Honey has been shown to have bioactivity. Fynbos (FB) honey was investigated for its bioactivity as this vegetation type is from a unique bio diverse region in the Cape Floristic Kingdom. Six FB and one medical grade Manuka (MAN) UMF 15+ honeys that were of quality grade (Codex Alimentarius) were used. Each honey sample was subjected to in vitro simulated gastro-duodenal digestion and the antioxidant and anti-inflammatory activity of each fraction was determined. These fractions were undigested/raw honey (UD), gastric digest (GD) and gastro-duodenal digest (GDD). Included were pH and digestive enzyme controls. The total polyphenol and the flavonoid content (TPC and TFC) were determined with the Folin-Ciocalteu (F-C) and aluminium chloride methods respectively. Antioxidant activity was measured with the trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays. Cellular antioxidant activity (CAA) in the Caco-2 and SC-1 cell lines using the dichloroflourescein diacetate (DCFH DA) assay was investigated. Nitric oxide (NO) scavenging activity was determined with the sodium nitroprusside (SNP) assay. Pro-inflammatory and anti-inflammatory effects of honey were evaluated in non-stimulated and stimulated with LPS/IFN γ murine macrophage RAW 264.7 cells, respectively. Cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was done. TPC and TFC of MAN were higher than that of FB honeys. With GD, TPC and TFC of MAN increased and following GDD, TPC decreased and TFC remained unchanged. In contrast TPC and TFC of FB honey were maintained with GD and GDD. TEAC assay revealed activity by MAN being higher than that of FB honeys. With GD digestion, the antioxidant activity of MAN was unchanged but following GDD, activity was reduced. For FB honeys, TEAC was maintained with GD and GDD. ORAC assay revealed that the activity of MAN was similar to that of FB. Digestion had no effect on activity of both MAN and FB honeys. CAA in the Caco-2 and SC-1 cell line was higher for MAN compared to FB honey. In both cell lines a similar trend was observed where with GD, CAA was unchanged while with GDD, CAA was reduced. This loss of CAA following GDD was found to be due to H2O2 formation as a result of polyphenol degradation in an alkaline environment containing sodium bicarbonate and pancreatin. NO scavenging activity of MAN was greater than FB. For both types of honey with GD, NO scavenging activity was unchanged and with GDD for MAN was reduced and for FB unchanged. Digestion showed an increased pro-inflammatory effect for MAN, FB1, FB2 and FB3. The UD fractions of MAN, FB1 and FB6 had anti-inflammatory effects. FB5 and FB6 honeys showed increased anti-inflammatory activity after GD and GDD. All honey fractions did not show any cytotoxicity. In conclusion, FB honey has antioxidant, pro- and anti-inflammatory properties. With digestion, GD activity was either increased or unchanged while with GDD activity was reduced, lost or unchanged. Observed effects were either due to pH and/or digestive enzyme activity. FB honey with its shown bioactivity could be an important local nutraceutical product. / Dissertation (MSc)--University of Pretoria, 2017. / Anatomy / MSc / Unrestricted

Este trabalho apresenta os estudos decorrentes da identificação de espécies moleculares associadas aos elementos Ca, Cu, Fe, Mg e Zn em amostras de castanha de caju (Anacardium Ocidentale), os quais foram realizados por: (1) digestão total da amostra, (2) digestão simulada in vitro, (3) extração de metais e proteínas empregando água, NaOH e Tampão Tris-HCl, (4) precipitação de proteínas com HCl ou acetona, (5) avaliação da associação entre espécies elementares e moleculares presentes nos diferentes meios extratores utilizando o acoplamento entre SEC-UV-SIMAAS e SEC-UV-ICP OES e (6) especiação de Fe em amostra de água de diálise empregando método espectrofotométrico. As determinações elementares nas amostras de castanha de caju demonstraram que os teores de Ca foram de (0,031 ± 0,001)%; Cu (19,1 ± 0,1) mg kg-1; Fe (53,5 ± 0,2) mg kg-1; Mg (0,22 ± 0,01) % e Zn (40 ± 3) mg kg-1, respectivamente. O emprego de soluções