| ObjectiveThis study from supporting Yinhuang preparation treatment of the upper respiratory tract infection,the preparation of chlorogenic acid,baicalin were nano nasal in situ gel preparation Using the star design,process optimization,prepared to meet the requirements of nasal chlorogenic acid and baicalin in nano nasal in situ gel,through to the nasal mucous membrane permeability and the degree of release in vitro and nasal mucous membrane irritation The safety and efficacy of nasal mucosal immune response were preliminarily investigated to provide a safe,convenient and long-lasting preparation for enhancing nasal mucosal immunity.Method（1）Performance liquid chromatography（HPLC）method is adopted,the setting up of chlorogenic acid and baicalin content determination method of nanoparticles nanoparticles prepared by ionic crosslinking method,coating rate as the evaluation index,the optimum of chitosan acetic acid solution p H,the supply of drugs and the concentration of chitosan,the preparation of chlorogenic acid and baicalin were loaded nanoparticles,and quality inspection.（2）Preparation of temperature-sensitive gel,using phase transition temperature as evaluation index and P407 and P188 as variables,process optimization and quality evaluation were carried out.（3）Dialysis bag method was used to investigate the release in vitro,HPLC method was used to determine the concentration of drugs in the solution and draw the curve.Membraneless dissolution method was used to investigate the effect of gel on the release of drugs under the action of dissolution,and draw the dissolution curve.（4）The mucosal morphology and ciliary movement of the upper jaw of the frog were observed,the safety of the nanoparticle in situ gel was investigated to establish the immunocompromised mouse model,the content of SIg A and lysozyme in saliva of the mice were determined,and the effect of chlorogenic acid-baicalin co-loaded nano-nasal gel on the immune response was investigated.Result（1）The chromatographic peaks of both chlorogenic acid and baicalin were more than 8000,the peak pattern was stable,and the trailing factor ranged from 0.95 to 1.05.The separation between the two peaks was good,and the method was not interfered by the heteropeaks,which indicated that the method was highly specific and met the experimental requirements The RSD%of precision and stability were less than 2%,indicating that the instrument and method had good precision,good repeatability and good drug stability,which met the requirements of sample analysis.（2）The optimal formulation of chlorogenic acid-baicalin co-loaded nanoparticles was as follows:chitosan（CS）was dissolved in 2%acetic acid aqueous solution（25m L）,dissolved evenly by magnetic stirrers,and prepared into a chitosan solution of 2.8mg·m L-1.11mg of baicalin and chlorogenic acid were added into PEG400 10m L to dissolve into yellow transparent solution,which was slowly dropped into chitosan solution and stirred in a magnetic stirrer at the rate of 1000r·min-1.After adding,the mixture was stirred for 10min,and p H5 was adjusted with 1mol·L-1Na OH solution.10m L 1g·L-1 sodium tripolyphosphate（TPP）solution was added with 2 drops/s and stirred at 1000r·min-1.After the addition of sodium tripolyphosphate solution,the co-loaded nanoparticles of chlorogenic acid and baicalin were obtained by stirring for 20min.The predicted encapsulation rate of chlorogenic acid was 44.72%,and baicalin was 55.32%.The optimal prescription was repeated 3 times.The encapsulation rates were44.32%,44.53%and 45.15%for chlorogenic acid and 55.22%,55.87%and54.65%for baicalin,respectively.The average particle size of the nanoparticles was（536.1±16.24）nm,and the polydispersion coefficient was（0.310±0.053）.And observed under electron microscope,the shape of the nanoparticles was uniform and full,and the Zeta potential（24.39±0.27）m V was measured.（3）The preparation process of chlorogenic acid-baicalin co-loaded nanoparticles in nasal cavity in-situ gel was as following:18.5%P407 was weighed and added into the chlorogenic acid-baicalin co-loaded nanoparticles suspension,which was dispersed evenly under magnetic stirring,and left overnight in 4 refrigerator until P407 was fully swollen and dispersed evenly 1%P188 was slowly added to the system,dispersed and integrated under magnetic agitation,and placed in 4 refrigerators overnight until P188 was completely expanded and dispersed evenly.Finally,1mol/L Na OH was added to adjust the p H of the system to about 5.5 under magnetic agitation.The optimized process was repeated for three times,and the Tgel results were 32.1℃,respectively 31.7℃32.3℃,good repeatability chlorogenic acid-baicalin co-loaded nano-nasal gel in situ without stratification after centrifugation,the measured p H was 5.5,and the phase transformation speed was fast at 32℃.（4）The dialysis bag method simulated the drug release process in vitro,and the membraneless dissolution method simulated the gel dissolution process in nasal mucus.The experimental results showed that the release of chlorogenic acid-baicalin was mainly affected by the gel dissolution in nasal mucus,and part of the release was released by diffusion.（5）In the upper jaw cilia experiment of bullfrog,the mucosal surface of 1%sodium deoxycholate group was fragmented and the edge of cilia completely fell off after 30 min,indicating that the cilia were toxic to the mucosal tissue.In the blank gel group and the chlorogenic acid-baicalin co-loaded nano-nasal gel preparation group,the mucosal surface was neat and the number of cilia was large,indicating that both blank gel and drug-containing gel had good safety.In the determination of bullfrogs percentage is continuous movement of the cilia and ciliated conveying rate of the results showed that the chlorogenic acid and baicalin in continuous movement of nano nasal gel preparation group percentage and cilia oxygen transportation rate is much higher than 1%to cholic acid sodium group,description of chlorogenic acid and baicalin were cilia of nano nasal gel has no obvious toxicity,can be applied in the nasal cavity.Compared with the control group,the content of SIg A in saliva of mice in the model group was significantly decreased（P<0.05）lysozyme content was significantly decreased（P<0.01）,indicating that continuous cold stimulation can reduce the mucosal immune function of upper respiratory tract,indicating that the high dose chlorogenic acid-baicalin co-loaded nano-nasal gel preparation group SIg A content and lysozyme content were often significantly higher than the model（P<0.05）,there was no significant difference between low dose group and model group（P>0.05）;SIg A content in F.forsythia group was significantly higher than that in model group（P<0.05）The results showed that the co-loading of chlorogenic acid-baicalin nano-nasal gel preparation could improve the SIg A decreased by cold stimulation and the lysozyme content,indicating that the co-loading of chlorogenic acid-baicalin nano-nasal gel preparation could enhance the mucosal immune function of upper respiratory tract.ConclusionUsing the star design,process optimization,prepared to meet the requirements of nasal chlorogenic acid and baicalin in nano nasal in situ gel,nasal residence time can be improved,for nasal mucosa SIg A can improve the low immune model mice,and lysozyme content for later research for theoretical basis is provided for the prevention and treatment of upper respiratory tract infection... |
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