The antioxidant action of Polypodium leucotomos extract and kojic acid: reactions with reactive oxygen species

Two natural products Polypodium leucotomos extract (PL) and kojic acid (KA) were tested for their ability to scavenge reactive oxygen species (·OH, ·O2-, H2O2, ¹O2) in phosphate buffer. Hydroxyl radicals were generated by the Fenton reaction, and the rate constants of scavenging were 1.6 x 10(9) M-1 s-1 for KA and 1.0 x 10(9) M-1 s-1 for PL, similar to that of ethanol (1.4 x 10(9) M-1 s-1). With superoxide anions generated by the xanthine/hypoxanthine system, KA and PL (0.2-1.0 mg/ml) inhibited ·O2-dependent reduction of nitroblue tetrazolium by up to 30 and 31%, respectively. In the detection of ¹O2 by rose bengal irradiation, PL at 1.0 mg/ml quenched singlet oxygen by 43% relative to azide and KA by 36%. The present study demonstrates that PL showed an antioxidant effect, scavenging three of four reactive oxygen species tested here. Unlike KA, PL did not significantly scavenge hydrogen peroxide.

Lämpötilan vaikutus oksaalihapon hapetukseen pulssitetulla koronapurkauksella

Tutkimuksen tarkoituksena oli kartoittaa lämpötilan vaikutusta veden orgaanisten haitta-aineiden hapetuksessa PCD-menetelmällä. Kokeita tehtiin näytteiden eri alkulämpötiloilla. Malliyhdisteenä kokeissa käytettiin oksaalihappoa. Teoriaosuudessa käsiteltiin pulssittaista koronapurkausta ilmiönä. Lisäksi tarkasteltiin, kuinka PCD-menetelmällä muodostuu hapettimia neste-kaasufaasissa. Syntyvistä hapettimista keskityttiin otsoniin ja hydroksyyliradikaaliin. Kokeellisessa osuudessa esiteltiin käytetty PCD-laitteisto. Esittelyn jälkeen siirryttiin hapetuskokeiden kuvaamiseen ja analyysin suorittamiseen titrauksella. Lopuksi koottiin tulokset. Tutkimuksissa prosessin hapetustehon havaittiin heikentyvän lämpötilan noustessa tutkitulla lämpötila-alueella, mikä voi selittyä kaasufaasissa muodostuvan otsonin heikentyvällä liukoisuudella. Tuloksia voidaan pitää viitteellisinä, ja selkeän mallin muodostamiseksi tarvitaan jatkotutkimuksia laajemmalla lämpötila-alueella tarkasti toistettavilla koejärjestelyillä.

Formation of nitrates in water in presence of formate and oxalate treated with pulsed corona discharge (PCD)

Tavallisten hapetusmenetelmien sijasta kehittyneitä hapetusmenetelmiä (AOP) on kehitetty yhä enemmän, jotta hapetusprosessista tulisi kannattavampi, tehokkaampi, ympäristöystävällisempi ja sitä voitaisiin hyödyntää laajalti eri paikoissa. Uusi teknologia, joka käyttää otsonia ja hydroksyyliradikaalia sähköimpulssien kanssa, on yksi mahdollinen tehokkaampi vedenkäsittelymentelmä. Kyseistä menetelmää kutsutaa pulsed corona discharge (PCD) -menetelmäksi, joka käyttää prosessissa muodostuvia otsonia ja hydroksyyliradikaalia hapettavina tekijöinä. Tässä työssä tutkittiin nitraatin muodostumista vedessä, kun vettä käsiteltiin PCD-laitteessa ja, kun oksalaatti- ja formaatti-ioneja oli sekoittuneina veteen. Nitraatteja muodostuu PCD–laitteessa veteen, kun ilman typpi reagoi hapettimina toimivien otsonin ja hydroksyyliradikaalin kanssa. Aiemmissa tutkimuksissa nitraatin muodostumisen on todistettu parantuvan, kun karboksyylihapot muurahais- ja oksaalihappo ovat sekoittuneina veteen. Tässä tutkimuksessa tarkoituksena oli tutkia, miten formaatti- ja oksalaatti-ionien, joiden pitoisuudet olivat 0 ppm, 50 ppm ja 100 ppm, läsnäolo vedessä vaikuttaa nitraatin muodostumiseen. PCD-kokeista saadut näytteet analysoitiin ionikromatografilla. Kyseisessä tutkimuksessa nitraatin muodostuminen oli samansuuruista jokaisessa kokeessa hapetusajan kasvaessa samalla, kun otettujen näytteiden pH-arvot laskivat. Tuloksena voitiin pitää sitä, ettei formaatti- tai oksalaatti-ioneilla ollut vaikutusta nitraatti-ionien muodostumiseen.

