Biodisponibilidad de clorofilas
|Director||Roca López-Cepero, María de la Gracia|
|Department||Universidad de Sevilla. Departamento de Nutrición y Bromatología, Toxicología y Medicina Legal|
|Abstract||The present doctoral thesis focuses on the main group of natural pigments, the chlorophylls. Although years of investigation has provided a vast information about the chemistry and biochemistry of chlorophylls, the new ...
The present doctoral thesis focuses on the main group of natural pigments, the chlorophylls. Although years of investigation has provided a vast information about the chemistry and biochemistry of chlorophylls, the new trends in food science give directions to human health and nutritional properties of the different phytochemicals present in our daily diet. For chlorophylls pigments such area of investigation is relatively new and consequently the reason of the present research. The first result of the thesis is a complete review of the main characteristics of the chlorophylls pigments. This chapter includes aspects as structures, function and localization, biosynthesis and degradation, biological actions, methods of analysis and roles as food additives. To develop the thesis it was necessary first to develop specific methods to obtain the complete set of chlorophyll standards that can be present in processed, storage or ripened foods. The 32 chlorophyll derivatives were analyzed in depth by high resolution time-of-flight mass spectrometry and studied their behavior during the MS2 fragmentation with powerful postprocessing software. It is found that, while MS2-based reactions of phytylated chlorophyll derivatives point to fragmentations at the phytyl and propionic chains, dephytylated chlorophyll derivatives behave different as the absence of phytyl makes β-keto ester group and E ring more prone to fragmentation. The introduction of an oxygenated function at E ring enhances the progress of fragmentation reactions through the β-keto ester group, developing also exclusive product ions for 132-hydroxy derivatives and for 151-hydroxy-lactone ones. Native chlorophyllides and pheophorbides mainly exhibit product ions that involve the fragmentation of D ring, as well as additional exclusive product ions. It is noteworthy that all b derivatives, except 151-hydroxy-lactone compounds, undergo specific CO losses. It has been proposed a new reaction mechanism based on the structural configuration of a and b chlorophyll derivatives that explains the exclusive CO fragmentation. Proposals of the key reaction mechanisms underlying the origin of new product ions have been made. The same methodological approach has been applied for the determination of chlorophyll profile in the five major edible seaweeds. Seven new chlorophyll epimers at C132 position including chlorophyll c1’, 132-OH pheophorbide a’, pheophorbide a’, 132-OH chlorophyll b’, 132-OH pheophytin a’, pheophytin a’, and 151-OH-lactone pheophorbide a’ were identified for the first time in edible seaweeds and they show the same fragmentation pattern as their parent chlorophyll. In addition, eight new chlorophyll derivatives including 132-OH chlorophyllide c2, chlorophyll c2, chlorophyll c1, chlorophyll c1´, pheophorbide d, purpurin-18 a, pheophytin d and phytyl-purpurin-18 a were identified for the first time in edible macro algae and it is found that additional unsaturated position at C171-C172 impedes α-cleavage reaction in MS2 analysis of chlorophyll c and the double keto rearrangement at the E ring in phytol purpurin-18 a and purpurin-18 a easily displaces the ion charge from remote positions and allows the fragmentation of the keto-lactone group. Highly surprising was the discovery of chlorophyll d derivatives in macro seaweeds. Chlorophyll d have been scarcely characterized (only in specific cyanobacteria), and pheophorbide d has never been reported previously. Such findings allowed by first time the complete MS2 characterization of d derivatives. Chlorophyll profile in edible seaweeds differs with species. Red algae (Nori) contain mainly a series of chlorophyll, mainly pheophorbides and pheophytins; green algae (Sea Lettuce and Aonori), a and b series, mainly chlorophyll a and b; brown algae (Kombu and Wakame), principally a and c series, mainly pheophytins. The study of the cooking effects on the chlorophyll profile in edible seaweeds revealed that they are associated with the processing parameters, the structure of edible seaweeds and their respective chlorophyll molecules. Pheophytinization and decarboxymethylation at C132 are favored in cooking process. Pheophytinization degree is higher for a series than b series. Oxidation reactions occur mainly for chlorophyll a and b, not for pheophytins. Due to seaweed differences in extracellular composition and different thermal sensitivity of chlorophyll structures, generally, cooking has no effect on Nori seaweeds, and induces similar chlorophyll losses during boiling or microwaving for Kombu seaweeds. On the contrary, for green seaweeds microwaving methods is softer than boiling. After the characterization of the chlorophyll profile of seaweeds, fresh dried and cooked, the three main macro algae were submitted to an in vitro digestion process, including the oral, gastric and intestinal phases to evaluate the stability of chlorophylls. In summary, three principal types of reactions were prompted: (1) oxidation reactions to produce 132-OH and 151-OHlactone derivatives; (2) pheophytinization reaction that favors a series than b and c