| Animal Medicine?AM?is a special part of the Traditional Chinese Medicine?TCM?,it has significant clinical efficacy.However,the drug effect ingredient is generally unclear.The quality control and evaluation methods of AM is not as good as the Plant Medicine and is unable to reflect the effectiveness and safety,which restrict the application and development of AM.In this study,the traditional effect of the sika deer velvet?Sika Deer Antler?is beneficial for the kidney,blood and skeleton.In order to establish a quantitative indicator for the quality evaluation of Sika Deer Antler,we choose the Sika Deer Antler with outstanding traditional effect and exact clinical efficacy but not clear substance as the research object.Researching the vitro activity of proliferative activity of testicular stromal cells?TM3?,acetylcholinesterase?AchE?inhibitory activity,and osteoblast?MC3T3-E1?proliferation activity,which is closely related with the traditional effect of the Sika Deer Antler,and detecting the influence of the vitro activity of Sika Deer Antler in different specifications and tissues to AchE inhibitory activity and proliferation activity of TM3 and MC3T3-E1,then selecting and building the biological titer evaluation method of Sika Deer Antler in different specifications and tissues.Moreover,Calcium and phosphorus contents in different Sika Deer Antler specimens and parts were determined.The correlation between in vitro bioactivity and calcium and phosphorus contents of Sika Deer Antler with different sika deer specimens and parts was studied by SAS software mathematical statistical analysis program.In the end,the method of High Performance Liquid Chromatography?HPLC?was used to determinate the ingredients of Sika Deer Antler,the HPLC specific chromatogram of Sika Deer Antler was established,and the method of Grey Relation Analysis?GRA?was used to analyze the relation degree between the common peaks and the activity of TM3,AchE,and MC3T3-E1.The main results are as follows:?1?The quality of different specifications of Sika Deer Antler can be evaluated and distinguished roughly via determining the biopotency of different specification samples.Ellman method was used to detect the inhibitory effect of water extraction of Sika Deer Antler to acetylcholinesterase.When the concentration of water extraction was 25?g·mL-1,the inhibition effect was the most significant?P<0.01?.The results showed that Sika Deer Antler had good acetylcholinesterase inhibitory activity,and the data obtained were used for further activity correlation study.?2?The effect of Sika Deer Antler with different specifications and different parts on the proliferation of MC3T3-E1 and TM3 were studied,which provided a basis for detecting the cell activity on the traditional efficacy of Sika Deer Antler.The method of Cell Counting Kit-8?CCK-8?was used to determine the effect of water extraction from Sika Deer Antler on the proliferation of MC3T3-E1 and TM3 cells.When the concentration of water extraction was 5 mg·mL-1and 10 mg·mL-1,the effect of promoting cell proliferation was significant?P<0.01?.The results showed that Sika Deer Antler could promote the proliferation of MC3T3-E1 and TM3 cells,and the data were used to further study the correlation of activity.?3?The content of calcium and phosphorus in Sika Deer Antler of different specifications and parts was determined by spectrophotometry and atomic absorption spectrometry.The results showed that the distribution of calcium and phosphorus in Sika Deer Antler was regular.The content of calcium and phosphorus of Sika Deer Antler of the same specifications was different,the lower part was the highest,followed by the middle part and the lowest in the upper part?P<0.01?;the content of calcium and phosphorus in tricot Velvet was higher than that in the second bark velvet.The contents of calcium and phosphorus in the second crop were slightly higher than those in the first crop?P<0.05?.The data obtained were used for further activity correlation analysis.?4?SAS software was used to study the correlation among the inhibitory activity of Sika Deer Antler on acetylcholinesterase and the effect of Sika Deer Antler on the proliferation of MC3T3-E1 and TM3 and the content of calcium and phosphorus.The results showed that the content of calcium and phosphorus was positively correlated with the osteoblast proliferation of MC3T3-E1 under the action of Sika Deer Antler?P<0.01?.The content of calcium and phosphorus was positive correlated with the proliferation of TM3 under the action of Sika Deer Antler.?P<0.01?.However,the change of calcium and phosphorus content was negatively correlated with the action of acetylcholinesterase in Sika Deer Antler?P<0.01?.It is suggested that calcium and phosphorus can be used as indirect quantitative indicators for evaluation of Sika Deer Antler grade.?5?The result of GRA has shown that the common peaks of Sika deer have the correlation with the proliferation activity of MC3T3-E1,the descending order of grey relation degree is chromatographic peak 2,chromatographic peak 4,chromatographic peak 5,chromatographic peak 7,chromatographic peak 3,chromatographic peak 1,chromatographic peak 6,chromatographic peak 9,chromatographic peak 8,chromatographic peak 10.Moreover,the common peaks of Sika deer have the correlation with the proliferation activity of TM3,the descending order of grey relation degree is chromatographic peak 2,chromatographic peak 6,chromatographic peak 1,chromatographic peak 5,chromatographic peak 4,chromatographic peak 3,chromatographic peak 7,chromatographic peak 9,chromatographic peak 8,chromatographic peak 10,furthermore,the common peaks of Sika deer have the correlation with the AchE inhibitory activity,the descending order of grey relation degree is chromatographic peak 7,chromatographic peak 2,chromatographic peak 10,chromatographic peak 8,chromatographic peak 9,chromatographic peak 4,chromatographic peak 1,chromatographic peak 3,chromatographic peak 5,chromatographic peak 6. |
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