| The paper serve anti-diabetic effective fraction in leaves of Olea europaea L.as research object,and study on the inhibitory activities of anti-diabetic effective fraction in leaves of Olea europaea L.to α-glucose enzymes and α-amylase.Meanwhile isolation and preparetion of anti-diabetic effective fraction in leaves of Olea europaea L.with off-line 2-dimension high-speed counter-current chromatography,and the obtained three compounds were analyzed by comparison with standard substances by HPLC.The purities all above 90%.The main research contents include the following aspects:1.Macroporous adsorption resin BMKX-4 was used to enrichment the active ingredient of the preliminary screening of anti-diabetic effective fraction in leaves of Olea europaea,and then gradient eluted with 20%,40%,60%,80% ethanol–aqueous solution(v/v)in turn.Different fractions(Frs.A-D)were prepared,and α-glucose enzymes system and α-amylase system were used to evaluate the inhibition to the different fractions.The four common compounds(Hydroxytyrosol acetate X1,Oleuropein X2,Ligstrosidein X3,2″-epifraxamoside X4)in different fractions were analyzed by HPLC.In addition,the correlation between the inhibitory activity and the peak areas was illuminated.As a result,the inhibitory activity of fraction A to α-glucose enzymes was strongest,and the inhibitory activity of fraction C to α-amylase was strongest.Compound X1,X3 were positively correlated with inhibition to α-glucose enzymes,and compound X2,X4 were negatively correlated with inhibition to α-glucose enzymes.Compound X2,X4 were positively correlated with inhibition to α-amylase,and compound X1,X3 were negatively correlated with inhibition to α-amylase.The study verifies that preliminary screening of anti-diabetic effective fraction in leaves of Olea europaea has better inhibitory effect on key enzymes for glucose metabolism.The activities of α-glucose enzymes and α-amylase could be inhibited by different fractions of Olea europaea leaf extract.Meanwhile found that Hydroxytyrosol acetate and Ligstrosidein possible were the main compounds to inhibit the activities of α-glucose enzymes,and 2″-epifraxamoside and Oleuropein possible were the main compounds to inhibit the activities of α-amylase.2.Application of 2-dimension high-speed counter-current chromatography preparative and separation of compounds from anti-diabetic effective fraction in leaves of Olea europaea.The first step of 2-dimension high-speed counter-current chromatography was to establish a rapid method to separate oleuropein from anti-diabetic effective fraction in leaves of Olea europaea by high-speed counter-current chromatography,n-butyl alcohol–ethyl acetate–methanol–water–chloroform(1:19:1:19:0.4,v/v)was selected as the two-phase solvent system for the separation.The upper phase was used as the stationary phase and lower phase was used as the mobile phase.The revolution speed was 900 r·min-1.Meanwhile,the flow rate of the mobile phase and sample loading capacity were studied and confirmed that the two factors can influence the resolution of oleuropein.The purity of oleuropein which was separated by this HSCCC method can reach 90.5% when the flow rate is 1.5 mL·min-1 and the concentration of sample loading is 20 mg·m L-1.In addition,this method can be used to separate oleuropein in abundance.The next step was to separate compounds from the remaining fractions which contain only a little of oleuropein.The optimization of solvent system was n-butyl alcohol–ethyl acetate-water(1:2:1:3,v/v),the upper phase was used as the stationary phase and lower phase was used as the mobile phase.Both the tail-head and head-tail elution modes were used with the revolution speed was 900 r·min-1,the flow rate is 2.0 mL·min-1 and the concentration of sample loading is 2 mg·m L-1.The purities of hydroxyl tyrosol and mignonette element-7-O-β-D-glycosidase were determined by peak area normalization method,and they were 95.3% and 91.2%,respectively. |
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