| ObjectiveAs the quality of life is increasing day by day,the expectations for health of people is higher and higher.People not only position the health goal in the ill treatment,but also pay attention to the disease prevention and keep in good health in ordinary days.So People come to realize that to strengthen their own immune system function,to enhance the function of resistance to disease,in order to make themselves far away from the pain of the disease.Naturally,Chinese herbal medicine that can strengthen the immune function is increasingly valued,and trusted by people gradually,is also gradually come to the ordinary people.The experiment is to investigate the best experimental conditions with new tetrazolium salt MTS colorimetric method in T and B lymphocyte proliferation and NK cell activity experiment.Using serum pharmacology method to assess the optimal dose of Rhodiolasachalinensisextract?RSE?and Codonopsispilosulaextract?CPE?combination as well as the effects of the combination on the immune function of mice.Admesasurement the content of functional factor using Phenol-sulfuric acid method and HPLC in medicinal materials in order to state Rhodiolasachalinensisextract?RSE?and Codonopsispilosulaextract?CPE?can enhance the immune function?Methods1.The experiment of determining the splee cell proliferation and NK cell activity using the method of MTS-PMS:BALB/c mice spleen was obtained in sterile condition,prepare different concentrations of spleen cell suspension,then added lipopolysaccharide?LPS?and concanavalin?ConA?to induce mice spleen lymphocyte proliferation;added mouse lymphoma YAC-1 cell in the logarithmic growth and mouse hepatocarcinoma H22 cells as a target cell to detect the killing activity of NK cells.Cultivating in 96 well culture plate,then added MTS-PMS reagent and measuring OD value at 492 run.According to the different cell concentration and the OD value relations curve to determine optimal cell concentration and optimal testing time with the method of the MTS-PMS.2.The experiment of serum pharmacology in vitro:Using the 3×3 facterialdesign,nine medicated serum of CPE and RSE mixture were made to assessthe optimum combinational dose of CPE and RSE by detecting the T,B lymphocyte proliferation abilities and NK cells killing activity in vitro.3.The experiment in vivo:Using the optimum combinational dose and reducing 2.5,5 and10 times as high,middle and low doses of RSE+CPE groups,and theT,B lymphocyte proliferation abilities and the killing activity of NK cells in mice were detected in vivo.4.The experiment of delayeding type hypersensitivity in mice:Feeding mice 30 days according above conditions to conduct delayeding type hypersensitivity.5.The experiment of measuring functional factor content:Admesasurement the content of Codonopsis pilosula,.salidroside and tyrosol using Phenol-sulfuric acid method and HPLC,next to conduct the experiment of precision,stability analysis,repeatability analysis and standard recovery.Results1.Confirmming the mouse spleen lymphocyte proliferation ability best when the cell concentration is 3.2×106/mL using MTS-PMS method.When the target cell was YAC-1,the best splenocyte concentration was 8×105 cell/mL,as well as the effector-target ratio was 20:1 the NK cell activity was highest.When the target cell was H22,the best concentration of splencyte was 1.6×106 cell/mL,as well as the effector-target ratio was 40:1 the NK cell activity was the highest.OD value was maximum at 8 hour after addition of MTS-PMS,OD value at 16 ho'ur was no significant with that of 8 hour.2.The result of serum pharmacology indicates that compared with control group,the proliferation abilities of T,B lymphocytes and the killing activity of NK cells inducing by ConA and LPS in 200mg·kg-1 RSE+790mg·kg-1 CPE group were increased?P<0.05?.3.The result of mice in vivo experiment indicated that compared with cyclophosphamide group,the spleen indexesin middle and high dosesof RSE+CPE group were significantly increased?P<0.05?;compared with low dose of RSE+CPE group,the WBC number in middle dose of RSE+CPEgroup was significantly increased?P<0.05?.Compared with cyclophosphamide group,the T,B cell proliferationabilities induced with ConA and LPS and killing activities of NK cellsin low,middle and high dose of RSE+CPE group and positive CVT-200group were significantly increased?P<0.05?.Compared withCVT-200 group,low and high doses of RSE+CPE group,the T,B cell proliferation abilitiesinduced with ConA and LPS and killing activity of NK cells in middle dose of RSE+CPEwere significantly increased.Middle dose of RSE+CPE group canenhan the content of IL-2,6,10,IFN-y and TNF-a.4.Comparing with blank control group,the ear swelling degree of the rest are markedly decreasing?P<0.05?.The ear swelling degree of high dose of RSE+CPE group and low dose of RSE+CPE group were exceeding the group of CTX?P<0.05?.The ear swelling degree of middle dose of RSE+CPE group was markedly exceeding high dose of RSE+CPE group and low dose of RSE+CPE group?P<0.05?.5.The result of Phenol-sulfuric acid method showing that the average congtent of Codonopsis pilosula was 26.21 mg/g;Salidroside and tyrosol content were 0.484mg/g and 1.087mg/g respectively using HPLC.Conclusion1.The best cell concentration to detect lymphocyte proliferation with MTS-PMS method is 3.2×106?/mL,the best detection time is 8-16 hour after MTS-PMS addition.As detection of NK cell activity the effector-target ratio is different dependent on target cell.2.The optimal dose of Rhodiolasachalinensisextract?RSE?and Codonopsispilosulaextract?CPE?mixture is RSE 40 mg·kg-1+CPE 158mg·kg-1 in vivo immunoregulation.3.RSE and CPEmixture can enhance the immune function of mice. |
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