The Study On The Effect Of The Osthole Extracts Microcapsules Agsinst The Trichomonas Gallinaes

It is common to use metronidazole and tinidazole to prevent and cure the trichomonas gallinaes preciouly,the effect of these two drug is very good,but after the long-term use,it has some severe toxic and side effects,so these two drug has been classified into the category of veterinary drugs which is banned to use in the food animals.Thus,it is of great significance to study the safe drug against the trichomonas gallinaes with high efficiency,low toxicity and minimum side effects.Some Chinese herbs in traditional medicine has obvious effects against the trichomonas gallinaes in vitro,such as osthole,the root of Chinese Pulsatilla,trollious chinensis Bunge,SopHora flavescens,pepper,antifebrile dichroa,Artemisia apiacea and so on.However,the current researches are focused on the trichomonas vaginalis in vivo,and the research on the Chinese herbs against the trichomonas gallinaes is very limited as well as all the compound preparation.In view of the trichomonas gallinaes and trichomonas vaginalis in vivo are in the same species,so it can be inferred that the Chinese herbs which can control the trichomonas vaginalis will also can against the trichomonas gallinaes.There are some scholars have proved that the osthole extracts has the function to control the trichomonas gallinaes,although the effect is really good and has been put into practice,there is no research on the effect of single effective ingredient of the osthole against the trichomonas gallinaes and the shapes of the drug currently.So,in this study,the single ingredient osthole were selected to investigate the mortality of the trichomonas gallinaes and the changes of its shapes under the light microscopy and scanning electron microscopy by cultivation of the trichomonas gallinaes in vitro culture,which has laid a basis for the study on the complementary and alternative medicine against the trichomonas gallinaes.Moreover,the development of the formulations of Chinese herbs is still a hotspot,especially which with a sustained release effect and easily to take are popular among all the farmers.Once Chinese herbs are made into the microcapsules,it has the advantage to cover its former odour,improve the stability of drug and its working time and decrease the times of taking drug and so on.Mixing the oral microcapsules with animal fodder and drinking water not only can achieve the effect of treatment,but also can reduce the labor force in the farm.Therefore,this study also focused on the preparation process of osthole microcapsules and the pHarmacodynamics of resistance to the trichomonas gallinaes in vitro and vivo as well as its pHarmacokinetics in animals.Test I The effect on osthole against the trichomonas gallinaes in vitro and morpHological observation:Objective:In order to study the inhibitory of osthole on the cultivation of the trichomonas gallinaes in,vitro and its effect on the morpHology of trichomonas.Method:The test has set up 4 different osthole concentration groups,0（blank control）,1,3,5 mg/mL respectively and the samples were sampled at 2,4,6,8,12 and 24 h after the cultivation from different groups respectively.The mortality rate of the trichomonas gallinaes were observed by light microscope with its morpHological changes by scanning electron microscopy（TEM）.Results:With the increase of the osthole concentration,the mortality rate of trichomonas gallinaes was also improved,that is to say,the ratio between the two was proportional,with the death time shortened,which means that the concentration of osthol and the death time of the trichomonas gallinaes is inversely proportional.According to the test,the 5 mg/mL group has the best effect,which can kill the whole trichomonas gallinaes after the 12h treatment,while the 1mg/mL concentration of the drug group did not kill all the trichomonas gallinaes until 24h of treatment,which failed to reach the expectation to cure the illness.The 3 mg/mL concentration group kill all the trichomonas gallinaes at the 24h treatment,which has the slower effect compared to 5 mg/mL group.Take the trichomonas gallinaes under the treatment of the 5 mg/mL concentration as a sample and observe under the scanning electron microscopy,it can be observed that the longer time of the drug treatment,the more incomplete of the