Study On Screening Effective Parts For Anti-allergic Rhinitis In Centipedae Herba And Preparation Of Nasal Spray

Objective:Centipedae Herba is a dry-flowered whole grass of Compositae genus Goose.It`s a relieving exterior syndrome with pungent and warm natured drugs,which is widely regarded as an essential medicine for the treatment of rhinitis and cold headache.This project mainly studied the extraction process,purification process and fingerprint of the volatile oil and total flavonoids from Centipedae Herba,and screened the effective parts with pharmacodynamic experiments,then prepared appropriate clinical application forms,in order to provide reference for clinical treatment of AR with Centipedae Herba.Methods:?1?The single-factor test combined with orthogonal experimental design was used to optimize the steam distillation extraction process of the volatile oil of Centipedae Herba,and the single factor test combined with the Box-Behnken Response Surface Methodology was used to determine the total flavonoids reflux extraction process of Centipedae Herba.?2?The method of determining the total flavonoids content of Centipedae Herba by UV was established,which was an indicator of the extraction process and purification process.?3?The rat allergic rhinitis model was made by intraperitoneal injection combined with nasal stimulation,when Ovalbumin and aluminum hydroxide were used as the main allergen and the immunoadjuvant,respectively.?4?The volatile oil and total flavonoids of Centipedae Herba were extracted according to the optimal extraction process,respectively.Triamcinolone acetonide spray was used as a positive control.Animal behavioral scores,plasma cytokines?including IFN-?,IL-4,TNF-?,IgE?and HE staining of nasal septal mucosa were selected as indicators to study the therapeutic effect of volatile oil and total flavonoids on AR.?5?HPLC method for determination of rutin content in total flavonoids of Centipedae Herba was established,which was an indicator for purification of total flavonoids.?6?The optimal process for the purification of total flavonoids from Centipedae Herba by D101 macroporous adsorption resin was optimized,and then the fingerprint of the purified liquid was established.?7?The preparation of nasal spray was screened,when the purified flavonoids from Centipedae Herba was used as the main active ingredient.Results:?1?The best process for extracting volatile oil from Centipedae Herba by steam distillation was as follows:crushing Centipedae Herba,passing 50 mesh sieve,adding 8 times of water,soaking for 4h and extracting for 6h,and the content is about 0.17%.The best process for extracting total flavonoids from Centipedae Herba by ethanol reflux method was as follows:crushing Centipedae Herba,passing through a 60 mesh sieve,degreasing with petroleum ether,refluxing with 65%ethanol,the ratio of material to liquid is16 times,extracting twice,2.8 h each time,and the total flavonoids content extracted is about 31.59 mg/g.?2?The UV method of determining the total flavonoids content of Centipedae Herba was established.The sample was dyed by NaNO₂-Al?NO₃?₃-NaOH method,the absorbance value of the dyed sample was detected at 505 nm.The sample concentration?mg/mL?was taken as the abscissa,and the absorbance was plotted on the ordinate and the linear equation was Y=9.7925x+0.0644,R²=0.9985.?3?The behavioral scores of the model group were significantly higher than those of the blank group?P<0.01?.The behavioral scores of the rats in each group were significantly lower than those of the model group?P<0.01?.Compared with the blank group,the plasma IFN-?content in rats of the model group was significantly decreased?P<0.01?,and the levels of IL-4,TNF-?and IgE were significantly increased.P<0.01).Compared with the model group,plasma IFN-?levels were significantly increased after treatment?P<0.01,P<0.05?,IL-4,TNF-?,IgE levels were significantly decreased?P<0.01?,there was no significant difference in the effect of low-and middle-dose volatile oil group,low-dose total flavonoids group and triamcinolone acetonide group on IFN-?content.Histological sections showed that the total flavonoids and volatile oil from Centipedae Herba could repair the pseudo-stratified columnar ciliated epithelial structure,eliminate vascular congestion and edema,and restore the morphology of epithelial cells.The therapeutic effect was within the designed dose range and was positively correlated with the dose.?4?The HPLC method of determining rutin content in flavonoids from Centipedae Herba was established to draw the standard curve with the concentration??g/mL?as the abscissa and the peak area?mAU?as the ordinate.The linear equation is Y=45.21x-58.04,R²=0.9994.?5?The best process for purification of total flavonoids from Centipedae Herba by D101 macroporous adsorption resin was as follows:the concentration of the sample solution is 4.00 mg/mL of total flavonoids?the concentration of rutin is 20.45?g/mL?,and the volume of the sample solution is 5 BV?15 mL?.The sample flow rate is 2BV/h,and the water-soluble impurities are washed away with 5BV pure water after loading.The eluent was 50%ethanol,the eluent is 8BV?24mL?,and the elution rate is3BV/h.?6?The fingerprint of the purified total flavonoid was established.The results showed that there were 10 common peaks in the matched peaks.All the common peaks peaked within 5 to 60 minutes,the baseline was stable,and the peaks were well separated.?7?The best preparation method of nasal spray was as follows:crushing flavonoids of Centipedae Herba,passing 60 mesh sieve.Adding the prescribed amount of propylene glycol,benzalkonium chloride and the prescribed amount of pure water in container A,stirring to dissolve.Adding the prescribed amount of total flavonoids and Tween-80 in container B,fully mixed and evenly dispersed.Adding a small amount of A in B,mixing for5min,continuing to add A to 50mL,stirring evenly,filtering and packing.Conclusion:?1?The RSD of the best extraction process verification test for the volatile oil of Centipedae Herba is 2.83%,when that of the total flavonoids is 3.10%,indicating that both extraction processes were stable and feasible,and can be used for the extraction respectively.?2?The total flavonoids and volatile oils of Centipedae Herba can up-regulate the expression of IFN-?,down-regulate the expression of IL-4,TNF-?and IgE,balance the Th1/Th2 immune response,repair the nasal mucosal inflammatory symptoms,and then exert the therapeutic effect of allergic rhinitis.The efficacy of total flavonoids and volatile oils of Centipedae Herba is better than that of triamcinolone acetonide.And the effect of total flavonoids is slightly better than volatile oil.?3?The total flavonoids content is changed from 17.51%to 61.29%after purification,the purification multiple is 3.50 times,the transfer rate is 87.09%.And the content of rutin is changed from 0.072%to0.241%after purification.,the purification ratio is 3.35 times,the transfer rate is 82.58%.The purification efficiency is higher,and the RSD of the verification test is less than 2%,indicating that the purification process is stable and feasible,and can be used for the purification of total flavonoids from Centipedae Herba.?4?The similarity of 10 batches of Centipedae Herba total flavonoids is greater than 0.9,indicating that the similarity of 10 batches of purified liquid is high,which further indicates that the purification process in this study is stable and reliable.Threr was certain guiding significance for the fingerprint to control quality of total flavonoids.?5?The total spray,the amount of spray and the difference of the volume of each spray are checked,and the results are stable and reliable.The preparation process is relatively simple and meets the basic requirements of industrial production.