Elevated levels of erythrocyte-conjugated dienes indicate increased lipid peroxidation in schistosomiasis mansoni patients

Schistosoma mansoni causes liver disease by inducing granulomatous inflammation. This favors formation of reactive oxygen species, including superoxide ions, hydrogen peroxide and hydroxyl radicals all of which may induce lipid peroxidation. We have evaluated lipid peroxidation in 18 patients with hepatosplenic schistosomiasis mansoni previously treated with oxamniquine followed by splenectomy, ligature of the left gastric vein and auto-implantation of spleen tissue, by measuring levels of erythrocyte-conjugated dienes and plasma malondialdehyde (MDA). Age-matched, healthy individuals (N = 18) formed the control group. Erythrocyte-conjugated dienes were extracted with dichloromethane/methanol and quantified by UV spectrophotometry, while plasma MDA was measured by reaction with thiobarbituric acid. Patient erythrocytes contained two times more conjugated dienes than control cells (584.5 ± 67.8 vs 271.7 ± 20.1 µmol/l, P < 0.001), whereas the increase in plasma MDA concentration (about 10%) was not statistically significant. These elevated conjugated dienes in patients infected by S. mansoni suggest increased lipid peroxidation in cell membranes, although this was not evident when a common marker of oxidative stress, plasma MDA, was measured. Nevertheless, these two markers of lipid peroxidation, circulating MDA and erythrocyte-conjugated dienes, correlated significantly in both patient (r = 0.62; P < 0.01) and control (r = 0.57; P < 0.05) groups. Our data show that patients with schistosomiasis have abnormal lipid peroxidation, with elevated erythrocyte-conjugated dienes implying dysfunctional cell membranes, and also imply that this may be attenuated by the redox capacity of antioxidant agents, which prevent accumulation of plasma MDA.

Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses

Molecular oxygen (O2) is the premier biological electron acceptor that serves vital roles in fundamental cellular functions. However, with the beneficial properties of O2 comes the inadvertent formation of reactive oxygen species (ROS) such as superoxide (O2·-), hydrogen peroxide, and hydroxyl radical (OH·). If unabated, ROS pose a serious threat to or cause the death of aerobic cells. To minimize the damaging effects of ROS, aerobic organisms evolved non-enzymatic and enzymatic antioxidant defenses. The latter include catalases, peroxidases, superoxide dismutases, and glutathione S-transferases (GST). Cellular ROS-sensing mechanisms are not well understood, but a number of transcription factors that regulate the expression of antioxidant genes are well characterized in prokaryotes and in yeast. In higher eukaryotes, oxidative stress responses are more complex and modulated by several regulators. In mammalian systems, two classes of transcription factors, nuclear factor kB and activator protein-1, are involved in the oxidative stress response. Antioxidant-specific gene induction, involved in xenobiotic metabolism, is mediated by the "antioxidant responsive element" (ARE) commonly found in the promoter region of such genes. ARE is present in mammalian GST, metallothioneine-I and MnSod genes, but has not been found in plant Gst genes. However, ARE is present in the promoter region of the three maize catalase (Cat) genes. In plants, ROS have been implicated in the damaging effects of various environmental stress conditions. Many plant defense genes are activated in response to these conditions, including the three maize Cat and some of the superoxide dismutase (Sod) genes.

Antioxidant and antimicrobial activities of Bauhinia racemosa L. stem bark

The present study was carried out to evaluate the antioxidant and antimicrobial activities of a methanol extract of Bauhinia racemosa (MEBR) (Caesalpiniaceae) stem bark in various systems. 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) radical, superoxide anion radical, nitric oxide radical, and hydroxyl radical scavenging assays were carried out to evaluate the antioxidant potential of the extract. The antioxidant activity of the methanol extract increased in a concentration-dependent manner. About 50, 100, 250, and 500 µg MEBR inhibited the peroxidation of a linoleic acid emulsion by 62.43, 67.21, 71.04, and 76.83%, respectively. Similarly, the effect of MEBR on reducing power increased in a concentration-dependent manner. In DPPH radical scavenging assays the IC50 value of the extract was 152.29 µg/ml. MEBR inhibited the nitric oxide radicals generated from sodium nitroprusside with an IC50 of 78.34 µg/ml, as opposed to 20.4 µg/ml for curcumin. Moreover, MEBR scavenged the superoxide generated by the PMS/NADH-NBT system. MEBR also inhibited the hydroxyl radical generated by Fenton's reaction, with an IC50 value of more than 1000 µg/ml, as compared to 5 µg/ml for catechin. The amounts of total phenolic compounds were also determined and 64.7 µg pyrocatechol phenol equivalents were detected in MEBR (1 mg). The antimicrobial activities of MEBR were determined by disc diffusion with five Gram-positive, four Gram-negative and four fungal species. MEBR showed broad-spectrum antimicrobial activity against all tested microorganisms. The results obtained in the present study indicate that MEBR can be a potential source of natural antioxidant and antimicrobial agents.