morpHology of trichomonas gallinaes.When the treatment of drug was at 0.5h,the trichomonas gallinaes was shaped from pyriform to circular,while the shape was complete;when the time was at 2h,the edge of the body was blurred with the wrinkles on its surface and some depression on its part.When 4h,the depression area was expanded with some cavity in it,the insect shape becomes irregular.Finally,the body parasites was split into pieces.The results showed that osthole had obvious effect against the trichomonas gallinaes,which is deserved a further study.Test Ⅱ The preparation of the osthole microcapsules:Objective:In order to obtain the osthol microcapsules with sustained release effect,the osthol was prepared into microcapsules by using different processes.Method 1:the preparation of the sodium alginate-osthol microcapsules,using Sodium alginate as the capsule materials as well as the CaCl₂ aqueous solution as the curing agent to product the osthol microcapsules.This method is used to measure the effect of the rate of sodium alginate and osthol on the entrapment efficiency and drug loading capacity of the microcapsules.Method 2:Using the gelatin/acacia-osthol curing agent to prouduct the osthol microcapsules.This method uses the orthogonal design L9（34）to investigate the effect of the rate of the microcapsules materials and drugs（A）,the amount of PEG-400（B）and glutaraldehyde（C）and the temperature at the period of being the microcapsules（D）on the the entrapment efficiency and drug loading capacity of the microcapsules.There are three different degrees which is set to do the above investigation about each factor.Results:According to the method 1,the changes on the the rate of the microcapsules materials and drugs will determine the entrapment efficiency and drug loading capacity of the microcapsules.The higher rate of the microcapsules materials and drugs,the higher entrapment efficiency and drug loading capacity of the microcapsules.When the rate of the microcapsules materials and drugs is 1:3,the entrapment efficiency and drug loading capacity are the highest,62.22%and 20.17%respectively.According to the method 2,the four factors,the rate of the microcapsules materials and drugs（A）,the amount of PEG-400（B）and glutaraldehyde（C）,the capsule temperature（D）have different influences on the entrapment efficiency and drug loading capacity.The influence on the entrapment efficiency of microcapsules was A>C>D>B and the optimal combination was A₃B₃C₃D₁.The influence on the drug loading capacity was A>C=D>B and the optimal combination was A₃B₃C₃D₁.By comparing the drug loading capacity,entrapment efficiency and microcapsule shapes completely,the optimal combination is the fourth,A₂B₃C₂D₃（on the orthogonal test designs table）,the ratio of the drug and microcapsules material is 1:2,the amount of PEG-400 is 5%,and the amount of aldehyde was 0.8% and the bagging temperature was 65℃.Test Ⅲ The quality evaluation of the osthol microcapsules:Microcapsules prepared in Test Ⅱ were subjected to measure the partical size of microcapsules and the releasing rate in vitro,which is used to provide the sale and stable microcapsules with a certain sustained release for the following test.Methods:Using the gelatin/acacia-osthol microcapsules prepared in the Test Ⅱ to do the measurement about the the shapes of microcapsules,the partical size and the releasing rate in vitro.Results:Most of the microcapsules were circular with a few of them were spindle and even irregular,the membrane of capsules were clear and transparent and the internal drug had the black dotted shadow.The bigger the shadow was,the higher amount of drug in the microcapsules.The partical size of the microcapsules ranged from 77 to 350μm.The stability test showed that the preservation rates of osthole at the microcapsules group were 99.79%,99.12%and 98.97%,respectively,after 4,6 and 8 months storage at room temperature,while that of drug microcapsules was 89.91%,78.43%and 66.77%,respectively,which indicated that producing the osthole into the drug microcapsules could improve the stability of the osthole significantly and avoid the decomposition in the air and under light.The release data in vitro showed that the release in vitro of osthole microcapsules was more slowly than that of medicine in artificial intestinal juice,and when treatment was at 0.25 h,the release rate of drug