Frozen Fresh Horse Deer Antler Study On The Isolation And Activity Of Protein Components

The antler is made of the hairy newly developing horn, which hasn't been ossified, from the Cervus Nippon Temminck buck or the Cervus elaphus Linnaeus buck of the mammalian, the phylum Chordata. In Chinese ancient medicine, the vital essence of a deer is in the horn. The antler is the horn's burgeon, whose energy is full without any leakage, so it benefits most to the blood and in reinforcing Yang. It is a nourishing medicine to reinforce Yang and promote longevity. Modern research also shows that the antler is pharmacological active in various aspects.In 1997, for the first time, Prof. Huo Yushu from China developed the antler freeze drying power, which turned the chemical research of the antler to the biological research. Many experts and scholars in and out of China has analyzed the component of the freezed fresh antler and tested its activeness. It is found that the advantage of the freezed fresh antler is the effective active components are well preserved without treated under high temperature.In this article, using the freeze fresh antler from the tarim red deer as the raw material, exchange chromatography of Q-Sepharose and SP-Sepharose ions is applied to make the prefractionation of the antler WSP. After capillary separation of the C18 high efficiency liquid chromatography, best conditions for HPLC is decided. At the same time, every fraction is qualitative diagnosed with 3, and cell-based assay of cell active factors is made in exchange chromatography fraction and gel filtration chromatography fraction. Details as below:The first chapter is about single factor study. Different homogenate method, ultra high pressure method under different condition, the impact of different pH value and ionic strength to the extraction percentage of the total antler WSP, all of these are studied to decide the best extraction condition. To prove that to freeze is better than to fry in preserving the antler WSP, especially the hydrone protein, SDS-PAGE electrophoresis method is applied to study the crude extract of freeze fresh antler, antler freeze drying powder, the protein molecular weight of antler prepared slice.The second chapter is various methods for crude separation of antler WSP on molecular sieve theory basis. Methods like roll film ultra-filtration, ultrafiltration centrifugation, alcohol sedimentation, the extraction percentage of G-50 gel filtration chromatography and Superdex 75 gel filtration chromatography to antler WSP whose molecular weight is less than 20kDa are studied. It is found that these methods don't give high extraction percentage and cause large loss to extract the antler hydrone protein. Then another way is considered to study the crude separation of ion exchange chromatography to antler protein on charge theory basis. This time strong anion exchange chromatography Q-sepharose F.F. is chosen to take crude separation of antler acidic protein and basic protein. The protein recovery rate and electrophoresis result shows that this method is working well in crude separation of antler protein, and the recovery rate is high. Thus method of ion exchange chromatography is decided for crude separation of antler protein.The third chapter is an important part. Strong anion exchange chromatography Q-Sepharose F.F. and strong cation exchange chromatography SP-Sepharose F.F. is separately used to take systematic separation of total antler protein. The impact that the original buffer with different pH and ion strength would have in antler protein recovery rate is separately studied. The best conditions for original buffer and elution is decided and 11 fractions of antler acidic protein Q1-Q6 and basic protein SP1-SP5 are obtained. C18 HPLC separation is applied separately to the 11 fractions, the best condition for liquid phase separation is chosen. We obtain a few materials which appears a single peak in RP-HPLC and a pure protein material.The fourth chapter is about ELISA kit assay to EGF of the antler acidic protein Q1-Q6 and Superdex 75 gel fraction S1-S6, ELISA kit assay toβ-NGF and IGF-1 of SP1-SP5 and Superdex 75 gel fraction S1-S6. The result is that IGF-1 activity is found between fraction S3-S5 segment.The specialty of this paper is mainly in:1. To take systematic separation of antler in freeze and fresh.2. For the first time, using ion exchange method to systematically separate antler WSP,and studying the best separation conditions.3. After C18 HPLC separation for each ion exchange segment, the best condition for liquid phase separation is chosen. We obtain a few materials which appears a single peak in RP-HPLC and a pure protein material.4. After cell-based assay to each antler fraction, IGF-1 activity is found between fraction S3-S5 segment of Superdex 75 gel chromatography.