Određivanje fenolnih jedinjenja u plodovima nekih biljaka iz familije Rosaceae i antioksidativne aktivnosti njihovih sintetičkih derivata

U poslednjih nekoliko decenija, interes za izučavanjem poznatih i pronalaženjem novih vrsta koje mogu imati značajan antioksidativni potencijal sve više raste. Imajući u vidu da su fenolna jedinjenja poznata kao jako dobri antioksidansi, a da su glavni izvori ovih jedinjenja biljke, konstantno se izučavaju biljne vrste koje potencijalno poseduju visok sadržaj prirodnih antioksidanasa. Familija Rosaceae obuhvata veliki broj biljnih vrsta koje ljudi od davnina koriste u svojoj ishrani, ali su mnoge divlje vrste nedovoljno proučavane, pa je deo ove disertacije baziran na analizi fenolnih jedinjenja u divljim plodovima jabuke, maline i trešnje. U cilju dobijanja maksimalnog prinosa ovih jedinjenja iz biljnog materijala, izvršena je optimizacija metode ekstrakcije fenolnih jedinjenja, i definisani su optimalni uslovi za ekstrakciju, koji obuhvataju pravilan odabir rastvarača (i njegove koncentracije), odnos mase uzorka i zapremine rastvarača, vreme i temperaturu ekstrakcije. U ovako dobijenim ekstraktima, analiziran je ukupan sadržaj fenolnih jedinjenja (ukupnih fenola, fenolnih kiselina, flavonoida i antocijanina), a rezultati su omogućili određivanje optimalnih uslova ekstrakcije ovih jedinjenja u uzorcima primenom softvera Design-Expert® i metodologije odziva površine (eng. Response Surface Methodology). Kvantifikacija pojedinačnih fenolnih jedinjenja u uzorcima dobijenim ekstrakcijom pod optimalnim uslovima izvršena je HPLC metodom, pri čemu su dobijeni rezultati potvrdili da su ovi plodovi bogati sadržajem biološki važnih fenolnih jedinjenja. Dobijene vrednosti koncentracija pojedinačnih fenolnih jedinjenja ukazale su da plodovi ispitivanih samoniklih vrsta familije Rosaceae mogu biti značajan izvor ove grupe jedinjenja u ishrani ljudi. Rezultati i metodologija analize fenolnog sastava ovih plodova, mogu biti od značaja i za buduća ispitivanja i analizu sadržaja fenolnih jedinjenja u plodovima biljaka sličnih karakteristika. Ispitana je i stabilnost i biodostupnost ovih komponenti u organizmu, simulacijom uslova in vitro Ugastrointestinalne digestije. Da bi se rasvetlio antioksidativni potencijal testiranih uzoraka, primenjen je niz metoda koje ispituju njihovu sposobnost da neutrališu štetne slobodno-radikalske vrste. Iako postoji sve veća težnja za nalaženjem i upotrebom prirodnih antioksidanasa, mogućnosti njihove univerzalne primene su često ograničene, pa se pribegava dobijanju antioksidativnih jedinjenja sintetičkim putem. U tom smislu, u ovom radu je izvršena sintetička modifikacija odabranih fenolnih kiselina, sa ciljem nalaženja novih jedinjenja sa boljim biološkim aktivnostima, pre svega antioksidativnom. Na ovaj način su dobijene dve serije jedinjenja, 1,2,4- triazol-3-tioni i 1,3,4-oksadiazoli. Dobijeni rezultati daju doprinos analizi fenolnih jedinjenja u prirodnim proizvodima, njihovoj funkcionalizaciji i primeniIn the past several decades, the interest in studying well-known and discovery of new species that may have significant antioxidant potential is constantly growing. Taking into account that phenolic compounds are known as good antioxidants, and that the main sources of these compounds are plants, there is a constant pursuit for plant species potentially possessing a high content of natural antioxidants. The Rosaceae family includes a large number of plant species that humans have used in their diet for a long time, but many wild species are insufficiently studied, therefore part of this PhD thesis is based on the analysis of phenolic compounds in wild apples, raspberries, and cherries (family Rosaceae). In order to obtain the maximum yield of these compounds, optimization of the extraction method of phenolic compounds from plant material was performed, and optimal conditions for extraction, including the appropriate choice of the solvent (and its concentration), the solid to solvent ratio, the time and temperature of the extraction, were defined. The total content of phenolic compounds (total phenols, phenolic acids, flavonoids, and anthocyanins) was analyzed in the obtained extracts, and the obtained results were used in determination of optimal conditions for the extraction of these compounds in the samples using the DesignExpert® software and the response surface methodology. The quantification of individual phenolic compounds in the samples obtained by extraction under optimal conditions was performed by HPLC method and the results confirmed that these fruits are rich in the biologically important phenolic compounds. The obtained concentrations of individual phenolic compounds indicated that the studied wild fruit species of the Rosaceae family could be a significant source of phenolic compounds in human nutrition. The results and methodology of analyzing the phenolic composition of these fruits can be of importance for future research and analysis of phenolic compounds content in fruit or plants with similar characteristics. Furthermore, the stability and bioaccessibility of these components in the organism were examined by the simulation of the in vitro gastrointestinal digestion conditions. In order to clarify the Iantioxidant potential of the tested samples, a number of methods for determination of the ability of obtained extracts to neutralize harmful free-radical species have been applied. Although there is a growing tendency to find and use natural antioxidants, the possibilities of their universal application are often limited, therefore antioxidant compounds are often synthetically obtained. The synthetic modification of selected phenolic acids was carried out with the aim of finding a new series of compounds with better biological activities, mainly antioxidant activity. Two series of novel compounds were synthesized, 1,2,4-triazol-3-thiones and 1,3,4-oxadiazoles. The obtained results in this PhD thesis contribute to the analysis of phenolic compounds in natural products, their functionalization, and application

