Inulinski kristali u živim primjercima biljke Centaurea rupestris L. (Asteraceae)

During anatomical investigations of Centaurea rupestris L., an Illyrian-Adriatic endemic species, the presence of inulin crystals was established not only in plant material preserved in fixing fluid FAA, but unexpectedly also in tissues of living plants. The latter phenomenon was rather rare, because the crystals were observed only in some of the samples of C. rupestris. In addition, inulin was found only in the fresh stem but not in the leaf tissue. In those stems, crystallization of inulin in the form of grains, of larger irregular crystals or spherocrystals, was noticed in the pith and pith ray cells, and inside the tracheary elements. The appearance of inulin crystals in some of the living specimens could be explained by desiccation of the plants growing in particularly dry places inside the habitat under the Mediterranean climate and soil conditions. In the investigated Balkan endemic C. fritschii Hayek, with area distribution, in continental climate, inulin crystals were never detected in living plants.Pri anatomskim istraživanjima ilirsko-jadranske endemične vrste Centaurea rupestris L. prisutnost inulinskih kristala utvrđena je ne samo u fiksiranom (konzerviranom) biljnom materijalu nego također i u tkivu živih biljaka. Pojava kristala inulina u živim primjercima vrste C. rupestris bila je prilično rijetka. Naime, kristali inulina zapaženi su samo u nekim svježim stabljikama analiziranih primjeraka biljke C. rupestris, a dolazili su u stanicama srčike i zraka srčike, te unutar provodnih elemenata ksilema. Inulin je kristalizirao u obliku zrnaca, većih nepravilnih kristala i u obliku sferokristala. Kristali inulina nisu zapaženi u svježim listovima biljke. Pojava inulinskih kristala u nekim živim primjercima vrste C. rupestris može se objasniti znatnim isušivanjem onih biljaka koje su rasle na osobito suhim staništima a pod mediteranskim klimatskim i edafskim uvjetima. U istraživanog balkanskog endema C. jritschii Hayek, koji ima areal rasprostranjeni a u područjima s kontinentalnom klimom, inulinski kristali nisu nikad zapaženi u živom biljnom mar terijalu

Anatomska istraživanja endemičnih vrsta Centaurea rupestris L. i C. fritschii Hayek (Asteraceae)

The paper deals with the anatomy of the shoot and the root of the species Centaurea rupestris L. and C. fritschii Hayek, endemic in the Balkans. The species show great correspondence in the internal structure indicating their close interrelationship. Some anatomical phenomena not described so far were established in the course of these studies. These are: the presence of a bundle sheat in medullary bundles, formation of stem pith hollow by lysigeny, and occurrence of inulin crystals in living plants.Endemične vrste roda Centaurea balkanske regije u anatomskom su i kemijskom pogledu gotovo potpuno neistražene. Anatomska istraživanja ilirsko-jadranske endemične vrste C. rupestris i balkanskog endema C. fritschii poduzeta su kao uvod fitokemijskim istraživanjima i obuhvaćaju analizu anatomije izdanka i korijena navedenih vrsta. Rezultati tih istraživanja pružaju cjelovitu sliku o unutarnjoj građi istraživanih vrsta, ukazujući na njezinu gotovo potpunu podudarnost, što predstavlja još jednu potvrdu srodnosti dviju navedenih vrsta. Malobrojne razlike u pogledu anatomske građe između vrste C. rupestris i vrste C. fritschii odraz su različitih ekoloških uvjeta u područjima u kojima te biljke imaju areal svog rasprostranjenja. Pri ovim istraživanjima utvrđene su i neke anatomske strukture i pojave koje dosad nisu zabilježene u literaturi, pa stoga predstavljaju doprinos i anatomiji bilja u cjelini. To su: pojava mehaničkog žilnog ovoja kod medularnih žila, postanak centralne šupljine u srčiki stabljike na lizigeni način te pojava kristala inulina u živim biljkama

