| Objective:To establish a method for extracting hyperoside from Honghanlian by ionic liquid aqueous two-phase system（IL-ATPs）,and use it in the study of the quality standard of Honghanlian comprehensive improvement.Methods:The binary aqueous phase systems of[Cnmim]Br（n=2,4,6,8）,[Cnmim]BF4（n=2,4,6,8）,[C4mim]N（CN）2 and Na2SO4,（NH4）2SO4,Na2HPO4were plotted binodal curve and tie line respectively,the effects of cationic side chain length,anionic species and inorganic salt types on the phase forming ability of ionic liquids were investigated.The ionic liquid species and inorganic salts with the strongest phase forming ability were preferred.The aqueous two-phase system consisting of[Cnmim]BF4（n=2,4,6,8）and（NH4）2SO4,Na2SO4was optimized.The extraction efficiency E and partition coefficient Kd of Hyperoside solution were determined.According to the experimental results,the ionic liquid two-phase system consisting of[C8mim]BF4 and（NH4）2SO4 was selected for the extraction of hyperoside.This system was used to extract Hyperoside from Honghanlian.By single factor investigation results such as ionic liquid concentration,salt concentration,dosage of medicinal materials,ultrasound extraction temperature,pH value and ultrasound extraction time,and Response surface test was carried out.The optimum extraction process of Hyperoside from Honghanlian by ionic liquid aqueous two-phase system was selected and applied to the quality standard of Honghanlian.Finally,the solid-liquid adsorption method was used to recover ionic liquids.TLC was used for qualitative identification;The determination of water,ash and extracts was carried out according to the corresponding general rules of the four parts of the Chinese Pharmacopoeia 2015 edition,and the limits were stipulated.The content of hyperoside in the Honghanlian and the content of hyperoside in different parts of Honghanlian were determined by HPLC.Results:The ionic liquid two-phase system consisting of[C8mim]BF41.0ml,（NH4）2SO41.50g and distilled water 2ml had the best extraction efficiency of Hyperoside in 0.1g Honghanlian,up to 96%.Under this condition,Hyperoside showed a good linear relationship between 4.956 and 74.346g/ml（r=0.9997）,and the method was specific and reproducible.The content of Hyperoside was 1.11mg/g by this method,and the content of Hyperoside was1.15mg/g and RSD was 2.5%by traditional extraction method.There was no significant difference between the two methods.The source of medicinal materials was revised in the draft quality standard;Added identification items including microscopic identification,TLC identification,inspection items,and leachables,etc.Stipulated that leaves should not be less than 15.0%,the water content shall not exceed 13.0%,and the total ash content shall not exceed 6.0%.When dilute ethanol is used as solvent,the alcohol-soluble extract shall not be less than 18.0%,According to the calculation of dried products,Hyperoside content(C21H20O12)should not be less than 0.040%.Conclusion:The composition of aqueous two-phase system for extracting Hyperoside from Honghanlian was determined by drawing different ionic liquids and inorganic salt plotted binodal curve and tie line,and determining the extraction rate and partition coefficient of Hyperoside solution.Based on the results of single factor investigation such as ionic liquid concentration and salt concentration,Response surface test was carried out to optimize the extraction process of Hyperoside from Honghanlian by ionic liquid two-phase system.A method for the determination of Hyperoside in Honghanlian by ionic liquid two-phase system（IL-ATPs）coupled with HPLC-UV was established and applied to improvement of quality standards of Honghanlian.At the same time,qualitative research was carried out to improve the quality standard of Honghanlian in an all-round way.This study provides a new method for extracting hyperoside from red lotus,and establishes a new model for the extraction and separation of target components of Chinese herbal medicine. |
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