Preparation Of Melilotus Extracts And Their External Gel Formulations

The oral tablet of melilotus extract(SETUS-M)developed and manufactured by Japanese pharmaceutical company entered the Chinese pharmaceutical market.It has been clinically used in hemorrhoids and soft tissue swelling.There is no domestic related drug on the market.The gel preparation of melilotus extract for external use has good market prospects and is worth developing.In this paper,preliminary exploration and research on the preparation process of melilotus extract and its external gel preparation were carried out to provide data support for the subsequent industrial development and production.In this paper,the analytical methods of coumarins in the plant of melilotus and its gel preparations for external use were established by high performance liquid chromatography(HPLC).The coumarins,which is main active component,with high content in the plant of melilotus were used as index components.The device and materials used in the analysis method are the commonly used ultraviolet-visible absorption detector(UVD),C18 chromatographic column,acetonitrile:water:acetic acid(42:58:0.2)as mobile phase.The preparation processes of coumarin from melilotus extract and its external gel preparation were studied.The extraction and concentration processes were optimized.solvent extraction,ultrasonic assisted extraction,and surfactant assisted extraction were compared.And compare evaporation concentration and membrane concentration processes to explore and study the process methods for improving the extraction and concentration efficiency of coumarin from melilotus.The methods for separating and purifying the melilotus extract were liquid-liquid extraction and macroporous adsorption resin.In this paper,the prescription and preparation method of the melilotus gel for external use were studied,and the bacteriostasis of the gel(inhibition of Staphylococcus aureus,Escherichia coli and Candida albicans)was investigated,and non-clinical safety tests(multiple complete skin irritation tests on rabbits,vaginal mucosa irritation tests and skin sensitization tests on guinea pigs)were carried out.Main results are as follows.The analysis of coumarin in melilotus and external gel preparations by HPLC in this paper,linearity,precision,accuracy and so on,all meet the evaluation requirements of analytical methods.The optimized process parameters of extraction of melilotus are confirmed.Melilotus coarser powders add 50%ethanol with 1%polysorbate-80 in order to extract the active components.The ratio of material to liquid is1:12(g/L).Extract for 1 hour at 60℃ for 2 times.The extraction rate of coumarin is 0.85%,and the purity of coumarin is 4.01%.Surface active agent assisted extraction has certain advantages.The ultrasonic assisted extraction process is not suitable for industrial production,but it can be used in the laboratory.The membrane concentration can be operated at room temperature conditions.After concentration,the content of coumarin is high with the membrane concentration,and the transfer rate is higher than the evaporation concentration.The membrane concentration,of which process time is short,can be considered as a concentration method for future industrialization.The crude extract of melilotus is conducted by liquid-liquid extraction with Ethyl acetate.The extract of melilotus is purified with macroporous adsorbing resin,using 60%ethanol as eluting solvent.The gel preparation for external use utilizes purified melilotus extract as the active ingredient,and carbomer as the matrix.The antibacterial effect of the gel on Staphylococcus aureus,Escherichia coli and Candida albicans can reach more than 90%.It has strong antibacterial effects.Animal tests showed that the gel had no irritation to the intact skin and vaginal mucosa of rabbits,and no sensitization to the skin of guinea pigs.In summary,the preparation method of melilotus extract,surfactant assisted extraction and membrane concentration can be selected.After separation and purification with extraction and macroporous resin,the purity of coumarin can reach 67.38%.the content of coumarin in the refined extract of melilotus not less than 0.5 mg/mL.After the optimization of the process of the external preparation of the gel of melilotus,the quality standard of the external gel preparation of melilotus can be formulated.The content of coumarin(C9H6O2)in the initially proposed gel should not be less than 20 μg/g.