gástrica e intestinal simuladas revelou que 10% de Ca, 29% de Zn, 44% de Mg, 80% de Fe e 90% de Cu são extraídos durante a digestão. Após o processo de diálise, verificou-se que 100% do Zn e 90% do Ca, Fe e Mg passaram através da membrana de ester de celulose (tamanho de poro = 12 kDa). Porém, o cobre apresentou uma porcentagem de diálise de 70%. O processo de extração de proteínas indicou que a solução alcalina possui elevada capacidade de solubilização dos analitos, quando comparada com os demais extratos. A análise molecular das soluções extratoras demonstrou que em meio alcalino e tamponado há uma maior solubilização de compostos de alto peso molecular e o meio aquoso solubiliza espécies de baixo peso molecular. A precipitação realizada a partir dos extratos proteícos utilizando HCl propiciou uma alteração no perfil de distribuição molecular dos compostos presentes nos extratos aquoso e tamponado. Porém, o uso da precipitação em meio de acetona revela um perfil de separação diferente, pois em meio orgânico ocorre a seleção de compostos de baixo peso molecular (< 6,5 kDa). As análises por SEC-UV-ICP OES em soluções gastrointestinais revelou que os elementos estão distribuidos entre proteínas de alto e baixo peso molecular. Mas o processo de diálise demonstrou que algumas interações elementares e moleculares foram desfeitas após esta etapa de precipitação com HCl ou acetona. Porém, estes dados podem fornecer o primeiro indício da presença de metaloproteínas em amostras de castanha de caju. O monitoramento das espécies de ferro em amostras de água de diálise demonstra que apenas o íon trivalente (Fe3+) foi identificado. Sendo que, ao realizar a quantificação desta espécie, verifica-se que o teor de ferro livre presente na solução foi de 15%. Portanto, esta solução continha, aproximadamente, 85% de espécies químicas de ferro presentes no extrato gastrointestinal, as quais estão associadas a outros compostos presentes na amostra / In this work studies were done for the identification of molecular species associated to Ca, Cu, Fe, Mg and Zn in cashew nuts (Anacardium Occidental) using the following procedures: (1) sample acid digestion for total element determination; (2) in vitro digestion; (3) metals and proteins extraction using water, NaOH and Tris-HCl; (4) protein precipitation with HCl or acetone; (5) evaluation of association between elemental and molecular species in different extractors using SEC-UV-SIMAAS and SEC-UV-ICP OES and (6) iron speciation analysis in dialysis water using a spectrophotometric method. The elemental analysis showed that in cashew nuts there is a great quantity of Ca (0.031 ± 0.001) % and Mg (0.22 ± 0.01) %, but minor quantity of Cu (19.1 ± 0.1) mg kg-1, Fe (53.5 ± 0.2) mg kg-1 and Zn (40 ± 3) mg kg-1. The bioavailability studies reveal that Ca (10%), Zn (29%), Mg (44%), Fe (80%) and Cu (90%) were extracted during the in vitro digestion. After dialysis, 100% of Zn and 90% of Ca, Fe and Mg passed through the membrane, however, only 70% of Cu was dialysed in this step. The protein extraction procedures indicate that alkaline media has a great capacity for solubilization of analytes, if compared with other extracts. The molecular analysis of extracts showed that in alkaline and buffered media there is a presence of high molecular weight compounds and water extracted low molecular weight compounds. The HCl precipitation in water and buffered extractors change the molecular distribution profile and acetone precipitation selected low molecular weight compounds (< 6.5 kDa) for all extractors. The SEC-UV-ICP OES analysis in gastric intestinal solutions showed that the elements were distributed between high and low molecular weight compounds, but the dialysis procedure revealed the elemental and molecular correlations disappear to some metals. The monitoring of iron species by spectrophotometric method demonstrated that only 15% of trivalent iron was identified in the water dialysis. Therefore, this solution contain approximately 85% of iron species present in the gastrointestinal extract which are associated with others compounds in the sample.

