Functional genomics for proving the existence of calcium binding proteins in finger millet and their facilitating role in calcium uptake under intestinal mimicking environment
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Date
2016-08
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G.B. Pant University of Agriculture and Technology, Pantnagar (Uttarakhand)
Abstract
The science of nutritional biology has progressed in recent years to develop food-based nutraceuticals as a form of highly personalized medicine or therapeutic agent. Finger millet [Eleusine coracana (L.) Gaertn.] is a crop with potential but under-explored source of nutraceutical properties as compared to other cereals. Finger millet is a reasonably good source of calcium with up to 450 mg/100 g in the seeds, which is 5–10 times higher than other cereals. Therefore, products derived from finger millet can be utilized in bone mass development in growing children, other bone ailments in adults and for preventing osteoporosis. The nutritional significance of finger millet must be properly translated to nutraceutical development based on scientific rationales and applied to other staple crops for their possible enrichment. The present study was centered on to prove the existence of calcium binding proteins in the finger millet and their facilitating role in calcium uptake using various biochemical and molecular approaches (Transcriptomics & Proteomics), besides biomarker(s) based validation under in vitro intestinal mimicking environment. Peptide mass finger printing of Stains-All stained protein separated by native and SDS-PAGE electrophoresis identified the presence of calreticulin and calcineurin B like proteins in finger millet grains. These genes showed >90% similarity with rice genes. To further validate the presence of such proteins, transcriptome data of developing spikes was used to identify 4 calreticulin and 5 calcineurin B like protein genes selected based on high FPKM values. These were also abundantly expressed in high calcium (GPHCPB-45) compared to low calcium (GPHCPB-1) genotype as also evident from real time PCR analysis. Higher expression of such calcium binding proteins in developing spikes envisaged their role in high accumulation of calcium in plants. In order to establish the facilitating role of finger millet ingredients in calcium uptake, two preparations viz. citrate buffered extract (CBE) and in vitro gastro-intestinal digest (IGID) of finger millet flour of high grain calcium genotype were used for challenging CaCo-2 human intestinal cells. Cytotoxic analysis revealed that higher doses above 100µg/µl for CBE and 5µg/µl of IGID were toxic. The IC0, IC10 and IC75 values were 25, 75, 250µg/µl for CBE, 0.5, 1, 5µg/µl for IGID and 25, 50 and 100mM for the CaCl2 respectively. The cytotoxicity was also evaluated the presence of combination of CBE with CaCl2: 75+25, 75+50, 75µg/µl +75mM and in combination IGID with CaCl2: 25+25, 5+50, 5 µg/µl +75mM. There were remarkable changes in the morphological features of CaCo-2 cells challenged with treatments. The formation of early dome shaped structures, increase in villi formation with differential polarity indicated that finger millet ingredients induced the differentiation of human intestinal Caco-2 cells for better uptake of nutrients. The IGID showed maximum morphological differentiation even at low concentrations of 1µg/µl. The facilitation of calcium uptake was further demonstrated using fluorescence imaging techniques: UV-fluorescent microscope and fluorescence plate reader using Fura-2 AM dye. The IGID was more effective than CBE. The expression analysis of selected biomarkers (CALB, S100, ATPASE, SLC8A, TRPV) of saturable and non-saturable calcium uptake pathways using real time PCR showed differential transcript abundance and more prominent effect was observed in IGID treatment either alone or in combination with CaCl2. The results obtained under in vitro intestinal mimicking environment indicate the presence of calcium uptake facilitators in finger millet flour digested with intestinal enzymes. The most plausible explanations of such facilitating role are either the release of bioactive peptides or free accesses of calcium in IGID which facilitated the calcium uptake in human Caco-2 cells. Such molecular insight derived from nutritional biology research led to development of potential nutraceuticals which can be employed for minimizing the calcium deficiency disorders.
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