Formulacije s limunovim sokom moduliraju dostupnost fitokemikalija špinata u in vitro modelu probave

Research background. Citrus limon (L.) Burm lemon juice is rich in many important natural chemical components (flavonoids, citric acid and vitamin C) and its use in traditional medicine is well known. Formulations of lemon juice with fruit polyphenols in beverages have been investigated, but there is very little information about their ability to modulate the digestive behaviour of polyphenols. The goal of this study is to determine the stability and digestive availability of spinach (Spinacia oleracea L.) polyphenols by adding different volume fractions of lemon juice (0, 2, 5, 10 and 20%) during in vitro digestion. Experimental approach. The content of polyphenols and other abundant compounds including nitrates, oxalic acid and l-ascorbic acid in spinach formulation with various volume fractions of lemon juice were measured in predigested and digested samples using in vitro human digestion model. Antioxidant and α-amylase inhibitory activities of spinach lemon juice formulation were also measured. Results and conclusions. The highest increases in total polyphenols, total flavonoids, total phenolic acids, oxalic acid and nitrate content were noted in predigested and almost all digested spinach samples formulated with the highest volume fraction of lemon juice. In the same sample, the content of individual compounds significantly increased after salivary (l-ascorbic acid), initial (p-coumaric acid) and intestinal (quercetin) phase of digestion. High bioaccessibility of polyphenols and l-ascorbic acid in all phases of digestion was observed in almost all spinach lemon juice formulations, with the exception of nitrates in gastric and intestinal phases and oxalic acid in the intestinal phase, which had moderate bioaccessibility. Novelty and scientific contribution. For the first time the stability and digestive availability of spinach polyphenols, oxalic acid, nitrates and l-ascorbic acid were tested with the addition of different volume fractions of lemon juice. The pH of lemon juice and its l-ascorbic acid content increase the stability and availability of polyphenols in spinach lemon juice formulation during in vitro digestion. Antioxidant and α-amylase inhibitory activities increase in dose-dependent manner after lemon juice addition. Accordingly, spinach formulated with 20% of lemon juice appears as the best source of dietary polyphenols with antioxidant and antidiabetic activities and nitrates that may be used as a functional drink.Pozadina istraživanja. Limunov (Citrus limon (L.) Burm) sok bogat je mnogim važnim prirodnim kemijskim spojevima (flavonoidima, limunskom kiselinom i vitaminom C) i njegova je upotreba u tradicionalnoj medicini dobro poznata. Formulacije limunovog soka s voćnim polifenolima u pićima su istražene, ali postoji vrlo malo informacija o njihovoj sposobnosti moduliranja dostupnosti polifenola u probavi. Svrha je ovog istraživanja bila utvrditi stabilnost i dostupnost polifenola špinata (Spinacia oleracea L.) u probavi dodavanjem različitih volumnih udjela soka limuna (0, 2, 5, 10 i 20 %). Eksperimentalni pristup. Sadržaj polifenola i drugih spojeva (nitrata, oksalne kiseline i L-askorbinske kiseline) izmjeren je u formulacijiama špinata s različitim volumnim udjelima limunovog soka u uzorcima prije i nakon in vitro modela ljudske probave. Također su mjerene antioksidacijska sposobnost i svojstvo inhibicije aktivnosti α-amilaze u formulacijama špinata s limunovim sokom. Rezultati i zaključci. Najizraženije povećanje udjela ukupnih polifenola, flavonoida i fenolnih kiselina, te oksalne kiseline i nitrata zabilježeno je prije probave i u gotovo svim probavljenim uzorcima špinata formuliranim s najvećim volumnim udjelom limunovog soka. U istom uzorku udio pojedinih spojeva značajno se povećao nakon faze probave u ustima (L-askorbinska kiselina), inicijalnoj (p-kumarinska kiselina) i intestinalnoj fazi (kvercetin). Visoka biodostupnost polifenola i L-askorbinske kiseline u svim fazama probave primijećena je u gotovo svim formulacijama špinata s limunovim sokom, s izuzetkom nitrata u želučanoj i crijevnoj fazi te oksalne kiseline u crijevnoj fazi, koji su imali umjerenu biodostupnost. Novina i znanstveni doprinos. Po prvi put je ispitana stabilnost i dostupnost u probavi polifenola špinata, oksalne kiseline, nitrata i L-askorbinske kiseline uz dodatak različitih volumnih udjela soka limuna. pH-vrijednost i L-askorbinska kiselina limunovog soka povećavaju stabilnost i dostupnost polifenola u formulaciji špinata s limunovim sokom tijekom in vitro probave. Antioksidacijska i sposobnost inhibicije aktivnosti α-amilaze povećavaju se ovisno o povećanju doze limunovog soka. Sukladno tome, formulacija špinata s 20 % limunovog soka predstavlja najbolji izvor dijetetskih polifenola s antioksidacijskim i antidijabetičkim djelovanjem te nitrata koji se mogu koristiti kao funkcionalni napitak

