Study On Quality Standards Of Active Components And Extracts Of Rhizoma Coptidis For Atopic Dermatitis

Objective: In this project,alkaloids were isolated and purified from Rhizoma coptidis,and active ingredients that inhibited keratinocyte apoptosis were screened to relieve skin barrier damage to achieve the treatment of atopic dermatitis.The mechanism of action and preliminary quality standards of Coptis chinensis Franch extracts were studied.The research provides a theoretical basis for the research and development of related products of Rhizoma coptidis in the later period.Methods and Results: 1.In this project,alkaloids were isolated and extracted from Rhizoma coptidis by means of separation and purification methods such as alcohol extraction,extraction,silica gel column,macroporous resin,and HPLC,and the structure was confirmed.As a result,7 known alkaloids were obtained.They are jatrorrhizine,epiberberine,coptisine,magnoflorine,palmatine,columbamine and berberine.2.Next,to find the active components that can inhibit the apoptosis of keratinocytes(Ha Ca T),the active components were screened in vitro.According to the results of MTT,we knowed that magnoflorine(MAG)has a lower cytotoxic effect,and based on this,the appropriate concentrations of the drugs were screened.Another,to promote the apoptosis of stratum corneum cells in patients with atipoc dermatitis(AD),IFN-? and TNF-? were used for in vitro model to make Ha Ca T cells apoptosis.Active ingredients were screened and confirmed using flow cytometry detection technology and Hoechst 33342 staining technology.Finally,MAG was found to inhibit the apoptosis of Ha Ca T cells in a dose-dependent form(500,1000,1500 ?g/m L).3.In order to further study the effect of MAG and Rhizoma coptidis extract on AD,Female BALB/c mice were selected for in vivo experiment verfication.In this experiment,the control group(NC),the model group(AD),the positive drug group(WDO),the magnoflorine group(MAG),and the Rhizoma coptidis extract group(RCE)were designed.Using 0.5% and 2% 2,4-DNCB reagent to model the skin on the back of mice.The evaluation of skin lesion severity,behavioral index evaluation,H&E tissue staining,immunohistochemical examination,serum cytokine content,and organ index were selected for experimental investigations in mice.The results showed that after a period of treatment,the WDO group,the MAG group and the RCE group have a decrease in skin damage,itching,and serum IL-4 and Ig E levels compared with the AD group.Among them,the H&E tissue in the MAG group staining results showed the MAG group reduced stratum corneum thickness,decreased inflammatory cell infiltration,and hair follicle recovery,etc.The immunohistochemical result of Caspase-3 showed that AD group(1054.9 � 74.9)has the most positive results,and MAG group has the least(509.8 � 60.0,p <0.01 VS.AD).4.Subsequently,according to the results of the above experiments,it was found that MAG had the effect of inhibiting keratinocytes apoptosis in vivo and in vitro,and then the next step was to study the mechanism of action.Firstly,HT-docking,CTD,and KEGG and other bioinformatics techniques were used to find the targets of apoptosis-related effects of MAG in the treatment of AD as a possible direction for mechanism research.Then,the mitochondrial membrane potential of the cells was measured by flow cytometry,and the results showed that MAG reduced the low membrane potential of mitochondria;the expressions of Caspase-3?Caspase-7?Caspase-8?Caspase-9,Bid,Cytochrome C and Cathepsin B were measured by western blot.The results showed that MAG inhibited the release of Cytochrome C from the mitochondrial inner membrane and the expressions of other proteins were down-regulated,indicating that the mechanism of action of MAG may be related to mitochondria.5.Finally,it was proved through animal experiments that RCE also played a role in relieving AD.In order to provide a theoretical basis for the subsequent development of Rhizoma coptidis,referring to the 2015 version of the Chinese Pharmacopoeia quality standard research methods,three batches of Rhizoma coptidis extract were subjected to quality standards.The study examined systemic adaptability and methodological verification.The characteristic peaks were magnoflorine,palmatine,and berberine.The results showed that precision,repeatability,and stability all met the requirements.The total average content of magnoflorine,palmatine and berberine were 3.216%,14.090%,5.893% respectively.Conclusion: In summary,this subject demonstrated that the MAG isolated from Rhizoma coptidis inhibited Ha Ca T cell apoptosis in vitro and in vivo.The primary mechanism might be that under the action of MAG,Bid was cleaved by cathepsin after lysosome destabilization,and then transferred to mitochondria to reduce the expressions of Caspase-3\7\8\9,Bid and CTSB proteins,and inhibited the release of Cytochrome C from the inner membrane of mitochondria,thus alleviating the skin damage of AD.In addition,the quality of Rhizoma coptidis extract was improved.Standard research provides a theoretical basis for subsequent product development.