| This study explored IRPS purification procedure, screening the best process to prepare IRPS MPEG-PLA copolymers microspheres by using orthogonal process, then mice were immunized with the best microspheres and measured indicators of blood total IgG levels and phagocytic activity of peritoneal macrophages and some other specific and non-specific indicators.1. Extraction and purification of IRPSDepartment of Radix Isatis was selected, then got crushed for the purpose of Radix powder with 200 mesh by the machine. Using improved methods of water extraction and alcohol precipitation to extract the IRPS, was combined with anthrone sulfuric acid method and visible spectrophotometer calculate the extraction rate of IRPS. Sephadex G-75 gel column was used to purify IRPS, through the columns of molecular sieve effect, IRPS can be divided into 5 polysaccharide components. The results showed that IRPS multiple extraction rate was about 11% to 12%;The polysaccharide extracted from purified polysaccharides collection contains 5 peaks.2.Preparation of IRPS MPEG-PLA copolymer microspheresMPEG (2000)-PLA and MPEG (6000)-PLA polymers were selectedand prepared the IRPS MPEG-PLA copolymer microspheres by double emulsion method and orthogonal design, to set out preliminary exploration optimal process of preparation of IRPS MPEG-PLA block copolymer microspheres.obtained by calculating the process group of the entrapment efficiency of microspheres, comparing microsphere particle size and distribution, observing morphological appearance of each group. The results showed that the molecular weight should be selected MPEG (2000)-PLA block copolymers, MPEG (2000)-PLA block copolymer concentration 100ug/ml, MPEG (2000)-PLA-dichloromethane as the oil phase was fixed volume of 20ml; IRPS volume of 10ml, concentration 0.06g/ml; PVA concentration of 0.5%. PVA for the external phase volume 100ml; stirring speed was 16000rpm.3. Influnce on the immunity of mice after administration with IRPS MPEG (2000)-PLA copolymer microspheres32 female Kunming mice were randomly divided into 8 groups, Group 1 to Group 8 were injected with 0.2ml contained IRPS MPEG (2000)-PLA block copolymer microspheres and blank MPEG (2000)-PLA block copolymer microspheres high concentration group(200mg/ml), concentration group (100mg/ml), the low concentration group (50mg/ml), IRPS group (2mg/ml)and saline group, Which IRPS and continuous administration of normal saline for 3 days, and blank microspheres containing sugaradministered only for 1 day. Comprehensive survey was administered by mice in the 7th and 14th day of the blood and got the results of the total IgG level, the total number of lymphocytes of IRPS microspheres with 3 different concentrations and the level of IgG were significantly higher than blank microspheres group and the saline control group, and with time, the IRPS microspheres concentrations (100mg/ml) was better than the immunnized only with IRPS group:Random choice of the 32 mice with the same method of groups was used to test macrophage function.The results showed that the effect of 3 concentrations(groups) of microspheres containing sugarwere lower than IRPS groups, but significantly higher than the blank microspheres (groups) and saline groups.4. Influnce on proliferation of chicken splenic lymphocytes in vitro after administration with IRPS MPEG (2000)-PLA copolymer microspheresThe test groups were divided into 7 groups of IRPS MPEG (2000)-PL A block copolymer microspheres 600ug/ml,300ug/ml,150ug/ml, blank microspheres 300ug/ml, IRPS 300ug/ml, concanavalin A (ConA) 10ug/ml and saline, respectively played role in the same concentration of chicken splenic lymphocytes, each repeated to 5 holes. The results showed that, with the concentration of IRPS microspheres increasing, OD450 value was also higher, IRPS microspheres was significantly higher than the blank microspheres and the saline groups. |
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