Study On The Quality Standards,stability And Preliminary Safety Of Peganum Harmala Alkaloids Creams

Objective:To establish the quality standard draft for peganum harmala alkaloids effective components and peganum harmala alkaloids cream?PHAC?,study on the stability and animal safety of PHAC,and provide a research basis for its new drug application.Methods:?1?The quality standard draft and stability for peganum harmala alkaloids effective components were studied.ICP-OES was used to determine the content of As,Cd,Cu and Pb,atomic absorption spectrometry was used to determine the content of Hg.The qualitative identification was carried out by TLC and the content determination was carried out by HPLC.The stability study was carried out by influencing factors test,classic isothermal kinetic method,t_0.9 method and weibull distribution method.?2?The quality standard draft and stability for PHAC were studied.The general quality of PHAC was inspected according to the general notices described in Chinese Pharmacopoeia?volume?,2010 edition?.The qualitative identification was carried out by TLC and the content determination was carried out by HPLC.The stability study was carried out by influencing factors test,accelerated test and long term test.Classic isothermal kinetic method,t_0.9 methods and weibull distribution method were used to estimate the validity of PHAC.?3?Skin acute toxicity test and long term toxicity test were used to estimate the skin safety of PHAC preliminarily.Results:?1?The quality standard draft for peganum harmala alkaloids effective components was established.The content of As is not more than 2mg?kg^-1,the content of Pb is not more than 2mg?kg^-1,the content of Cd is not more than 0.3mg?kg^-1,the content of Cu is not more than 15.99mg?kg^-1,the content of Hg is not more than 0.01mg?kg^-1.TLC was used to identified harmine and harmaline in peganum harmala alkaloids effective components,developing agent was the mixture of ethyl acetate,methanol and ammoniawater?10:1:0.6?,sampling volume was 1?L,and the silica gel G plate was detected at 254nm.The RP-HPLC analysis of harmine and harmaline in peganum harmala alkaloids effective components was carried out on Agilent TC-C₁₈?2??5?m,4.6mm×250mm?,elution solvents was the mixture of methanol and0.01mol·L^-1 ammonium sulfate solution-diethylamine,the pH was adjust to?4.00±0.01?by adding phosphoric acid,the flow rate was 0.5 mL·min^-1,column temperature was 25?,the detective wave length was 320nm for harmine and 372nm for harmaline.The RP-HPLC analysis of vasicine in peganum harmala alkaloids effective components was carried out on Agilent TC-C₁₈?5?m,4.6mm×250mm?,elution solvents was the mixture of methanol and 0.1%trifluoroacetic acid?12:88?,the flow rate was 1mL·min^-1,column temperature was 30?,the detective wave length was 280nm.The contents of known component in 10 batches of peganum harmala alkaloids effective components were more than 50%,which meet the requirements of new medicines registration with the total effective components.The results of influencing factors test of peganum harmala alkaloids effective components showes that it is stable under the high light and high temperature conditions,but is sensitive to humidity.The contents decline of harmaline corresponded with the first-order kinetics models.Calculated validity?25??is 15.5 years by classic isothermal kinetic method,calculated validity?25??is 16.2 years by t_0.9methods,and calculated validity?25??is 42.1 years by weibull distribution method.?2?The quality standard draft for PHAC was set up.The inspection items were all met the requirements.TLC was used to identified harmine and harmaline in PHAC,developing agent was the mixture of ethyl acetate,methanol and ammoniawater?10:1:0.6?,sampling volume of reference solution was 2?L,sampling volume of sample solution was 6?L,the silica gel G plate was dected at 254nm.The Rp-HPLC analysis of harmine and harmaline in PHAC was carried out on Agilent TC-C₁₈?2??5?m,4.6mm×250mm?,and elution solvents was the mixture of methanol and 0.01mol·L^-1 ammonium sulfate solution-diethylamine,the pH was adjust to?4.00±0.01?by adding phosphoric acid,the flow rate was 0.5 mL·min^-1,column temperature was 25?,the detective wave length was 320nm for harmine and 372nm for harmaline.The Rp-HPLC analysis of vasicine in PHAC was carried out on Agilent TC-C₁₈?5?m,4.6mm×250mm?,elution solvents was the mixtue of methanol and 0.1%trifluoroacetic acid?12:88?,the flow rate was 1mL·min^-1,column temperature was 30?,the detective wave length was 280nm.The results of influencing factors test of PHAC showes that it is stable under high light and humidity conditions,harmine have a good stability under high temperature conditions,but harmaline is unstable under high temperature conditions.The results of 6 months accelerated test and 9 months long term test showes that inspection items were all met the requirements,the content of harmine has no obvious changes,but harmaline is unstable,its content gradually decreased over time.The results of accelerated test showes that the content of total alkaloids decreased 8.13%,the results of long term test showes that the content of total alkaloids decreased 3.38%.In the classic isothermal kinetic test,the descend of contents of harmaline corresponded with the first-order kinetics models.Caculated validity?6??is5.8 months by classic isothermal kinetic method,caculated validity?6??is 5.8 months by t_0.9 method,caculated validity?6??is 4.5 months by weibull distribution method.?3?PHAC had no acute toxicity or irritation to the intact skin or damaged skin and had no sensitization or overt toxicity to the intact skin.Conclusion:?1?The established quality control methods of peganum harmala alkaloids effective components meet the requirements of methodology.It should be stored under room temperature,airtight and dry conditions.?2?The established quality control methods of PHAC meet the requirements of methodology,and PHAC should be stored under 6?,airtight and dry conditions.?3?The animal skin safety of PHAC is good.