A Study On The Effective Substances Of The Classical Prescription Huanglian Ointment And The Mechanism Of The Treatment Of Eczema

Purpose:Based on the theory of traditional Chinese medicine,the extraction process and molding process were optimized to study the efficacy and mechanism of Huanglian ointment,so as to develop it into a safe and effective external preparation with clear components,controllable quality,stable curative effect.Methods:1 Extraction process researchBased on the traditional extraction method of Huanglian ointment,the best extraction process parameters were optimized by using modern technology and single factor test combined with response surface design.2 Formability process researchUse modern traditional Chinese medicine pharmaceutical technology,a response surface optimization test was carried out on the basis of single factor test.The stability of Huanglian ointment,the uniformity of curcumin as an indicator and the comprehensive score of cumulative berberine hydrochloride permeation were used as evaluation indicators.Optimum process parameters for moldability are selected.3 Study on the effective components of Huanglian ointmentUPLC-Q-TOF-MS was used to screen the data according to mzcloud,mzvavel and chemspider databases.According to the information of molecular weight,characteristic ions,mass spectrometry fragmentation law,chromatographic retention time and other information of each component,and combined with literature reports,the effective components in Huanglian ointment were clarified.4 Establishment of fingerprint of Huanglian ointmentHPLC was used to screen mobile phase,detection wavelength and extraction solvent.The similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine was used to establish the fingerprint method of Huanglian ointment.At the same time,multivariate statistical analysis,such as principal component analysis（PCA）,orthogonal partial least squares discriminant analysis（OPLS-DA）and other methods were combined to fully integrate the complex chemical data for reference.The quality difference of 21 batches of Huanglian ointment was observed.5 Study on efficacy and mechanism of Huanglian ointmentThe therapeutic effect of Huanglian ointment on eczema was investigated by detecting the skin healing,ear swelling,pawing behavior and inflammatory cell infiltration in mice skin;On this basis,the expression of substance P in skin tissue of mice in each group was detected by immunohistochemistry;The serum levels of Ig E,INF-γ,TNF-α,IL-6,IL-4 and IL-2 were detected by ELISA;Western blot was used to detect the expression of p-p38/p38,p-ERK_1/2/ERK_1/2,p-JNK/JNK,NF-KBp65 protein in the skin tissue of mice in each group,so as to explore the mechanism of Huanglian ointment in the treatment of eczema.Results:1 Extraction process researchUsing single factor experiment combined with response surface design experiment,the best extraction process is obtained:frying time（A）59.52min,frying temperature（B）141.98℃,soaking time（C）23.79h.In order to facilitate the actual operation,the verification conditions were revised to the frying time of 60min,the frying temperature of 140℃,and the soaking time of 24h.Frying extraction was carried out under this technological condition,and 3 sets of verification experiments were done in parallel.The results:the average value of the comprehensive scores of the 3 groups was 96.19%>95.34%.The results showed that the extraction process of Huanglian Ointment was stable and feasible.2 Formability process researchUsing single factor experiment combined with response surface design experiment,the best formability process is obtained:the ratio of medicinal oil:beeswax is 7:1,the emulsification temperature is 70℃,and the heating time is 10min.The screening process was verified,and 3 batches of preparations were repeatedly prepared according to the optimized moldability process conditions.The results show that the measured value of the comprehensive score of Huanglian Ointment prepared with good process parameters is similar to the theoretical value and greater than the theoretical value.The prepared ointment has uniform color,fine texture,suitable viscosity,and is easy to be applied to the skin and lubricated.The above results show that the preferred molding process is stable and feasible,and suitable for industrial production.3 Study on the effective components of Huanglian ointmentUPLC-Q-TOF-MS technology was used to identify and analyze the main chemical components in Huanglian ointment.By obtaining the quasi-molecular ion peak of the chromatographic peak and the fragment ion of the secondary mass spectrum.The methanol extract of Huanglian Ointment can be qualitatively analyzed.Through the analysis of mass spectrometry information and elemental composition in ESI positive and negative ion modes,and comparison with literature data,a total of 43compounds have been identified,including phenylpropionic acids.Ingredients ferulic acid;alkaloids berberine,berberine,berberine,phellodendron,palmatine,etc.;flavonoids:curcumin,curcumone,and lignans such as sesamin,sesamol and sesalin Ingredients,amide chemical components such as erucamide.4 