Extraction And Isolation Of Activity Component From Velvet Antler And Research Of Its Anti-Tumor Effect

Objective: Velvet antler is a kind of rarely tradional medicine,it has extensive effect on curing disease.As a special cartilage,velvet anlter is a natural cytokine'storeroom',and has lots of protein and proteoglycan.It is indicated that peoteoglycan has well effect of anti-tumor lately. Previous research on velvet antler is about ethanol extraction mostly, but less about protein and polysaccharide which dissolve in water,and least of them on anti-tumor research systematicly. Therefore we will extract protein and proteoglycan which is dissolvable in water using Tris buffer,and study their anti-tumor effect and mechanism systematicly .In addition,we not only study the cooperative effect of using proteoglycan and chemical medicine ,but also anti-tumor effect of crude extraction comparing to proteoglycan.Method: we extract the component of water dissolvable with Tris buffer,and determine the content of protein and proteoglycan in the extraction with Bradford method and Azure I method respectively ,isolate proteoglycan in the extraction by ion exchange chromatography,and determine the content of protein and proteoglycan in the same way,and estimate the effection of isolation to build a fast ,simple and convenient way for isolation.Extraction and isolation are freeze-dried .Build transplantional sarcoma 180 model in mice and then divide them into seven groups randomly.It's negative control group(cacer model),positive control group(cydophosphamide,CTX,25mg/kg),low dosage of proteoglycan group (40mg/kg),middle dosage of proteoglycan group(80mg/kg),high dosage of proteoglycan group (160mg/kg),crude extraction group (400mg/kg),cooperation group (40mg/kg proteoglycan and 25mg/kg CTX).After inoculating tumor cells 24 hours ,give different treatment to every group for 10 days,negative control group are given physiological saline by gavage and positive control group are administrated by injection in traperitonealy,other group are treated by gavage .After 10 days,peel off tumor tissue and weigh,make celiac macrophage mixture and spleen lymphocyte mixture,measure macrophage function of absorbing Neutral red T,B lymphocyte transformation function ,spleen index ,tumor inhibiting index and life time prolongation index.Result: The content of protein and proteoglycan in the top of velvet antler is 13.13% and 1.67%. There were three bars of proteoglycan when eluting by DEAE Sepharose and the corresponding concentration of NaCl is 0.7mol/L,0.8mol/L,0.9mol/L, conbined and concentrate three proteoglycan fractions according as eluting bar. Each fraction was 45.25%,25.15%,18.26% of total proteoglycans respectively.The rate of proteoglycan reclaimant is 88.66%.The immunoregulating experiment of transplantion S180 sarcamo indicates that,there is no sinifigant diffence between high dosage of proteoglycan,crude extraction,cooperation group and tumor-loded mice with daily weight growth,but spleen index,T,B lymphocyte transformation function and macrophage absorbance index is higher than negative control group(P < 0.05). T,B lymphocyte transformation function and macrophage absorbance index of high dosage of proteoglycan is higher than positive control group(P<0.05) ,but no signifigant difference with spleen index. T lymphocyte transformation function and macrophage absorbance index of crude extraction is signifigant diffrent with positive control group(P<0.05),but no signifigant diffrent wiht B lymphocyte transformation function and spleen index .Spleen index,T,B lymphocyte transformation function is higher than middle dosage and CTX group ,but there is no signifigant different except T lymphocyte transformation function(P<0.05). Tumor inhibiting experiment indicates that the tumor inhibiting index of crude extraction group is 78.15%,and has signifigant diffrence with positive control group(39.87%)(P<0.01),and of cooperation group is 50.16%,higher than positive control group and middle dosage of proteoglycan,but has no signifigant difference with positive control group(39.87%).The tumor inhibiting index of high dosage of proteoglycan is 23.47%,lower than 30%. life time prolongation index of high dosage of proteoglycan group and crude extracion group are both 119.79% ,higher than positive control group but no signifigant different,and of cooperation group is higher than middle dosage(103.13%)and positive control group(P<0.01).Conclusion: It's easy to extract water dissolvable component with Tris buffer, The content of protein and proteoglycan in the top of velvet antler is 13.13% and 1.67%. relatively. Proteoglycan can be isolated by DEAE Sepharose F.F anion exchang chromatography,also this method is fast and convenient.The effect of tumor inhibiting (78.15%)of crude extraction is best of each group, The life time prolongation (212.50%) of cooperation is highest of all.There is dosage dependment of every dosage of proteoglycan of the effect of immunregulating,and when it's dosage is 160mg/kg,the effect is better than crude extracion group and cooperation group.immune regulation function of proteoglycan is better than crude extraction,and the cooperation of proteoglycan and chemical medicine can enhance endurance of tumor-loded mice to chemical medicine.