DNA Damage and Oxidative DNA Damage in Inflammatory Bowel Disease

Background and aims: Inflammation has long been regarded as a major contributor to cellular oxidative damage and to be involved in the promotion of carcinogenesis. Methods: We aimed to investigate the oxidative damage in inflammatory bowel disease [IBD] patients through a case–control and prospective study involving 344 IBD patients and 294 healthy controls. DNA damage and oxidative DNA damage were measured by comet assay techniques, and oxidative stress by plasmatic lipid peroxidation, protein carbonyls, and total antioxidant capacity. Results: Higher DNA damage [p < 0.001] was found both in Crohn’s disease [CD] (9.7 arbitrary units [AU]; interquartile range [IQR]: 6.2–14.0) and ulcerative colitis [UC] [7.1 AU; IQR: 4.4–11.7], when compared with controls [5.4 AU; IQR: 3.8–6.8], and this was also the case with oxidative DNA damage [p < 0.001] [CD: 3.6 AU; IQR: 1.8–6.8; UC: 4.6 AU; IQR: 2.4–8.1], when compared with controls: 2.3 AU; IQR: 1.2–4.2]. Stratifying patients into groups according to therapy (5-aminosalicylic acid [5-ASA], azathioprine, anti-TNF, and combined therapy [azathioprine and anti-TNF]) revealed significant between-group differences in the level of DNA damage, both in CD and UC, with the combined therapy exhibiting the highest DNA damage levels [11.6 AU; IQR: 9.5–14.3, and 12.4 AU; IQR: 10.6–15.0, respectively]. Among CD patients, disease behaviour [B1 and B2], and age at diagnosis over 40 years [A3] stand as risk factors for DNA damage. For UC patients, the risk factors found for DNA damage were disease activity, treatment, age at diagnosis under 40 years [A1 + A2] and disease locations [E2 and E3]. Conclusions: In IBD there is an increase in DNA damage, and treatment, age at diagnosis and inflammatory burden seem to be risk factors.

HPLC-DAD-ESI/MS analysis of phenolic compounds in four tomato varieties and evaluation of the antioxidant capacity

Tomato (Lycopersicon esculentum L.) is the second most important vegetable crop worldwide and a key component in the so-called “Mediterranean diet”. In the Northeastern region of Portugal, local populations still prefer to consume traditional tomato varieties which they find very tasty and healthy, as they are grown using extensive farming techniques. A previous study of our research team described the nutritional value of the round (batateiro), long (comprido), heart (coração) and yellow (amarelo) tomato varieties [1], but the phenolic profile was unknown until now. Thus, the objective of this study was to characterize the phenolic profiles of these four tomato farmers’ varieties by using HPLC-DAD-ESI/MS and evaluate its antioxidant capacity through four in vitro assays based on different reaction mechanisms. A cis p-coumaric acid derivative was the most abundant compound in yellow and round tomato varieties, while 4-O-caffeolyquinic acid was the most abundant in long and heart varieties. The most abundant flavonoid was quercetin pentosylrutinoside in the four tomato varieties. Yellow tomato presented the highest levels of phenolic compounds, including phenolic acids and flavonoids, but the lowest antioxidant activity. In turn, the round tomato gave the best results in all the antioxidant activity assays. This study demonstrated that these tomato farmers’ varieties are a source of phenolic compounds, mainly phenolic acid derivatives [2], and possess high antioxidant capacity [1]; being thus key elements in the diet to prevent chronic degenerative diseases associated to oxidative stress, such as cancer and coronary artery disease.

