and phorate by myeloperoxidase

The present paper deals with the investigations of optimal conditions for the myeloperoxidase (MPO) mediated oxidation of chlorpyrifos, azinphos-methyl and phorate, organophosphorous pesticides (OPs) containing phosphorothionate group, from thio- to oxo-forms, in the presence of hydrogen peroxide. The aim of the work was to apply this oxidation method in the AChE based bioanalytical tests for OPs determination. The maximum concentration of oxo-forms for all tested pesticides was achieved after 10 min incubation of OPs in 50 mM phosphate buffer (pH 6.0) with 100 nM MPO in the presence of 50 mu M H(2)O(2). Optimal temperature for obtaining maximal concentration of oxo-forms was 37 degrees C. Only the parent compounds and their oxo-forms were identified chromatographically in the OPs samples after their exposure to MPO. Moreover, no hydrolysis products were detected in the time interval of 1 h after the MPO catalyzed reaction was stopped by catalase. The efficiency of OPs transformation from thio- to oxo-forms was measured using acethylcholinesterase (AChE) test, by comparison of percent of AChE inhibition before and after exposure to the oxidized sample. (C) 2011 Elsevier Inc. All rights reserved

Inhibition of Aldose Reductase by Gentiana lutea Extracts

Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2) activity has been implicated in the development of various secondary complications of diabetes. Thus, ALR2 inhibition could be an effective strategy in the prevention or delay of certain diabetic complications. Gentiana lutea grows naturally in the central and southern areas of Europe. Its roots are commonly consumed as a beverage in some European countries and are also known to have medicinal properties. The water, ethanol, methanol, and ether extracts of the roots of G. lutea were subjected to in vitro bioassay to evaluate their inhibitory activity on the ALR2. While the ether and methanol extracts showed greater inhibitory activities against both rat lens and human ALR2, the water and ethanol extracts showed moderate inhibitory activities. Moreover, the ether and methanol extracts of G. lutea roots significantly and dose-dependently inhibited sorbitol accumulation in human erythrocytes under high glucose conditions. Molecular docking studies with the constituents commonly present in the roots of G. lutea indicate that a secoiridoid glycoside, amarogentin, may be a potential inhibitor of ALR2. This is the first paper that shows G. lutea extracts exhibit inhibitory activity towards ALR2 and these results suggest that Gentiana or its constituents might be useful to prevent or treat diabetic complications

The Antiradical, Anti-Inflammatory and Anti-Genotoxic Potential of Herbal Preparation Chlamyfin

Herbal preparation Chlamyfin was investigated for its total polyphenol content, 2,2-diphenyl-1picrylhydrazyl (DPPH) scavenging activity, and anti-inflammatory and genotoxic properties. A high total polyphenol content provided evidence of high DPPH radical scavenging activity (IC50 = 4.96 +/- 0.23 mu g/ml). Analysis of the electrochemical behavior of Chlamyfin indicated high reducing ability, i.e., high antioxidant capacity, in agreement with the DPPH test. Analysis of myeloperoxidase (MPO) inhibition by Chlamyfin suggested anti-inflammatory action (IC50 values of 5.40 mu g/ml and 4.45 i_tg/m1 for an incubation time of 10 and 30 min, respectively). For genotoxic assessment, oxidative stress was induced by irradiation of peripheral whole blood with gamma-radiation in vitro. In the presence of Chlamyfin, reduced incidence of micronuclei without disturbance to the proliferative potential of cells was evidenced in both irradiated and unirradiated samples, indicating its genoprotective properties. It was shown that Chlamyfm, in addition to its bactericidal effect, also possesses strong antioxidant, anti-inflammatory and anti-genotoxic properties