series; (3) pheophorbidation reaction that occurs obviously only when the initial chlorophyll profile for digestion is mainly composed of pheophytins. Cooking does not introduce significant modifications of chlorophyll profiles during in vitro digestion. Due to their polarity, chlorophyll compounds (as many other phytochemicals) requires the incorporation in micelles previous to the absorption by the intestinal cells. Consequently, the micellarization process is an estimation of which proportion of a compound after the digestion is theoretically ready for the enterocyte in the form of micelles. The efficiency of the compound transferred from the digesta to aqueous micellar fraction (AMF) is defined as the percentage of micellarization. The research concluded that dephytylated chlorophylls are easier micellarizated than phytylated ones. The analysis of seaweeds material allowed by first time comparing three series of chlorophylls, resulting that a and c series are favored than b series. Finally, the detailed analysis of chlorophyll derivatives determined that oxidized chlorophyll derivatives are favored than parent chlorophylls, especially for phytylated ones. The analysis of the cooking effects on digestive and micellarization properties of chlorophyll pigments from edible seaweeds, established that cooking improves the recovery of chlorophyll pigments in edible seaweeds, especially for Sea Lettuce and Kombu, but decreases the micellarization rate of chlorophyll pigments in Nori and Kombu with that of Sea Lettuce unchanged. To obtain a general view of a combination of all the process analyzed in the thesis (cooking, in vitro digestion and micellarization) on the chlorophyll profile of seaweeds, an index of processing bioaccessibility has been proposed. It showed that in the respect of chlorophyll pigments, Nori is recommended to be consumed fresh dried; while Sea Lettuce and Kombu seaweeds are better to ingest microwaved. Followed with the physiological process, aqueous micellar fractions of chlorophyll pigments from fresh dried and cooked seaweeds were combined with DMEM and subjected to Caco-2 cell absorption. After the incubation and consequent analysis, it was observed that dephytylated chlorophylls are better absorbed than phytylated chlorophylls and that oxidation reactions are promoted during the absorption process. Generally, cooking does not affect the cell absorption of chlorophyll derivatives. Once demonstrated that chlorophylls are absorbed by human intestinal cells, it was necessary to investigate the absorption process. It was mandatory to set up the protocol to incorporate the required concentrations of chlorophyll derivatives into the micelles. Applying the methodology, pheophorbide and pheophytin a-rich micelles were formulated in an absorption assay with highly polarized Caco-2 membrane cells growing on transwell plates that can present physiological intestinal environment for nutrient absorption. Pheophorbide a has a higher ability of incorporation into mixed micelles and higher absorption rate compared with pheophytin a. At 37 oC, absorption rate of pheophorbide a increases with increasing concentration in the mixed micelles and saturated at higher concentrations implying a facilitated transportation involved. Meanwhile the absorption rate of pheophytin a seems to be linearly increasing with higher concentration. At 4oC, absorption rate of pheophorbide a and pheophytin a increases totally linearly, although absorption rate of both pheophorbide a and pheophytin a is obviously higher at 37oC than at 4oC. In conclusion, it was shown that pheophorbide a is actively transported into the intestinal cell while for pheophytin a, the most likely mechanism implied in its cell absorption process should be a passive transport. Finally, to characterize the complete chlorophyll intestinal absorption process, other delivery protocols were investigated. These experiments permit the evaluation of transportation characteristics related with apical and basolateral surface, directionality or even efllux. Results showed that pheophorbide a molecule can be transported across the Caco-2 cell membrane from the basolateral side to apical side but not for the opposite direction. At difference, pheophytin a cannot cross the cell membrane regardless of transport direction. Hence, absorption rate of pheophytin a was significantly lowered by reversing the transport direction, but for pheophorbide a, not so significantly. In low concentrations, absorption rate of pheophorbide a is even faster from basolateral to apical than from apical to basolateral. Efflux experiment revealed that the majority of absorbed pheophorbide a and pheophytin a are delivered back to apical side, and others are still in the cell monolayer. Nearly none were found in basolateral side. To conclude the thesis, taking into account the absorption process of pheophorbide a is mediated by a carrier, attempts to identify such transporters were made. SR-BI and NPC1L1 transporters are two of the best characterized carriers implied in the carotenoid absorption process. Subsequently, experiments of antibodies and specific inhibitor treatment against SR-BI and NPC1L1 transporters found that SR-BI, but not NPC1L1 seems to be partially involved in the intestinal absorption of pheophorbide a by Caco-2 cells. It is the first time that a transporter of chlorophyll derivative is identified.
|Citation||Chen, K. (2016). Biodisponibilidad de clorofilas. (Tesis Doctoral Inédita). Universidad de Sevilla, Sevilla.|