Uticaj proređene sadnje na obrazovanje prinosa nadzemne biomase Miscanthus x giganteus

A simple field trial was performed to investigate the influence of less successful miscanthus culture establishment due to scarce planting on the yield of aboveground biomass. The trial was carried out on highly productive soil of non-carbonate chernozem type. Two months old miscanthus sprouting rhizomes with 1-2 developed stems were planted. The experiment included three levels of planting density: 0.50, 0.65 and 1.00 sprouting rhizomes/m2. At the lowest planting density (0.50), the average yield of 4-8 years old plantation, which is the stage with the highest aboveground biomass yield, amounted to 13.5±2.9 t dry matter (d.m.)/ha, while at the planting density of 1.00, the achieved yield was 21.2±4.5 t d.m./ha. Our results show that, at the recommended planting density of 15000 rhizomes/ha, on a highly productive soil, even with low survival of planted seedlings (> 43 %), satisfying yields of aboveground miscanthus biomass may be obtained.Cilj istraživanja je da se jednostavnim ogledom u poljskim uslovima ustanovi uticaj nižih nivoa uspešnosti zasnivanja miskantusa kroz proređenu sadnju (presađivanje) biljčica miskantusa na obrazovanje prinosa nadzemne biomase. Poljski ogled je zasnovan na visoko-produktivnom zemljištu tipa izluženi černozem. Sađeni su busenovi miskantusa starosti 2 meseca, sa 1-2 razvijena stabla. Ogled je obuhvatio 3 gustine sadnje: 0,50; 0,65 i 1,00 busenova miskantusa po m2. Faza zasnivanja useva trajala je 3 godine. Pri najnižoj gustini sadnje (0,50) prosečan prinos useva od 4-8 godine gajenja, kada se dostižu maksimumi prinosa nadzemne biomase, iznosio je 13,5±2,9 tona suve materije (s.m.)/ha, naspram 21,2±4,5 tona s.m./ha pri gustini sadnje 1,00. Rezultatipokazaju da se kod preporučene gustine sadnje rizoma miskantusa od 15000 rizoma/ha, na visoko produktivnom zemljištu, u povoljnim agroekološkim uslovima i pri relativno niskom prijemu (>43%) mogu dobiti zadovoljavajući prinosi nadzemne biomase miskantusa

Uticaj endotelina i L-NAME na koncentraciju mokraćne kiseline i uree u plazmi kod Wistar pacova