Caracterização estrutural e avaliação das atividades farmacológicas da fucana B extraída da alga Dictyota menstrualis

Seaweeds are a major source of biologically active compounds . In the extracellular matrix of these organisms are sulfated polysaccharides that functions as structural components preventing it against dehydration. The fraction 0.9 (FucB) rich in sulfated fucans obtained from brown seaweed Dictyota menstrualis was chemical characterized and evaluated for pharmacological activity by testing anticoagulant activity, stimulatory action on the synthesis of an antithrombotic heparan sulfate, antioxidant activity and its effects in cell proliferation. The main components were FucB carbohydrates (49.80 ± 0.10 %) and sulfate (42.30 ± 0.015 %), with phenolic compounds ( 3.86 ± 0.016 %) and low protein contamination ( 0.58 ± 0.001 % ) . FucB showed polydisperse profile and analysis of signals in the infrared at 1262, 1074 and 930 cm -1 and 840 assigned to S = O bonds sulfate esters , CO bond presence of 3,6- anhydrogalactose , β -D- galactose non- sulfated sulfate and the axial position of fucose C4 , respectively. FucB exhibited moderate anticoagulant activity , the polysaccharides prolonged time (aPTT ) 200 ug ( > 90s ) partial thromboplastin FucB no effect on prothrombin time (PT), which corresponds to the extrinsic pathway of coagulation was observed. This stimulation promoted fraction of about 3.6 times the synthesis of heparan sulfate (HS) by endothelial cells of the rabbit aorta ( RAEC ) in culture compared with cells not treated with FucB . This has also been shown to compete for the binding site with heparin. The rich fraction sulfated fucans exhibited strong antioxidant activity assays on total antioxidant (109.7 and 89.5 % compared with BHT and ascorbic acid standards ) , reducing power ( 71 % compared to ascorbic acid ) and ferric chelation ( 71 , comparing with 5 % ascorbic acid). The fraction of algae showed cytostatic activity on the RAEC cells revealed that the increase of the synthesis of heparan sulfate is not related to proliferation. FucB showed antiproliferative action on cell lines modified as Hela and Hep G2 by MTT assay . These results suggest that FucB Dictyota menstrualis have anticoagulant , antithrombotic , antioxidant potential as well as a possible antitumor action, promoting the stimulation of the synthesis of antithrombotic HS by endothelial cells and is useful in the prevention of thrombosis, also due to its inhibitory action on species reactive oxygen ( ROS ) in some in vitro systems , being involved in promoting a hypercoagulable state

Phenolic composition of four sage species: salvia farinacea, salvia mexico, salvia greggii and salvia officinalis

Salvia species are used worldwide for medicine purposes. In general, these medicinal plants have high amounts of flavonoids and phenolic acids, that are thought to be closely related to their health properties [1,2]. In this work, the aerial parts of Salvia farinacea, Salvia mexico, Salvia greggii and Salvia officinalis were extracted with hot water [3]. Extracts were evaluated for their total phenolic content by an adaptation of the Folin-Ciocalteu method and further analysed by high performance liquid chromatography associated with electrospray mass spectrometry (HPLC-DAD-ESI-MSn) in the negative ion mode [4], in order to identify their individual phenolic constituents. The aqueous extracts of S. farinacea, S. mexico, S. officinalis and S. greggii contained, respectively, 106±13, 159±38, 175±46 and 136±1 μg GAE/mg of total phenolics. These four species were characterized by a clear prevalence of caffeic acid derivatives, in particular of rosmarinic acid (MW 360), that is generally the most abundant phenolic compound in Salvia species [2,3]. In addition, S. mexico and S. officinalis contained moderate amounts of salvianolic acid B (MW 718). Among these two, S. mexico was richer in O-caffeoylquinic acid (MW 354), while the latter presented high amounts of salvianolic acid K (MW 556) and moderate amounts of its structural isomer. All the extracts were enriched in flavones: S. farinacea and S. officinalis contained high amounts of luteolin-O-glucuronide while S. mexico contained luteolin-C-glucoside with respective characteristic mass spectrometry fragmentation pattern m/z at 461→285 and m/z at 447→357, 327. Similarly, S. greggii extract presented high content of luteolin-7-O-glucoside ([M-H]− at m/z 447→ 285) and luteolin-C-glucoside and moderate quantities of apigenin-C-hexoside ([M-H]− at m/z 431→341, 311). Further studies are being undertaken in order to understand the contribution of these phenolic constituents in the biological activities of Salvia plants.

