| Objective:Ginseng forest frog oil soft capsule is a kind of health food using ginseng and frog oil as main raw materials,which can enhance the immune function of the body.In this paper,the ginseng forest frog oil soft capsule was as the research object,evaluated its toxicological safety,and further to explore its effect oncellular and humoral immune function,and provide experimental basis for the development of ginseng forest frog oil soft capsule.Methods:1.toxicological safety assessmentAcute oral toxicity test in mice: Maximum tolerated dose method was adopted,20 SPFgrade ICR mice were selected,half male and half female,18.6g/kg BW ginseng frog oil soft capsule was administrated orally by a single exposure,the observation period was 14 days,the toxic symptoms and death of mice were observed.Genetic toxicity test:Micronucleus test of mouse bone marrow cells: Oral 30 h test method was adopted,1000 polychromatic erythrocytes(PCE)were counted each mouse,micronucleus rate was calculated.Mouse sperm abnormality test: Adult male mice were selected.The test samples were administered by oral gavage once a day,lasted for 5 days.On the 30 day after last gavage,bilateral epididymides were taken out,sperm suspension smears were prepared,the sperm abnormality rate in mice was calculated.Ames test: Flat plate incorporation mehod was adopted,four strains of salmonella typhimurium were selected,including TA97,TA98,TA100,TA102.The experiment was done with and without S9 metabolic activity system,respectively.The experiment included 5 dose groups,three parallel sampleseach dose group.And the number of colony per dish was counted after culture for 48 h.28 day feeding test in rats:SPFgrade Wistar rats were selected,40maleand40 female,body weight of 65 g ~ 85 g,The experiment term lasted for 28 days.During theexperiment,the general status,poisoning symptoms and deaths of the animals were observed,the weight and intake were recorded once a week,food utility ratewas calculated.On the twenty-ninth day of the experiment,the rats were dissected and hematology,blood biochemistry and histopathology were examined.2.Study on enhancing immune functionThymus and spleen index of mice: The mice were orally administered test samples for 30 days.On the 31 day of the experiment,the thymus and spleens of the mice were removed and examined,organ-body ratiowas calculated.Con A-induced splenic lymphocyte transformation in mice: The mice were orally administered for test samples 30 days,on the 31 day of the experiment,spleen cells suspension was prepared,spleen lymphocyteswere cultured,MTT was added,then were culturedfor 4h,OD570 nm value is measured.Delayed type hypersensitivity(DTH): The mice were orally administered test samples for 30 days.On the 31 day of the experiment,2%(V/V)SRBC was intraperitoneally injectedfor immunization.On the 4 day after immunization,the thickness of the left hind foot wasmeasured.Mouse antibody-producing cells counting: The mice were orally administered test samples for 30 days.On the 31 day of the experiment,2%(V/V)SRBC was intraperitoneally injectedfor immunization.On the 4 day after immunization,the spleens were removed and the spleen cell suspension was prepared,mixed with a certain amount of SRBC and then were added into agarose gel,hemolytic plaque number was measured.Half hemolytic value(HC50): The mice were orally administered test samples for30 days.On the 31 day of the experiment,2%(V/V)SRBC was intraperitoneally injectedfor immunization.The serum was separated 4 days after immunization.SRBC,complement and all’s reagent were added into serum after dilution.OD570 nm value was measured.Results:1.Toxicological safety evaluationIn the acute oral toxicity test,the oral MTD of the mice was more than 18.6g/kg BW,and the acute toxicity was classified as the actual non-toxic level.The results of genetic toxicity test showed that micronucleus test of mouse bone marrow cells,mouse sperm abnormality test and Ames test were negative.Experimental results of 28 d feeding in rats showed that during experimental period,the growth and development of rats were in good condition,there were no significant differences in body weight,total food utility rate,blood index,blood biochemical index,and organ-body ratio(P>0.05)between the dose groups and control group.The oathological examination showed that no abnormal pathologic changes were found in the liver,kidney,spleen,testes and other organs of rats in the high dose group and the control group.The oral NOAEL of rats of ginseng forest frog oil soft capsule was more than 5.33g/kg BW(dose of high dose group),equivalent to100 times the daily intake of adults.2.Study on enhancing immune functionIn the cellular immunity and humoral immunity test,there was significant difference between the high dose group and the negative control group(P < 0.05),ginseng forest frog oil soft capsule could enhance the degree of DTH,promote the proliferation of spleen lymphocytes induced by Con A,increase the number of antibody-producing cells in mice,and increase the content of serum hemolysin(HC50).Compared with the negative control group,there was no significant difference in thymus / body weight and spleen / body weight in each dose group.Conclusion:1.The mice oral MTD of ginseng forest frog oil soft capsule was more than18.6g/kg BW,and the acute toxicity grade was the actual non-toxic level.Three genetic toxicity tests(micronucleus test of mouse bone marrow cells,mouse sperm abnormality test,Ames test)were negative.In the 28 day feeding test,there is no obvious toxic effect,NOAEL was more than 5.33g/kg,equivalent to 100 timesthe daily intake of adults.2.In a certain dose,Ginseng forest frog oil soft capsule can enhance cellular immune function and humoral immune function. |
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