Effects of endothelins, which include vasoconstriction and vasodilatation are possible by different molecular mechanisms. Endothelins induce increased synthesis of nitric oxide which in turn inhibits the synthesis of endothelin. Changes in biochemical parameters connected with the increased and decreased synthesis of nitric oxide are not thoroughly examined yet. In a series of experiments we compared the effects of endothelin (ET-1) on the inhibition of nitric oxide synthase (NOS), as well as the effects of L-nitro arginin metal estar (L-NAME) on plasma urea and uric acid concentration. Experiments were performed on anesthetized female adult Wistar rats. The experimental groups received an i. v. bolus of ET-1, L-NAME or saline. All parameters, heamodynamic and biochemical, were measured before the animals were sacrificed. L-NAME increased the mean arterial pressure, decreased renal blood flow and increased vascular resistance in carotid and renal arteries and increased in plasma uric acid and urea concentrations. ET- 1 significantly decreased uric acid concentration in the plasma but did not effect plasma urea concentration compared to the control group. These differences show complex relations of nitric oxide with cellular signalization compared to the basic NO-cGMP pathway.Efekti endotelina koji uključuju vazokonstrikciju I vazodilataciju ostvaruju se različitim molekularnim mehanizmima. Endotelin indukuje povećanu sintezu azotnog oksida za koji se smatra da povratno inhibira sintezu endotelina. Promene biohemijskih parametara vezanih za povećanu i smanjenu sintezu NO nisu dovoljno proučene. U ovom radu su upoređeni efekti endotelina (ET-1) i inhibitora NOS-a, L-NAME na koncentraciju mokraćne kiseline i uree u plazmi. Eksperimenti su izvedeni na anesteziranim odraslim NW pacovima, ženskog pola. Eksperimentalne životinje su u i.v. bolusu primale ET-1, L-NAME ili fiziološki rastvor. Svi hemodinamski i biohemijski parametri su mereni pre žrtvovanja. L-NAME dovodi do povećanja srednjeg arterijskog pritiska smanjenja renalnog protoka i povećanja vaskularnog otpora u karotidnoj i renalnoj arteriji i povećanja koncentracije mokraćne kiseline i uree u plazmi. ET-1 značajno smanjuje koncentraciju mokraćne kiseline u plazmi ali ne utiče na koncentraciju uree u odnosu na kontrolu. Ove razlike pokazuju kompleksnije relacije NO-a sa ćelijskom signalizacijom u odnosu na osnovni NO-cGMP put

Effects of high dose olive leaf extract on the hemodynamic and oxidative stress parameters in normotensive and spontaneously hypertensive rats

The antihypertensive activity of olive leaf extract (OLE), a natural antioxidant is recognized, but its influence on the cardiovascular system when administered in a high dose has not yet been investigated. The aim of the present study was to determine the acute effects of excessive intake of standardized OLE on blood pressure, heart rate and oxidative status in both spontaneously hypertensive rats and normotensive Wistar rats. The systolic arterial pressure and heart rate were measured using a tail-cuff and pneumatic pulse detector before and 60 and 120 min after intragastric OLE administration. The activities of catalase, glutathione peroxidase, superoxide dismutase (SOD) and glutathione reductase in erythrocytes, as well as lipid peroxidation in plasma (pTBARS) were measured spectrophotometrically at the same time points. A high-dose of OLE did not influence blood pressure, heart rate or pTBARS in normotensive rats, while the SOD, catalase and glutathione reductase activities were significantly increased. The same dose significantly decreased blood pressure in hypertensive rats, but increased the pTBARS and SOD activity. Excessive oral intake of OLE induced moderate hypotensive effects only in spontaneously hypertensive rats, suggesting the absence of harmful hemodynamic effects after an oral overdose in both rat strains. However, its pro-oxidative role when given in a high dose in hypertensive organisms should not be neglected

Synthesis and antioxidant activity of 1,3,4-oxadiazoles and their diacylhydrazine precursors derived from phenolic acids

Eight 1,3,4-oxadiazole derivatives containing phenolic acid moieties (7a-h) and eight of their diacylhydrazine precursors (6a-h) were synthesized, characterized using spectroscopic methods and examined by scavenging of stable DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals. The most potent phenolic 1,3,4-oxadiazoles showed better DPPH scavenging activity in comparison with their corresponding diacylhydrazine precursors as a result of participation of both aromatic rings and a 1,3,4-oxadiazole moiety in resonance stabilization of the formed phenoxyl radical. Four diacylhydrazines (6d, 6e, 6g, and 6h) and four 1,3,4-oxadiazoles (7d, 7e, 7g and 7h) with the best DPPH scavenging activity, were chosen for further evaluation of their antioxidant potential through various assays. The investigated compounds exerted pronounced ABTS radical scavenging capacity, moderate to good H2O2 scavenging properties and strong ferric ion reducing capacity. Further in vitro evaluation of the antioxidant properties of the most active compounds demonstrated their protective effects in normal lung fibroblasts MRC-5 against hydrogen peroxide induced oxidative stress. Diacylhydrazine 6h increased two times the activity of glutathione peroxidase in treated cells in comparison with a control sample and did not affect the superoxide dismutase activity

Trace metals in soils and several brassicaceae plant species from serpentine sites of Serbia

