Screening Of 5-LOX Inhibitor In Leaves Of Lonicera Japonica And Its Anti-inflammatory Effect Research

Inflammation is an important pathological process,which is closely related to many diseases.Natural products have attracted the attention of many scholars because of its safety,reliable medicinal value,and low toxicity.There are many active ingredients in leaves of Lonicera japonica?LLJ?,and its pharmacological activity is comparable to flower.Therefore,it is of great significance to study the compounds with anti-inflammatory effects in LLJ.This thesis established a high effective approach to screening 5-lipoxygenase?5-LOX?inhibitors from LLJ using enzyme-immobilized TiO₂ nanotubes and versatilely studied its anti-inflammatory effects via enzyme model,cell model,and anti-microbial test.The main research content is divided into the following four parts:1.Screening of 5-LOX inhibitor from Lonicera japonica leaves using enzyme-immobilized TiO₂ nanotubesThe model of Enzyme-immobilized TiO₂ nanotubes was constructed and the optimal conditions for immobilization were determined to be 6%of glutaraldehyde concentration,90 min of immobilized time,and the 1.44?g of enzyme amount.Then the model was successfully applied to screen potential 5-LOX inhibitor from the extract of LLJ.Four compounds confirmed as Luteolin,luteoside,lonicerin,and isochlorogenic acid C were selected to be potent ligands of 5-LOX using HPLC/MS.Meanwhile,a mixture of other potential ligands M1 was also screened out and prepared by semi-preparing liquid chromatography.The results provided a fast,efficient and specific method which provides a theoretical basis for the development of anti-inflammatory drugs.2.Study on the inhibion of active ingredients from Lonicera japonica leaves on 5-LOXThe interaction of active ingredients and M1 with 5-LOX was researched using Fe²⁺-xylenol orange method,FT-IR spectrum and CD spectrum.Luteolin,quercetin,luteoside,lonicerin,isochlorogenic acid C and M1 showed inhibition on 5-LOX.The results corresponded with the results of enzyme-immobilized screening method,except quercetin which content was extremely low in LLJ.These further indicated that the sceening meothd was feasible and accurate.The enzyme kinetics results manifested the inhibitory mechanisms of luteolin on 5-LOX was reversible non-competitive inhibition.In addition,FT-IR and CD results all showed that the secondary structure of 5-LOX was changed respectively after interaction with luteolin,quercetin,luteoside,lonicerin and isochlorogenic acid C with?-helix content increased and?-sheet content decreased significantly.3.Anti-inflammatory activity of active ingredients from LLJ on LPS-stimulated RAW 264.7 cellsAn RAW 264.7 cells inflammation model induced by lipopolysaccharide?LPS?was established to research the effect of 5-LOX inhibitors?luteolin,quercetin,luteoside,lonicerin,isochlorogenic acid C,M1?from LLJ on the accumulation of nitric oxide.It turned out that lonicerin,isochlorogenic acid C,M1 were nontoxicity under experimental dose and could significantly suppressing the production of NO in cells,they all had good anti-inflammatory activity.4.Anti-inflammatory and anti-microbial activity of nano-silver synthesized using LLJ extractNano-silver reduced by leaves of Lonicera japonica?NS-LLJ?was prepared by green synthesis method using LLJ extract and its effect on 5-LOX was stueied.Meanwhile,choosing escherichia coli?EC?and staphlococcus aureus?SA?as experimental strains,the antibacterial activity of NS-LLJ was tested by punch method and broth dilution method.The results revealed that the synthesized nanoparticles are round particles with good dispersity and average particle size of 20-30 nm.NS-LLJ showed significant inhibitory effect on 5-LOX with IC₅₀=5.08?g/mL and exhibited antibacterial activity against EA and SA with the minimal inhibitory concentration?MIC?value of 7.81 and 1.95?g/mL,respectively.