Separation And Purification Of Chemical Components From Leaves Of Custard Apple And Tubers Of Gymnadenia Conopsea By CCC And Their Biological Activitie

Natural products are rich in resources,diverse in chemical structure and strong in biological activity,which are important resources for research and development of food,health food and medicine.However,the composition and structure of natural products are complex and diverse.It is of great significance for the further development of natural products to separate and purify their chemical components,identify their structures and study their pharmacology.leaves of custard apple and tubers of Gymnadenia conopsea,as traditional medicine and food homologous plants,have a long history of application and exact efficacy,and have a broad application and development prospect in food and drug industry.As a preparation and separation technology,counter-current chromatography has the advantages of high efficiency,large amount of preparation and high purity,which is widely used in the separation and purification of natural products,dyes,drugs and other fields.In this paper,the main chemical components of leaves of custard apple and tubers of Gymnadenia conopsea were separated and purified by counter-current chromatography,and the pharmacological activities of some components were studied.Two methods for the separation of the main chemical components of leaves of custard apple extract were established by CCC.First,the conventional counter-current chromatography mode with ethyl acetate:n-butanol:water=4:1:5（v/v）as the solvent system and the separation method with the cyclic counter-current chromatography mode;second,the ethyl acetate:n-But-anol:water as the solvent system,the ethyl acetate and water solvent system as the initial gradient,the lower phase as the stationary phase,the best operation mode as"FWD-OUT",the best speed as 800 rpm,the best flow rate is 2.0 m L/min,the separation method of LGCCC mode combined with IRCCC mode.The separated components were analyzed by HPLC,and identified as Rutin and Quercetin-3-O-robinobioside,Quercetin-3-O-β-D-Glucoside,Kaem Pfe-rol-3-O-robinobioside,Kaempferol-3-O-rutinoside by MS,¹H and ¹³C-NMR.Both methods have achieved the separation and purification of five flavonoids in leaves of custard apple,the purity of which is more than 98%.It is to establish a method suitable for the separation of the main chemical components of tubers of Gymnadenia conopsea extract by CCC.Ethyl acetate-n-butanol-water was used as the solvent system,and ethyl acetate phase of saturated water was used as the initial mobile phase A,the operation mode as"FWD-OUT",the speed as 800 rpm,the flow rate is 5.0m L/min,the gradient condition and injection amount were optimized in the separation process.The optimal conditions of LGCCC were:0 min,100%A;50 min,100%A;68 min,80%A;88min,80%A;150 min,45%A;230 min,0%A;280 min,0%A;280-340min cleaning mode;340-360min ejection mode,with the injection volume of 500mg.The two pump gradient elution sweep ejection countercurrent separation mode combined with the preparation of the liquid phase,the purity of the separated substances was more than 98%by HPLC,and they were identified as P-hydroxybenzylethyl ether,P-hydroxybenzyl alcohol,Dactylorhin A,Dactylorhin B,P-hydroxybenzaldehyde,Lorogiossin,and mililarine by ¹H and ¹³C-NMR.It realizes the efficient,accurate and fast separation of 7 complex components of tubers of Gymnadenia conopsea.The research also discovers,the antioxidant and hypoglycemic activities of the crude extract and the monomer compounds were studied,which proved that the leaves of custard apple had better antioxidant and potential hypoglycemic activities.The crude flavonoid extract after extraction and purification has better hypoglycemic activity than the crude ethanol extract.Quercetin-3-O-robinobioside,Quercetin-3-O-β-D-glucoside and Rutin,have better hypo Glyce-mic activity than Kaempferol-3-O-robinobioside and Kaempferol-3-O-rutinoside.which is consistent with the antioxidant effect.From the structural analysis,it can be concluded that the hydroxy group at 3’position can enhance the hypoglycemic activity of flavonoids It is of great significance to study the hypoglycemic mechanism of flavonoids.This experiment provides a guidance for the directional separation of effective active ingredients in leaves of custard apple,and provides a theoretical basis for the efficient development and utilization of food,health products and plant drugs with leaves of custard apple as raw materials.