Study On The Mechanism Of Hemolysis Induced By Alfalfa Saponins

This experiment mainly study the hemolytic characteristics of Alfalfa Saponins（AS）in vivo and in vitro.By filling the stomach of mice with Alfalfa Saponins to investigate whether ingest saponins would cause hemolysis,the hemolysis test in vitro to investigate the mechanism of hemolysis of alfalfa saponins.The results are as follows:Text 1 In vivo hemolysis test of Alfalfa SaponinsIn order to explore whether the intake of saponins can cause intravascular hemolysis.A total of 180 mice were selected and divided into 6 treatments according to their body weight and sex,with 3 replicates in each treatment,and with 10 mice in each replicats.1m L of 0.0125,0.025,0.05,0.1,0.2g/m L saponins were respectively given a gavage to the mice of I,II,III,IV,V group,the control group was given normal saline（1m L）,the test period was 30 days.Test shows:（1）After intragastric administration of alfalfa saponins,routine blood test,the number of red blood cells and red blood cell hematocrit were decreased and group V was significantly lower than that of the control group（P < 0.05）,the average corpuscular hemoglobin concentration was higher than the control group and group IV,V and the control group were significantly different（P < 0.05）.（2）The plasma free hemoglobin levels in V group were significantly higher than those in the control group（P < 0.01）.Group IV was significantly higher than the control group and group I,II（P < 0.01）.（3）With the increasing of AS the size of the red blood cells were mild unequal,some of the light staining area of the red blood cells in group V was disappeared,the cell staining was deeper,and the morphology was abnormal.（4）The maximum resistance value of red cell osmotic fragility was significantly increased（P < 0.05）.（5）In the hematopoietic system of the bone marrow,the red blood cells were active,and the ratio of grain to red was significantly lower（P < 0.05）.（6）The effect of AS on the tissue structure of parenchyma was not affected.Test 2 Alfalfa Saponins in vitro hemolysis testThe effect of AS on red cell morphology was observed by fluorescence electron microscope and scanning electron microscope,the former was used to observe the action of AS and red blood cells,the latter was to observe the effect of different concentrations on the morphology of cells.The spectrophotometric method was used to detect enzyme activity of Na ⁺-K ⁺-ATP and Ca²⁺-Mg²⁺ATP in vitro hemolysis the main contents of the three aspects of AS hemolysis were designed,which were affected by the glucose channel,the anion channel,the ATP enzyme related solution and the blocking agent.Results show: Under the microscope,the red blood cells with AS treated were severe deformation,and there were a large number of spherical red blood cells and vesicles.The activity of Na+-K+ATP and Ca²⁺-Mg²⁺ATP were significantly decreased（P < 0.01）.AS in vitro hemolysis showed that the 10%hypertonic glucose significantly antagonize atherosclerosis hemolysis（P < 0.01）;high concentration glucose channel inhibitor cell relaxation element B and dehydrogenation DHEA can inhibit atherosclerosis hemolysis（P < 0.01）;Li Cl and Na Cl were significantly inhibited AS in chloride solution（P < 0.01）,and high concentration of blocking agent NPPB and DIDIS significantly promoted AS hemolysis（P < 0.01）;sodium ion salt solution can inhibit hemolysis in different degrees and high concentration of potassium salt solution and Na ⁺-K ⁺ATP enzyme inhibitor strophanthus glycoside K significantly increased hemolysis（P < 0.01）;Mg SO4?7H2O and Ca Cl2 can significantly inhibit atherosclerosis hemolysis（P < 0.01）.Cinobufagin has the synergistic effect of the hemolysis of atherosclerosis,the high concentration of calcium channel blockers verapamil can inhibit atherosclerosis hemolysis（P < 0.01）.These results showed that the mechanism of AS in vitro hemolysis may be related to the role of GLUT1 as the site of action,by changing the sodium pump,enzyme Ca²⁺-Mg²⁺ATP and anion transport activity and conformation and eventually the osmotic pressure of the cells increased,red cell hemolysis.In clinical application,to provide theoretical basis for help to detoxify of the effect of alfalfa saponin hemolysis.