Studies On Antimicrobial And Antioxidant Activities Of Polymethoxylated Flavoens From Jinchen Orange Peels

Jinchen orange is a native sweet crop of Citrus sinensis Osbeck in China, and the peels of fruit is considered as an important source of polymethoxylated flavones (PMFs) of high concentration. PMFs are known to have physiological effects on organ systems. This study was established in the systemic discussion of PMFs from Jinchen orange peels. At the function of isolation and identification of PMFs, the levels of PMFs in peels from different cultivars of citrus fruits grown in the middle & upper regions of the Yangtze River were analyzed. The inhibitory activities of PMFs against food spoilage organisms and pathogenic bacterias in vitro were examined and the action mechanism against Pseudomonas was also evaluated. Otherwise, the antioxidant of PMFs extracts was evaluated, and the synergistic effects of PMFs extracts with organic acids and amino acids on superoxide anion were also determined. In addtion, the effect of preservation on chilled meat coated by PMFs extracts was studied. Theae researches would give references for exploitation of potential natural food antioxidant and preservative.The main research subjects and results are listed as follows:1, The extraction technique about flavonoids from Jinchen orange peels by means of enzyme hydrolysis and alcohol-extraction was studied. The yield of flavonoids increased distinctly, comparing with the traditional technique of extraction. The influences of extraction time, extraction temperature, pH value and the mass ratio of cellulase to material on the yield of total flavonoids from jinchen orange peels with the enzyme-assisted extraction technique were studied, according to the orthogonal test design based on the single factor experiments. The optimum extraction conditions were obtained as follows:the added ratio of cellulose 5%, the extraction time 2h, pH value 4 and the temperature 60℃, and the yield of flavonoids was up to 2.67±0.06%, which incresed by 14%, comparing with the the yield of flavonoids from the single factor experiments.2, The solution of total flavonoids from Jinchen orange peels were extracted with diethyl ether, and then loaded on a sephadex LH-20 gel column (2.5 cm×30 cm) with an ultraviolet detector set at 340 nm. The peaks with maximum absorbance were collected to give a purified PMFs mixture. PMFs extracts were characterized by chromatographic and spectroscopic techniques, such as LC-MS、NMR and UV. Seven individual PMF were identified. Quercetogetin (QUE), nobiletin (NOB), heptamethoxyflavone (HEP), 5-demethylnobiletin (5-DN) and tangeretin (TAN) were characterized through electrospray ionization mass spectrometry (ESI-MS) in positive mode of protonated molecular ions [M+H]+, the diagnostic fragment ions, together with the UV-Vis spectra and HPLC elution order from literature data. Sinensetin (SIN) and tetramethyl-o-scutellarein (SCU) were isolated and identified through their MS,1H NMR and UV-Vis spectral studies. The levels of PMFs in peels from different cultivars of citrus fruits grown in the middle & upper regions of the Yangtze River were determined for the first time. The results showed that PMFs levels were dependent on the cultivar, C. aurantium’Bitter orange’to be the most valuable citrus variety for isolating NOB and TAN, while C. unshiu’Owari satuma’is the most appropriate variety for HEP. C. sinensis contains seven individual PMFs, with significant levels of SIN and SCU.3, Antimicrobial activity of PMFs extracts against bacteria and fungi by Oxford plate assay and by growth rate method, respectively. The minimum inhibitory concentration (MIC) were tested by two-fold serial dilution method. The results showed that PMFs extracts had strong inhibition on microorganism with broad-spectrum. Gram positive bacterias were more sensitive to PMFs extracts than gram negative bacterias. It could also inhibit the growth of fungi. The MIC test results illustrated that the higher inhibition diameter did not always correspond to the minor value obtained with the MIC test. The inhibitory effects of PMFs extracts against Aspergillus. niger was stronger than Penicillium. digitatum. PMFs extracts appeared obviously inhibitory effect and efficacy at a dose-dependent manner.4, The inhibitory activities of NOB and TAN against Pseudomonas in vitro were examined. The effects of NOB and TAN on cell morphology, the release of cell constituents, the synthesis of proteins and activities of key dehydrogenase were evaluated with the aim of elucidating its antibacterial mechanism. The results showed that the concentrations of transaminase and reducing sugar in bacteria solution increased when treated with NOB and TAN. It was observed by electron microscopy that the structure of bacteria cells were destroyed seriously and induced cells plasmolysis. In addition, NOB and TAN could significantly inhibit the activities of succinate dehydrogenase (SDH), malata dehydrogenase (MDH) and ATPase, as well as the proteins synthesis in Pseudomonas. Therefore, the study suggested that NOB and TAN could destroy the permeability of cell membrane with the great release of cell constituents, lead to metabolic function dysfunction and inhibit the synthesis of proteins, result in cell pyknosis. Eventually growth and reproduction of Pseudomonas cells were inhibited and death.5, The antioxidant of PMFs extracts of Jinchen orange peels was evaluated by using a chemiluminescence (CL) method in vitro. The scavenging ability of PMFs on superoxide anion, hydroxide radial, and hydrogen peroxide were determined by the Pyrogallol-Luminol system, CuSO4-Phen-Vc-H2O2 system, and Luminol-H2O2 system, respectively. DNA damage preventing effect of PMFs was evaluated by the CuSO4-Phen-Vc-H2O2-DNA CL system. The results showed that PMFs extracts exhibited effective antioxidant activity to remove superoxide anion, hydroxide radial, hydrogen peroxide and prevent DNA from being damaged in the tested method. The IC50 was 6.1, 7.8,0.6 and 4.0μg/ml, respectively. PMFs extracts had antioxidative effect in a dose-dependent manner. In addition, the synergistic effects of PMFs extracts with organic acids (citric acid, oxalic acid and malic acid) and amino acids (alanine, lysine, serine, threonine) on superoxide anion were determined by the pyrogallol-luminol system. Mixtures of citric acid and lysine with PMFs extracts had higher synergistic effects than any other tested mixtures, and the optimum concentrations were exhibited by combinations of 7μg/mL PMFs+120μg/mL citric acid (1.2, P<0.05) and 7μg/mL PMFs+42μg/mL lysine (1.1, P< 0.05). For the other amino acids and organic acids, the values of the measured inhibition effect (% MIE) of the mixtures with PMFs extracts were not significantly different from their expected inhibition effect (% EIE), which did not show synergism.6, The effect of preservation on chilled meat coated by PMFs extracts was studied. The colour, pH value, TVB-N, total plate counts and Pseudomonas number were evaluation target. The result showed that the evaluation targets of chilled meat coated by PMFs extracts was obviously better than control group. Also, there is no bad effect on the colour of chilled meat coated by PMFs extracts. The shelf life was 8d.