Chemical Resource And Exploration On The Resource Value Of Glycyrrhiza Inflata Flavonoids And Lycium Barbarum Phenolamides

Glycyrrhiza inflata is the main material for the extraction of glycyrrhizin in the pharmaceutical and food industries.Currently,the demands for glycyrrhizin are increasing due to its wild utilization not only in medical industry but also in food and household products industry.There are still abundant flavonoids in the residues,contributed to the limitation of extraction process.However,these residues are mainly treated by landfill,combustion,and other extensive ways.In addition,a large number of non-medicinal parts which are produced in the harvesting process of G.inflata are not effectively utilized,resulting in a serious waste of G.inflata resources.Therefore,this study aims to assess the resource value and promote the resource utilization of G.inflata based on comprehensive chemical evaluations of flavonoids in G.inflata.Meanwhile,we have reported that phenolamides in L.barbarum exhibited potent biological activities such as antioxidant and anti-inflammatory,etc.However,the low content of those components in L.barbarum limits its wide application in the medical health industry.Therefore,to solve the problem of insufficient resources of phenolamides in L.barbarum,we will explore the green biosynthesis strategies of phenolamides in this study to promote the resource utilization of these components.The content of this research mainly includes the following aspects:Chapter One,Study on the chemical resources of G.inflata1.Analysis and identification of flavonoids in G.inflataStudies have shown that there were significant differences in flavonoids in three different varieties of licorice cultivars,and there were also some differences in the bioactivities of these components.For the further exploration of the value of G.inflata,it is necessary to analyze and evaluate the flavonoids in G.inflata.In present study,a approach based on UPLC-MS/MS was used for the directional separation of flavonoids in G.inflata.The mass spectrum cracking law showed that the main flavonoids in G.inflata will generate characteristic ions such as ³A⁺ and 4^B+through RDA reaction.The neutral losses of 42 Da（C₃H₆）and 56 Da（C₄H₈）were yielded when isopentenyl groups presented on the side chain.Meanwhile,the neutral losses of 42 Da（C₃H₆）and 54 Da（C₄H₆）,and 70 Da（C₄H₆O）will occur when the pyran ring exists on the side chain.The characteristic peaks in the different parts of G.inflata were isolated and identified by using 1DNMR,2D-NMR,and LC-MS methods.Results showed that the main compounds separated from G.inflata were chalcones,flavanones,and dihydrostilbenes,of which four new compounds were isolated from the aboveground parts and all of them were isopentenyl dihydrostilbenes.2.Dynamic analysis and evaluation of flavonoid accumulation in different parts of G.inflata at different growth stagesIn the present study,the content of flavonoids in the root,stems,and leaves of G.inflata was analyzed and evaluated by using the HPLC method.The results showed that there were significant differences in flavonoids between the aboveground and underground parts.Licochalcone A,licochalcone B,licochalcone D,and isoliquiritigenin are mainly distributed in the underground part while 6-prenyleriodictyol,8-prenyleriodictyol and 8-prenylnaringenin are mainly distributed in aboveground part.By analyzing the content of flavonoids in September,October and November,it was found that the content of flavonoids in aboveground and underground parts was the highest in October,reaching about 1.3%and 1.7%,respectively.Chapter two,Exploration on the value of G.inflata flavonoids1.Study on antibacterial activity and related mechanism of flavonoids in G.inflataβ-lactam antibiotics could inhibit the cell wall synthesis of Gram-positive bacteria.However,with the abuse of antibiotics in recent years,a large number of drug-resistant bacteria have appeared leading to the failure of most antibiotics which target bacteria cell walls.Therefore,it is urgent to search for new antibiotics or new antimicrobial targets.We have reported that flavonoids in G.inflata had potent activities against Gram-positive bacteria.Thus,in present study,it is mainly aimed to illustrate the main antibacterial substances and related mechanism of flavonoids in G.inflata for promoting the rational use of flavonoids as antibacterial agents.The antibacterial spectrum of flavonoids in G.inflata was studied.It was found that flavonoids in G.inflata showed potent activities against gram-positive bacteria such as Staphylococcus