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A. R. C. Braga and V. V. de Rosso
separated using a semi-preparative C-18 column connected with a HPLC system.
Further, each chromopeptide fraction (with absorbance at 615 nm) was collected
and analyzed by mass spectrometry. For the bioactivity assays, the solvent was
evaporated, and the resulting pellet was dissolved in 20% DMSO. Rechromatography of each fraction was performed under analytical conditions. Additionally,
cytotoxicity of each chromopeptide fraction on Human cervical adenocarcinoma
(HeLa) and human epithelial colonic carcinoma (Caco-2) cells was evaluated by 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay
(Mosmann 1983). Data collected was expressed as percentage of viability with
untreated cells taken as 100%. The authors showed that the released chromopeptides
varied in size from 2 to13 amino acid residues and were identified in both subunits
of C-phycocyanin. In addition, all five chromopeptide fractions obtained presented
significant antioxidant and metal-chelating activities and showed cytotoxic effects
on human cervical adenocarcinoma and epithelial colonic cancer cell lines. There
was a positive correlation between the antioxidative potency and the other biological activities of chromopeptides. Finally, they concluded that the digestion by pepsin
releases biologically active chromopeptides from C-phycocyanin, and the bioactivity
is mostly related to the antioxidative potency provided by the chromophore.
In the same line of work, Wu et al. (2015) evaluated purified R-phycoerythrin
(R-PE) after in vitro-simulated GI digestion to investigate the antioxidant activity
of the resulting peptides. The results indicated that digestion-resistant antioxidant
peptides of R-PE may be obtained by in vitro GI proteinases degradation.
Regardless of the protocol used to determine the behavior of microalgal pigments
throughout the in vitro digestion process, the steps are very similar, but some differences related to enzyme concentrations, agitation speed, reagent concentrations,
among others, are observed in the different studies.
Despite this diversity in the methods and applications, one can draw a few general
conclusions about the design of gastrointestinal (GI) models. Firstly, the model
should be as simple as possible, but not so simple that the results do not provide
relevant information to the “real life” situation. Secondly, what has been done previously is not always the best or the most relevant approach. Finally, digestion is not a
goal in itself and the way that digestate samples are collected is very dependent on
the type of measurement to be made.
10.4 In Vivo Bioavailability Methods
Rats and mice models have been used to understand the microalgal carotenoid distribution on different organs and tissues, mainly in liver, plasma, and feces. Carotenoids
were evaluated in rats after administration of microalgal biomass. The quantification of carotenoids was carried out by the absorbance of the microalgal extracts,
previously obtained from Spirulina platensis, Haematococcus pluvialis, and Botryococcus braunii, measured at 450, 470, 645, and 661.5 nm to estimate the content
of total carotenoids and chlorophyll using Lichtenthaler equations. Astaxanthin was
A. R. C. Braga and V. V. de Rosso
separated using a semi-preparative C-18 column connected with a HPLC system.
Further, each chromopeptide fraction (with absorbance at 615 nm) was collected
and analyzed by mass spectrometry. For the bioactivity assays, the solvent was
evaporated, and the resulting pellet was dissolved in 20% DMSO. Rechromatography of each fraction was performed under analytical conditions. Additionally,
cytotoxicity of each chromopeptide fraction on Human cervical adenocarcinoma
(HeLa) and human epithelial colonic carcinoma (Caco-2) cells was evaluated by 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay
(Mosmann 1983). Data collected was expressed as percentage of viability with
untreated cells taken as 100%. The authors showed that the released chromopeptides
varied in size from 2 to13 amino acid residues and were identified in both subunits
of C-phycocyanin. In addition, all five chromopeptide fractions obtained presented
significant antioxidant and metal-chelating activities and showed cytotoxic effects
on human cervical adenocarcinoma and epithelial colonic cancer cell lines. There
was a positive correlation between the antioxidative potency and the other biological activities of chromopeptides. Finally, they concluded that the digestion by pepsin
releases biologically active chromopeptides from C-phycocyanin, and the bioactivity
is mostly related to the antioxidative potency provided by the chromophore.
In the same line of work, Wu et al. (2015) evaluated purified R-phycoerythrin
(R-PE) after in vitro-simulated GI digestion to investigate the antioxidant activity
of the resulting peptides. The results indicated that digestion-resistant antioxidant
peptides of R-PE may be obtained by in vitro GI proteinases degradation.
Regardless of the protocol used to determine the behavior of microalgal pigments
throughout the in vitro digestion process, the steps are very similar, but some differences related to enzyme concentrations, agitation speed, reagent concentrations,
among others, are observed in the different studies.
Despite this diversity in the methods and applications, one can draw a few general
conclusions about the design of gastrointestinal (GI) models. Firstly, the model
should be as simple as possible, but not so simple that the results do not provide
relevant information to the “real life” situation. Secondly, what has been done previously is not always the best or the most relevant approach. Finally, digestion is not a
goal in itself and the way that digestate samples are collected is very dependent on
the type of measurement to be made.
10.4 In Vivo Bioavailability Methods
Rats and mice models have been used to understand the microalgal carotenoid distribution on different organs and tissues, mainly in liver, plasma, and feces. Carotenoids
were evaluated in rats after administration of microalgal biomass. The quantification of carotenoids was carried out by the absorbance of the microalgal extracts,
previously obtained from Spirulina platensis, Haematococcus pluvialis, and Botryococcus braunii, measured at 450, 470, 645, and 661.5 nm to estimate the content
of total carotenoids and chlorophyll using Lichtenthaler equations. Astaxanthin was