Screening Of Lipase Inhibitors In Artemisia Argyi Leaves And Preparation And Release Properties Of Rice Starch Inclusion Complex

As a traditional food,Artemisia argyi leaves has abundant active ingredients and potential efficacys.Today,obesity has become a global public health problem,and its serious trend cannot be ignored.The previous experimental results in our laboratory confirmed that the extracts of Artemisia argyi leaves had a significant inhibitory effect on lipase,and lipase is a key enzyme in dietary fat absorption.The current popular method for rapid screening of target enzyme inhibitors is undoubtedly the ultrafiltration method.There is no report on the use of this method to screen lipase inhibitors from Artemisia argyi leaves.The ultrafiltration method is mostly carried out in the system of water as the medium,and the screening of fat-soluble components is limited,so it is urgent to establish a screening method suitable for fat-soluble active components,so as to realize the systematic screening of lipase inhibitors in Artemisia argyi leaves extracts.Some natural terpenoids and flavonoids,as common lipase inhibitors,have poor stability in gastric acid and cannot target specific sites to play their role,resulting in low bioavailability,which ultimately limits their use in food,etc.Therefore,it is very important to scientifically and effectively protect the activity of molecules.Therefore,this paper is based on the establishment of a screening method suitable for lipid-soluble lipase inhibitors,combined with ultrafiltration centrifugation mass spectrometry,to systematically and comprehensively screen,identify and evaluate lipase inhibitors in Artemisia argyi leaves.And based on the targeted delivery system of inclusion complex,rice starch is used to protect the activity of small molecules,enhance its stability in gastric acid,and make it targeted to reach the small intestine to exert its effect,thereby improving its bioavailability.The main research contents and results are as follows:（1）Extraction of different polar components of Artemisia argyi leaves.The small,medium and large polar components of Artemisia argyi leaves were extracted step by step by solvent extraction method,and there were obvious differences in the extraction rate,total flavonoid content and total polyphenols of each polar component.The results of the in vitro enzyme activity of the extract showed that the small polar component and medium polar component of the Artemisia argyi leaves had significant lipase inhibitory activities,and their inhibition rates were 89.30±8.14% and 88.05±8.09%,respectively,which were valuable to screen the active components in the next step.While the large polar extract of Artemisia argyi leaves did not show inhibitory activity on lipase.（2）Screening of lipase inhibitors in the small polar fractions of Artemisia argyi leaves.A screening method suitable for lipid-soluble lipase inhibitors was established,namely the two-phase mixing method.By exploring the screening conditions of the two-phase mixing method,the suitable screening conditions were obtained with the lipase concentration of 0.4 mg/mL,the volume ratio of sample to lipase of 300 μL:300μL,and the two-phase mixing time of 30 s.Two-phase mixed method combined with gas chromatography-mass spectrometry（GC-MS）,screened and identified two lipid-soluble lipase inhibitors from the small polar fractions of Artemisia argyi leaves,namely β-caryophyllene and 2,4,6-Tri-tert-butylphenol.Finally,the active molecules were evaluated and verified by in vitro enzyme activity assay and fluorescence quenching assay.The results showed that both β-caryophyllene and2,4,6-tri-tert-butylphenol exhibited inhibitory effects on lipase,and the highest inhibition rate can reach 51.23±0.56% and 57.02±2.99%,respectively.The two active compounds and lipase were mainly combined by hydrophobic interaction to form a complex.At the same time,the feasibility of the two-phase mixing method was also proved.（3）Screening of lipase inhibitors in polar components of Artemisia argyi leaves.Using ultrafiltration centrifugation mass spectrometry combined with ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry（UPLC-Q-TOF-MS）technology,6 kinds of lipase inhibitors were rapidly and effectively screened from medium polar extracts of Artemisia argyi leaves.The chemical structures of two lipase inhibitors,7-hydroxycoumarin and luteolin,were identified.Finally,in vitro enzyme activity experiments and fluorescence quenching experiments were used to evaluate and verify the active molecules.The results showed that both 7-hydroxycoumarin and luteolin could effectively inhibit the activity of lipase.And the highest inhibition rate can reach 75.82±1.97% and 87.72±5.41%,respectively.7-hydroxycoumarin binds to lipase by electrostatic interaction,while luteolin binds to lipase through hydrophobic interaction.At the same time,it is proved that ultrafiltration centrifugation mass spectrometry combined with UPLC-Q-TOF-MS was a reliable method for screening biologically active molecules,and its inhibitors can be further studied.（4）Inclusion of active ingredients of Artemisia argyi leaves and rice starch.The luteolin-rice starch inclusion complex was prepared by self-assembly method,and characterized by scanning electron microscopy,Fourier transform infrared spectroscopy and X-ray diffraction techniques,which proved that luteolin was successfully incorporated into rice starch.The encapsulation efficiency was 96.67±0.09%,and the sample loading was 20.12±0.02 mg/g.Through the simulated digestion experiment in vitro of the inclusion complex,it is proved that the luteolin in the inclusion complex would not be released rapidly in the gastric stage,but can reach the small intestine smoothly,realizing the targeted release of luteolin,thereby improving the bioavailability of luteolin.