Phenolic composition of four sage species: salvia farinacea, salvia mexico, salvia greggii and salvia officinalis

Salvia species are used worldwide for medicine purposes. In general, these medicinal plants have high amounts of flavonoids and phenolic acids, that are thought to be closely related to their health properties [1,2]. In this work, the aerial parts of Salvia farinacea, Salvia mexico, Salvia greggii and Salvia officinalis were extracted with hot water [3]. Extracts were evaluated for their total phenolic content by an adaptation of the Folin-Ciocalteu method and further analysed by high performance liquid chromatography associated with electrospray mass spectrometry (HPLC-DAD-ESI-MSn) in the negative ion mode [4], in order to identify their individual phenolic constituents. The aqueous extracts of S. farinacea, S. mexico, S. officinalis and S. greggii contained, respectively, 106±13, 159±38, 175±46 and 136±1 μg GAE/mg of total phenolics. These four species were characterized by a clear prevalence of caffeic acid derivatives, in particular of rosmarinic acid (MW 360), that is generally the most abundant phenolic compound in Salvia species [2,3]. In addition, S. mexico and S. officinalis contained moderate amounts of salvianolic acid B (MW 718). Among these two, S. mexico was richer in O-caffeoylquinic acid (MW 354), while the latter presented high amounts of salvianolic acid K (MW 556) and moderate amounts of its structural isomer. All the extracts were enriched in flavones: S. farinacea and S. officinalis contained high amounts of luteolin-O-glucuronide while S. mexico contained luteolin-C-glucoside with respective characteristic mass spectrometry fragmentation pattern m/z at 461→285 and m/z at 447→357, 327. Similarly, S. greggii extract presented high content of luteolin-7-O-glucoside ([M-H]− at m/z 447→ 285) and luteolin-C-glucoside and moderate quantities of apigenin-C-hexoside ([M-H]− at m/z 431→341, 311). Further studies are being undertaken in order to understand the contribution of these phenolic constituents in the biological activities of Salvia plants.

Antioxidant potential of two Apiaceae plant extracts: a comparative study focused on the phenolic composition

The present study aimed to characterize the extracts prepared from Pimpinella anisum L. (anise) and Coriandrum sativum L. (coriander) (Apiaceae plants) seeds in terms of phenolic composition, and to correlate the obtained profiles with the antioxidant activity. Anise gave the highest abundance in phenolic compounds (42.09± 0.11 mg/g extract), mainly flavonoids (28.08±0.17 mg/g extract) and phenolic acids (14.01±0.06 mg/g extract), and also the highest antioxidant potential, measured by the ability to inhibit lipid peroxidation and β-carotene bleaching, the reducing power and the free radical scavenging activity. Apigenin and luteolin derivatives, as also caffeoylquinic acid derivatives seem to be directly related with the higher in vitro antioxidant potential of the anise extract. In contrast, the lower antioxidant potential of coriander seems to be due to its lower abundance in phenolic compounds (2.24±0.01 mg/g extract). Further studies are necessary to evaluate the in vivo antioxidant potential of the tested extracts, but the in vitro experiments already performed highlight them as potential health promoters.

Pesquisa de fungos endofíticos presentes em videira (Vitis vinifera L.) com capacidade para inibir o crescimento do agente causal da podridão negra (Guignardia bidwellii)

