Screening For Inhibitors Of Acetylcholinesterase

Acetylcholinesterase(AChE)has attracted wide attention as a key enzyme for treatment of Alzheimer’s disease(AD),and most drugs used clinically to treat Alzheimer’s disease(AD)are based on the cholinergic hypothesis.The existing drugs have limitations,such as low activities and severe side effects of existing drugs.In this article,the orthogonal test was used to optimize the extraction conditions of flavonoids in Albiziae Flos.The effects of reflux extraction and ultrasonic extraction on the extraction rate of flavonoids in Albiziae Flos were compared.The single factor experiment and response surface method were used to optimize the extraction conditions of flavonoids in Lamiophlomis rotate.The active components of 12 kinds of plants were extracted and crude separated.Moreover,modified Ellman method was used to screen acetylcholinesterase inhibitors(AChEIs),and half inhibition concentration(IC50),Km value and AChE inhibition type of plants were determined.Firstly,orthogonal test was used to optimize the extraction conditions of flavonoids in Albiziae Flos,and the extraction rate of flavonoids in Albiziae Flos through reflux extraction method was compared with that through ultrasonic extraction method.Among them,the optimal extraction conditions for the reflux method were: extraction time was 1 h,solid-liquid ratio was 1: 20(g/mL),and ethanol concentration was 40%.The optimal extraction conditions for the ultrasonic method were: extraction time was 10 min,solid-liquid ratio was 1: 10(g/mL),and ethanol concentration was 60%.In addition,compared with the reflux extraction method,the ultrasonic extraction method has a higher extraction rate of total flavonoids in Albiziae Flos.Secondly,the extraction parameters of the Lamiophlomis rotate were optimized byresponse surface methodology.The optimal parameters for extracting the flavonoids in Lamiophlomis rotate were determined as follows,the soaking time was 5 h,the ethanol concentration was 57%,and the solid-liquid ratio was 1: 57(g/mL).The verification tests were performed under the optimal conditions,.The three extraction rates were obtained as 7.203%,7.109% and 7.172%,respectively.The RSD is less than 3%.Then,12 natural plants were selected for extraction and crude separation,including 8 flavonoid-rich plants and 4 alkaloids-rich plants.For the flavonoids,extraction was performed with organic solvents(chloroform,ethyl acetate,n-butanol)with different polarities to obtain chloroform layer,ethyl acetate layer,n-butanol layer and water layer.For alkaloids,extraction with chloroform yields strong alkaline,medium strong alkaline and weak alkaline alkaloids,respectively.Finally,12 kinds of plants were tested for anti-AChE activity by the modified Ellman method.The strong alkaline alkaloids of Cortex Phellodendri,n-butanol layer of Apocynum venetum L,ethyl acetate layer of Apocynum venetum L,ethyl acetate layer of Abelmoschi corolla,n-butanol layer of Bistortae rhizome,n-butanol layer and ethyl acetate layer of Spatholobus suberectus Dunn have obvious AChE inhibitory activity(inhibition rate>50%).The extracts of the other plants have weak AChE inhibitory activity(inhibition rate of 20%-50%)or almost no inhibitory activity(inhibition rate <20%).The plant with an inhibition rate greater than 50% at the final concentration 1 mg/mL was selected for measurement of IC50 value and enzyme reaction kinetics.The IC50 values of different plants were obtained.Among them,the inhibition types of AChE by the ethyl acetate layer of Apocynum venetum L and ethyl acetate layer of Bistortae rhizome were mixed inhibition.