Study On Preparation And Biological Activity Of Polyphenol Actives From Olive Pomace

Olive oil is favored by consumers due to its rich nutritional value and health benefits.At present,olive oil is obtained mainly by pressing olive fruit and two phase centrifugal separation.A large amount olive two phase centrifugal pomace（OTPCP）has been produced in this process.Because a large number of phenolic compounds still remain in OTPCP,it will not only cause the waste of resources,but also lead to environment pollution.In this paper,OTPCP was taken as the main research object.The composition of chemical components and kinetics and mechanism of pyrolysis for OTPCP were analyzed,and the extraction,enrichment and purification technology of polyphenols from OTPCP were study.At the same time,the fragmentation pathway,metabolic mechanism,preparation of derivatives and biological activity for the characteristic phenolic compounds were also explored.This study will provide a theoretical basis for efficient development and comprehensive utilization of OTPCP.（1）Study on HPLC characteristics and fragmentation pathway of mass spectrometry of polyphenols from olive pomaceThe processing methods on OTPCP removed stone of direct water extraction and ultrasound assisted enzyme hydrolysis（UAEH）were compared.The results showed that the solid content of direct aqueous extract was 11.60 mg/g,the total polyphenol content was 0.75 mg/g.While the solid content of the sampled treated by theUAEH was 34.55 mg/g,the total polyphenol content was 1.20 mg/g.In addition,a HPLC-MS method was developed for the simultaneous determination of the main phenolic compounds in the OTPCP,such as gallic acid,hydroxytyrosol,tyrosol,vanillic acid,caffeic acid,syringic acid,ferulic acid and oleuropein.And the possible mass spectrometric fragmentation patterns have been proposed in positive and negative ion modes.In the negative ion mode,the fragmentation pathways of gallic acid and caffic acid was similar,mainly with the loss of-COOH.The cracking routes of syringic acid,ferulic acid and vanillic acid was also consistent,and the process is dominated by the-OH、-COOH and-CH₃.The fragmentation ions of main phenolic compounds in positive ion mode were obviously different from negative ion mode,and a large amount of protonated,potassium and sodium product fragmentation ions were observed.（2）Study on extraction kinetics of UAEH and extraction characteristics of polyphenols from olive pomaceThe polyphenols were extracted by UAEH from OTPCP without stones,and the optimized conditions obtained from response surface methodology（RSM）were pH 5.75,treatment 40 min and temperature 55℃,the yield was 3.99%±0.81（n=3）,and the verify experimental result was close to predictions value.The extraction kinetics model was fitted well to the ln（C_t/C₀）=kt,the influence of temperature on the extraction process was obvious,and the rate constants（k）increased with the increased in the extraction temperature.The activation energy of UAEH was lower.Furthermore,compared with the extraction of petroleum ether and n-butanol,the total polyphenol content in ethyl acetate fraction was the highest,the content was 64.43 mg/g.Meanwhile,the contents of main phenolic compounds from high to low in turn for hydroxytyrosol,tyrosol,gallic acid,caffeic acid,ferulic acid and vanillic acid.And the ethyl acetate extract had good antioxidant activity.（3）The relationship between changes of polyphenols and pyrolysis characteristics of olive pomace with and without UAEHThe methods of fourier transform infrared spectra（FT-IR）,scanning electron microscope（SEM）,differential scanning calorimetry（DSC）and thermogravimetry（TG）were used to analyze sample changes and pyrolysis characteritics.The results from FT-IR,SEM and DSC showed that the structure and composition of samples were changes by UAEH.The pyrolysis characteristic of OTPCP untreated and treated by UAEH was investigated using the thermogravimetric analysis at different heating rates.The thermal decomposition was divided into three stages in the thermograph curve.Although TG curve showed the same decomposition trend for two samples,the temperature interval and peak temperature were different.The Kissinger method,Kissinger-Akahira-Sunose（KAS）method and Flynn-Wall-Ozawa（FWO）method showed the avtivation energy of sample treated by UAEH was lower than that of sample untreated during the pyrolysis process.The KAS and FWO methods revealed the similar kinetic parameters characteristic for the conversion degree from 0.1 to 0.9,and the average values of activation energy were 201.42 kJ/mol and 162.97 kJ/mol,respectively.The Coats Redfern（CR）method suggested the second-order model（F₂,f（α）=（1-α）²）could be used to better describe the main thermal decomposition mechanism of OTPCP untreated and treated.