Cucumber Mosaic Virus obtained from Pepper (Capsicum annuum L.) in Bosnia and Herzegovina

Iz zaraženih biljaka paprike (Capsicum annuum L.) uzgajanih u okolici Bijeljine (Bosna i Hercegovina) izoliran je dosta nestabilan virusni izolat koji pripada skupini cucumovirusi. Po svojim biološkim svojstvima, tj. krugu eksperimentalnih domaćina i njihovoj reakciji, te svojstvima u sirovom soku, virus je sličan tipičnim sojevima virusa mozaika krastavca (VMK; cucumber mosaic virus), dok njegovo ponašanje u serološkim pokusima dvostruke radijalne imunodifuzije ukazuje na to da pripada nestabilnim izolatima toga virusa.From infected pepper (Capsicum annuurn L.) plants grown in the surroundings of the town of Bijeljina a rather unstable cucumovirus isolate was obtained. Concerning the host range reactions and its stability in sap, the virus was similar to the typical strains of cucumber mosiac virus (CMV). Its behaviour in experiments of double radial immunodiffusion showed the affiliation to unstable CMV isolates

Sample Preparation Methods for the Determination of the Antioxidative Capacity of Apple Juices

The elevated ecological awareness nowadays led to a higher consumption of apples juices. Apples juices are rich in dietary fiber, pectin, potassium, and vitamins A and C as well as in different classes of phenolic compounds, which can protect the human body against oxidative stress by scavenging oxygen free radicals. During production and storage some bioactive compounds might decompose resulting in a reduced antioxidative capacity. Common methods for the determination of the antioxidative capacity of other food stuff are based on the extraction of the compounds under study using acetone, which is senseless in aqueous juices. But acetone might have an influence on the compounds responsible for the antioxidative capacity. Thus, self made apple juice samples were analyzed without organic solvent as well as mixed with acetone to see differences caused by the solvent. The results obtained with both procedures were compared using a paired t-test in order to see statistically significant differences in the results. No statistically significant differences were found between the testing with and without acetone. Thus the time of analysis, the amount of solvents needed, and the required labor force can be reduced without loosing analytical quality. Total content of phenolic compounds ranged from 400 to 650 mg gallic acid equivalent /L (Folin-Ciocalteu method) and antioxidative capacity from 1.0 to 1.6 mmol Trolox®/L (ABTS assay). (doi: 10.5562/cca1756

Phenolic Compounds in Centaurea rupestris Tissues and Their Antiphytoviral Activity