Nucleotides as Antiviral Compounds. On the Feasibility of an Esterase-dependent Prodrug Strategy for 2-5A

Nukleotidien ja oligonukleotidien analogeilla on merkittävä rooli virusten aiheuttamien tautien hoidossa. Tämän kaltaiset yhdisteet voivat estää spesifisesti virusten proteiineja tai aktivoida luontaista immuunijärjestelmää, jossa 2-5A:ksi kutsutut lyhyet 2´,5´-sitoutuneet oligomeerit ovat keskeisiä tekijöitä. Nukleotideihin ja oligonukleotideihin pohjautuvien lääkkeiden tehokkuus riippuu pääasiassa aihiolääkestrategiasta, jolla niiden sisäänottoa soluun tehostetaan. Tavanomaisessa aihiolääkestrategiassa negatiivisesti varautuneet fosfaattiryhmät suojataan rasvaliukoisilla biohajoavilla suojaryhmillä, jotta molekyyli läpäisee solukalvon helpommin. Solun sisällä aihiolääke muuttuu aktiiviseksi lääkeaineeksi, kun suojaryhmät irtoavat solun entsyymien, kuten esteraasien vaikutuksesta. Väitöskirjassa arvioitiin esteraasin katalysoiman aihiolääkestrategian soveltuvuutta 2-5A-trimeerille syntetisoimalla kaksi erilaista 2-5A-aihiolääkekandidaattia ja tutkimalla 2-5A:n purkautumista karboksiesteraasi-entsyymin vaikutuksesta. Suojaryhmäsuunnitelma perustui esteraasilabiileihin 2,2-disubstituoituihin asyylioksipropyyliryhmiin ja asyylioksimetyyliryhmiin, joilla suojattiin trimeerien fosfaatti- ja 3´-hydroksyyliryhmät. Tulokset osoittivat, että esteraasilabiilien suojaryhmien irtoaminen 2-5A:sta hidastui merkittävästi, kun yhdisteeseen kertyi negatiivista varausta. Lisäksi suojaryhmien hajotessa muodostui elektrofiilisiä alkyloivia aineita, jotka ovat mahdollisesti toksisia. Näistä syistä johtuen kehitettiin kuusi uudenlaista 2,2,-disubstituoitua 4-asyylitio- 3-oksobutyyliryhmää fosfodiestereiden suojaamiseksi. Suojaryhmät irtoavat sekä esteraasin katalysoimana, että lämpötilan vaikutuksesta. Tämä on hyödyllinen ominaisuus silloin, kun entsyymin affiniteetti negatiivisesti varattuun substraattiin heikkenee. Suojaryhmien hydrolyyttinen ja entsymaattinen stabiilisuus on helposti säädeltävissä, jotta suojauksen purkautumisen nopeus voidaan optimoida. Vapautuneet suojaryhmät eivät ole merkittävästi alkyloivia, sillä niiden ei havaittu alkyloivan glutationia.

Characterization of the interdependency between residues that bind the substrate in a β-glycosidase

The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the β-glycosidase from Spodoptera frugiperda (Sfβgly), both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfβgly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl β-galactoside and p-nitrophenyl β-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfβgly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ES‡ (enzyme-transition state complex) of the double mutations (∆∆G‡xy) is not the sum of the effects resulting from the single mutations (∆∆G‡x and ∆∆G‡y). This difference in ∆∆G‡ indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in ∆∆G‡xy. Crystallographic data on β-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.