Neste trabalho testou-se o potencial antagonista de 16 fungos endofíticos isolados de videiras (Vitis vinifera L.), de castas representativas do Alentejo produzidas em modo de proteção integrada e em modo biológico, contra Guignardia bidwellii. Os isolados identificados após ITS-PCR e sequenciação pertencem aos géneros Epicoccum, Alternaria, Botrytis, Athelia, Phoma e Gibberella. Os isolados testados mostraram atividade antagonista contra G. bidwellii quer por inibição direta, quer através da produção de compostos voláteis, à exceção dos dois isolados de B. cinerea. No entanto, todos os isolados produziram alguns compostos voláteis com reconhecida atividade antimicrobiana, tais como benzaldeído, 3-metil-1-butanol e derivados de ácido propanoico. Foi ainda observado que seis dos isolados produziram também metabolitos não voláteis com capacidade de inibir o crescimento de G. bidwellii. Os resultados obtidos vêm mostrar o potencial dos fungos endofíticos como agentes de luta biológica no controlo de G. bidwellii, podendo constituir novas alternativas no âmbito de Proteção de Plantas; ABSTRACT: Endophytic fungi present in grapevines (Vitis vinifera L.) with the ability to inhibit the growth of the causal agent of black rot (Guignardia bidwellii) In this work the antagonistic potential of 16 endophytic grapevine fungi isolates (Vitis vinifera L.), from representative cultivars of the Alentejo region produced either under integrated pest management or organic mode, was tested against Guignardia bidwellii. Isolates were identified through ITS-PCR and sequencing, as belonging to the genera Epicoccum, Alternaria, Botrytis, Athelia, Phoma and Gibberella. Isolates showed antagonist activity against G. bidwellii either by direct inhibition or through the production of volatile compounds, with the exception of two isolates of B. cinerea. Nevertheless, all isolates produced volatile compounds with known antimicrobial activity such as benzaldehyde, 3-methyl-1-butanol and propionic acid derivatives. Additionally, six isolates produced non-volatile metabolites with the ability to inhibit G. bidwellii growth. These results show the potential that endophytic fungi have as agents for biological control of G. bidwellii, opening new options in the field of Plant Protection.

Remote Substituent Effects on the Stereoselectivity and Organocatalytic Activity of Densely Substituted Unnatural Proline Esters in Aldol Reactions

The organocatalytic activities of highly substituted proline esters obtained through asymmetric [3+2] cycloadditions of azomethine ylides derived from glycine iminoesters have been analyzed by 19F NMR and through kinetic isotope effects. Kinetic rate constants have been determined for unnatural proline esters incorporating different substituents. It has been found that exo-L and endo-L unnatural proline methyl esters yield opposite enantiomers in aldol reactions between cyclic ketones and aromatic aldehydes. The combined results reported in this study show subtle and remote effects that determine the organocatalytic behavior of these synthetic but readily available amino acid derivatives. These data can be used as design criteria for the development of new pyrrolidine-based organocatalysts.

Avaliação microbiológica e bromatológica da silagem ácida obtida de resíduos da indústria de filetagem de tilápia do Nilo (Oreochromis niloticus)

The experiment constituted in evaluating the acid silage prepared from Nile tilapia (Oreochromis niloticus) filletage industry residues. This silage was prepared in a filetage industry through the residues milling and 5% of ascetic acid addition and stock piling in a period up to until 201 days. Storaged silages in a period of 7, 12, 22, 27, 41, 48, 61, 75, 84, 91, 96, 110, 140, 151, 181, 187 and 201dias were evaluated. Bromatologics analyses of rude protein(PB), etereal extract (EE), humidity (UM), ashes (CZ) and microbiological analyses of Salmonella, total coliforms, faecals and Escherichia coli of the storaged silages in a period of 7, 91 and 201 days were realized. The pH was evaluated in all the storaged periods. Differences (P > 0.05) in the substance of PB, EE, UM and CZ were not observed. A linear increase in PH was observed, being stabilizing in plateau of 4.74. In relation to the microbiological analysis the presence of Salmonella, total coliforms, faecals and E. Coli were not found. Was concluded that the acid silage gotten from tilapia filetage residues produced with the use of 5% ascetic acid can be stored during 201 days without having Salmonella, total coliforms and faecals proliferation.

