| Impatiens balsamina L.is an annnual herbaceous plant.As the main producing area of Impatiens balsam which contains flavonoids,naphthraquinones and organic acids,China has rich resources of Impatiens balsam.From the classic ancient books to the folk methods,to the folk methods and to the modern clinical application,which all documented that the Impatiens have a significant effect on the treatment of leuconychia,tinea pedis,and onychia laterails.At the same time,domestic and foreign studies have showed that 1,4-naphthraquinones in Impatiens balsamina L.have strong antifungal activity.Therefore,The subject was to analysis the composition of Impatiens balsamina L.and study the antifungal activity in vitro of 1,4naphthraquinones,and preliminarily explore relationship between the significant effect of Impatiens flower in the treatment of dermatophyte and the antifungal activity of 1,4-naphthquinones,so as to provide more powerful evidence for the drug use value of Impatiens balsamina L.Objectives:(1)To establish HPLC method of lawsone(HNQ)and 2-methoxy-1,4-naphthoquinone(MeONQ)qualitative and quantitative analysis.To establish the HPLC fingerprints of chemical constituents in Impatiens balsamina L.and provide the basic for the quality evaluate of it.With the content of HNQ and MeONQ as a reference,HNQ and MeONQ in Impatiens balsamina L.from different areas and different medicinal parts were determined.The quality difference of Impatiens balsamina L.from different areas and the content of two 1,4-naphthraquinones in different medicinal parts,which were compared.The extraction process of HNQ and MeONQ was optimized to improve the extraction rate.(2)To determine the content of lawsone(HNQ)and 2-methoxy-1,4-naphthoquinone(MeONQ)and antifungal activities of different solvent fractions of Impatiens balsamina L.and analyze relationship between content and antifungal activities,in order to define antifungal active constituents of Impatiens balsamina L.(3)To determine antifungal activity of petroleum ether extract on Trichophyton rubrum,and to analyze the effect of combination of petroleum ether extract and miconazole nitrate on Trichophyton rubrum.Methods:(1)The ethanol extracts of samples were analyzed on a Agilent TC-C18(2)column(250 mm×4.6 mm,5 μm)eluted with the mobile phase of acetonitrile(A)and 1%acetic acid/water solution(B).The flow rate of the mobile phase,the column temperature,and the detective wavelength were set at 1.0 mL/min,30℃,and 280 nm,respectively.(2)To extract HNQ and MeONQ in Impatiens balsamina L.by using ultrasonic-assisted extraction,and the extraction rate of 1,4-naphthoquinone was the evaluation index.On the basis of single factor experiment,screening liquid-material ratio,ultrasonic power,temperature as influence factor,the optimum extraction condition was determined by utilizing Box-Behnken response surface analysis method.(3)The content and transfer rate of HNQ and MeONQ in different extraction parts was simultaneously determined by using HPLC method.(4)The fungicidal activities of extracts of five fractions against Trichophyton rubrum were studied by filter paper diffusion method.(5)By microdilution method,we examined minimal inhibitory concentration(MIC)of petroleum ether extraction.The fractionation inhibitory concentration(FIC)of the petroleum ether extract and miconazole nitrate was determined,and the interaction between two kinds of bacteriostat was analyzed.Results:(1)The method for lawsone(HNQ)and 2-methoxy-1,4-naphthoquinone(MeONQ)in Impatiens balsamina L.were established by HPLC.The regression equation of lawsone was:y=52628699.87x+106418.24(r=0.9993)and the linear range was 0.004～0.400 mg/mL.The linear regression equation of the obtained 2-methoxy-1,4-naphthoquinone was:y=48612540.85x+305337.13(r=0.9996),and the linear range was 0.008～0.800 mg/mL,and the mean recoveries were 98.56%and 102.75%,with the RSD(n=6)of 1.92%and 1.96%,respectively..(2)Through preliminary analysis and identification,there be likely to ten chemical constituents in Impatiens balsamina L.including five flavonoids,such as rutin,quercetin;and three naphthone quinones,such as index components lawsone and 2-methoxy-1,4-naphthoquinone;and one coumarins,scopoletin or 6-Hydroxy-7-methoxy-2H-chromen-2-one;and one naphthalenes.At the same time,the related analysis and identification work is still in progress.