Target-directed catalytic metallodrugs

Most drugs function by binding reversibly to specific biological targets, and therapeutic effects generally require saturation of these targets. One means of decreasing required drug concentrations is incorporation of reactive metal centers that elicit irreversible modification of targets. A common approach has been the design of artificial proteases/nucleases containing metal centers capable of hydrolyzing targeted proteins or nucleic acids. However, these hydrolytic catalysts typically provide relatively low rate constants for target inactivation. Recently, various catalysts were synthesized that use oxidative mechanisms to selectively cleave/inactivate therapeutic targets, including HIV RRE RNA or angiotensin converting enzyme (ACE). These oxidative mechanisms, which typically involve reactive oxygen species (ROS), provide access to comparatively high rate constants for target inactivation. Target-binding affinity, co-reactant selectivity, reduction potential, coordination unsaturation, ROS products (metal-associated vsmetal-dissociated; hydroxyl vs superoxide), and multiple-turnover redox chemistry were studied for each catalyst, and these parameters were related to the efficiency, selectivity, and mechanism(s) of inactivation/cleavage of the corresponding target for each catalyst. Important factors for future oxidative catalyst development are 1) positioning of catalyst reduction potential and redox reactivity to match the physiological environment of use, 2) maintenance of catalyst stability by use of chelates with either high denticity or other means of stabilization, such as the square planar geometric stabilization of Ni- and Cu-ATCUN complexes, 3) optimal rate of inactivation of targets relative to the rate of generation of diffusible ROS, 4) targeting and linker domains that afford better control of catalyst orientation, and 5) general bio-availability and drug delivery requirements.

Antioxidant effect of 4-nerolidylcatechol and α-tocopherol in erythrocyte ghost membranes and phospholipid bilayers

4-Nerolidylcatechol (4-NC) is found in Pothomorphe umbellataroot extracts and is reported to have a topical protective effect against UVB radiation-induced skin damage, toxicity in melanoma cell lines, and antimalarial activity. We report a comparative study of the antioxidant activity of 4-NC and α-tocopherol against lipid peroxidation initiated by two free radical-generating systems: 2,2′-azobis(2-aminopropane) hydrochloride (AAPH) and FeSO4/H2O2, in red blood cell ghost membranes and in egg phosphatidylcholine (PC) vesicles. Lipid peroxidation was monitored by membrane fluidity changes assessed by electron paramagnetic resonance spectroscopy of a spin-labeled lipid and by the formation of thiobarbituric acid-reactive substances. When lipoperoxidation was initiated by the hydroxyl radical in erythrocyte ghost membranes, both 4-NC and α-tocopherol acted in a very efficient manner. However, lower activities were observed when lipoperoxidation was initiated by the peroxyl radical; and, in this case, the protective effect of α-tocopherol was lower than that of 4-NC. In egg PC vesicles, malondialdehyde formation indicated that 4-NC was effective against lipoperoxidation initiated by both AAPH and FeSO4/H2O2, whereas α-tocopherol was less efficient in protecting against lipoperoxidation by AAPH, and behaved as a pro-oxidant for FeSO4/H2O2. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical assay indicated that two free radicals were scavenged per 4-NC molecule, and one free radical was scavenged per α-tocopherol molecule. These data provide new insights into the antioxidant capacity of 4-NC, which may have therapeutic applications for formulations designed to protect the skin from sunlight irradiation.

Bio and chemocatalysis for stereo and regioselective one-pot reaction applications

The development of cost efficient, selective and sustainable chemical processes for production of chiral building blocks is of great importance in synthetic and industrial organic chemistry. One way to reach these objectives is to carry out several reactions steps in one vessel at one time. Furthermore, when this kind of one-pot multi step reactions are catalyzed by heterogeneous chemo- and bio-catalysts, which can be separated from the reaction products by filtration, practical access to chiral small molecules for further utilization can be obtained. The initial reactions studied in this thesis are the two step dynamic kinetic resolution of rac-2-hydroxy-1-indanone and the regioselective hydrogenation of 1,2-indanedione. These reactions are then combined in a new heterogeneously catalyzed one-pot reaction sequence enabling simple recovery of the catalysts by filtration, facilitating simple reaction product isolation. Conclusively, the readily available 1,2-indanedione is by the presented one-pot sequence, utilizing heterogeneous enzyme and transition metal based catalysts, transferred with high regio- and stereoselectivity to a useful chiral vicinal hydroxyl ketone structure. Additional and complementary investigation of homogeneous half-sandwich ruthenium complexes for catalyzing the epimerization of chiral secondary alcohols of five natural products containing additional non-functionalized stereocenters was conducted. In principle, this kind of epimerization reactions of single stereocenters could be utilized for converting inexpensive starting materials, containing other stereogenic centers, into diastereomeric mixtures from which more valuable compounds can be isolated by traditional isolation techniques.