was 100%,while that of microcapsules was only 95%after 24h,that is to say the microencapsulation of osthole has a certain sustained release effect.Test Ⅳ The study on the pHarmacodynamics of the osthol microcapsules against the trichomonas gallinaes in vitro:This test is set to evaluate the pharmacodynamics effect of the osthol microcapsules against the trichomonas gallinaes in vitro.Methods:（1）Test in vitro:the osthole microcapsules were added into the trichomonas gallinaes hepar-peptone-glucose medium,which makes the concentration of osthole in the culture medium was 1 mg/ml,3 mg/ml and 5 mg/ml respectively,which were set up as 3 test groups.Simultaneously,the blank microcapsule control group was also set.The activity of Trichomonas gallinaes was observed by blood counting chambers after taking drug with the intervals of 2,4,6,8,12,24,36 h and the mortality of Trichomonas gallinaes was calculated by the standard cytometry.（2）Test in vivo:The forty 45-day-old young pigeons with severe trichomonas gallinaes illness were randomly divided into 5 groups,8 in each group and 5 days for treatment.Group 1:Each pigeon was fed with 50 mg osthole drug for 5 days;Group 2:each pigeon was fed with microcapsules containing 50 mg of osthole every day for 5 days;Group 3:each pigeon was fed with 50 mg osthole drug on the first day and the third day;Group 4:Each pigeon was fed with microcapsules containing 50 mg of osthole in the first day and the third day;Group5:The positive control group without treatment,besides,group 6 was set as a healthy negative control group.the death of Trichomonas gallinaes was examined on the 5th day and the 7th day after the first treatment by microscope respectively.Results:The results showed that in vitro insect,concentrations of 1 mg/ml,3mg/ml and 5 mg/ml Osthole microcapsule test group worms 36 hours the mortality was 78.18%,85.01%,100%,while the blank microcapsule group was 20.39%,control group mortality rate was 20.18%.Compared with the blank group,the osthole microcapsule group was significantly different from the blank group,indicating that the ostholic microcapsule had significant in vitro insect-resistant effect.The mortality rate of the caterpillar larvae and the mortality rate of the blank group were close Osthole microcapsule group compared with the control group significant difference even very significant,indicating osthol microcapsules have significant in vitro insect resistance;blank microcapsules mortality Trichomonas pigeon group and the control group mortality is closer,Indicating that the blank microcapsules do not kill the trichomoniasis doctrine.The results of test against trichomonas gallinaes in vivo showed that treating osthole drug and its microcapsules for continuous five days can kill all the parasites,the mortality rate was 100%,the infection rate of the microcapsules treatment group was significantly lower than the drug group on day 1 and day 3,the infection rates of ttrichomonas gallinaes in microcapsules and drug treatment groups were 75%and 62.5%on day 5,50%and 37.5%on day 7 respectively,which showed that the effect of the microcapsules treatment group against trichomonas gallinaes was superior to the drug group.The results showed that both the osthole group and its microcapsules treatment group have significant effect on healing trichomonas gallinaes for treating continuous 5 days,when the times of treating was lacking,the effect of microcapsules group was better than drug group.Test V PHarmacokinetics of osthol with microcapsules in vivoMethods:Five rabbits fed the same amount of self-made osthol microcapsules that equivalent to 150mg/kg osthole.After administration,collected plasma samples at 5,15,30,45,60,75,90,120min and determination of plasma osthol content by high performance liquid chromatograpHy.Result:Result showed that the peak value of t1/2 was（69.31±0.15）h,C_max was（43.67±3.07）μg/mL,the peak time T_max was（60.00±1.03）min,Ke was（0.01±0）h-1,AUC_0～t was（2767.15±18.10）mg h ml-1,AUC_0～∞ was（3207.5815 ±18）and the blood concentration in the piggen was 10.48μg/mL after 120min.The results showed that,if osthol preparation into ostrich microcapsules,and fed to the pigeon,Will be maintained in the pigeon a certain concentration of plasma longer.;t_max（60.00± 1.03）min;C_max（43.67±3.07）mg ml-1;Ke（0.01±0）h-1;AUC_o～t（2767.15±18.10）mg h ml-1;AUC_0～∞（3207.5815±18.00）.