(3)HPLC fingerprint of Impatiens balsamina L.was established,ten main marker peaks were selected,fingerprint similarities of 10 sample batches were distributed between 0.837 to 0.957.(4)The extraction technological parameters of component in Impatiens balsamina L.were as follow:liquid-solid of 50 mL/g,ultrasonic power of 180 W,ultrasonic temperature of 40℃.Under these conditions the extraction rate of HNQ and MeONQ was 9.2242 mg/g,compared with the predictive value of 8.8913 mg/g,the absolute values of deviations of the two less than 5%.(5)The quantitative results showed that the contents of HNQ and MeONQ were separately in the range of 0.011%～0.739%,0.178%～0.588%.Meanwhile,the contents of two 1,4-naphthoquinone in Impatiens balsamina L.cultivated in Beijing are higher than those cultivated in other distracts.(6)There were great differnences of content of HNQ and MeONQ in different medicinal parts of Impatiens balsamina L.The content of HNQ order of size:leaf>seed>stem>flower,the leaves were the highest,the petals were the lowest,however,the content of MeONQ order of size:seed>flower>leaf>stem,and seed were the highest,the lowest were stem.(7)Five different polar parts of petroleum ether,dichloromethane,ethyl acetate,n-butanol and water phase were obtained from ethanol extract of Impatiens balsamina L.the petroleum ether extract showed the highest amount of HNQ and MeONQ,their content were 42.0%and 39.5%,and transfer rate were 30.5%and 52.5%,respectively.Secondly,the dichloromethane extraction,their content of HNQ and MeONQ were 24.4%and 0.378%,and transfer rate were 5.85%and 0.49%,respectively.However,ethtyl acetate,n-butanol and water solvent fractions all were hard to detect HNQ and MeONQ.(8)Compared antifungal activity of different polar parts,the results showed,when the concentration of 8 mg/mL,petroleum ether extract was the best,the diameter of inhibition zone was 56.5 mm;dichloromethane extraction was low antifungal activities,the diameter of inhibition zone was 11.5 mm,and the other three phases exhibited no antifungal activities.(9)The results of microdilution method showed that MIC of petroleum ether extraction against Trichophyton rubrum was 1000 μg/mL,the MIC of miconazole nitrate was 400 μg/mL.The fractional inhibitory concentration(FIC)of combination therapy petroleum ether extraction with miconazole nitrate was 1.0.According to the criteria for fractional inhibitory concentration(FIC),there were additive effect between two kinds of drugs,namely the antifungal activity of combination therapy were greater than any single one.Conclusion:The Qualitative and quantitative analysis method of HNQ and MeONQ was established,this method was proved to be simple,accurate and repeatable.They could be used for the determination and quality control of the essential components in Impatiens balsamina L.There were obviously differences of fingerprint similarities,the contents of HNQ and MeONQ from different areas,which revealed the growing areas of this plant are closely linked to its quality.The model was in good agreement with verifying experiment,the predictability was satisfactory,the optimized technological parameters were reliable,so the model of response surface methodology had feasibility.As the index components,lawsone and 2-methoxy-1,4-naphthoquinone in Impatiens balsamina L.from different areas,which were great difference.In different medicinal parts,the content of HNQ and MeONQ also were different greatly.In petroleum ether extract,the amount of HNQ and MeONQ were the highest and few other components,and the activities against Trichophyton rubrum was the best,so the antifungal active part was petroleum ether extract and active constituents of Impatiens balsamina L.were naphthoquinones.There were additive effect between petroleum ether extract and miconazole nitrate,namely the antifungal activity of combination therapy were greater than any single one. |
PDF Full Text Request