| With the vigorous development of China’s manned space program, the bone-loss induced by space microgravity can not be ignored any more. There is ongoing concern for human health during spaceflights. The loss of bone calcium, the reduction of bone mineral density and the decline of bone mechanical strength during space flight can not be recovered completely after returning to ground in a short time, which not only affects the execution and development of space flight missions, but also even endangers the health and lives of astronauts. In this research, Auricularia auriacula peptides prepared after proteolysis were firstly chosen as carriers chelating with calcium ions and the protective effect of the chelate on bone-loss induced by simulated microgravity was further investigated. The chelate of peptide-calcium ions can accelerate the absorption and utilization of calcium ions by astronauts in space microgravity due to the special absorption mechanism of small peptides, which both improves the biology titer and provides a new idea or direction for natural products such as protein from plant as well. The main contents of research are as follows:The optimization of extraction conditions for Auricularia auriacula protein. The effect of pressure on the extraction for Auricularia auriacula protein was firstly studied by comparing the yields of protein under different pressure conditions, including high,low and normal pressure. As a result, high pressure homogenization showed the highest yield and was chosen as the best extraction method for Auricularia auriacula protein. The optimum extraction conditions were the ratio of solid material to liquid 1:104, homogeneous pressure 25 MPa, homogeneous time 25 min and the the yield of protein was up to 5.35%.The screening of proteolytic method and conditions. The best Auricularia auriacula peptides were determined by comparing the proliferative activities for osteoblast(OB) cells after different periods of time of enzymatic hydrolysis and alkaline hydrolysis, respectively. The different peptides and their corresponding chelate promoted the proliferation of OB cells in different degree. Repeated MTT experiments indicated Trypsin enzyme hydrolysis was a better method, and the conditions were p H8.0, 37 ℃ and hydrolysis time 30 min. The peptides prepared following the conditions above chelating with calcium ions showed the cell proliferation rate of OB was up to 90%.The purification of Auricularia auriacula peptides. In this research, semipreparative HPLC devices were adopted to isolate and purify peptides. The active compositions were determined and purified repeatedly by comparing the cell proliferation rates of OB of different compositions chelating with calcium ions which flowed at different peak times. After purification, its molecular weight and the amino acid compositions of AP-2 were analyzed and determined. The results indicated that AP-2 with the molecular weight of 10573 D contained all the necessary amino acids.The optimization of conditions for AP-2 chelating with calcium ions. The effect of peptide-calcium ions chelate was related to the ratio of peptides to calcium ions, p H, chelating time and temperature. The optimum chelating conditions were selected through response surface optimization by comparing the cell proliferation rates of OB. The results indicated that the ratio of peptides to calcium ions 2.4:1, p H 9.0, chelating time 63 min and chelating temperature 68 ℃ contributed the proliferation of OB cells the most and the proliferation rate was up to 98.2%.Research on the protective effect of chelate on bone-loss induced by simulated microgravity in vitro. The simulated microgravity was realized by 2D-RWVS. Results indicated that the content of calcium increased and the activity alkaline phosphatase enzyme(ALP) was promoted significantly in OB cells treated with peptide-calcium ions chelate which were cultivated in simulated microgravity for 48 h, which indicated that the chelate was able to protect bone-loss induced by simulated in some degree.Research on the protective effect of chelate on bone-loss induced by simulated microgravity in vivo. The simulated microgravity was to establish a tail-suspended mice model. The results indicated that AP-2-calcium ions chelate could recover the damage of immune organs including thymus and adrenal glands effectively and the loss of inorganic mineral salts content in bone was inhibited significantlt as well. Meanwhile, the chelate enhanced the ALP activity and reduced the tartrate resistant acid phosphatase(Str ACP) activity in serum, which both provided better protective effect on bone in simulated microgravity.This study possessed certain theoretical value and practical significance to study the protection approach on bone-loss and develop new counter-measures in space microgravity. |
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