（4）Enrichment technology and kinetic characteristics of polyphenols from olive pomaceThe nine different resins were selected to investigated the adsorption characteristics of polyphenols from olive pomace through static adsorption.The XDA-1 and NKA-Ⅱresins showed outstanding adsorption and desorption capacity.The adsorption rates were 83.33%and84.68%,respectively.when 40%,60%and 80%ethanol solutions were used as desorption solution.Moreover,the desorption rates were 76.62%and 73.70%,respectively.Adsorption kinetics and isothermal models results revealed that the adsorption of polyphenols by the two resins was in accordance with the pseudo second-order kinetic and the Freundlich adsorption isotherm model.The adsorption behaviors belonged to rapid adsorption.It showed that chemical adsorption played a dominant role in the adsorption process,and there might be electron transfer between the adsorbent and adsorbed material.Moreover,physical adsorption could not be neglected.At the same time,the results of the particle diffusion model illustrated that the adsorption of polyphenols by XDA-1 and NKA-Ⅱresins were complex and diverse,which were mainly controlled by boundary layer diffusion and particle internal diffusion.The calculation results of thermodynamic parameters also indicated that the adsorption of polyphenols by the resin selected were spontaneous and exothermic process.Subsequently,the experiment of gradient elution were carried out through treated XDA-1 resins column,the result indicated the total polyphenols were mainly obtained from the 40%and 60%ethanol fractions,and they had strong antioxidant capacity.Therefore,it could be considered as an active fraction.（5）Study on chemical constituents in polyphenol enriched products of XDA-1 from OTPCPThe active fraction from enriched products was extracted through petroleum ether,ethyl acetate and n-butanol,and the different extracts were investigated under the same GC-MS conditions.The petroleum ether extract was mainly composed of fatty acids.In addition to unsaturated fatty acid,alkanes,alkenes and heterocycles,some polyphenols,such as 4-hydroxy-phenylethanol,3,4-dihydroxyphenylethanol and 4-hydroxy-3-methoxycinnamic acid,were found in the ethyl acetate fraction.The extract of n-butanol mainly contained esters and aldehydes.The chemical composition of the ethyl acetate extract was further studied using different chromatographic techniques.A total of 14 compounds were identified by structure identification,which covered phenylpropanoids compound,flavonoids compounds and polyphenols compounds,including two compounds（catechin and ethyl gallate）isolated from the OTPCP for the first time.（6）Interaction between phenolic compounds from OTPCP and bovine serum albuminThe interaction between hydroxytyrosol and tyrosol and bovine serum albumin（BSA）were investigated by fluorescence spectrophotometry.The results showed the quenching rate constants of the two system were greater than 2.0×10¹⁰ L/（mol·s）,and it suggested that the quenching mechanism of BSA by phenolic compounds was static quenching.The binding constants（K_a）were higher than 1.0×10³ L/mol,the number of binding sites were close to 1.0.Meanwhile,the experiment results presented that there was non-radiative energy transfer between the donor and the acceptor.The binding distances between the two phenolic compounds and BSA were less than 7.0 nm.Therefore,the whole quenching process was in accord with F?rest energy transfer theory.The results from thermodynamic parameters showed that the interaction between these phenolic compounds and BSA were mainly hydrogen bonds,van der Waals force and hydrophobic forces.UV-visible absorption spectra and synchronous fluorescence revealed that the peptide chain structure or microenvironment of amino acid residues might be changed when the different types of quenchers interact with BSA.When Cu²⁺,Zn²⁺and Fe³⁺were involved in the reaction system of phenolic compounds and BSA,the changes were caused in quenching rate constants,binding constants,binding distances and interaction forces due to different quenching system.（7）Preparation and activity evaluation on derivatives of tyrosol from OTPCPTyrosol was used as a reactant to productβ-ketoester by transesterification,afterwards,the3,4-dihydropyrimidinone derivative was prepared using the above target compound.And it was more conducive to the preparation of the target product when the amount of catalyst（Yb（OTf）₃）was 0.3 mmol.The reaction mechanism might be related to a complex system between Yb³⁺and reactants.Furthermore,the results showed the product yield was higher when the reactant n（benzaldehyde）∶n（urea）∶n（ketone ester）was 1∶1.2∶1.2 and the Biginelli reaction was carried out at 90℃for 3.0 h under the acidic condition.The major step in this process might be nucleophilic addition reaction of N-acylimine ions with carbonyl isomers.In addition,theβ-ketoester derivatives prepared using tyrosol with benzyl had a greater inhibitory effect onα-glucosidase andα-amylase than the product of direct transesterification.It was worth noting that the inhibition of enayme activity for 3,4-dihydropyrimidinone derivatives prepared using thisβ-ketoester derivatives was imporved significantly.Meanwhile,compared with other substituent groups,fluorine-containing these derivatives had more significant inhibition effect on both enzyme.