Quantitative and qualitative analysis of phenolic compounds in tissue extracts from Centaurea rupestris L. as well as their antiphytoviral activity against Tomato bushy stunt virus was performed. Extracts of flowers, leaves and roots from C. rupestris growing in the wild, as well as extracts from shoots, tissue consisting of callus and shoots and undifferentiated callus grown in vitro, were tested. Between tested extracts predominantly quantitative and only several qualitative differences in phenolics were detected by high performance liquid chromatography. The highest amounts of quercetagetin 3’-methylether-7-O-ß-D-glucopyranoside and quercetin were detected in flowers while in leaves the high-est amounts of luteolin, caffeic and p-coumaric acid were detected. Except for roots, antiphytoviral activi-ty of all other extracts was high, inducing virus inhibition, ranging from 43 to 90 %. Simultaneous appli-cation of quercetin, caffeic or p-coumaric acid with virus decreased the number of lesions indicating that these substances contribute to the antiphytoviral activity of C. rupestris extracts. (doi: 10.5562/cca2272

Utjecaj strukturno srodnih flavonoida na ekspresiju hsp gena u ljudskim promieloidnim leukemijskim stanicama

Quercetin is a known specific inhibitor of hsp70 synthesis and thus might be a potent agent for enhancing the selective cytotoxicity of heat on tumour cells. A comparative analysis of the effects of quercetin and five structurally related flavonoids on hsp90α, hsp70A, hsp60 and hsp27 gene expression was carried out using human myeloid leukaemia cells (HL-60). The cells were preincubated with 50 μM quercetin, kaempferol, myricetin, taxifolin, isorhamnetin, methylquercetagetin or 0.1 % DMSO (controls) for 24 h at 37 °C before heat shock treatment (43 °C for 30 min). Total RNA was isolated from heat-stressed and unstressed cells and analysed by RT PCR. Hsp27 gene expression was inhibited by flavonoids more strongly than other hsp genes investigated in heat stressed as well as in unstressed cells. Among the hsp genes tested, only hsp60 was expressed above control level under the influence of taxifolin. Members of the hsp70 and hsp27 families are highly expressed in breast and lung cancer and leukaemias and they play a role in the acquired resistance to chemotherapy or radiation therapy combined with hyperthermia. Therefore, hsps present potential targets for cancer diagnosis and treatment. The present structure/activity study indicates that position, number and substitution of hydroxyl groups of the B ring and saturation of the C2-C3 bond are important factors affecting flavonoid activity on hsp gene expression. This study could help provide a basis for further design of specific inhibitors of hsp gene expression.Kvercetin je poznati specifični inhibitor sinteze hsp70 i kao takav mogući čimbenik povećanja selektivnog citotoksičnog učinka topline na tumorske stanice. Provedena je komparativna analiza učinka kvercetina i njemu strukturno srodnih flavonoida na ekspresiju hsp90α, hsp70A, hsp60 i hsp27 gena u stanicama humane mijeloidne leukemije (HL-60). Stanice su inkubirane s 50 μM kvercetina, kempferola, miricetina, taksifolina, izoramnetina, metilkvercetagetina ili 0,1% DMSO (kontrola) tijekom 24 h na 37 °C prije obrade toplinskim šokom (43 °C tijekom 30 minuta). Izolirana je ukupna RNA iz stanica koje su bile, kao i one koje nisu bile, podvrgnute toplinskom stresu te je provedena RT-PCR analiza. Ekspresija gena hsp27 bila je jače inhibirana flavonoidima nego ostali istraživani hsp geni, i to podjednako u stanicama podvrgnutim kao i u onima koje nisu bile podvrgnute toplinskom stresu. Među istraživanim hsp genima jedino je ekspresija hsp60 bila iznad kontrolne razine pod utjecajem taksifolina. Članovi porodica hsp70 i hsp27 snažno su ekspresirani kod raka dojke, pluća i leukemija te imaju bitnu ulogu u stečenoj rezistenciji stanica pri kemoterapiji ili terapiji zračenjem u kombinaciji s hipertermijom. Stoga bi hsp proteine trebalo istraživati pri dijagnosticiranju i liječenju raka. Prikazana studija strukture i aktivnosti upućuje na to da su položaj, broj i supstitucija hidroksilnih skupina na prstenu B kao i zasićeni vez C2–C3 kod flavonoida bitni čimbenici koji utječu na ekspresiju hsp gena. Ova bi studija mogla poslužiti kao osnova za daljnje strukturiranje specifičnih inhibitora ekspresije hsp gena

