Isolation of polyphenol compounds from olive waste and inhibition of their derivatives for α -glucosidase and α -amylase

: Olive waste was used as a sustainable resource because it contained a variety of valuable compounds. The polyphenols active fraction from enrichment by microporous resin and extraction with ethyl acetate were analysed by different chromatographic methods. A total of 14 polyphenolic compounds were isolated and identified by structure elucidation. Based on the above obtained compounds, tyrosol was selected as a characteristic polyphenol and participated in transesterification reaction to synthesise β -ketoester using Yb(OTf) 3 . Then the Biginelli reaction with benzaldehyde, urea and ketoester (1:1.2:1.2) was performed at 90 ℃ for 3.0 h under the acidic condition. In addition, the β -ketoester prepared using tyrosol with benzyl had a greater inhibitory effect on α -glucosidase and α -amylase, and the inhibition of enzyme activity for 3, 4-dihydropyrimidinone derivatives prepared using abovementioned β -ketoester was improved significantly. Meanwhile, fluorine-containing dihydropyrimidinone derivatives were considerable inhibitors for both enzymes.


Enzyme inhibition activity assay
The α-glucosidase inhibition activity assay was conducted based on the method described by Mangala Gowri et al. and Tan et al. with some modifications. The potency of α-glucosidase inhibitor was evaluated by detecting the formation of 4-nitrophenol by α-glucosidase after reaction with 4-nitrophenyl α-D-glucopyranoside (PNPG), which was considered as reaction substance.
And α-glucosidase solution (0.5 mg/mL) was prepared in phosphate buffer and stored at low temperature before use. Briefly, 200 μL of 0.5 mg/mL α-Glucosidase solution and 150 μL of target compound solutions at various concentrations were added to each tube containing 2.0 mL phosphate buffer. Before the 150 μL of 5.0 mmol/L PNPG solution was added to initiate reaction, the mixture systems need to be incubated at 37 o C for 10 min, then reaction samples were again subjected to incubation at 37 o C for another 10 min. Followed by adding 2.5 mL of Na 2 CO 3 (0.1 moL/L) to stop the reaction. The absorbance of each tube was measured at 405 nm by a UV-visible spectrophotometer. For all tests, the inhibition assay was performed in triplicate.
Acarbose was used as a positive control. A reaction mixture using equal amount of DMSO reagent to replace the sample solution was used as the control.
The α-amylase inhibition activity assay was performed following the method of previous reported with a slight modification. The solution of starch in phosphate buffer was heated at 100 o C until transparent and α-amylase solution was prepared using phosphate buffer before the experiment. Briefly, at the beginning of test, the mixture system was composed of α-amylase solution and sample solution at different concentrations (in DMSO). Following pre-incubated at 37 o C for 10 min. After that, 300 μL of soluble starch as a substrate was pipetted into each tube to start the reaction. Subsequently, the reaction system was incubated at 37 o C for 10 min. Then 1.0 mL of 96 mM 3, 5-dinitrosalicylic acid (DNS) color reagent was added for reaction termination.
And the test tubes were placed in a boiling water to incubate for 5.0 min and cooled to room temperature. The final volume of reaction solution was made up to 10 mL using distilled water, and the absorbance was measured at 540 nm through the UV-visible spectrophotometer. Acarbose was used as a positive control. The value of IC 50 was calculated to evaluate the effect of enzyme inhibitors. All assays were carried out in triplicate and values were presented as IC 50 .