Screening Of Anti-wart Active Ingredients And Preparation Of Ointment On Salvia Miltiorrhiza And Sophora Flavescens Empirical Formula

Verruca vulgaris is a skin disease caused by human papillomavirus(HPV),which has the characteristics of long-term and recurrence.At present,there are technical problems of recurrence rate and side effects in the clinical use of chemical corrosives,broad-spectrum antiviral agents,immunomodulators,liquid nitrogen freezing method,laser method,etc.;traditional Chinese medicine through multi-target,all-round treatment of verruca vulgaris,with high safety and cure rate,recurrence rate and small toxic effect.Salvia miltiorrhiza and sophora flavescens empirical formula is the prescription of decoction summarized by the clinician’s experience in treating verruca vulgaris for many years.According to the basic research and clinical data of the enterprise,the empirical formula has a short treatment cycle and alleviates the sufferings of patients.However,there are problems in the use and preservation of the decoction.Entrusted by the company to screen and refine the active ingredients of the prescription,and prepare external preparations to provide ointments for clinical treatment of common warts.The pathological phenomenon of verruca vulgaris is that the abnormal proliferation of epidermal echinoderm cells stimulated by HPV causes the papilloma like process in the skin.Human immortalized keratinocytes(HaCaT)are used to simulate the abnormal proliferation of cells in verruca vulgaris tissues.Take inhibition rate of empirical formula,Salviae Miltiorrhizae,Sophora flavescens,and main components as an indicator,investigate the effect of CCK-8 on HaCaT cell proliferation while screening effective active ingredients.Further combine with flow cytometry,Hoechst33258 DNA staining,and Western blotting to investigate the effects of effective active ingredients on HaCaT cell cycle distribution,apoptosis,and expression of related proteins.The experimental results showed that all of the empirical formula,Salviae Miltiorrhizae,Sophorae Flavescentis and main component had an inhibitory effect on the proliferation of HaCaT cells;Salvianolic acid B and oxymatrine,the main components of salvia miltiorrhiza and matrine,can significantly inhibit the proliferation of HaCaT cells at low concentrations.Salvianolic acid B and Oxymatrine can block cells staying in G0/G1 phase,stop DNA replication,and cause early apoptosis of cells.Hoechst33258 DNA staining method can directly see that salvianolic acid B and oxymatrine have a concentration and time effect relationship and induce apoptosis.Western blot showed that salvianolic acid B and Oxymatrine can significantly up-regulate the expression of caspase 3,caspase 9 and Bax proteins,down-regulate the expression of Bcl-2 protein,and promote the apoptosis of HaCaT cells.It is suggested that Salvia miltiorrhiza and sophora flavescens empirical formula can prevent the abnormal proliferation of echinoderm cells and promote their apoptosis,so as to achieve the purpose of treatment.The extraction parameters and separation parameters of macroporous resin were investigated by orthogonal test.The transfer rate of salvianolic acid B and Oxymatrine was taken as the extraction index,and the elution rate of components was taken as the resin refining index to select the best purification process.The best extraction and separation process of Total phenolic acid is to use 10 times of 50%ethanol reflux to extract Salvia miltiorrhiza pieces for 2 times,and the extraction time is 1 h;the volume ratio of salvianolic acid B and AB-8 resin is 1:2,respectively,using 7.5%ethanol and 35%ethanol of 5 times of the column volume for impurity removal and elution,and the flow rate is 4 BV·h-1.The best extraction and separation process of total alkaloids is to soak it in 0.5h and then extract it twice with 10 times of water for 2h;the volume ratio of medicinal material and AB-8 resin is 1:2.5,and then remove impurities and elute it with 6%ethanol and 25%ethanol with 4 times of column volume respectively,the flow rate is 2 BV·h-1.The purification process of salvianolic acid B and Oxymatrine meets the requirements of the enterprise,and the content of the extract is high,which can save cost and facilitate industrial production.In the study of ointment,taking the stability of salvianolic acid B as the premise,ensuring the stability and homogeneity of the preparation,taking the decline rate of salvianolic acid B content as the index,the best dosage form was selected from the three dosage forms of O/W,W/O and ointment.The experimental results showed that the best dosage form was salvianolic acid B,Oxymatrine extract,freeze-dried into powder,No.9 screen into fine powder,a small amount of more the mixture of beeswax and linseed oil(the ratio of drug to substrate is 1:10)melted at 110℃ was added,and 2%azone was added.The mixture was dispersed evenly and cooled to room temperature by high shear emulsification machine.Salvia miltiorrhiza and sophora flavescens empirical anti verruca vulgaris ointment is a semi-solid preparation with light brown,fine texture,easy coating and good stability.The transdermal absorption of salvianolic acid B and oxymatrine in vitro was linear with time,and the transdermal effect was good.