Praćenje metabolizma flavonoida u humanim stanicama na temelju fluorescencije izazvane interakcijom kvercetina s proteinima

Despite the wealth of information concerning biological effects of flavonoids, a systematic approach to analyzing the molecular targets is still lacking and, for this reason, a rational evaluation of the risks or benefits of flavonoid-containing foods or of possible pharmaceutical applications is difficult. We have exploited the property of quercetin to elicit fluorescence when bound to specific target proteins and assayed several flavonoids with different modifications (methylation, hydroxylation, glycosylation). Quercetin target proteins can be visualized in living cells, but in vital human leukaemia cells (HL-60) the fluorescence decreases rapidly after labelling, while metabolically inactive apoptotic cells retain the fluorescence. These cytological differences were apparent under the fluorescent microscope and were quantified using flow cytometry. Metabolic conversion of quercetin in vital cells was confirmed and quantified by HPLC analysis. While apoptotic cells still contained considerable amounts of quercetin, vital cells rapidly metabolized the flavonoid (e.g., by methylation or glycosylation). Biochemical results are consistent with the cytological observations and support the conclusion that quercetin becomes rapidly converted to non-fluorogenic metabolites in vital cells. Loss of fluorescence in vital cells allows convenient monitoring and quantifying of the dynamics of quercetin metabolism in human cells.Unatoč mnoštvu informacija koje se odnose na biološke učinke flavonoida, sustavni pristup analizi njihovih ciljnih molekula još uvijek nedostaje. Iz toga razloga vrlo je teško racionalno vrednovati opasnosti ili koristi koje donosi hrana koja sadrži flavonoide kao i njihovu moguću farmakološku primjenu. Iskoristili smo svojstvo kvercetina da izazove fluorescenciju kada se veže za specifične ciljne proteine i analizirali nekoliko različito modificiranih flavonoida (metilacija, hidroksilacija, glikozilacija). Ciljni proteini za koje se kvercetin veže u živim stanicama mogu se vizualizirati na temelju fluorescencije. U živim stanicama humane leukemije (HL-60) fluorescencija naglo pada nakon označavanja flavonoidima, dok metabolički inaktivne apoptotične stanice zadržavaju fluorescenciju. Te su citološke razlike jasno zapažene pod fluorescencijskim mikroskopom, a kvantificirane su pomoću protočne citometrije. Metabolička pretvorba kvercetina u živim stanicama potvr|ena je i kvantificirana pomoću HPLC analiza. Dok apoptotične stanice zadržavaju značajnu količinu kvercetina, žive ga stanice brzo metaboliziraju (npr. metilacijom ili glikozilacijom). Ti su biokemijski rezultati u skladu s citološkim promatranjima i podupiru zaključak da se kvercetin u živim stanicama brzo pretvara u nefluorogene metabolite. Gubitak fluorescencije u živim stanicama omogućava praćenje i kvantifikaciju dinamike metabolizma kvercetina u humanim stanicama

Utjecaj flavonoida na razinu glutationa, peroksidaciju lipida i ekspresiju citokroma P450 CYP1A1 u staničnim linijama karcinoma grkljana