Establishment of fingerprint of Huanglian ointmentThe fingerprints of Huanglian Ointment were established by high performance liquid chromatography.The HPLC fingerprints of 21 batches of Huanglian Ointment were combined with chemical recognition pattern analysis.A total of 17chromatographic peaks were calibrated,and the similarity of each chromatographic peak was greater than 0.950,which passed the standard.The control indicates that:peak 3 is phellodendrine hydrochloride,peak 4 is sesamol,peak 5 is ferulic acid,peak7 is berberine hydrochloride,peak 8 is berberine hydrochloride,and peak 9 is barberine hydrochloride Martin,peak 15 is curcumin,peak 16 is sesamin.By investigating the principal component analysis,cluster analysis,and partial least square discriminant analysis of 21 batches of Huanglian Ointment,it is concluded that there are slight differences in different batches of Huanglian Ointment.The 21batches of Huanglian Ointment can be divided into 3 categories.The first category is S1～S7;the second category is S8～S14;the third category is S15～S21,which is consistent with the actual sample source.A total of 3 different quality markers were screened,namely peak 5（ferulic acid）and 4 Peak（Sesamol）and Peak 16（Sesamin）.Therefore,when formulating the quality standard of Huanglian Ointment,these three markers can be focused on,and the quality of Dihuanglian Ointment can be more scientific and comprehensive.5 Study on efficacy and mechanism of Huanglian ointmentIn this paper,the animal model and related detection indicators that can reflect the original indications of Huanglian Ointment（eczema）were selected to carry out the research on the activity of Huanglian Ointment in the treatment of eczema.The results showed that after continuous use of Huanglian Ointment,the scabs on the backs of mice gradually fell off,and on the 18th day of administration,the scabs of the mice in the high-dose Huanglian Ointment group were completely off.Each dose group of Huanglian Ointment has an inhibitory effect on the ear swelling of mice.The high-dose group has a very significant inhibitory effect on mouse ear swelling（p<0.01）,and the middle-dose group can significantly inhibit ear swelling in mice（P<0.05）;HE staining experiment results show that Huanglian Ointment can reduce inflammation and improve skin edema by inhibiting the infiltration of inflammatory cells.The above results show that Huanglian Ointment retains its traditional functions and main treatments,and has a good therapeutic effect on eczema.The results of mouse scratching behavior detection and immunohistochemical experiments showed that the skin tissue of the model group showed abundant effective staining of substance P,and the model group had severe scratching behavior;The high and medium dose groups of Huanglian Ointment had only a small amount of expression and the coloring is lighter.the scratching frequency of mice in the high and medium dose groups of Huanglian Ointment is significantly reduced;The low dose group has a certain inhibitory effect on substance P in skin tissues,but the effect is not obvious.ELISA results:It is found that Huanglian Ointment can achieve anti-inflammatory effects by inhibiting the expression of Ig E,INF-γ,TNF-α,IL-6,IL-4 and IL-2 cytokines in the serum.The results of Western Blot experiments showed that the high and medium dose groups of Huanglian Ointment had a significant inhibitory effect on the expression of p-p38 and p-JNK（p<0.01;p<0.05）;the middle and low dose groups of Huanglian Ointment had a significant effect on the expression of p-ERK,It has a significant inhibitory effect（p<0.01;p<0.05）;Each dose group of Huanglian Ointment has a certain inhibitory effect on the expression of NF-k B p65.Based on the above experimental results,the mechanism of action of Huanglian Ointment is speculated as"Huanglian Ointment can inhibit the production of substance P,thereby inhibiting the scratching behavior of mice and alleviating the symptoms of itching;Huanglian Ointment can inhibit the production of inflammatory factors in the serum,thereby inhibiting skin tissue.On this basis,Huanglian Ointment can affect the inflammatory process of eczema by inhibiting the activation of the MAPK/NF-k B pathway."Conclusion:In this study,the traditional preparation process of"Fried Emulsification Method"of Huanglian Ointment was followed,and the extraction process and molding process parameters of Huanglian Ointment were clarified using modern Chinese medicine pharmaceutical technology.The liquid-mass spectrometry technology is used to comprehensively identify and analyze the main chemical components in Huanglian Ointment.On this basis,the fingerprint map of Huanglian Ointment was established combined with multivariate statistical analysis to provide a theoretical basis for the quality control of Huanglian Ointment.At the same time,modern molecular biology techniques such as ELISA experiment,immunohistochemistry experiment and Western blot experiment were used to clarify the performance of Huanglian Ointment.The mechanism of action provide data support to reveal the scientific connotation of the classic Huanglian ointment.