Production of Recombinant Trichoderma Reesei Endoglucanase Protein CEL7B by Using Kluyveromyces Lactis

This research is about producing recombinant Trichoderma reesei endoglucanase Cel7B by using Kluyveromyces lactis, transformed with chromosomally integrated Cel7B cDNA, as a host cell (K. lactis Cel7B). Cel7B is one of the glycoside hydrolyze family of proteins that are produced by T. reesei. Cel7B together with other endoglucanases, exoglucanases, and â-glucosidases hydrolyze cellulose to glucose, which can then be fermented to biofuels or other value-added products. The research objective of this MS project is to examine favorable fermentation conditions for recombinant Cel7B enzyme production and improved activity. Production of enzyme on different types of media was examined, and the activity of the enzyme was measured by using different tools or procedures. The first condition tested for was using different concentrations of galactose as a carbon and energy source; however galactose also acts as a potent promoter of recombinant Cel7B expression in K. lactis Cel7B. The purpose of this method is to determine the relationship between production of enzyme with increasing sugar concentration. The second culture condition test was using different types of media: a complex medium-yeast extract, peptone, galactose (YPGal); a minimal medium-yeast nitrogen base (YNB) with galactose; and a minimal medium with supplement-yeast nitrogen base with casamino acid (YBC), a nitrogen source, with galactose. The third condition was using different types of reactors or fermenters: a small reactor (shake flask) and a larger automated bioreactor (BioFlo 3000 fermenter). The purpose of this method is to determine the quantity of the protein produced by using different environments of production. Different tools to determine the presence and activity of Cel7B enzyme were used. For the presence of enzyme, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used. Secondly, to detect enzyme activity, the carboxymethyl cellulose- 3,5-dinitrosalicylic acid (CMC- DNS) assay was employed. SDS-PAGE showed that the enzyme band was at 67 kDa, which is larger than native Cel7B (52 kDa.), likely due to over glycolylation during post-translational processing in K. lactis. For the different types of media used in our fermentation, recombinant Cel7B was produced from yeast extract peptone galactose (YPGal), and yeast nitrogen base with casamino acid (YBC), but was not produced and no activity was detected from yeast nitrogen base (YNB). This experiment concluded that the Cel7B production requires the amino acid resources as part of fermentation medium. In experiments where recombinant Cel7B net activity was measured at 1% galactose initial concentration in YPGal and YBC media, higher enzyme activity was detected for the complex medium YPGal. Higher activity of recombinant Cel7B was detected for flask culture in 2% galactose compared to 1% galactose for YBC medium. Two bioreactor experiments were conducted under these culture conditions at 30°C, pH 7.0, dissolved oxygen of 50% of saturation, and 250 rpm agitation (variable depending on DO control) K. lactis-Cel7B yeast growth curves were quite reproducible with maximum optical density (O.D) at 600 nm of between 7 and 8 (when factoring dilution of 10:1). Galactose was consumed rapidly during the first 15 hours of bioreactor culture and recombinant Cel7B started to appear in the culture at 10-15 hours and increased thereafter up to a maximum of between 0.9 and 1.6 mg/mL/hr in these experiments. These bioreactor enzyme activity results are much higher than comparable experiments conducted with flask-scale culture (0.5 mg/mL/hr). In order to achieve the highest recombinant Cel7B activity from batch culture of K. lactis-Cel7B, based on this research it is best to use a complex medium, 2% initial galactose concentration, and an automated bioreactor where good control of temperature, pH, and dissolved oxygen can be achieved.

Total synthesis of 3,5-O-dicaffeoylquinic acid and its derivatives

We report the first total synthesis of 3,5-O-dicaffeoylquinic acid and its derivatives, 3,5-O-diferuloylqui- nic acid and 3,5-(3,4-dimethoxycinnamyl)quinic acid, in a nine-step sequence. The key step involves Knoevenagel condensations between vanillin, 3,4-dimethoxybenzaldehyde or 4-hydroxy-3-methoxy- benzaldehyde and the dimalonate ester of quinic acid.

Generation and Analysis of Interaction Energy Maps of p-Substituted Benzoic Acid [(5-Nitro-thiophen-2-yl)-methylene]-Hydrazides Active against MRSA

In this preliminary study eighteen p-substituted benzoic acid [(5-nitro-thiophen-2-yl)-methylene]-hydrazides with antimicrobial activity were evaluated against multidrug-resistant Staphylococcus aureus, correlating the three-dimensional characteristics of the ligands with their respective bioactivities. The computer programs Sybyl and CORINA were used, respectively, for the design and three-dimensional conversion of the ligands. Molecular interaction fields were calculated using GRID program. Calculations using Volsurf resulted in a statistically consistent model with 48 structural descriptors showing that hydrophobicity is a fundamental property in the analyzed biological response.

