Study On The Analgesic,Anti-inflammatory And Uric Acid-lowering Active Components Of Coix Seed

With the improvement of living standards and changes in dietary habits,the increasing consumption of high-purine foods has led to an annual increase in the prevalence and incidence of gout,whose main symptoms include acute gouty arthritis and hyperuricemia（HUA）.Rapid and effective relief of inflammatory pain during acute attacks and improvement of uric acid levels are key to treating gout.Coix seed is a kind of medicinal and edible homologous food,with anti-tumour,analgesic,anti-inflammatory,and immunomodulatory effects.However,there are few studies on the active ingredients related to the prevention and treatment of HUA and gout with Coix seed.In order to comprehensively understand the substance basis of Coix seed to play an analgesic,anti-inflammatory and good uric acid-lowering effect,this paper used multiple-column chromatography to separate the anti-gout active components of Coix seed,screened and explored the active substances through in vitro and in vivo experiments.The main research content and results are as follows:1.Coarse powder of Coix seed was sequentially extracted with 70%ethanol and deionized water to obtain Coix seed ethanol extract（yield 1.52%）and water extract,respectively.The water extract was further precipitated by ethanol to obtain alcohol precipitation（yield 8.23%）and alcohol solution（yield 0.61%）.The analgesic,anti-inflammatory,and uric acid-lowering activities of different solvent extracts of Coix seed were evaluated using the acetic acid-induced writhing test in mice,xylene-induced mouse ear swelling model,and HUA mouse model induced by potassium oxonate and hypoxanthine.The results showed that alcohol precipitation of Coix seed significantly reduced the number of writhing movements in mice and ear swelling（P<0.01）,and the inhibition rate of writhing response and ear swelling were 49.01%and59.65%,respectively.It indicated that the alcohol precipitation of Coix seed had analgesic and anti-inflammatory activities,while Coix seed ethanol extract had significant uric acid-lowering activity（P<0.01）.2.The water extract of Coix seed was fractionated using ethanol precipitation to obtain two kinds of precipitates.Further screening of the analgesic and anti-inflammatory activity components was performed using the acetic acid writhing test,hot plate test,xylene-induced ear swelling and carrageenan-induced paw swelling in mice.The results showed that CSP-A had significant effects on reducing the number of writhing responses and increasing the pain threshold（P<0.01）.Moreover,CSP-A could significantly decrease the serum PGE2 level in mice after acetic acid stimulation,indicating that the analgesic ingredient of Coix seed was mainly in CSP-A.Both CSP-B and CSP-A showed good anti-inflammatory effects,and the anti-inflammatory effect of CSP-B was better than that of CSP-A.After separation and purification by DEAE-52 cellulose column,HW-55F gel column,HW-40F gel column,and Sephacryl S-400 gel column,pure polysaccharide CSP-Ⅰand oligosaccharide CSP-Ⅱwere obtained.The chemical structures were identified by high-performance liquid chromatography（HPLC）,ion chromatography,nuclear magnetic resonance（NMR）and other analytical methods.CSP-Ⅰwas（1→6）-α-glucan.The sequence of sugar residues in CSP-II wasβ-Frup（2→[1）-α-Glcp（6]₅→1）-α-Glcp（4→1）-α-Glcp.In vitro anti-inflammatory activity studies showed that both CSP-I and CSP-II significantly inhibited the accumulation of nitric oxide（NO）in RAW264.7 macrophage cells stimulated by lipopolysaccharide（LPS）in a dose-dependent manner,as well as the production of tumour necrosis factor-α（TNF-α）,interleukin（IL）-6,and IL-1β.CSP-II exhibited a stronger inhibitory effect than CSP-I.3.In order to further explore the substances with uric acid-lowering activity of Coix seed,the ethanol extract was separated into four components using MCI column chromatography and further screened for uric acid-lowering activity using an HUA mouse model.It was found that the 50%ethanolic eluate significantly reduced the serum uric acid and creatinine levels in mice（P<0.01）,and had a certain improvement effect on mouse kidney damage caused by high uric acid.Four monomeric compounds were obtained by ODS column isolation and purification,and their structures were identified by NMR analysis as follows:1-phenyl-1,3,3-trimethyl ureaa（1）,dihydrocaffeic acid（2）,6-phenethyl glucoside（3）,and phenethyl-6’-O-α-pyranosyl-arabino-O-β-pyranosyl-glucoside（4）.Compounds 1 and 2 were isolated from Coix seed for the first time.In vitro enzyme inhibition experiments showed that both compounds 1 and 2 could effectively inhibit the activity of xanthine oxidase,and the half-maximal inhibitory concentration(IC₅₀)of compound 1 was lower than that of compound 2.Both compounds were reversible mixed-type inhibitors,which reduced the fluorescence intensity of xanthine oxidase through static quenching and could cause changes in the secondary structure of enzyme.Hydrogen bonding and hydrophobic interactions were the main binding forces between xanthine oxidase and compounds 1 and 2,respectively.