Study On The Extraction, Isolation Of Polyphenols From Mulberry Leaves And Its Activities

The extraction of polyphenols from mulberry leaves was optimized. The polyphenols from mulberry leaves were divided into fractions using ethanol (MP), fractions using petroleum ether (MPP), fractions using ethyl acetate (MPE), fractions using butanol (MPB) and fractions using water (MPW). A RP-HPLC method of determining polyphenols in mulberry leaves extracts was established. Using LC-MS, the main molecular weight of polyphenols from mulberry leaves were determined. The antioxidant activity, antimicrobial activity and inhibition mechanism of mulberry leaves extracts were also explored.By single-factor experiments and response surface analysis, the optimum extraction condition was obtained as follows:extraction time of 2 h, extraction temperature of 60℃, ethanol concentration of 46%, the solid-liquid ratio of 1:25(m/v), and the yield of polyphenols from mulberry leaves could be up to 2.44%with three times extraction.A RP-HPLC method of determining the contents of polyphenols from mulberry leaves extracts was established. Using this method, the contents of polyphenols in different fractions from mulberry leaves were determined. The results showed that in MP, the highest content was chlorogenic acid, followed by rutin, hupi glycosides, quercetin, gallic acid, morin. In MPP, quercetin was detected, and no presence of gallic acid, chlorgenic acid, rutin, Hupi glycosides, morin. In MPE, the content of chlorgenic acid was the highest, followed by rutin, Hupi glycosides, gallic acid, quercetin, morin. In MPB, the content of rutin was highest, followed by quercetin, Hupi glycosides, gallic acid, morin, and no presence of chlorgenic acid. In MPW, the content of Hupi glycosides was the highest, followed by chlorgenic acid, gallic acid, rutin, and no presence of morin, quercetin. Comparing of the contents and types of polyphenols in different fractions of mullberry leaves, the polyphenols were more concentrated in medium-polar fractions using ethyl acetate (MPE) and ethanol (MP). According to the figures of ESI-MS and the qualities of molecular ion peaks, the possible molecular formula were determined by polyphenols controls and references. In MPE, it may contain chlorgenic acid, coffee quinine acid, rutin, naringin and so on.The results of antioxidant experiments in vitro showed that the MPP, MPE, MP had equivalent reduction capacities, which was much higher than the MPB; the MPE had the highest ability of scavenging DPPH radical, which IC50 was 3.47 mg/mL,while the MPB almost had no ability of scavenging DPPH radical; the capacity of scavenging ABTS radical of MP, MPE, MPP was much higher than the MPB, and the IC50 of MP, MPE, MPP was 1.14 mg/mL,1.34 mg/mL,1.34 mg/mL respectively; the capacity of scavenging superoxide radical of MP, MPP was much higher than the others; the capacity of scavenging hydroxyl radical of MP was the highest, which IC50 was 10.09 mg/mL. In summary, the MPE and MP had strongly antioxdiant activities. It may be corresponding with the quantities and qualities of phonenols from mullberry leaves.By the oxford cup method and the double dilution method, the antibacterial effects of mulberry leaves extracts were investigated. The results showed that the MPP and the MPW had no inhibition on the experimental strains, while MPE had antibacterial effects on Micrococcus luteus, Staphylococcus aureus, Pseudomonas aeruginosa, Serratia marcescens, which minimum inhibitory (MIC) values were 25.0 mg/mL 12.5 mg/mL,100.0 mg/mL,25.0 mg/mL respectively; MP had antibacterial effects on Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Serratia marcescens, which MIC values were 12.5 mg/mL,12.5 mg/mL,25.0 mg/mL,50.0 mg/mL respectively. In the initial inhibition mechanism studies, it found that MPE, MP had greater impacts on the bacterial cell walls, cell membrane and protein synthesis than MPP, MPW. MPE's influence was greater than the MP. From the environmental scanning and transmission electron microscopy figures, it showed that with MPE processing, Lactobacillus brevis's cell shape did not change, but cell membrane, cell wall ruptured, cell integrity were destroyed, and the cell contents escaped, which leading to the lose of metabolism and proliferation of cells.