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- Inhibition of bladder tumor growth by chitooligosaccharides in an experimental carcinogenesis modelPublication . Fernandes, João C.; Sereno, José; Garrido, Patricia; Parada, Belmiro; Cunha, Maria F. X.; Reis, Flávio; Pintado, Manuela E.; Santos-Silva, AliceUrinary bladder cancer is one of the most common cancers worldwide, with the highest incidence in industrialized countries. Patients with cancer commonly use unconventional and complementary therapy including nutraceuticals. In this study we evaluated the efficacy of chitooligosaccharides (in orange juice) in rat bladder cancer chemoprevention and as therapeutic agent, on a rat model of urinary bladder carcinogenesis induced with N-butyl-N-(4-hydroxybutyl) nitrosamine. Results indicate that chitooligosaccharides may have a preventive effect on bladder cancer development and a curative effect upon established bladder tumors, dependent on the concentration ingested 500 mg/kg b.w., every three days, showed capacity to inhibit and prevent the proliferation of bladder cancer; however, this was associated with secondary effects such as hypercholesterolemia and hypertriglyceridemia. The use of lower doses (50 and 250 mg/kg b.w.) showed only therapeutic effects. It is further suggested that this antitumor effect might be due to its expected anti-inflammatory action, as well as by mechanisms not directly dependent of COX-2 inhibition, such as cellular proliferation control and improvement in antioxidant profile.
- Cytotoxicity and genotoxicity of chitooligosaccharides upon lymphocytesPublication . Fernandes, João C.; Borges, Margarida; Nascimento, Henrique; Bronze-da-Rocha, Elsa; Ramos, Oscar S.; Pintado, Manuela E.; Malcata, F. Xavier; Santos-Silva, AliceTwo COS mixtures and a low molecular weight chitosan (LMWC) were tested for potential cytotoxicity and genotoxicity upon human lymphocytes. Genotoxicity was evaluated in vitro by cytokinesis-blocked micronucleus and alkaline comet assays, while cytotoxicity was assessed by flow cytometry analysis. Our results suggest that COS do not exhibit any genotoxicity upon human lymphocytes, independently of MW or concentration. However, above 0.07 mg/mL COS induced strong cytotoxic effects. According to the concentration used, such cytotoxicity will induce cell death, essentially by necrosis (>0.10 mg/mL) and/or apoptosis (<0.10 mg/mL). The level of necrosis/apoptosis induced by high COS concentrations, suggests a promising use as apoptosis inducers in specific cancer situations.
- Plant aqueous extracts: antioxidant capacity via haemolysis and bacteriophage P22 protectionPublication . Gião, Maria S.; Leitão, Isabel; Pereira, Ana; Borges, André B.; Guedes, Catarina J.; Fernandes, João C.; Belo, Luís; Santos-Silva, Alice; Hogg, Tim A.; Pintado, Manuela E.; Malcata, F. XavierThe bacteriophage P22/Salmonella Typhimurium system, as well as human erythrocytes have been used to assay for protection, against forced oxidation caused by hydrogen peroxide, brought about by several aqueous extracts of selected adventitious plants grown in Portugal. This study proved, for the first time, that the aforementioned bacteriophage-based system is a suitable method to assess the antioxidant activity of plant extracts; among the 12 plants tested, raspberry (Rubus idaeus), sage (Salvia sp.), savory (Satureja montana) and yarrow (Achillea millefolium) were found to effectively protect against oxidative damage caused by H2O2. Haemolysis was inhibited via pre-treatment with every plant extract tested, except heath at 0.1% (w/v). The two analytical methods produced different results – and for some plants, there was a dependence (either direct or inverse) of the quantitative protection effect on extract concentration, whereas for others no significant dependence was found at all. Savory yielded the most promising results, using either method. Therefore, the P22/Salmonella system can be used as a suitable in vivo assay, and human erythrocytes as a suitable in vitro assay to confirm (or not) the antioxidant capacity of plant extracts in biological matrices.
- Evaluation of chitoligosaccharides effect upon probiotic bacteriaPublication . Fernandes, João C.; Eaton, Peter; Franco, Isabel; Ramos, Óscar S.; Sousa, Sérgio; Nascimento, Henrique; Gomes, Ana; Santos-Silva, Alice; Xavier, Malcata F.; Pintado, Manuela E.The main objective of the present study was to evaluate the antibacterial effect – through the determination of minimum inhibitory (and lethal) concentrations, as well as the possible prebiotic potential of chitooligosaccharides (COS) – through the determination of growth curves, on Bifidobacterium animalis Bb12, Bifidobacterium animalis Bo and Lactobacillus acidophilus Ki. Atomic force microscopy was further used to obtain high resolution images of COS effects upon the cell morphology. Our results demonstrate that COS do not stimulate the growth of those strains, neither the strains are capable of using COS as a primary source of carbon. Analysis of morphology when exposed to inhibitory/bactericidal concentrations, suggested that COS do not exert any direct damage upon the bacteria structure, instead the bacteria are apparently covered by COS, which likely prevent nutrient uptake.
- Antioxidant activity of chitooligosaccharides upon two biological systems: erythrocytes and bacteriophagesPublication . Fernandes, João C.; Eaton, Peter; Nascimento, Henrique; Gião, Maria S.; Ramos, Oscar. L.; Belo, Luís; Santos-Silva, Alice; Pintado, Manuela E.; Malcata, F. XavierMost of the reports to date on the antioxidant capacity of chitosans and chitooligosaccharides (COS) are based on strictly chemical methods. When studying antioxidants with potential in vivo applications, the method used to evaluate the antioxidant activity should be representative of the conditions in which the antioxidant might have a protective effect. In this work we evaluate the antioxidant activity of two COS mixtures and a low MW chitosan (LMWC) upon two biological oxidizable substrates – erythrocytes and phages, subjected to accelerated oxidation conditions. Our results suggest that COS/LMWC can be used as antioxidants in biological systems. All the tested compounds reduced either the hemolytic and DNA damage, by inhibiting H2O2- and AAPH-radicals. However, the results obtained for these biological assays did not reveal a dose dependence, contrary to the chemical assay, suggesting that the protective concentrations should be established, in order to prevent enhancement of the oxidative damage – i.e. a prooxidant effect.