Study On Extraction Process Of Polyphenols From Eucommia Ulmoides Leaves And Antioxidant And Antitumor Activities

Eucommia ulmoides Oliver,belonging to the Eucommiaceae,has a long history of planting,and has many effects such as anti-oxidation,blood pressure reduction,strong muscles and bones,and immunity enhancement.It is a valuable nourishing medicinal material.However,the research on Eucommia is not comprehensive,most of them are limited to Eucommia bark and Eucommia ulmoides,and the resources of Eucommia ulmoides leaves are not effectively used.Modern studies have shown that the medicinal value and effective ingredients of Eucommia Folium are basically the same as Eucommia Folium and the medicinal functions are basically the same.In this paper,Eucommia ulmoides leaves are used as raw materials to extract non-extractable polyphenols in Eucommia ulmoides leaves by high-pressure pulse-assisted water enzymatic method.Response surface method is used to optimize the extraction process,and its antioxidant activity,enzyme activity inhibition,anti-proliferation and anti-tumor activities are systematically studied.Evaluation.First,the non-extractable polyphenols（Non-extractable Polyphenols NEPP）in Eucommia ulmoides leaves were extracted by high-pressure pulsed electric field assisted water enzyme method.The three-factor and three-level response surface method was used to take cellulase dosage,field strength and pulse number as single factors.The extraction process was optimized,and the best extraction process was:the enzyme dosage was 18 mg,the field strength was 2.81 k V/cm,the number of pulses was 100,and the predicted value was 34.969 mg/g.Under this condition,the test was verified and NEPP was measured.The extraction amount is（34.961±0.052）mg/g.Using acetone as the extraction solvent,extract the extractable polyphenols（EPP）of Eucommia ulmoides leaves by ultrasonic method.The extraction conditions are:85%acetone,material-liquid ratio 1:10 g/v,ultrasonic 30 min（240W,room temperature）,Repeat extraction 3 times,mix the supernatant to obtain EPP extract.The EPP and NEPP extracts were purified by macroporous resin.After purification,the contents of EPP and NEPP were 609.7 mg/g and 80.84 mg/g.Secondly,the antioxidant activity of polyphenols was comprehensively evaluated by three different in vitro antioxidant assay methods（DPPH,ABTS and FRAP）.Full wavelength scanning to determine the optimal measurement wavelengths of DPPH and ABTS solutions are 514 nm and 734 nm.Measure the change in absorbance to determine the optimal reaction time of polyphenol samples with DPPH and ABTS solutions.On the basis of the above test,the antioxidants of polyphenols are tested active.The results showed that the maximum scavenging rate of DPPH and ABTS free radicals of 0.05 mg/m L EPP and 1mg/m L NEPP to DPPH and ABTS reached 91.5%,89%and 68.2%,96.1%.In the FRAP test,the measured FRAP values of EPP and NEPP were 2.90 m Mol/mg and 0.675 m Mol/mg,respectively.The above results show that EPP and NEPP have strong antioxidant activity and iron ion reducing ability,and because the content of EPP is higher than NEPP,the ability of EPP to scavenge free radicals and iron ion reducing ability is much higher than that of NEPP,indicating antioxidant The capacity is directly proportional to the polyphenol content.Through two different enzyme activity inhibition tests（tyrosinase andα-glucosidase）to evaluate the inhibitory effect of Eucommia ulmoides polyphenols on enzyme activity,The results showed that the inhibitory effect of EPP on enzyme activity was much higher than that of NEPP,and no inhibitory effect of NEPP onα-glucosidase was detected.Finally,the antiproliferative effect of Eucommia polyphenols on Caco-2 colorectal cancer cells was evaluated by MTT method.When the concentrations of EPP and NEPP reach a certain value,they have a certain inhibitory effect on cell growth,and the inhibition rate increases with the increase of the sample concentration,and the inhibitory effect of EPP is stronger than NEPP.At the same time,microscopic observation of the cell morphology after EPP treatment revealed that 98%of the cells were dead when the EPP concentration was 1 mg/m L.Next,the content of ROS in the cells was measured,and it was found that the content of ROS in the cells treated with polyphenol+H₂O₂ was higher than that in the polyphenol treatment group and higher than that in the H₂O₂ treatment group,which showed that Eucommia polyphenols may have certain regulatory factors in the cells.The effect of this is to increase the permeability of the cell membrane and increase the ROS content.