Screening Of Natural Products Against Toxoplasma Gondii And The Anti-T. Gondii Active Of Licochalcone A

Toxoplasma gondii?T.gondii?is an obligate intracellular and apicomplexan protozoan parasite with a complex life cycle;it can infect a wide range of warm-blooded animals,including humans,which can cause serious public health problems.It is important to recognize the role of natural products as a huge compound library for the treatment of toxoplasmosis.In this experiment,we selected a series of Chinese medicine monomers with anticarcinogenic and antiparasitic activities to screen the activity of anti-toxoplasma,and revealed the mechanism,which provided a theoretical basis for the clinical treatment and the development of anti-toxoplasma drug.The possible toxic effects of baicalin,wogonoside,liquiritin,licochalcone A,arecoline hydrobromide,toosendanin,tuberostemonine,bruceine D,chlorogenic acid,L-mandelic acid and D-mandelic acid on HFF cells were estimated based on the reduction of MTT.The minimum inhibitory concentration of each compound was used for preliminary screening of anti-T.gondii in vitro,and Giemsa staining was used to observe the effect.The results of cytotoxicity test showed that the highest concentration of 11 kinds of traditional Chinese medicine monomers on HFF cells were:baicalin 200?g/m L,wogonoside 80?g/m L,liquiritin 70?g/mL,licochalcone A 10?g/m L,arecoline hydrobromide 20?g/mL,toosendanin 5?g/mL,tuberostemonine 40?g/m L,bruceine D1?g/m L,chlorogenic acid 400?g/m L,L-mandelic acid 80?g/m L and D-mandelic acid 80?g/m L.Among them,baicalin and licochalcone A has a significant effect on promoting the proliferation of HFF cells at the concentration of 60-150?g/mL and 4-8?g/m L,and it has the strongest effect at about 100?g/m L and 5?g/m L.Preliminary screening tests showed that only licochalcone A had obvious anti-T.gondii effect.The concentration of licochalcone A in the cell culture medium was determined by HPLC,and the specificity,linear relationship,precision and recovery rate of the method were investigated.Then,the time curve of licochalcone A in the medium was measured.The established method is highly specific.licochalcone A has good linearity in the concentration range of 0.5-2.0?g/mL,and the recovery rate is high and the precision is good.The concentration of licochalcone A in the medium decreased with time,and the licochalcone A of three concentrations?1.3?g/m L,1.2?g/m L,0.8?g/m L?at 48 h was not detected.To investigate the effect of licochalcone A on the invasion of T.gondii to HFF cells,licochalcone A was added at different concentrations,then tachyzoites was added culture for 24 h.The anti-T.gondii invasion test showed that when the concentration of licochalcone A was higher than 1?g/mL,no tachyzoites and parasitophorous vesicles were observed.With the decrease of licochalcone A concentration,the more tachyzoites invaded into the cells.For measurements of parasite burden the method of qPCR was established.The precision and recovery rate of the quantitative method were determined.Objective to investigate the antiproliferative effect of Licochalcone A on T.gondii tachyzoites and calculate the IC₅₀.Meanwhile,Giemsa staining was used to observe the effect of different concentrations of Licochalcone A on the proliferation of T.gondii.The RSD value of precision and recovery rate was less than 5 when establishing the qPCR method to evaluate parasite burden.The results of anti-proliferation test showed that as the concentration of Licochalcone A increased,the number of parasitophorous vesicles,the number of tachyzoites,and the average number of tachyzoites in the parasitophorous vesicles were decreased.The amount of tacyzoites load at each concentration of Licochalcone A was measured by qPCR.The results agreed with the results of Giemsa staining,and the IC₅₀ of licochalcone A was 0.848?g/mL.The effect of Licochalcone A on the ultrastructure of tacyzoites was observed by SEM and TEM.The lipid accumulation was observed by confocal laser scanning microscope,and the fluorescence intensity was measured by flow cytometry.SEM showed that tachyzoites decreased in size and became rounded,with surface depressions after treatment with different concentrations of licochalcone A.The TEM showed that,with the time increased,the liqid body were observed in the cytoplasm,and the membrane structure of each organelle disappeared and eventually the tachyzoites died.Nile red staining also showed that the accumulation of neutral lipids in tacyzoites increased with time.In order to evaluate the anti-T.gondii activity of licochalcone A,we first determined the maximum safe dose of orally and intraperitoneal injection in mice.An acute T.gondii infection model of mouse was established,and to evaluated the treatment effect of licochacone A.licochalcone A in different concentrations were treated the infected mouse by oral and intraperitoneal injection and draw the survival curve.To determine the safety of the treatment dose,the mice were treated with the same treatment as the previous experiment,and the survival rate of the mice was observed.The tissues were taken on the fortieth day,and the changes of the tissues were observed by HE staining.The maximal dose of oral licochalcone A reached 1000 mg/kg,and without any side effects.When the dose reached 386 mg/kg.bw,the mouse died.After injected intraperitoneally for 3 times in 24 h no death occurred at 1000 mg/m L,but slight ataxia occurred in some cases.The survival rate of the low dose intraperitoneal injection group was 80%,in the middle dose intraperitoneal injection group was 90%,and in the high dose intraperitoneal injection group was 100%,and the mouse in the oral dose group all died,but the average survival time was prolonged,and no significant difference between the various dose groups.The results showed that the protective rate of licochalcone A treated acute T.gondii infection mouse was reached 100%by intraperitoneal injection.In conclusion,licochalcone A can inhibit the invasion and intracellular replication of T.gondii and protect the mice model of acute infection.Ultrastructural observation shows that the inhibitory effect of licochalcone A on T.gondii may be related to lipid metabolism.These experiments show that licochalcone A is a potential anti-T.gondii and is expected to become a drug against T.gondii.