Extraction Optimization Process Of Phenolic Compounds From Malus ‘Royalty’ And Analysis Of Its Antibacterial Activity

Malus spp.is a famous ornamental tree in spring from China.Its leaves are rich in phenolic active substances.At present,the treatment of Malus leaves in China’s landscaping is mostly burning or discarding.It is of great significance to explore the extraction technology of phenolic substances in Malus leaves and their antioxidant,bacteriostatic and rust prevention characteristcs for its production,planting,processing and utilization.In this study,Malus ‘Royalty’ was used as the experimental material,the response surface method is used to optimize the leaves phenolic compounds extraction technology,through the high performance liquid chromatography(HPLC-DAD)method for identification and quantitative analysis.On this basis,the antioxidant activity of Malus leaves in vitro and its inhibitory effect on foodborne bacteria were explored,the mechanism of polyphenols inhibiting the spore germination of plant-derived fungi was studied,and the reference genes suitable for polyphenol stress were screened.The results are as follows:(1)Response surface optimization experimental analysis showed that the optimum conditions for the extraction of polyphenols from the leaves of Malus ‘Royalty’ were methanol concentration of 79%,material-liquid ratio of 1:23 g/m L,extraction time of 39 min,and extraction times of 2.Under the optimized process conditions,the predicted maximum extraction yield of total polyphenols in Malus ‘Royalty’ leaves was 30.50 mg/g,which verified that the average extraction yield of polyphenols under this condition was 30.37±0.11 mg/g.(2)The identification results of HPLC-DAD showed that 14 substances were detected in the polyphenol extract of Malus ‘Royalty’ leaves.Among them,cyanidin is the main pigment color for forming colors in ‘Royalty’ leaves.(3)The results of antioxidant test showed that the polyphenol extract from the leaves of Malus ‘Royalty’ had strong ability to scavenge DPPH and ABTS free radicals,and also had a certain reducing ability to iron ions,which indicated that its antioxidant activity was good in vitro.(4)The results of bacteriostatic test showed that the minimum inhibitory concentrations of polyphenols from Malus ‘Royalty’ leaves against Escherichia coli,Salmonella typhimurium and Staphylococcus aureus were 3.00,0.70 and 0.50 mg/m L,and the minimum bactericidal concentrations were 3.40,1.00 and 0.80 mg/m L.The inhibitory effect on Gram-positive bacteria was better than that on Gram-negative bacteria.(5)The results of teliospores germination inhibition test showed that after continuous culture for 12 h,the germination inhibition rate of 2.50 mg/m L polyphenol extract group reached 47.55 ± 3.95%,indicating that the polyphenols from Malus ‘Royalty’ leaves had good inhibitory activity against plant fungi.The results of reference gene screening showed that ARF1 and EF1 could be used as appropriate reference genes for quantitative study under polyphenol stress.