Correlation, by multivariate statistical analysis, between the scavenging capacity against reactive oxygen species and the bioactive compounds from frozen fruit pulps

The contents of total phenolic compounds (TPC), total flavonoids (TF), and ascorbic acid (AA) of 18 frozen fruit pulps and their scavenging capacities against peroxyl radical (ROO•), hydrogen peroxide (H2O2), and hydroxyl radical (•OH) were determined. Principal Component Analysis (PCA) showed that TPC (total phenolic compounds) and AA (ascorbic acid) presented positive correlation with the scavenging capacity against ROO•, and TF (total flavonoids) showed positive correlation with the scavenging capacity against •OH and ROO• However, the scavenging capacity against H2O2 presented low correlation with TF (total flavonoids), TPC (total phenolic compounds), and AA (ascorbic acid). The Hierarchical Cluster Analysis (HCA) allowed the classification of the fruit pulps into three groups: one group was formed by the açai pulp with high TF, total flavonoids, content (134.02 mg CE/100 g pulp) and the highest scavenging capacity against ROO•, •OH and H2O2; the second group was formed by the acerola pulp with high TPC, total phenolic compounds, (658.40 mg GAE/100 g pulp) and AA , ascorbic acid, (506.27 mg/100 g pulp) contents; and the third group was formed by pineapple, cacao, caja, cashew-apple, coconut, cupuaçu, guava, orange, lemon, mango, passion fruit, watermelon, pitanga, tamarind, tangerine, and umbu pulps, which could not be separated considering only the contents of bioactive compounds and the scavenging properties.

Antioxidant activities of kombucha prepared from three different substrates and changes in content of probiotics during storage

Kombucha is a health-promoting fermented beverage worldwide. The present study compared the free-radical scavenging abilities and total reducing power (TRP) of kombucha prepared from low-cost green tea (LGTK), black tea (BTK), and tea powder (TPK). LGTK had the highest scavenging abilities against 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion and hydroxyl radicals, while BTK showed the highest TRP. Changes in content of probiotics in LGTK were investigated during storage as well. The number of acetic acid bacteria decreased moderately up to 10 days of storage. The number of lactic acid bacteria (LAB) decreased significantly, and their survival rate was only 0.98% at the 8th day of storage.

Antioxidant activities of fractions from longan pericarps

The antioxidant activities of ethanolic crude extract (LPCE) and its four different solvent sub-fractions (namely, diethyl ether fraction (LPDF), ethyl acetate fraction (LPEF), n-butyl alcohol fraction (LPBF) and residue fraction (LPR)) from longan pericarps were investigated employing various systems including 2,2-diphenyl-1-picrylhydrazyl (DPPH)/ 2,2'-amino-di(2-ethyl-benzothiazoline sulphonic acid-6)ammonium salt (ABTS)/hydroxyl radical scavenging activity, total phenolic content and reducing power. Each extract showed concentration-dependent antioxidant activity. LPEF showed the highest scavenging activity against DPPH, ABTS and hydroxyl radicals with EC50 values of 0.506, 0.228 and 4.489 mg/mL, respectively. LPEF showed the highest reducing power with EC50 values of 0.253 mg/mL. The next was LPDF with EC50 values of 0.260 mg/mL. LPEF possessed the highest total phenolic content (230.816 mg/g, expressed as gallic acid equivalents), followed by LPDF, LPBF, LPCE and LPR. The results suggested that longan pericarp fractions possessed significant antioxidant activities and could be a promising source of natural antioxidant.

Extraction optimization of antioxidant polysaccharides from leaves of Gynura bicolor (Roxb. & Willd.) DC

Orthogonal design was employed to study the effect of extraction time, temperature and liquid-to-solid ratio on the production of antioxidant polysaccharides from leaves of Gynura bicolor (PLG). Analysis of variance was performed on the data obtained. The most relevant variable was extraction time. A liquid-solid ratio of 30:1 (v/w), a temperature of 80 °C and an extraction time of 3 h were found to be optimal for PLG. The optimal extraction yield of 4.9% was obtained through additional verification test. Hydroxyl radical-scavenging activity, reducing power and ferrous ion chelating ability of PLG were determined. PLG possess concentration-dependent antioxidant potency and IC50 of PLG was 4.67, 0.24 and 4.31 mg/mL for hydroxyl radical-scavenging and ferric ion chelating abilities as well as reducing power, respectively. The results suggest that G. bicolor polysaccharides could be potential source of natural antioxidant and be contributor to the health benefits of G. bicolor.