The Inhibitory Effect And Mechanism Of Quinoa Saponins On Oral Pathongens

Quinoa is the main food crop in the Andes,its excellent nutritional value promotes cultivation throughout the world.Quinoa produces about 20%-35% bran in the process of processing quinoa,which is usually wasted with no commercial value,and it is found that these bran usually contain about 3%-8% of quinoa saponins.Saponins are natural compound with various biological activities,and it is reported that quinoa saponins have anti-inflammatory and anti-fungal effects,but the inhibitory effect of quinoa saponins on badge-related microorganisms remains unclear.The main research contents of this subject are to determine the inhibitory effect of saponins,which were isolated and purified from quinoa bran and alkali treated,against oral pathogens including Porphyromonas gingivalis,Clostridium perfringens and Fusobacterium nucleatum.And we also studied the inhibitory mechanism of saponins,which would provide a theoretical basis for exploring novel bad breath products.The minimal inhibitory concentration(MIC)and the minimum bactericidal concentration(MBC)of the samples were determined by the method of microdistribution gradient dilution method to evaluate the antibacterial activity of saponins after separation,purification and alkali treatment.The samples included crude quinoa saponins(QS),alkali-treated crude quinoa saponins(ATS),30% ethanol-eluted saponin(QS-30),alkali-treated 30% ethanol-eluted saponin(ATS-30),80% ethanol-eluted saponin(QS-80),alkali-treated 80% ethanol-eluted saponin(ATS-80),the results showed that the ATS-80 had a significant effect on the three : Porphyromonas gingivalis was 62.5?g/mL(MIC)and 250?g/mL(MBC);Clostridium perfringens was 31.3?g/mL(MIC)and 250?g/mL(MBC);Fusobacterium nucleatum 31.3?g/mL was(MIC)and 125?g/mL(MBC).ATS-80 were identified by high performance liquid chromatography(HPLC)and liquid chromatography-mass spectrometry(LC/ESI-MS/MS).The results showed that the fraction of QS-80 had a large change between the saponin monomer and the monomer content before and after the alkali treatment,and even the peak 1,2,3 and 4 in QS-80 component are not detected in ATS-80 component.The content of high polarity saponins in the QS-80 component was higher than that of ATS-80 at 284.5 mg/g,while the content of low polarity saponin in ATS-80 was higher than that of QS-80 was 341.87 mg/g.The change in the polarity of the saponins after this alkali treatment plays a key role in inhibiting microbial activity.The mechanism of the inhibitory effect of quinoa saponins on oral pathogens after alkali treatment was investigated by measuring the integrity of bacterial cell membrane,membrane potential change,inhibition rate of biofilm,biofilm activity and bacterial morphology.The results showed that ATS-80 could cause the increase of nucleic acid and protein content in the culture medium.After treatment with ATS-80 for 32 hours,the content of nucleic acids and proteins in the culture medium between 0 and 24 h increased steadily and then became stable.The Fusobacterium nucleatum was most sensitive to ATS-80.Compared with the control group,after treated by ATS-80 at the concentration of MIC and MBC,the multiplication of nucleic acid in Fusobacterium nucleatum(OD260nm)were 4.30 and 6.19 respectively,and the multiplication of protein were 12.22 and 21.36 respectively,indicating that different concentration of quinoa saponin ATS-80 could markedly influence the integrity of cell membrane.Simultaneously,quinoa saponin ATS-80 could significantly reduce the bacterial cell membrane potential.When Porphyromonas gingivalis,Clostridium perfringens and Clostridium perfringens were treated by ATS-80 at the concentration of MIC and MBC,the change of cell potential were as follows: 48.40%(MIC)and 75.07%(MBC);58.44%(MIC)and 80.13%(MBC);54.33%(MIC)and 80.38%(MBC),respectively.According to the results,the inhibition rate of the biofilm was 38.56% at the MIC concentration and 89.73% at the concentration of MBC,suggesting that varied concentration of ATS-80 had a significant effect on the inhibition rate of Fusobacterium nucleatum biofilm.Compared with the biofilm activity of the blank control group(70.435%),the biofilm activity of Fusobacterium nucleatum were 40.231%(MIC)and 10.569%(MBC)when treated by ATS-80,indicating ATS-80 had great influence on the biofilm activity of Fusobacterium nucleatum.Adopting lens electron microscope to observe the bacterial morphology of Fusobacterium nucleatum,we found the normal bacterial morphology of Fusobacterium nucleatum has complete cell structure,cell wall and cell membrane integrity evenly distributed.However,the cell wall ruptured,the cell contents gathered around the cell membrane when treated by ATS-80 at the concentration of MIC.What's more,the cells didn't remain complete morphology,cell wall and cell membrane ruptured and the contents of the dissolved into the culture medium when treated by ATS-80 at the concentration of MBC.All those results directly reflected the antibacterial mechanism of ATS-80.In conclusion,we can apply 80% ethanol-eluting saponins after alkali treatment as antibacterial agents to treat bad breath.