Preparation Of A Lotus Leaf Extract With Dual Functions,Screening Of Key Active Compounds And Preliminary Study Of Its Hypouricemic Effect

Lotus leaf,a medicinal and edible plant widely distributed in China,possesses strong antioxidant and xanthine oxidase inhibitory(XOI)activities.It can be used for developing plant-based food ingredients with dual antioxidant and XOI activities and is a kind of highquality raw material for developing plant-based healthy food to regulate hyperuricemia.Due to the complex chemical profile of the lotus leaf and the unknown of key compounds contributing to activities,there is a lack of the preparation methods and quality control standards of lotus leaf ingredient with dual functions and the basic theory of lotus leaf-based food improving hyperuricemia is weak.In the present study,various lotus leaf extracts were prepared by multiple methods followed by the evaluation of superoxide anion scavenging(OS)and XOI activities and the comprehensive analysis of chemical profiles,which realized the accurate classification of lotus leaf extracts and confirmed the most favorable extraction approach for preparing lotus leaf-based food ingredient with dual OS and XOI activities.Quality control standards for lotus leaf extracts with dual functions were defined.Network pharmacology was used to predict targets and pathways by which lotus leaf extract with dual functions ameliorated hyperuricemia.The mechanism by which lotus leaf alkaloids and flavonoid inhibited xanthine oxidase(XO)and their interactions on XOI activity were further investigated.The results will provide method guidance for the industrialized batch manufacturing of stable lotus leaf extracts with dual functions and effective strategies for the development of lotus leaf-based hypouricemic functional food and theoretical supports for the development of new XO inhibitors and the precise preparation of multi-factor hypouricemic functional food ingredients.The main results were listed as follows:(1)Thirty-five lotus leaf extracts were prepared by 4 extraction methods including ultrasound-assisted extraction,reflux extraction,maceration and enzymatic hydrolysis coupled with ultrasound-assisted extraction methods using 12 kinds of solvents followed by the evaluation of their OS and XOI activities,which confirmed that 50% ethanol combined with enzymatic hydrolysis coupled with ultrasound-assisted extraction was the most favorable extraction approach for preparing lotus leaf-based food ingredient with dual OS and XOI activities.Alkaloid and flavonoid profiles of 31 lotus leaf extracts were qualitatively and quantitatively analysed via UPLC-MS/MS.A total of 17 alkaloids and 23 flavonoids were identified and it was the first time that hesperetin had been identified in lotus leaf.Lotus leaf extracts were accurately classified based on their OS and XOI activities and chemical profiles.27 representative lotus leaf extracts with abundant characteristic compounds were screened.The alkaloid profiles of lotus leaf extracts were mainly influenced by the extraction solvents and the flavonoid profiles of lotus leaf extracts were influenced by the extraction solvent and enzymatic hydrolysis coupled with ultrasound-assisted extraction.The chemical profiles of lotus leaf extracts prepared by 50% methanol,50% ethanol and 50% acetone via enzymatic hydrolysis coupled with ultrasound-assisted extraction were similar and they all possessed 12 characteristic alkaloids,high contents of flavonoid aglycones and flavonoid glycosides and strong OS and XOI activities.(2)Four typical lotus leaf extracts with different chemical profiles and different OS and XOI activities were selected screened out as research objects from 27 representative lotus leaf extracts.Recombinant models were constructed to explore the contribution of characteristic compounds to activities.The results indicated that the flavonoids mainly contributed to OS activity of typical lotus leaf extracts with abundant flavonoids while alkaloids had almost no contribution to OS activity and both alkaloids and flavonoids played important roles in the XOI activity of typical lotus leaf extracts.Besides,the interaction between alkaloids and flavonoids could significantly affect the OS and XOI activities of typical lotus leaf extracts.On the basis,quality control standards for lotus leaf extracts with dual functions were screened by omission experiment as: norcoclaurine,O-nornuciferine,N-nornuciferine,nuciferine,luteolin,diosmetin,quercetin and quercetin-3-O-glucuronide.Based on network pharmacology,pathways by which lotus leaf extract with dual functions prepared by 50% ethanol combined with enzymatic hydrolysis coupled with ultrasound-assisted extraction improved hyperuricemia were investigated.The results showed that the extract could improve hyperuricemia and its complications by multiple actions on uric acid synthesis and transport,inflammation response,oxidative stress and lipid metabolism.(3)The XOI activities of 5 benzylisoquinoline alkaloids,6 apophine alkaloids and 1flavonoid(diosmetin)in lotus leaf were determined,among which diosmetin possessed the strongest XOI activity while norcoclaurine and N-nornuciferine were benzylisoquinoline and apophine alkaloid possessing the strongest XOI activities,respectively.XOI activities of benzylisoquinoline alkaloids were closely associated with the substitution of methoxy/hydroxy group on C-6/7 and the substitution of methyl group/hydrogen atom on N-2.The XOI activities of apophine alkaloids were closely associated with the substitution of methoxy/hydroxy group on C-2 and the substitution of methyl group/hydrogen atoms on N-6.The mechanism by which lotus leaf alkaloids and diosmetin inhibited XO were further investigated via kinetics analysis of XO inhibition,fluorescence quenching assay and molecular docking.The result indicated that norcoclaurine,armepavine,N-nornuciferine,nuciferine were mixed-type inhibitors of XO,which could partially enter the active site of XO and formed complexes with XO while diosmetin inhibited XO competitively and could enter the active site of XO and formed complex with XO.These five compounds had only one binding site with XO and interacted with Leu648,Phe649,Val1011,Phe1013 and other amino acid residues at the catalytic site of XO.CI index was used to evaluate the interactions between compounds.When the molar ratio of norcoclaurine,armepavine,N-nornuciferine,nuciferine and diosmetin was 7:21:147:49:32 and the XOI rate was less than 85%,the synergy effect was the strongest.