Serpentine soils from 16 sample points in Serbia as well as the roots and shoots of eight Brassicaceae family species: Aethionema saxatile, Alyssum montanum, Alyssum repens, Cardamine plumieri, Erysimum linariifolium, Erysimum carniolicum, Isatis tinctoria, Rorippa lippizensis, were analyzed with regard to their concentrations of P, K, Fe, Ca, Mg, Ni, Zn, Mn, Cu, Cr, Cd, and Pb. Most of the soil samples were typical of ultramafic sites with low concentrations of P, K and Ca and high concentrations of Mg, Fe, Ni and Zn. Ca/Mg ratio was lt 1 in most soil samples and Brassicaceae plants. Only in A. montanum, A. repens, E. linariifolium and R. lippizensis was the Ca/Mg ratio >1. The levels of P, K, Fe and Zn were high, Mn and Cu occurred in low amounts, whereas Cr, Cd, Co and Pb were only traceable. In the roots and shoots of A. montanum and A. repens the measured concentrations of Ni were 657 mg kg(-1) and 676 mg kg(-1) respectively, which is the first instance that such high concentrations of Ni were detected in these two species

Transcription factor p53 exhibits increased binding to the A2-macroglobulin gene promoter and decreased glycosylation in fetal and adult rat liver during the acute-phase response

The binding affinity of p53 for the MG promoter was assessed by DNA-affinity chromatography with the extended α2-macroglobulin (MG) gene promoter (-852/+12) and immunoblot analysis. During the increased MG gene transcription observed in the fetus and the acute-phase (AP) response in both the fetus and the adult, p53 exhibited increased binding to the MG promoter. This increase was accompanied by decreased O-linked N-acetyl glucosamine glycosylation of p53. We suggest that the enzymatic removal of sugar moieties in vivo serves to activate the MG gene promoter binding potential of p53 and its participation in upregulated MG gene transcription.DNK afinitetna hromatografija, sa promotorskim regionom gena za alfa2-makroglobulin (MG) (-852/+12), i imunoblot analiza su pokazale povećan afinitet vezivanja p53 za promotorski region gena za MG u kontrolnoj jetri fetusa, kao i ulogup53 u transkripcionoj regulaciji gena za MG i u fetalnoj i adultnoj jetri tokom akutno-faznog odgovora (AFO). Povećanje vezivanja p53 za promotorski region gena za MG je praćeno smanjenjem stepena glikozilacije p53. Rezultati sugerišu da u in vivo uslovima uklanjanje šećernih ostataka sa p53 omogućava njegovo vezivanje za promotorski region MG gena, kao i ulogu u transkripcionoj aktivaciji ispitivanog gena tokom AFO.Projekat ministarstva br. 143002

The radioprotective efficacy of the rat acute-phase protein alpha2-macroglobulin on bone marrow cells

The rat acute phase protein α2-macroglobulin (α2M) plays an important role in the restoration of disrupted homeostasis by inhibiting different types of non-specific proteases and facilitating the transport of cytokines, growth factors and hormones. Previously, we observed that administration of α2M to experimental animals prior to the infliction of life- threatening trauma in the form of scalding or total-body irradiation, significantly improved their survival rates. The aim of the present work was to evaluate the radioprotective effect on blood cells of α2M that, when administered 30 min before irradiation with 6.7 Gy (LD50/30), provides 100% survival of experimental animals where in unprotected irradiated rats the said dose results in 50% lethality. We observed that rats pretreated with α2M, after an initial decline, exhibited complete recovery of the leukocyte count due to the preservation of bone marrow cells, observed as a stable mitotic index. In untreated irradiated rats the decrease of the mitotic index reflected the significant destruction of bone marrow cells that resulted in a protracted decline in the leukocyte count. We conclude that the radioprotection provided by α2M was in part mediated through cytoprotection of new blood cells produced in the bone marrow.Pacovski alfa2-makroglobulin (α2M) ima važnu ulogu u uspostavljanju narušene homeostaze inhibicijom različitih tipova nespecifičnih proteaza i olakšavajući transport citokina, hormona rasta i hormona. Naša ranija istraživanja su pokazala da administracija α2M eksperimentalnim životinjama u traumama tipa opekotine ili ozračivanja celog organizma značajno povećava njihovu stopu preživljavanja. Cilj ove studije je bio izučavanje radioprotektivne uloge α2M na ćelije kosne srži. α2M je apliciran 30 minuta pre ozračivanja pacova dozom od 6.7 Gy (LD50/30) X-zraka i omogućio je 100% preživljavanje pacova za razliku od ozračenih pacova bez tretmana kod kojih je smrtnost bila 50%. Rezultati su pokazali da tretiranje životinja sa α2M, nakon inicijalnog pada, omogućavaju potpuni oporavak broja leukocita kao posledica očuvanja ćelija kosne srži, što se uočava preko stabilnog mitotskog indeksa. Kod ozračenih pacova bez tretmana signifikantno smanjenje mitotskog indeksa, kao posledica narušavanja ćelija kosne srži, rezultuje i u prolongiranom padu broja leukocita. Na osnovu ovih rezultata može se zaključiti da se radioprotektivna uloga α2M delom odvija putem citoprotekcije novih krvnih ćelija u kosnoj srži.Projekat ministarstva br. 143002

