| Angong Niuhuang Wan,a clinically acute drug,has the effects of clearing heat and detoxifying toxicityand,and clearing phlegm and resuscitation,It is mainly used in the treatment of stroke,coma,high fever syncope,and so on.Niuhuang is monarch medicine,containing ingredients of bile acid(cholic acid,bezoar bile acid,etc.),modern research shows that bile acids have better anti-inflammatory and antibacterial pharmacological effects.Baicalin(BA),belongs to flavonoids,is the main component of Scutellaria baicalensis Georgi of the traditional Chinese medicine and has strong pharmacological activity: anti-inflammatory,anti-oxidant and scavenging free radicals,and protective effects on the cardiovascular system and nervous system.However,due to its own poor water-soluble & fat-soluble and the protective effect of the blood-brain barrier(BBB),affecting the application of baicalin in brain diseases.Object To improve stability and brain targeting of baicalin,taking the advantages of liposome drug delivery system and “unification of medicines and excipients” of cholic acid,prepare the liposome with cholic acid and baicalin.;Through observing the pharmacodynamics of cholic acid-baicalin liposomes(CA-BA-LP)in cerebral ischemia-reperfusion,evaluate its efficacy,and it was verified that after the preparation of liposome with cholic acid and baicalin,the cholic acid had the characteristics of "unification of medicines and excipient",and the two could synergistically alleviate cerebral ischemia reperfusion injury to a certain extent.The toxic effects of CA-BA-LP on nasal cilia by nasal administration was used to evaluate the toxic effects of CA-BA-LP.Method1.With the encapsulation rate and drug loading as evaluation indexes,the best preparation process was selected by membrane dispersion method,reverse evaporation method and foam dispersion method;single factor was used to investigate the order of bile acid addition,the content of bile acid,and the proportionof phospholipid durg,the proportion of phospholipid cholesterol,hydration volume and other factors on the encapsulation efficiency and drug loading.2.Based on the single factor,the preparation process is further optimized by the response surface Box-behnken Design.3.The Zetasizer 3000 particle size analyzer was used to determine the particle size and Zeta potential of the liposomes,and the appearance and structure of the liposomes were observed by transmission electron microscope.The in vitro release curves of baicalin solution and CA-BA-LP were fitted with origin8.5 using different model equations.Storaging 45 d at 4 °C,observe the stability of liposomes.4.Rats were divided into sham operation group,model group(MACO),baicalin monomer group(BA),cholic acid + baicalin monomer group(CA-BA),cholic acid baicalin liposome group(CA-BA-LP),evaluating the effect of CA-BA-LP in cerebral ischemia-reperfusion injury in rats from neurobehavioral scores,brain edema content,TTC staining,cerebral infarct size,and HE staining.5.Through the nasal ciliary toxicity experiment on frogs,evaluate preliminarily the nasal toxicity of CA-BA-LP.Results1.The preparation methods screening results show that the encapsulation efficiency and drug loading of the liposome were the highest by the reverse evaporation method.2.Through the single factor experiment and response surface Box-behnken Design optimization,the best preparation process: phospholipid cholesterol ratio 4.55:1(w /w),phospholipid drug ratio 7.23:1(w / w),cholic acid 9.06 mg,The method of adding bile acid is to add bile acid to the mixed phase of phospholipid and cholesterol,the ultrasonic time is 10 min,the ultrasonic temperature is 20±5 ° C,and the hydration volume is 1 mL.3.The appearance of CA-BA-LP is light yellow suspension.After a certain dilution,it shows light blue opalescence under illumination.It is observed by transmission electron microscopy as spherical or spheroidal nanoparticles with an average encapsulation of 44.1%.The drug loading was 5.05%,the particle size was130~160nm,and the Zeta potential was-8.53~-7.28 mv.The in vitro release accorded with the biphasic kinetic equation,indicating that CA-BA-LP has certain sustainedrelease effect;The storage rate at 4°C was not included in the encapsulation rate and drug loading within 5 days.There are significant changes,both of which have declined on the 20 th day.4.Pharmacodynamic results: after administration,BA group,CA-BA group,CABA-LP group in neurobehavioral score,brain edema content,and cerebral infarct size all decreased,indicating that baicalin has a certain protective effect on brain injury;effects of CA-BA-LP group was better than BA group and CA-BA group(P<0.05);HE staining results showed that baicalin can improve the pathological condition of cerebral ischemia,and the treatment effect could be enhanced after making the liposomes of choleric acid and baicalin together.5.Results of nasal cilia experiment: CA-BA-LP has a relative percentage of ciliary exercise time greater than 85%,and it has no obvious toxicity to nasal cilia and has certain safety.Conclusion The encapsulation efficiency and drug loading of CA-BA-LP prepared by reverse evaporation method are the best.The in vitro release curve conforms to the biphasic kinetics and has a sustained release effect.The pharmacodynamics experiment of CA-BA-LP showed that baicalin has protective effect on ischemic brain injury.After preparing liposome with cholic acid and baicalin together,the therapeutic effect is better than that of baicalin monomer.To a certain extent,two durgs has a synergistic effect.The nasal cilia toxicity test showed that CA-BA-LP has no obvious toxicity to nasal cilia and can be prepared for later nasal administration studies. |
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