aureus,methicillinresistant Staphylococcus aureus（MRSA）,while showed the weak activity against gram-negative bacteria.Licochalcone A,licochalcone B,licochalcone D and licochalcone E are the major bioactive compounds in the underground part,and 6-prenyleriodictyol,8-prenyleriodictyol,eriodictyol and 8-prenylnaringenin are the main bioactive compounds in the overground part.Importantly,the results of structural-activity relationship（SAR）revealed that the presence of isopentenyl could significantly enhance the antimicrobial activities of chalcones,flavanones,and dihydrostilbenes.The antibacterial mechanism of LA and TPB was revealed by using the multi-omics technique.The results also showed that LA and TPB may inhibit MRSA through multiple targets,including targeting the MRSA membrane,causing changes in membrane permeability,membrane fluidity,and membrane potential,inducing oxidative stress damage in MRSA cells,inhibiting glucose phosphorylation.We also found that the antibacterial activity of TPB was significantly reduced by adding metal ions Mn2+ and Fe3+ into the medium.It is speculated that TPB may interfere with the absorption and transport of metal ions by MRSA.2.Screening bioactive compounds against pancreatic lipase from G.inflata and structureactivity relationship analysisPancreatic lipase is mainly responsible for the hydrolysis of triglycerides in food,so it has been considered as the main target for the inhibition of obesity.Previous studies revealed that chalcones showed significant anti-pancreatic lipase activity.In this study,the ligand fishing method was thus used to screen out the bioactive components against pancreatic lipase in G.inflata,and the structure-activity relationship was then analyzed.The results showed that chalcones including licochalcone A,licochalcone B,licochalcone D and licochalcone E were the major bioactive components in G.inflata.The results of SAR revealed that the presence of 5-position isopentenyl on B-ring or 3-position isopentenyl on B-ring and the presence of 3-OH on A ring could significantly improve the anti-pancreatic lipase activity of chalcone compounds3.Preparation and bioassay evaluation of flavonoid/starch composite filmsThe degradable and edible starch film has been widely used in the food preservation industry.However,it cannot prolong the shelf life of food effectively,due to its poor antibacterial and antioxidant activities.In present study,the effects of G.inflata flavonoids（GIFs）on the physicochemical properties and bioactivities of starch film were thus evaluated,to prepare bioactive films.Fourier transform infrared spectroscopy（IR）,X single-crystal diffractometer（XRD）,and scanning electron microscopy（SEM）were used to characterize the structure of films.The results indicated that the interaction between flavonoids and starch was mainly through intermolecular forces.The physicochemical properties of the films showed that the flavonoids could significantly increase the elongation at break and decrease the transmittance of light together with water vapor.In addition,the bioassay results presented that the addition of flavonoids could effectively improve the antibacterial and antioxidant properties of starch film.Chapter three,Exploration of the resource value of phenolamides1.Synthesis of phenolamides by using immobilized enzymeIn this study,a two-step strategy was used to synthesize phenolamides.The first step was to synthesize aromatic esters using solid acid catalytic resin,and phenolamides were then synthesized by using immobilized lipase.The structures of the products were identified using 1D-NMR and LC-MS.The results of 1H-NMR showed that the stereoscopic configurations of the synthesized products were all trans,indicating the high stereoselectivity of reaction.The effects of different solvents,the dosage of the nucleophile,and the dosage of enzyme on the reaction conversion were evaluated.The results showed that the optimal reaction solvent is MTBE and the optimal Nu/S is 2:1,the optimal E/S is 20:1,and the optimal temperature is 50℃.Under the optimal reaction conditions,the yield was still maintained at over 60.6%,when the enzyme was reused 5 times.In addition,it was found that the conversion rates of all substrates were above 65%under the optimum conditions.2.The potential of phenolamides against α-glucosidase and structure activity relationship analysisThe results of SAR revealed that tyramine derivatives had better activity against αglucosidase compared with phenethylamine derivatives.The presence of 4-OH,3-OH or 3-OCH₃ could significantly improve the activities of tyramine derivatives against α-glucosidase.