| In this article, how to control the quality standard of the goat embryo immunoregulatory factor(GEIF) of low molecular weight has been systematic studied. And we have developed a Chromatographic conditions and ways to detect the Chromatographic Fingerprints, We also have analysed attributes and stabilities of main componentes.1 , The standard of raw material : according to the comparative study of the characteristics of different stage of goat embryo development , the goat embryo about 2 month old has been identified, That is: embryo length for 5.0-15.0 cm, embryo weight for 10-50 g, head length for 1.5-3.5 cm, neck length for 0.4-2.0 cm, tail length for 1-2.5 cm, foreleg length for 1.5-3.5 cm, pinna length for 0.3-0.6 cm.2, The standard of main technology: through comparing the results of dialysis with ultrafiltration ,we found the latter may obtain more compositions with high activity. So the ultrafiltration has been choosed as the main craft.3, The standard of identification: it has been identified according to physiochemistry , UV spectrum , molecular weight , Chromatographic Fingerprints and amino acid analysis.(1) The standard of physiochemistry: According to methods from the Chinese pharmacopoeia (2000 version) and discipline of biological substance (2000 version) , we identified the colon diaphaneity, pH, the content of polypeptide and nucleic acid, protein reactionThe results showed that the GEIF is a clear and yellow liquid , pH is 6.5~ 7.0. and has the characteristic of dialysis and ultrafiltration, the content of polypeptides and nucleic acids are more than 600 ug / ml respectively. Sulfosalicylic acid Reaction appeared no protein.(2) The standard of UV spectrum: GEIF of ten samples obtained from different time have the character absorption peak at 251 ? nm and the ratio of A260nm to A28onm is higher than 2.0.(3) The standard of the molecular weight: the molecular weight of GEIF of ten samples obtained from different time was all under 3000 Da.(4) The standard of the Chromatographic Fingerprints: We have analysed GEIF by Reversed-Phase High-performance Liquid Chromatography (RP-HPLC) and studied the influence of mobile phase, gradient flow-rate, temperature and wavelength to separate unknown composition and developed the best condition of HPLC; Under this condition, we have analysised the Chromatographic Fingerprints of ten samples; The purity and characteristic of main components was analysised through spectrum scanning diagrams detected by Diode Array Detector(DAD), and Ninhydrin Reaction; Moreover, the Chromatographic Fingerprints at different times(2 h,4 h,6 h,8 h,12 h,24 h) under two temperatures(-20癈and 20癈) were analysised, respectively.Among the 17 separated peaks, we got the area above 5% of total area have 7 peaks. As to the precision , all the standard deviation of reserve-time can be controlled below 0.3%, The area and the height of peaks below 5.0% and 4.0% respectively ; Comparing the Chromatographic Fingerprints of ten samples , the similarities are near to 1; We found 8 pure peaks among these. Moreover, 8 peaks were recognized to have the compositions of peptide, 7 peaks have the nucleic acid and 2 peaks have nucleic acid peptide. Similarities of the Chromatographic Fingerprints obtained by stability experiments are all higher than 0.995.(5) The standard of amino acids: though analysed the amino acids of GEIF by Amino acids Analysis Meter, we found it contained 17 kinds of hydrolysed amino acides, including Asp, Thr, Ser, Glu, Pro, Glu, Ala, Val, Cys, Met, lle, Leu, Tyr, Phe, His, Lys and Arg.4, The standard of bioactivity: The cell-mediated immunoregulatoryfunction was identified by E rosette formation and T lymphocyte transformation5, The standard of reliability: according to the sterile experiment, theallergy experiment, the excrescent poison experiment, the pyrogen respondexperiment, proved that the GEIF of ten samples had no poison side effects.In conclusion, the experiment data for... |
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