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| Author(s): |
Pablo Gomes Ferreira
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2010-06-23 |
| Examining board members: |
Ione Salgado;
Heloisa Sobreiro Selistre de Araujo;
Marcia Regina Braga;
Claudio Chrysostomo Werneck;
Marcos José Salvador
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| Advisor: | Ione Salgado |
| Abstract | |
Although soybean isoflavones naturally accumulate in their glyco- and malonylconjugated forms, the beneficial effects that soybean containing foods have on human health have been credited to their aglycones. This study aimed to develop a methodology for producing soybean extracts enriched with isoflavones aglycones and evaluate their activities in the synthesis and degradation of nitric oxide (NO) and proliferation of breast carcinoma cells (MCF-7) cultured in vitro. The nitric oxide (NO) donor, sodium nitroprusside (SNP), was used as a promoter for the accumulation of isoflavones in soybean seeds, which were identified and quantified by high performance liquid chromatography and mass spectrometry. SNP treatment increased the synthesis of the aglycone isoflavones in a dose-dependent manner. The relative production of aglycone forms was approximately 75% in the exudates (promoter solution), compared to the extraction made from only the tissues (34%). After treatment with SNP, exudate from the embrionary axis accumulated the largest amount of free isoflavones (1.61 µg/mg) compared to cotyledon (1.1 µg/mg), whereas in controls the total production of isoflavones was 0.3 µg/mg, with 95 % in conjugated forms. The extracts (350 µg) of embrionary axis or cotyledon enriched in aglycones, but not the controls, stimulated the acid reduction of nitrite (measured with specific electrode) leading to production of 1.4 and 0.9 ?mol of NO at pH 2, 0, values that were 8 times and 5.1 times higher when compared to the reduction only in the presence of 50 µM nitrite. The nitrite reduction activity was decreased with increasing pH and these results were validated by detection of NO by chemiluminescence. The main isoflavones, free or conjugated, identified in the extracts did not stimulate the acid reduction of nitrite. However, we observed that treatment with SNP also caused the accumulation of other phenolic compounds such as gallic, caffeic and p-coumaric acids, which stimulated the acid reduction of nitrite. At neutral pH extracts showed scavenging activity of NO and the embryo again was the most active (0.85 ?mol NO degraded/min) when compared to the cotyledon (0.24 ?mol/min) and to the spontaneous degradation of the control (0.1 ?mol/min). The embryo and cotyledon extracts showed dose-dependent biphasic effects on viability and proliferation of MCF-7 cells. At lower doses (250-500 µg/mL) extracts promoted (10-25%) and at higher concentrations (750-1000 µg/mL) inhibited (15%) cell proliferation. At 500 µg/mL, the proliferative effect of the extracts was reversed by nitrite (100 µM) in 34-36%. The higher doses of the extracts also stimulated emission of NO (detected with a fluorescent indicator). The biphasic effect was attributed to the action of isoflavone aglycones in activating estrogen receptors and stimulating the production of nitric oxide, which was increased by nitrite. Overall, the results suggested that soybean extracts enriched in bioactive compounds interfere with the metabolism of NO and the proliferation of breast adenocarcinoma cells (AU) | |