| Compound Xinyi nasal drops is a hospital preparation of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine.Its dosage form is nasal drops,which is mainly used for the treatment of allergic rhinitis.The prescription consists of magnolia liliflora,moutan cortex,wine radix scutellariae,centipeda minima and cicada slough,etc.It has the effect of dissipating wind,clearing heat,cooling blood and drying dampness.Previous studies have shown that compound Xinyi nasal drops have anti-inflammatory,anti-allergic,immune regulation and other effects,and can significantly improve the symptoms of allergic rhinitis such as nasal itching,nasal congestion and runny nose,and the combined administration of other drugs has better therapeutic effect.This preparation has been clinically used in the First Affiliated Hospital of Anhui University of Chinese Medicine for more than 20 years,with definite curative effect and no obvious toxic side effects,and has obtained the registration approval number of preparation(Anhui pharmaceutical system word Z20080006).Since the application of hospital preparation compound Xinyi nasal drops was made before2008,the preparation process and quality evaluation of the preparation were relatively simple,and the components measured were relatively simple,which could not reflect the characteristics of the multi-component TCM compound,nor could it fully evaluate its intrinsic quality.Meanwhile,only preliminary pharmacodynamic study was conducted on its treatment of allergic rhinitis.The exact mechanism of action remains unclear.Objective:In order to improve the scientific rationality and quality control level of the preparation process of compound Xinyi nose drops,the Box-Behnken response surface method was used to optimize the water extraction process of compound Xinyi nose drops,and the single factor method was used to screen the alcohol precipitation process,the concentration of the extract and the p H value of the solution,and the best preparation process was optimized and verified.The fingerprint was established by UPLC method and the content was determined.Meanwhile,the quality markers of compound Xinyi nasal drops were predicted by network pharmacology,which provided the basis for the quality control and pharmacodynamic study.To explore the therapeutic effect of compound Xinyi nasal drops on guinea pigs with allergic rhinitis and the regulatory effect of STAT3/IL-17A/NF-κB signaling pathway in guinea pigs with allergic rhinitis,so as to provide reliable experimental basis for further research and development of this preparation.Methods:1 Optimum preparation process of Compound Xinyi nasal dropsThe comprehensive scores of magnoflorine,paeoniflorin,baicalin,wogonoside,baicalein,magnolin,brevilin A and polysaccharide contents and dry extract yield were used as evaluation indexes.The main influencing factors and levels were determined by single factor experiment,and then the extraction process of compound Xinyi nasal drops was optimized by Box Behnken experiment design.The single factor method was used to screen the alcohol precipitation process,the concentration of the extract and the p H value of the solution,and finally the best preparation process was obtained and verified.2 Study on quality control of Compound Xinyi nasal DropsTen batches of compound Xinyi nasal drops were prepared with the best preparation technology,and UPLC fingerprints were established by ultra high performance liquid chromatography.The candidate quality marker(Q-Marker)was preliminarily predicted by identifying related components with reference products,and the content of related components was determined.The “core ingredient-target-pathway”network was constructed by network pharmacology method to further predict Q-Marker and core target of compound Xinyi nasal drops,providing basis for quality control and pharmacodynamic study of compound Xinyi nasal drops.3 Regulation effect of Compound Xinyi nasal drops on STAT3/IL-17A/NF-κB signaling pathway in guinea pigs with allergic rhinitisThe guinea pig model of allergic rhinitis induced by OVA was established,and the guinea pigs were randomly divided into model group,compound Xinyi nasal drops 3.46mg/kg,10.37 mg/kg dose groups,Rhinocort group,and normal control group,and continuous nasal drops for 25 days.After treatment,guinea pigs were scored for nasal symptoms.HE staining was used to evaluate the inflammation of nasal mucosa.The expression levels of inflammatory cytokines IL-6,IL-17 A and TNF-α in guinea pig serum were detected by ELISA.The m RNA expression levels of STAT3,IL-17 A and NF-κB p65 in nasal mucosa tissues of guinea pigs were detected by q RT-PCR.The expression levels of STAT3,p-STAT3,IL-17 A,NF-κB p65 and p-NF-κB p65 were detected by Western blot.Results:1 Optimum preparation process of Compound Xinyi nasal dropsThe optimal water extraction process: extraction times were 2 times,extraction time was 90 min(the first extraction time was 55 min,the second extraction time was35 min),liquid-solid ratio 13:1(the first extraction liquid-solid ratio was 13:1;The liquid-solid ratio of the second extraction was 11:1),soaking time was 60 min;The optimal alcohol precipitation process: 70% ethanol volume fraction,alcohol precipitation time 48h;Other preparation conditions: A rotary evaporator was used for low-temperature decompression concentration,and the concentration temperature was kept at 65℃-78℃,the vacuum degree was between 0.060 MPa-0.096 MPa,and the optimal p H value of the liquid was 6.5-7.0.2 Study on quality control of Compound Xinyi nasal DropsThe UPLC fingerprints of 10 batches of compound Xinyi nasal drops were established by ultra-high performance liquid chromatography,15 common peaks were identified,and 7 components were identified,which were magnolflorine,paeoniflorin,baicalin,wogonoside,baicalein,magnolin and brevilin A,with the similarity greater than 0.992.The results of content determination showed that the average content of 7components was 0.0133-2.0865 mg/m L,RSDS were 5.03%-11.52%.Combined with the analysis of network pharmacology,118 targets were obtained by screening the above7 components acting on allergic rhinitis.The top 10 key targets were TNF,IL6,ALB,AKT1,VEGFA,STAT3,TP53,EGFR,MMP9 and PTGS2 in order from the highest degree to the lowest degree.GO analysis and KEGG signal pathway enrichment analysis showed that the treatment of allergic rhinitis with compound Xinyi nasal drops was mainly related to inhibiting the production of inflammatory factors and playing the role of immune regulation.Based on the analysis of key targets obtained from literature and previous screening,it was found that seven components play a role in the treatment of allergic rhinitis mainly through STAT3/IL-17A/NF-κB signaling pathway.3 Regulation effect of Compound Xinyi nasal drops on STAT3/IL-17A/NF-κB signaling pathway in guinea pigs with allergic rhinitisAfter OVA induction,guinea pigs showed continuous nose scratching,sneezing,runny nose and other symptoms.After treatment with compound Xinyi nasal drops and Rhinocort,the symptoms of allergic rhinitis in guinea pigs were significantly improved.HE staining of guinea pig nasal mucosa tissue showed that the upper epidermal cells of guinea pig nasal mucosa in model group were shed,accompanied by tissue edema and a large number of eosinophil infiltration,and the above symptoms were significantly relieved after drug treatment.ELISA assay showed that compound Xinyi nasal drops could significantly reduce the expression levels of IL-6,IL-17 A and TNF-α.q RT-PCR and Western blot tests showed that compound Xinyi nasal drops could significantly reduce the expression levels of STAT3,IL-17 A,NF-κB p65 m RNA and their phosphorylated proteins,and inhibit the STAT3/IL-17A/NF-κB signaling pathway.Conclusion:The optimum preparation technology of compound Xinyi nasal drops is reasonable and feasible.The establishment of fingerprint and multi-component content determination method were stable and feasible.Combined with network pharmacology,seven potential Q-markers in compound Xinyi nasal drops were predicted.It was found that STAT3/IL-17A/NF-κB pathway may be one of the main pathways for the treatment of allergic rhinitis.Pharmacodynamic experiments further verified that compound Xinyi nasal drops may play a therapeutic role in regulating the inflammatory response of nasal mucosa through STAT3/IL-17A/NF-κB pathway. |
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