| Ammopiptanthus is a leguminous xerophytic plant,the total flavonoids were extracted and separated from its seeds.Previous studies have shown that the total flavonoids from seeds of Ammopiptanthus has important antiviral activity.But so far,there is still a blank on the research about content determination method of total flavonoids from seeds of Ammopiptanthus and its immunomodulatory activity.In order to further explore the purification process of total flavonoids from seeds of Ammopiptanthus and its immunomodulatory activity,this studies were researched from the following aspects:(1)Establishing of a method about the detection of the total flavonoids from seeds of Ammopiptanthus content.Four chromogenic method were chose for determination of total flavonoids,the best absorption wavelengths were selected by wavelength scanning of quercetin standard and total flavonoids from seeds of Ammopiptanthus.Standard curves for quercetin standard by four chromogenic method were built and used it to determine the content total flavonoids from seeds of Ammopiptanthus.(2)Study on the purification process of total flavonoids from seeds of Ammopiptanthus with macroporous-resins.This experimentation choose3 kinds of macroporous-resins including AB-8?D101?S-8 to used for static adsorption and elution test.By dynamic adsorption and elution test with concentration of sample solution ? sample volume?sample flow rate?p H?ethanol concentration?elution flow rate and eluent volume to establish the optimum conditions for purifying total flavonoids from seeds of Ammopiptanthus with AB-8 macroporous-resins.And then extracted by ethyl acetate to further improve the total flavonoid purity.(3)Study on the immune activity of total flavonoids from seeds of Ammopiptanthus.Establishment of immunosuppression model in mice by cyclophosphamide(CTX)and give it to total flavonoids from seeds of Ammopiptanthus in different concentration of dose[High dose group(250mg/m L)? Medium dose group(150mg/m L)? Low dose group(50mg/m L)].Through the determination of immune organ organ index,cytokines(IL-2,IL-4),carbon particle clearance and observation of immune organ tissue structure to explore the immune regulation of mice in vivo.At the same time,the experiment explored the effects of different concentrations of total flavonoids from the seeds of Ilex Ilex on the proliferation of mouse spleen lymphocytes in vitro.The results showed,(1)the determination method of total flavonoids from seeds of Ammopiptanthus with Al Cl3-CH4 O chromogenic method have obviously maximum absorption at303 nm and 335 nm.The method validation showed that the precision(RSD=0.541%),reproducibility(RSD=1.05%),stability(RSD=0.62%)whith Al Cl3-CH4 O method at 335 nm are in the range of error and The recovery rate reached 102.57%.This method can be used on the content of total flavonoids of Ammopiptanthus seeds were effectively detected effectively.(2)The optimum adsorption and elution conditions for purifying total flavonoids was 5BV of 6 mg/m L sample solution at p H=5.5 was adsorbed with AB-8 macroporous-resins at rate of 3.5BV/h and then desorbed total flavonoids with 6BV of 70% ethanol at rate of 1.5BV/h.The purity of the purified product obtained under these conditions was 41.07%,compared to 5.69%for the crude total flavonoids extract.After purified with AB-8 resin,the total flavonoids were prepared into a certain concentration solution and extracted with ethyl acetate,the total flavonoids content reached76.15%.(3)Compared with the CY model group,the spleen index(4.431±0.574)and thymusindex(3.162±0.524)of the mice treated with middle dose group were extremely significant increased(P <0.01).(4)The levels of IL-2(541.3±33.41)and IL-4(44.819±2.257)in the mice treated with middle dose group were significantly increased(P<0.01).(5)Compared with the CY model group,the carbon clearance index K and the phagocytic index with middle dose group were significantly increased(P<0.01).(6)Compared with CTX,the total number of mature lymphocytes in spleen of mice were increased,the area of white pulp were increased,the germinal centers were relatively expanded,and the number of splenic corpuscle were increased.The number of mature lymphocytes in thymus was increased.The volume of thymus gland was increased and the germinal center was relatively enlarged(7)In vitro proliferation of spleen lymphocytes showed,the groups that were only added with the total flavonoids from seeds of Ammopiptanthus were compared with the negative control group,there was a positive correlation between cell proliferation and drug concentration in a certain concentration range(10 ~ 320?g/m L),and the number of cell proliferation was significantly increased(P <0.01),there was no significant increase in the number of viable cells when it was less than 10 ?g/m L(P >0.05).The groups that were added with the total flavonoids from seeds of Ammopiptanthus and Con A were compared with the negative control group,when Con A and the total flavonoids from seeds of Ammopiptanthus were added to the same group,the number of living cells were significantly increased compared with the negative control group(P <0.01).There was no significant difference in the number of viable cells in Con A and total flavonoids from seeds of Ammopiptanthus group compared with the positive control group with added Con A alone(P >0.05),when the concentration of total flavonoids from seeds of Ammopiptanthus was below 5 ?g/m L.The study have established the method of detection of the total flavonoids from seeds of Ammopiptanthus content,which provided a basis for the accurate detection of its content.At the same time,established purification process of total flavonoids from seeds of Ammopiptanthus,the purity of the dry matter was improved effectively by the method.The results showed that total flavonoids from seeds of Ammopiptanthus could promote the immune function of animals in a certain concentration range through the test of immune regulation in vivo and the proliferation of spleen lymphocytes in vitro,. |
PDF Full Text Request