Modification Of Phloretin By Aminoethyl And Its Biological Activity, Safety And Preservative Effect On Salmon Sliced

Phloretin is a kind of flavonoids with dihydrochalcone structure which distributed mainly in apple fruit and bark. Phloretin has many biological activities, such as bacteriostasis, antioxidant, anti-tyrosinase, anti-cancer, anti-inflammatory and so on. Based on its properties, phloretin has broad promising application in the food, pharmaceutical, cosmetics and other fields. However, phloretin has poor water solubility of only 20μg/mL due to its planar molecular structure, which has strong inter-molecular hydrogen bond. This is a main limiting factor in the applications of phloretin. To solve this defect, this study used young fruits of apple to prepare phloretin, then performed chemical modification to obtain aminoethyl-phloretin, a water-soluble derivative of phloretin. The activities of radical-scavenging, protection of biological macromolecules antibacterial activity and antibacterial mechanism of aminoethyl-phloretin were studied, respectively. Safety study (Mice acute oral toxicity and 30 days feeding subacut & toxicity test), preservation effect on refrigerated salmon were investigated.The main methods and results of this study are following:1. Preparation of phloretin and aminoethyl-phloretin.Phloretin was obtained by hydrolysis of phlorizin, which was isolated from the young apple fruit.7-hydroxyl replaced aminoethyl-phloretin was got by phloretin reacted with 2-bromo ethylamine hydrobromide. N, N-dimethyl formamide was used as reaction solvent, anhydrous potassium carbonate used as catalyst, reactant ratio was phloretin:2-bromine ethylamine hydrobromide:anhydrous potassium carbonate=1:1:2, then separation by silica gel column chromatography and recrystallization, the purity of aminoethyl-phloretin got to 98.1%. UV spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance spectrum were employed to characterized the new compound. The solubility of the compound was measured by HPLC method, the results showed that the solubility of aminoethyl-phloretin in water reached 9163.78± 0.50μg/mL at room temperature, which were 430 times than that of phloretin (23.13± 0.23μg/mL). This expands the application scope of phloretin in the food, drug and cosmetics industries.2. The research on biological activity and antibacterial mechanism of aminoethyl-phloretin.The activity of scavenging free radicals, protection of biological macromolecules, and bacteriostasis of aminoethyl-phloretin were measured, respectively. The main conclusions are as follows:Aminoethyl-phloretin has good free radical scavenging activity. The half radical-scavenging concentration of DPPH and ABTS were 68.00±0.67μM and 48.20 ±0.45μM. Aminoethyl-phloretin can protect half of plasmid DNA from oxidative damage caused by AAPH at a concentration of 230.67±1.67μM. Aminoethyl-phloretin has good antibacterial activity in an aqueous medium. Aminoethyl-phloretin showed good antibacterial effect on the six food related strains. The inhibition effect on Gram positive bacteria is better than Gram negative bacteria. The minimal inhibitory concentration (MIC) of AEP on Listeria monocytogenes, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens, Salmonella typhimurium were:90.23±0.45,90.35±0.87,40.13±0.65,900.98±0.22,600.87±0.34,1000.69± 0.65μg/mL, respectively.The antibacterial mechanism of aminoethyl-phloretin was researched by using Listeria monocytogenes as a bacterial model. Aminoethyl-phloretin can seriously inhibit the growth curve of L. monocytogenes, so the cells can not reach the logarithmic growth phase or delay logarithmic phase, thereby inhibiting cell growth and metabolism. Scanning electron microscopy, atomic absorption spectrophotometry, measuremet of A260nm UV-absorbing substances, suggesting aminoethyl-phloretin can cause damage to cell membranes so that cells aggregate, shrinkage, formation of holes, resulting in the leakage of bacterial cell contents, such as potassium that leading to cell death.3. Safety study on aminoethyl-phloretin.Acute oral toxicity test on mice,30 days feeding subacute toxicity test on Sprague Dawley rat of aminoethyl-phloretin were evaluated. Acute toxicity test results showed that the lethal dosage 50 (LD50) of aminoethyl-phloretin was greater than 8 g/kg-BW, so it belongs to the actual non-toxic. The results of 30 days feeding subacute toxicity test on Sprague Dawley rat showed that the SD rat ntragastric administration with aminoethyl-phloretin had no obvious symptoms of poisoning and deaths occurred, compared with the control group. Average daily food intake, weight gain, organs absolute weight and relative weight were no significant changes, routine blood and blood biochemical parameters had no significant abnormality. Pathological examination showed the liver, spleen, kidneys and ovaries (testicles) of high and 5-time high groups (The 100 and 500 times of daily possible intake of human body) had no significant histopathological lesions. The super-high dose group (The 1000 times of daily possible intake of human body) had no significant changes in daily performance and blood test, but had slight liver cell and reproductive cell damage. The maximum dose of no effect was 1000 mg/kg·BW, which was the 1000 times of the maximum daily intake for human body. So the compound belongs to the category of non-toxic.5. Preservation effect of aminoethyl-phloretin on refrigerated salmon.A comprehensive study of the preservation effects of aminoethyl-phloretin on salmon from sensory evaluation, physical and chemical indicators and microbial index were studied, respectively. The main conclusions are as follows:Aminoethyl-phloretin had strong protective effect for sensory quality of salmon by color, taste and elasticity of muscle. Aminoethyl-phloretin significantly reduced the fat oxidation levels of fish and significantly inhibited the formation of biogenic amines during storage. Aminoethyl-phloretin had strong inhibition effect on the total bacterial’ counts, total Pseudomonas bacteria counts. The salmon fish treated with 0.024% of aminoethyl-phloretin aqueous solution can reduce the level of fat oxidation by 3.35 times, the production of biogenic amines can be reduced by 10 times, the total number of bacteria can be suppressed by 2 logarithmic, total number of Pseudomonas bacteria can be suppressed by 1.5 logarithmic. The salmon treated with 0.024% of aminoethyl-phloretin can maintain a high quality for 4 days.The study obtained a novel water-soluble phloretin derivative with good bioactivities by chemical modification and investigated its inhibitory mechanism, safety and preservation effect. This study not only provided new ideas for phloretin application, but also gave a new preservation method for ready-to-use food.