Efficacy Evaluation Of Coumarin And Cedrol Against Toxoplasma Gondii

Toxoplasma gondii,as an opportunistic pathogenic protozoa,has a complex life history,a wide range of host types and a variety of transmission routes.Almost all warm-blooded animals,including humans,can become reservoirs of T.gondii.About one-third of the global population is infected with T.gondii,which can seriously affect livestock development and human health.At present,there is no effective vaccine against toxoplasmosis,and the control of toxoplasmosis infection depends on drugs to a large extent.However,the current anti-toxoplasmosis drugs and treatment programs are mainly antibiotics,which may cause bone marrow suppression and other related side effects,posing a great threat to the body’s health,and the drug resistance caused by the abuse of antibiotics brings certain difficulties to the treatment of toxoplasmosis infection.Therefore,it is urgent to find and develop new,low toxicity and high efficiency drugs against T.gondii.In recent years,natural compounds obtained from plants have been tried to be used in the study of disease resistance agents.Among them,coumarin and cedrol have good efficacy in anticancer,antibacterial and antiparasitic aspects,so it is speculated that they have good anti-toxoplasma effect.Therefore,this paper will evaluate the anti-toxoplasma effects of coumarin and cedrol,and preliminarily explore how they play their anti-toxoplasma effects,so as to provide theoretical basis and new ideas for the development of novel anti-toxoplasma drugs and treatment strategies.The effects of coumarin and cedrol on the cytotoxicity and toxoplasma activity of DF-1（chicken embryo fibroblasts）were determined by CCK-8 method.The safe concentration range and half inhibitory concentration(CC₅₀)for DF-1 cell growth were calculated.The results showed that the CC₅₀ values of coumarin and cedrol were 959.8μg/m L and 94.19μg/m L,respectively.The IC₅₀ values of coumarin and cedrol against toxoplasma were calculated and the IC₅₀ values were 445.7μg/m L and 80.26μg/m L,respectively.The selectivity indices（SI）of coumarin and cedrol against T.gondii were calculated to evaluate their anti-toxoplasma effects.The SI values of coumarin and cedrol were 2.15 and 1.17,respectively.In the range of safe concentration,IFA test was used to observe the anti-insect effect in vitro,and the invasion rate of T.gondii tachyzoites after stimulation with coumarin and cedrol was calculated.The results showed that different concentrations of coumarin and cedrol could significantly inhibit the invasion of host cells by T.gondii tachyzoites,and the influence of cedrol on the invasion ability of T.gondii was concentrate-dependent.To evaluate the effects of coumarin and cedrol on the proliferation of toxoplasma tachyzoites in host cells.The results showed that the ability of Toxoplasma to divide and proliferate was also affected,resulting in a significant decrease in the number of Toxoplasma in the cell.In order to determine whether coumarin and cedrol are effective against T.gondii infection in vivo and whether they can prolong the survival time of infected mice,mice were divided into groups.Mice in all groups except the blank group were intraperitoneally injected with 1×10² tachyzoites of T.gondii strain.The mice infected with T.gondii except the control group were given 50 mg/kg pyrimidine,200 mg/kg coumarin and 180 mg/kg cedrol intragastric administration,once a day.The clinical manifestations of the mice in each group were observed,the survival time of the mice in each group was calculated,and the death/survival curve was drawn.The results of coumarin and cedrol treatment of toxoplasma-infected mice showed that the infected mice showed clinical symptoms such as arching back,rough coat and urinary incontinence during the treatment.The feeding,mental and activity of the mice treated with pyrimidine,coumarin and cedrol were significantly stronger than that of the control group.The survival time of the infected mice was significantly prolonged,and the survival rate of the infected mice was increased.The inhibitory effects of coumarin and cedrol on toxoplasma tachyzoites in liver,spleen,lung,kidney of infected mice were detected by nested PCR.The test results showed that compared with the pyrimidine group,DNA bands of T.gondii tachyzoites in tissues and organs of mice treated with coumarin and cedrol were still visible,indicating that T.gondii was still present in mice and had not been completely eliminated.Furthermore,the standard curve of T.gondii tachyzoites was established by absolute fluorescence quantitative PCR method,and the parasite body load in each tissue of mice was calculated.The standard curve y=-0.3005x+11.273,coefficient of variation R²=0.9996,the results more intuitively showed that coumarin and cedrol treatment inhibited the proliferation of toxoplasma in mice,the treatment of mice liver,spleen,lung,kidney parasite load significantly reduced.Pathological tissue sections were made to observe the pathological changes of tissues and organs of infected mice treated with coumarin and cedrol.Pathological observation and analysis of liver,spleen,lung and brain tissue of mice in each group showed that there were no obvious lesions in liver,spleen,lung and brain of mice in the blank group without T.gondii infection.The liver tissues of mice in the control group showed different degrees of edema,congestion and bleeding,indicating that toxoplasma infection caused damage to tissues and organs of mice.No obvious lesion was found in liver and spleen of mice treated with pyrimidine,and slight congestion was observed in lung and brain.In coumarin-treated group,macrophage infiltration in liver,mild spleen congestion,mild interstitial pneumonia,and mild meningeal congestion were observed.Necrotic foci,congestion,hemorrhage,multinucleated macrophage infiltration,pulmonary interstitial congestion and hemorrhage occurred in the liver and spleen of cedrol treatment group.No obvious lesions were observed in the brain.In order to test the protective effect of coumarin and cedrol on the body of infected mice,mice in each group were weighed and labeled,and were weighed again 7 days after oral intragastric administration to calculate the weight gain.The results showed that mice in the infected control group lost weight without medication,but gained weight to varying degrees after treatment with coumarin and cedrol.The liver and spleen of mice in each group were collected and the liver and spleen index were calculated.The results showed that the liver and spleen of mice in the infected control group were significantly enlarged.The spleen enlargement was inhibited after coumarin and cedrol administration,but the inhibitory effect on liver enlargement was not obvious.Then,the activities of serum transaminase ALT and AST were determined to evaluate the protection degree of coumarin and cedrol on the liver of T.gondii infected mice,and the antioxidant damage ability of coumarin and cedrol was evaluated by GSH level.The increase of serum transaminase activity in the control group indicated that toxoplasma infection did cause liver damage,while the activity of serum transaminase in the liver of mice treated with coumarin and cedrol was significantly decreased,suggesting that the liver damage was alleviated,but the level of GSH was not significantly changed.In order to explore whether the anti-toxoplasma ability of coumarin and cedrol is related to oxidative stress,DCFH-DA was used to detect the effects of coumarin and cedrol on the changes of toxoplasma reactive oxygen species（ROS）.The results showed that compared with the control group,the number of worms stained by DCFH-DA after stimulation by coumarin and cedrol increased,which promoted the release of ROS.The expression level of antioxidative stress gene in T.gondii was detected by quantitative PCR.The results showed that compared with the infected control group,the expression of antioxidant damage genes in T.gondii was decreased in coumarin and cedrol.In conclusion,coumarin and cedrol both show good anti-toxoplasma effect in vivo and in vitro,and can effectively relieve the damage caused by toxoplasma infection.Moreover,the anti-toxoplasma action is related to the oxidative stress caused by toxoplasma infection.The results showed that coumarin and cedrol had potential anti-toxoplasma value and were expected to be developed as new anti-toxoplasma drugs.