Uticaj hronične promene vrednosti hematokrita na krvni pritisak i glomerulsku filtraciju kod pacova sa urođenom hipertenzijom

Many studies in hypertensive humans and animals have shown that increased blood viscosity is in direct relation with essential hypertension. The aim of our studies was to investigate the effects of chronic hematocrit value changes on arterial blood pressure and kidney function in genetically induced hypertension. To this end, we studied the effects of several interventions, designed to increase/decrease hematocrit, on hemodynamic parameters, vascular reactivity, glomerular filtration and renal function curve in spontaneously hypertensive rats (SHR). Results of our study show that chronic hematocrit value elevation increases blood pressure and peripheral vascular resistance in SHR. On the other hand, chronic hematocrit lowering elucidates blood pressure and peripheral vascular resistance decrease followed by cardiac output rising. Both hematocrit value changes significantly reduce vasodilatory vascular response. Hematocrit lowering induces acute renal failure. Sodium excretion is shifted to higher blood pressure values in high hematocrit value animals and opposite - lower blood pressure values in low hematocrit value animals. Repeated transfusions develop salt sensitive malignant hypertension in SHR. Further studies are necessary to evaluate the degree of kidney damage after chronic hematocrit value changes in SHR.Brojna ispitivanja izvedena na životinjama i ljudima su ukazala da je povećanje viskoznosti krvi u direktnoj vezi sa hipertenzijom. Cilj naše studije je bio da se istraži uticaj promene vrednosti hematokrita (Hct) na krvni pritisak i funkciju bubrega u urođenoj hipertenziji. U tom cilju, istraživan je uticaj hroničnog povećanja ili smanjenja vrednosti Hct na hemodinamske parametre, vaskularnu reaktivnost, glomerulsku filtraciju i krivu bubrežne funkcije kod pacova sa urođenom hipertenzijom (SHR). Rezultati naše studije su ukazali da hronično povećanje vrednosti Hct dovodi do povećanja arterijskog krvnog pritiska (AKP) i perifernog vaskularnog otpora (PVO) kod SHR . Nasuprot tome, hronično smanjenje vrednosti Hct dovodi do smanjenja AKP i PVO, praćenih povećanjem minutnog volumena srca. Obe promene vrednosti Hct dovode do smanjenja vaskularne reaktivnosti. Hronično smanjenje vrednosti Hct izaziva nastanak akutne bubrežne insuficijencije. Ekskrecija natrijuma je pomerena ka većim vrednostima krvnog pritiska kod pacova sa povećanim vrednostima Hct, a ka nižim kod pacova sa sniženim vrednostima Hct. Hronično povećanje vrednosti Hct dovodi do razvoja maligne hipertenzije kod SHR. Potrebne su dalje studije da bi se utvrdio tačan stepen i priroda oštećenja bubrega nakon hronične promene vrednosti Hct

Optimization of the extraction of antioxidants from stinging nettle leaf using response surface methodology

The aim of this study was to optimize the parameters for the extraction of total flavonoids from stinging nettle leaf. Comparison of the effects of different solvents on total flavonoid content showed that, regardless of extraction time, aqueous methanolicextracts had higher total flavonoid content than did aqueous ethanolic extracts. So, full factorial design and response surface methodology (RSM) were em-ployed to estimate the effects of methanol content (50, 75and 100%) and extraction time (30, 60and 90 min) on the total flavonoid content and antioxidant capacities of the extracts. RSM analysis showed that methanol content in the solvent influenced significantly total flavonoid content and FRAP (ferric-reducing antioxidant power) antioxidant capacity, while extraction time had no significant effect on either of these responses. Extraction parameters for maximal total flavonoid content were estimated to be 69% aqueous methanol and 67 min, and 65% aqueous methanol and 83 min for maximal FRAPantioxidantcapacity. DPPH (2,2-diphenyl-1-picrylhydrazyl) antioxidant capacity was not significantly affected by extraction time or methanol percentage in the solvent