Extraction And Purification Of Phenolic Compounds From Areca Seeds And Inhibition Mechanism Of Streptococcus Mutans And Its Biofilm

Areca catechu L.is a tropical palm crop,which is composed of Areca husk and Areca seed.Areca catechu L.is widely cultivated in Hainan,Taiwan and other places in China,and is an important economic and medicinal plant.As the first of the four southern medicines in China,areca has the functions of killing insects,eliminating storage,benefiting water and blocking malaria.However,the current research on areca catechu mainly focuses on alkaloid compounds,and there are few reports on non-alkaloid compounds.However,as one of the most abundant non-alkaloid compounds in areca nut,the research and functional evaluation of areca are less,which seriously restricts the high added value and industrial development of areca industry.Studies have shown that natural phenolic compounds can prevent caries and inhibit cariescausing bacteria,S.mutans,without drug resistance after long-term use.It has been reported that areca phenolic compounds have a good inhibitory effect on S.mutans,but its mechanism has not been systematically studied.Phenolic compounds in areca mainly distribute in areca seed.Therefore,in this paper,areca seeds were used as raw materials to extract and purify phenolic compounds,and the mechanism of inhibition of S.mutans and its biofilms by purified products was explored.The main research contents and results are as follows:Extraction of phenolic compounds from areca seed and determination of their inhibitory activity against S.mutans and its biofilm.Ultrasonic-microwave synergistic extraction(UMSE)was used to extract phenolic compounds(USP)from areca seed,and the optimal extraction conditions were determined by single factor and response surface experiments: The yield and content of phenolic compounds were 287.94 ± 8.32 mg/g and 45.54 ± 0.64%,respectively,when the extraction time was 584 s and microwave power was 278 W.Compared with conventional liquid-solid extraction(CLSE),UMSE showed higher efficiency and yield,and higher inhibition of S.mutans and its biofilm activity(MIC,MBC and MBIC were 2.50,5.00 and 1.00mg/m L,respectively).Isolation and purification of USP and determination of its inhibitory activity against S.mutans and its biofilm.AB-8 resin with better USP separation effect was screened by static and dynamic screening experiments.Dynamic adsorption/elution optimization experiment determined that the optimal technological conditions were as follows: the loading concentration was 10 mg/m L;The loading velocity was 1.0 BV/h.Elution flow rate was 1.0 BV/h.The elution volume was 6.0 BV,12.0 BV,4.0 BV,6.0 BV and 2.0 BV,respectively,in water and 20%,30%,40% and 90%(v/v)ethanol.The purity and recovery of 30% ethanol purified phase(30%EPH)were 96.66 ± 2.15% and 58.10 ± 2.36%,respectively.In addition,30%EPH had the best bactericidal effect on S.mutans,with MIC,MBC and MBIC of 0.31,0.63 and 0.13 mg/m L,respectively.Further,UPLC-Q-TOF-MS、FT-IR、MALDI-TOF-MS and thiolysis experiments were used to analyze 30%EPH.The results showed that 30%EPH was a group of phenolic compounds composed by B-type proanthocyanidins and their polymers,and the degree of polymerization(DP)was 13.Inhibition of S.mutans by 30%EPH of areca seed phenolic extract.Time-kill curve and glycolysis test showed that 30%EPH had high germicidal activity against S.mutans and inhibited the production of acid by sucrose experiment.At the same time,membrane permeability,membrane integrity,SEM,TEM,membrane fluidity,and the membrane protein experiments showed that 30%EPH can destroy the structure of cell membrane proteins.At the same time,30%EPH changed the lipid bilayer structure,resulting in decreased membrane fluidity,decreased membrane potential,hyperpolarization of the cell membrane,and the outflow of important components such as intracellular proteins and nucleic acids.Furthermore,30%EPH disrupted cell membrane integrity,which resulted in the death of S.mutans.In conclusion,with the increase of 30%EPH concentration,the activity of S.mutans could be effectively inhibited.Inhibition mechanism of 30%EPH on S.mutans biofilm.The inhibition rate of S.mutans biofilm at 30%EPH concentration was dose-dependent,and the inhibition rate of S.mutans biofilm at 30%EPH sub-concentration(1/3MIC)was 88.70 ± 3.18%.In addition,the growth experiment found that 1/3MIC 30%EPH did not affect the growth of bacteria,proving that1/3MIC 30%EPH did not inhibit the formation of biofilms by inhibiting the growth of bacteria.Adhesion test,hydrophobicity test and extracellular polysaccharide(EPS)test showed that1/3MIC 30%EPH significantly affected S.mutans.The inhibition rates of adhesion,waterinsoluble extracellular polysaccharide(IEPS)and water-soluble extracellular polysaccharide(SEPS)were 84.93 ± 0.74%,79.14 ± 0.98% and 54.57 ± 1.47%,respectively.GTF activity test found that 1/3MIC 30%EPH reached the maximum inhibition rate of GTF at 24 h,which was85.91 ± 3.48%.Therefore,sub concentration of 30%EPH can reduce the generation of IEPS and SEPS by inhibiting the activity of GTF,thus preventing bacterial adhesion or aggregation to form biofilms.SEM and CLSM experiments intuitively observed that 30%EPH of 1/3MIC concentration significantly inhibited the formation of S.mutans biofilm.