Este trabalho apresenta os estudos decorrentes da identificação de espécies moleculares associadas aos elementos Ca, Cu, Fe, Mg e Zn em amostras de castanha de caju (Anacardium Ocidentale), os quais foram realizados por: (1) digestão total da amostra, (2) digestão simulada in vitro, (3) extração de metais e proteínas empregando água, NaOH e Tampão Tris-HCl, (4) precipitação de proteínas com HCl ou acetona, (5) avaliação da associação entre espécies elementares e moleculares presentes nos diferentes meios extratores utilizando o acoplamento entre SEC-UV-SIMAAS e SEC-UV-ICP OES e (6) especiação de Fe em amostra de água de diálise empregando método espectrofotométrico. As determinações elementares nas amostras de castanha de caju demonstraram que os teores de Ca foram de (0,031 ± 0,001)%; Cu (19,1 ± 0,1) mg kg-1; Fe (53,5 ± 0,2) mg kg-1; Mg (0,22 ± 0,01) % e Zn (40 ± 3) mg kg-1, respectivamente. O emprego de soluções gástrica e intestinal simuladas revelou que 10% de Ca, 29% de Zn, 44% de Mg, 80% de Fe e 90% de Cu são extraídos durante a digestão. Após o processo de diálise, verificou-se que 100% do Zn e 90% do Ca, Fe e Mg passaram através da membrana de ester de celulose (tamanho de poro = 12 kDa). Porém, o cobre apresentou uma porcentagem de diálise de 70%. O processo de extração de proteínas indicou que a solução alcalina possui elevada capacidade de solubilização dos analitos, quando comparada com os demais extratos. A análise molecular das soluções extratoras demonstrou que em meio alcalino e tamponado há uma maior solubilização de compostos de alto peso molecular e o meio aquoso solubiliza espécies de baixo peso molecular. A precipitação realizada a partir dos extratos proteícos utilizando HCl propiciou uma alteração no perfil de distribuição molecular dos compostos presentes nos extratos aquoso e tamponado. Porém, o uso da precipitação em meio de acetona revela um perfil de separação diferente, pois em meio orgânico ocorre a seleção de compostos de baixo peso molecular (< 6,5 kDa). As análises por SEC-UV-ICP OES em soluções gastrointestinais revelou que os elementos estão distribuidos entre proteínas de alto e baixo peso molecular. Mas o processo de diálise demonstrou que algumas interações elementares e moleculares foram desfeitas após esta etapa de precipitação com HCl ou acetona. Porém, estes dados podem fornecer o primeiro indício da presença de metaloproteínas em amostras de castanha de caju. O monitoramento das espécies de ferro em amostras de água de diálise demonstra que apenas o íon trivalente (Fe3+) foi identificado. Sendo que, ao realizar a quantificação desta espécie, verifica-se que o teor de ferro livre presente na solução foi de 15%. Portanto, esta solução continha, aproximadamente, 85% de espécies químicas de ferro presentes no extrato gastrointestinal, as quais estão associadas a outros compostos presentes na amostra / In this work studies were done for the identification of molecular species associated to Ca, Cu, Fe, Mg and Zn in cashew nuts (Anacardium Occidental) using the following procedures: (1) sample acid digestion for total element determination; (2) in vitro digestion; (3) metals and proteins extraction using water, NaOH and Tris-HCl; (4) protein precipitation with HCl or acetone; (5) evaluation of association between elemental and molecular species in different extractors using SEC-UV-SIMAAS and SEC-UV-ICP OES and (6) iron speciation analysis in dialysis water using a spectrophotometric method. The elemental analysis showed that in cashew nuts there is a great quantity of Ca (0.031 ± 0.001) % and Mg (0.22 ± 0.01) %, but minor quantity of Cu (19.1 ± 0.1) mg kg-1, Fe (53.5 ± 0.2) mg kg-1 and Zn (40 ± 3) mg kg-1. The bioavailability studies reveal that Ca (10%), Zn (29%), Mg (44%), Fe (80%) and Cu (90%) were extracted during the in vitro digestion. After dialysis, 100% of Zn and 90% of Ca, Fe and Mg passed through the membrane, however, only 70% of Cu was dialysed in this step. The protein extraction procedures indicate that alkaline media has a great capacity for solubilization of analytes, if compared with other extracts. The molecular analysis of extracts showed that in alkaline and buffered media there is a presence of high molecular weight compounds and water extracted low molecular weight compounds. The HCl precipitation in water and buffered extractors change the molecular distribution profile and acetone precipitation selected low molecular weight compounds (< 6.5 kDa) for all extractors. The SEC-UV-ICP OES analysis in gastric intestinal solutions showed that the elements were distributed between high and low molecular weight compounds, but the dialysis procedure revealed the elemental and molecular correlations disappear to some metals. The monitoring of iron species by spectrophotometric method demonstrated that only 15% of trivalent iron was identified in the water dialysis. Therefore, this solution contain approximately 85% of iron species present in the gastrointestinal extract which are associated with others compounds in the sample.