Preparation Of Pilose Antler Oral Liquid And Effect On Immunological Function In Mice

Objective: Use conventional Chinese crude drug extraction methods as technique of impregnation extraction and recirculation extraction to extract biological components of pilose antler. Extraction methods were optimized with biological components of crude extract as the standard. Chitosan as clarificant, Preparation of pilose antler oral liquid, detection amino acid content of oral solution.Reference misture index in pharmacopoeia and then establish the quality standard for pilose antler oral liquid. Meamwhile, establish model of hypoimmunit in mice for observe effect of pilose antler oral solution in animal clinical test.Method: (1) Water and alcohol as dissolvent, we used technique of impregnation extraction and recirculation extraction to extract biological components from pilose antler. Optimize extraction methods according to components of crude extract as standard at different time, dissolvent volume and different concentration proportion. Protein was detected by ninhydrin reaction, biuret reaction and TLC. We determined content of protein with Brodford from several crude extract, and draw standard curve of protein content. (2) We used enzyme hydrolyzing and flocculating method cleaning dopant. Pilose antler oral liquid was prepared by conventional method, and determined content of protein by Coomassie brilliant blue kit. To analyse amino acid components of oral liquid so as to provide basis for the quality standard for pilose antler oral liquid by amino acid analyzer. (3)The models of mice on the hypoimmunity were induced by CTX. The weight of spleen and the function of abdominal cavity macrophage were measured. Spectrophotography was used to estimate levels of serum hemolysin antibody. The proliferation of T and B lymphocytes were observed by MTT assay, so as to investigate the effect of POAL on immunological function in mice.Result: (1) We calculated components of crude protein in two extraction methods on the basis of standard curve of protein content. The components of crude protein are 42.85μg/mL and 41.16μg/mL with technique of impregnation extraction, and 57.21μg/mL and 16.94μg/mL with recirculation extraction. We conclude the technique of recirculation extraction could be repeated and the level of crude extract is relative high. But this method takes more time and expend large bulk of solvent .In present study, the technique of recirculation get more crude extract use 75% alcohol for dissolvent. (2)We adopt conventional method of preparation, enzyme hydrolyzing and flocculating method to improve clarity. And the pilose antler oral liquid has good clarity and good stability. The quality consistent with misture standard from States Pharmacopoeia. Besides, we analyze total amino acid contents of oral liquid by amino acid analyzer. The total amino acid contents are 15.63mg/100mL, which is significantly higher than the contents of crude extract liquid 47.2mg/100mL when convert same level with crude extract liquid. (3)The PAOL could enhance the immunological function in normal mice and could significant recovery humoral immunity level of hypoimmunity in mice. In addition, the PAOL could improve cell immunity in mice. The PAOL could enhance the spleen index in immunosuppression mice (p<0.05). The 0.1mL PAOL could enhance the spleen index in normal mice. But the PAOL did not have the effect of improving phagocytosis function of PMΦin mice.Brief summary: The result of optimize extraction is that there is relative high crude protein level in recirculation extraction of 57.21μg/mL when using 75% alcohol for dissolvent. According to the method of oral liquid offered by States Pharmacopoeia, pilose antler oral liquid was prepared. Using enzyme hydrolyzingand flocculating method improve clarity, we have established quality standard for pilose antler oral liquid according to misture standard of 2005 states Pharmacopoeia. The pilose antler oral liquid was of good clarity and stability. The quality maintains well with little deposition, which indicates that it consistent with misture standard. The PAOL could enhance immunological function, and could recovery humoral immunity function and cell immunity function at the model of hypoimmunity, while it has not improved phagocytosis function of PMΦin mice.