Hepatic structure-pharmacokinetic relationships: The hepatic disposition and metabolite kinetics of a homologous series of O-acyl derivatives of salicylic acid

1 The hepatic disposition and metabolite kinetics of a homologous series of O-acyl (acetyl, propionyl, butanoyl, pentanoyl, hexanoyl and octanoyl) esters of salicylic acid (C2SA, C3SA, C4SA, C5SA, C6SA and C8SA, respectively) was determined using a single-pass, in-sills rat liver preparation. 2 The hepatic venous outflow profiles for the parent esters and the generated metabolite, salicylic acid (SA) were analysed by HPLC. Non-parametric moments analysis was used to determine the area under the curve (AUC'), mean transit time (MTT) and normalized variance (CV2) for the parent esters and generated SA. 3 Pregenerated SA ([C-14]-salicylic acid) was injected into each liver with the parent ester to determine its distribution characteristics. 4 The overall recovery of ester plus metabolite was 89% of the ester dose injected and independent of the ester carbon number, suggesting that ester extraction was due to hepatic metabolism to salicylic acid. 5 The metabolite AUC' value increased directly with the lipophilicity of the parent ester (from 0.12 for C2SA to 0.95 for C8SA). By contrast, the parent AUC' decreased with the lipophilicity (from 0.85 for C2SA to zero for C8SA). The metabolite MTT value also showed a trend to increase with the lipophilicity of the parent ester (from 15.72 s for C3SA to 61.97 s for C8SA). However, the parent MTT value shows no significant change across the series. 6 The two-compartment dispersion model was used to derive the kinetic parameters for parent ester, pregenerated SA and generated SA. Consequently, these parameters were used to estimate the values of AUG', MITT and CV2 for the parent ester and metabolite. The moments values obtained using the two-compartment dispersion model show similar trends to the corresponding moments values obtained from the outflow profiles using a non-parametric approach. 7 The more lipophilic aspirin analogues are more confined to the portal circulation after oral administration than aspirin due to their more extensive hepatic elimination avoiding systemic prostacyclin inhibition. Given that aspirin's selectivity as an anti-thrombotic agent has been postulated to be due to selective anti-platelet effects in the portal circulation, the more lipophilic and highly extracted analogues are potentially more selective anti-thrombotic agents than aspirin.

Comparison of the metabolic and endocrine effects of 3,5,3'-triiodothyroacetic acid and thyroxine.

To test the hypothesis that 3,5,3'-triiodothyroacetic acid (Triac) is more active as a TSH suppressor than on peripheral parameters of thyroid hormone action, the following parameters were studied: basal metabolic rate, sleeping energy expenditure (SEE), sex hormone-binding globulin, and cholesterol. In a double blind trial, 14 subjects received during 3 weeks (phase 1) 180 micrograms T4 or 1700 micrograms Triac daily, divided into 3 doses, to suppress thyroidal secretion. The dosage was doubled for the next 3 weeks (phase 2). Under T4 treatment, TSH reached 0.11 mU/L during phase 1 and less than 0.03 mU/L during phase 2. With Triac, a marked TSH inhibition occurred after 1 week (0.17 mU/L), followed by an escape during the following 2 weeks (0.63 mU/L). During phase 2, an almost complete TSH suppression was obtained (0.03 mU/L). Both Triac doses suppressed endogenous thyroid hormone secretion, as evidenced by T4 and rT3 levels. Both substances induced a 2-fold stimulation of sex hormone-binding globulin during phase 2. Serum cholesterol decreased similarly, without affecting the high/low density lipoprotein ratio. T4 increased SEE by 4.1% and 8.5% during phases 1 and 2. Triac failed to induce the expected peripheral metabolic responses of the thyroid hormones, as demonstrated by an unchanged SEE and basal metabolic rate. These results clearly show a preferential action of Triac on TSH suppression.