Study On Mechanisms Involved In Antimutagenesis Of Aqueous Extract Of Astragalus Mongholicus And Radix Glycyrrhizae

In the progress of research on antimutagenesis of Chinese herbalmedicine,the main study both here and abroad is concentrated on screening ofantimutagen, but exact reports on mechanisms involved in antimutagenesis ofChinese herbal medicine are hardly seen. There are many synergistic effectsbetween astragalus mongholicus and radix glycyrrhizae (AG), compatibility ofthe two medicines should have enormous clinical potentiality. So we compatethem to make a study of the influence and mechanisms involved inantimutagenesis of Chinese herbal medicine. Taking CP as mutagen and VC asreference of antimutagen, we carried out a series of tests on aqueous extract ofastragalus mongholicus and radix glycyrrhizae (AG) both inside and outsidemice, which included determinations on bone-marrow PCE micronucleus,lymphocyte proliferation, activity of GSH-PX and SOD in the whole blood,spleen index, thymus gland index, IL-2, function of macrophage, hemolysin inblood serum, PFC etc. This research provides an experiment basis for the newpath which develops Chinese herbal medicine to prevent and cure, havingimportant theories meaning and practical value, the results as follow.1 Definition of the best experiment dose of AG. The stomaches of mice werefilled by AG in 5 doses including 10 times, 20 times, 40 times, 50 times, 60times. Set up positive and negative control groups and filled stomaches for 14dconsistently. Tested micronucleus rate and stimulation index of lymphocyte, weconcluded after statistical analysis that 50 times is the best experiment dose ofAG.2 Experiment on lymphocyte transformation. Mice were Divided into 6groups at random: positive control group, AG group, AG+CP group, VC group,VC+CP group and negative control group. Infused stomach once everyday, 0.5ml for each mouse, for 14 days, the dose of VC is 40 mg/(kg﹒bw). 30h beforeexecution the positive control group, AG+CP group and VC +CP group wereinjected by CP once with the dose 40 mg/(kg﹒bw). Prepared the spleensuspension with germ free and carried out the experiment on lymphocytetransformation according to the MTT colorimetric method. The result showedthat AG can enhanced the function of both T and B lymphocytes, opposed theimmunosuppression and injury caused by CP, which was more powerful thanVC.3 Micronucleus test of bone marrow cells. The mice used were the same inexperiment two, took bone marrow to smear, observed 1000 PCE every mouseand calculated the micronucleus rate. The result showed that AG obviouslyinhibited micronucleus frequencies of mouse bone marrow cells induced by CP,which is better than VC. Inhibited mutagenesis of CP, diminished the damage ofDNA and enhanced the reparation potentia.4 Determination of the activity of GSH-PX and SOD in the whole blood.Used the mice in experiment two. Removaled the eyeballs to make blood andditermined according to method described in the kit. The result showed thatAG elevated the activity of GSH-PX and SOD in the whole blood and catalyzedresolution of hydrogen dioxide. It had the function to eliminate free radicals,breaking off and ending oxidizing reaction of free radicals. It promoted peroxideto resolve and become propotional Spirits, reducing the poison. So it enhancedthe antioxidation ability of body.5 Experiment on the measurement of spleen index and thymus gland index.Groups and administration were the same to experiment two. Each mouse wassacrificed after weighing, taking the spleen and thymus gland to weigh withgerm free. Calculated the spleen index and thymus gland index respectively. Theresult showed that AG increased the spleen index and thymus gland indexremarkably, raised the weight of immune organ, enhanced immune response,opposed the immunosuppression and injury caused by CP, which was morepowerful than VC.6 Experiment on the production and activity of IL-2. Groups andadministration were the same to experiment two. Prepared the supernatant ofspleen suspension that was cultivated with germ free and carried out theexperiment according the method of CTLL-2 cell's proliferation. The resultshowed that AG encourged the production and activity of IL-2, reversed the lowlevel of IL-2 caused by the CP.7 Experiment on the determination of phagocytosis ability of macrophage.Groups and administration were the same to experiment two. Each mouse wasgiven intraperitoneal injection by 10g/L starch solution 2 ml at the 13st day. 24hlater gave the intraperitoneal injection by 2% chicken red blood cell suspension 1ml, 30min later gave the intraperitoneal injection by heparin 0.2ml after puttingthe mice to death, imbibed the peritoneal fluid to smear , dried, stained andobserved under the immersion objective. The result assessment: observed theinformation that macrophage phagocytized chicken red blood cells, enumerated100 macrophages and the number of chicken red blood cells phagocytized bymacrophage, calculated the percentage of phagocytosis.Percentage of phagocytosis = ( the number of macrophages that phagocytizedchicken red blood cells /100 macrophages )×100%. The result showed that AGenhanced the ability of macrophage, increased the function to phagocytize,opposed the inhibition on cell immunity induced by CP.8 Experiment on hemolysin in blood serum. Groups and administration werethe same to experiment two, stamoches were filled for 14d. Each mouse wasgiven intraperitoneal injection by chicken red blood cell suspension 0.2 ml at thefirst day. 1h after the last filling removaled the eyeballs to make blood and tookblood serum, then diluted and took out 1 ml mixing with chicken red blood cellsuspension 0.5 ml and 10% cavia cobaya blood serum 0.5 ml, incubated in theattemperator at 37 ℃ for 30 min, ended the reaction in the refrigerator at 0℃ andcentrifuged, took the supernatant to make shade selection by 721spectrophotometer at 540 nm and determined the absorbance. The result showedthat AG promoted the production of hemolysin in blood serum allergized bychicken red blood cell and opposed the inhibition caused by CP dramatically.9 Test of PFC. Groups and administration were the same to experiment 8,stomachs were filled for 14d, used improved method of SRBC experiment tomeasure. The result showed that AG enhanced the ability of PFC, promotedsecreting function of B lymphocytes, strengthening its creation ability ofantibody, opposed the inhibition induced by CP obviously.Through the study on AG we can draw the conclusion that possiblemechanisms involved in antimutagenesis of Chinese herbal medicine show asbelow:1 AG can inhibit mutagenesis of mutagen, diminish the damage of DNA andenhance the reparation potentia. It maybe plays the constructive role through theprotection and reinforcement on Ts cell and the inhibition on mixed functionoxidase.2 It can enhance organism immune function to ectogenic mutagen obviouslyand exempt the destruction of chromosome by mutagen.3 It has the antioxidation and the function to eliminate free radicals.4 The high expression of P 53 genes and DNA polymerase β can acceleratethe reparation potentia of DNA's function. These mechanisms on molecule levellike above still need a further research..