Flavonoids are phytochemicals exhibiting a wide range of biological activities, among which are antioxidant activity, the ability to modulate activity of several enzymes or cell receptors and possibility to interfere with essential biochemical pathways. Using human laryngeal carcinoma HEp2 cells and their drug-resistant CK2 subline, we examined the effect of five flavonoids, three structurally related flavons (quercetin, fisetin, and myricetin), one flavonol (luteolin) and one glycosilated flavanone (naringin) for: (i) their ability to inhibit mitochondrial dehydrogenases as an indicator of cytotoxic effect, (ii) their influence on glutathione level, (iii) antioxidant/prooxidant effects and influence on cell membrane permeability, and (iv) effect on expression of cytochrome CYP1A1. Cytotoxic action of the investigated flavonoids after 72 hours of treatment follows this order: luteolin>quercetin>fisetin>naringin>myricetin. Our results show that CK2 were more resistant to toxic concentrations of flavonoids as compared to parental cells. Quercetin increased the total GSH level in both cell lines. CK2 cells are less perceptible to lipid peroxidation and damage caused by free radicals. Quercetin showed prooxidant effect in both cell lines, luteolin only in HEp2 cells, whereas other tested flavonoids did not cause lipid peroxidation in the tested cell lines. These data suggest that the same compound, quercetin, can act as a prooxidant, but also, it may prevent damage in cells caused by free radicals, due to the induction of GSH, by forming less harmful complex. Quercetin treatment damaged cell membranes in both cell lines. Fisetin caused higher cell membrane permeability only in HEp2 cells. However, these two compounds did not enhance the damage caused by hydrogen peroxide. Quercetin, naringin, myricetin and fisetin increased the expression of CYP1A1 in both cell lines, while luteolin decreased basal level of CYP1A1 only in HEp2 cells. In conclusion, small differences in chemical structure of flavonoids led to drastic change of their biological effects.Flavonoidi su fitokemikalije koje imaju široki raspon bioloških učinaka, između ostalih antioksidativnu aktivnost, sposobnost mijenjanja aktivnosti pojedinih enzima ili staničnih receptora i mogućnost utjecaja na osnovne biokemijske putove. Korištenjem staničnih linija karcinoma grkljana čovjeka HEp2 i njihovih rezistentnih podlinija CK2, istražena je sposobnost pet flavonoida, tri strukturno slična flavona (kvercetin, fisetin i miricetin), jednog flavonola (luteolin) i jednoga glikoziliranog flavanona (naringin) da: (i) inhibiraju mitohondrijske dehidrogenaze koje služe kao pokazatelj citotoksičnog učinka, (ii) utječu na razinu glutationa, (iii) imaju antioksidativno/prooksidativno djelovanje i utječu na propusnost stanične membrane i (iv) utječu na ekspresiju citokroma CYP1A1. Citotoksično djelovanje ispitanih flavonoida nakon 72 sata bilo je u slijedu: luteolin > kvercetin > fisetin > naringin > miricetin. Dobiveni rezultati pokazali su da su stanice CK2 bile rezistentnije na toksične koncentracije flavonoida u usporedbi s roditeljskim stanicama. Kvercetin je povećao ukupnu razinu glutationa (GSH) u obje stanične linije. Stanice CK2 bile su manje podložne peroksidaciji lipida i oštećenjima uzrokovanim slobodnim radikalima. Kvercetin je pokazao prooksidativno djelovanje u obje stanične linije, luteolin samo u HEp2 stanicama, a ostali ispitani flavonoidi nisu uzrokovali peroksidaciju lipida. Ti podaci pokazuju da isti spoj, kvercetin, može djelovati kao prooksidans, ali također može spriječiti oštećenje stanica uzrokovanih slobodnim radikalima, zbog indukcije GSH, formiranjem manje štetnog kompleksa. Kvercetin oštećuje stanične membrane u obje stanične linije, dok fisetin povećava propusnost stanične membrane samo u HEp2 stanicama. No, ta dva spoja ne pojačavaju oštećenje stanične membrane vodikovim peroksidom. Kvercetin, naringin, miricetin i fisetin povećali su ekspresiju CYP1A1 u obje stanične linije, dok je luteolin smanjio ekspresiju CYP 1A1 u HEp2 stanicama. Iz tih se rezultata može zaključiti da male razlike u kemijskoj strukturi flavonoida uzrokuju značajne razlike u njihovu biološkom učinku