| Functional factors of food play an important role in the fields of food,pharmacy and chemical industry,which have vital function to health and treatment of diseases for human.As a kind of nutritional supplement,L-amino acids are indispensable for their vital functions,such as adjusting metabolism of nutrient substances,maintaining the balance of environment in vivo,regulating gene express and cellular signal transduction,and so forth.The flavonoids are a class of natural antioxidant with good activity for scavenging radicals,which are important pathogenesis factors in living creatures.Copper is an essential element for most aerobic organisms,employed as a structural and catalytic cofactor,and consequently it is involved in many biological pathways.As is known to all,functional factors of food have some physiological functions,but they have some drawbacks,as low bioactivities,single function and limited application.These disadvantages could lead to the low bioavailability,the waste of raw materials and the enhancement of cost production for the flavonoids.Therefore,it's one of hot points for designing new functional compounds with good multiple biologic activities in food fields,and it will have important effects to food safety production and human health.Based on the theory of coordination chemistry,the function of organic compounds could be improved,and new functions might be formed when the compounds coordinate metal ions because of their synergistic effect.Hence,it's an important way for developing metal complexes with functional factors of food.In this paper,with L-amino acids as main ligands and 2-?2'-pyridyl?benzothiazole?PBT?as auxiliary ligand,five new ternary copper complexes have been synthesized and characterized.Based on reference methods,four copper complexes containing Genistein,Apigenin,Luteolin and Kaempferol have been prepared,respectively.The complexes were characterized by X-ray diffraction,elemental analysis,spectroscopy methods?IR,UV-vis,ESI-MS?and molar conductivity.The study on application for the complexes has been done by classical experimental methods and molecular docking technique.The main results were as follows:?1?According to L-amino acids as main ligands and PBT as auxiliary ligand,five new ternary copper complexes have been synthesized and characterized,and the general molecule formulas are[Cu?PBT??L-Ile?]NO3?1??L-Ile=L-Isoleucinate?,[Cu?PBT??L-Val?]-ClO4?2??L-Val=L-Valinate?,[Cu?PBT??L-Leu?]ClO4?3??L-Leu=L-Leucinate?,[Cu?PBT?-?L-Arg?]ClO4?4??L-Arg=L-Argininate?,[Cu?PBT??L-Ala?]n?ClO4?n?5??L-Ala=L-Alaninate?,respectively.The crystal structures of 14 were obtained through elemental analysis,IR,UV-vis,ESI-MS and X-crystal diffraction spectrometer,indicating the copper atoms of these complexes located in a distorted tetragonal pyramids.The coordination polymer 5 consists of the left-and right-hand helical chains arranging alternately.?2?The antibacterial activity of complexes 14 was determined by using the Oxford cup and the double dilution methods.These complexes showed stronger effects on gram-positive bacteria?S.aureus,B.subtilis,L.monocytogenes?and gram-negative bacteria?E.coli,Salmonella?.Aspergillus niger had also been inhibited in a certain extent.The complexes showed broad-spectrum bacteriostasis and positive synergistic effect.?3?Using the NBT light reduction method,Fenton reaction and DPPH method,the ability of 14 for scavenging superoxide anion O2-·,hydroxyl radical OH·and DPPH·was investigated.The results suggested that the complexes had excellent scavenging ability for O2-·with IC50 from 0.132 to 0.178?M.The scavenging ability to OH·was higher than that to DPPH·.In general,these complexes showed a good antioxidant activity in vitro.?4?The antitumor activity of complexes 14 in vitro was investigated by MTT method.The complexes exhibited cytotoxicity on Bel-7402 cell line(IC50=15.230.8?M)following the trend:4>3>2>1.The cytotoxicity of the complexes was determined by cell apoptosis,the reactive oxygen species?ROS?and mitochondrial membrane potential.Western blotting assays confirmed that cytochrome C was released,the expression of antiapoptotic protein bcl-2 was downregulated,and the expression of apoptotic protein Bax was upregulated.The results were consistent with the characteristics of mitochondrial action pathway.?5?The interactions of complexes 14 with CT DNA were studieds by multi-spectroscopic techniques?UV-vis,FS and CD?and viscometry measurement.These studies confirmed the binding mode of the complexes to CT DNA was an insertion with certain affinity,following the trend:4>3>2>1.The ability of the plasmid pBR233 DNA cleavage of the complexes was determined by agarogel electrophoresis,and the mechanism of the DNA cleavage was investigated.The oxidation species involved in the process were O2-·and OH·.Finally,the scavenging ability of complexes 14 to O2-·and OH·was verified.?6?Based on human serum albumin?HSA?as a drug delivery carrier,the interaction of14 with HSA was studied by fluorescence and UV-vis spectra,with the order of the binding constants 3>4>2>1.Based on the calculation of thermodynamic parameters,the complex-HSA complexes are formed with the molar ratio of 1:1,and hydrophobic and hydrogen interactions play a vital role among them.The conformation of HSA has been changed with the presence of complexes 14,which had been confirmed by synchronous fluorescence,three-dimensional fluorescence and CD spectra.The results suggested the complexes intercalated into a hydrophobic cavity of?A domain,resulting to fluorescence quenching for the 214-Trp amino acid residues and making?-helical secondary structure partially transforming into?-sheet and Random coil secondary structures.?7?Using molecular docking technology,the interaction between complexes 14 and DNA was studied,and the same thing was done to HSA.The results revealed that the complexes inserted into the base pairs DG-4 and DC-5 of DNA,and some hydrogen bonds were formed between the complexes and amino acide residues.The complexes intercalated into a hydrophobic cavity of?A domain of HSA,and some hydrogen bonds were formed between the complexes and amino acide residues too.The results of the molecular docking were consistent with that of the classical experiments.?8?According to reference motheds,four copper complexes containing flavonoid compounds had been prepared,which are Apigenin,Genistein,Luteolin and Genistein.The complexes were characterized by elemental analysis,IR,UV and molar conductivity.The possible formula are as follows:Cu?Apg?2?H2O?2?6?,[Cu?Gen?2?H2O?2]·3H2O?7?,[Cu?Lut??H2O?]n?8?,[Cu?Kae?2?H2O?2]·1.5H2O?9?.?9?NBT light reduction assay,salicylic acid method and DPPH way were used to determine the ability of complexes 69 for scavenging O2-·,OH·and DPPH.The complexes showed the excellent cleaning effect on O2-·with IC50 from 0.25?M to 0.85?M.The order of this effect is:7>6>9>8.As for OH·,IC50 of complex 8 is less than 25?M,and that of other three complexes are greater than 100?M,with the order of action:8>9>7>6.The complexes showed positive synergistic effect on the scavenging ability of O2-·and OH·.The order of IC500 values for DPPH·is:9>8>7>6.Therefore,flavonoid ligands played a major role in scavenging OH·and DPPH·,and the central copper ion is crucial in scavenging O2-·.?10?The main structure-activity relationship of the antioxidant activity of 69 is as follows:???The catechol hydroxyl groups are the most important active site in the B ring of flavonoids;???The hydroxyl substitution of C3 on C ring is beneficial for the improvement of antioxidant activity;???The C2=C3 double position on C ring,especially the active group in the C3 position,has an positive effect on the antioxidant activity of the complexes;???In general,the more number of substituted hydroxyl groups,the stronger the antioxidant activity of the complexes;????conjugative effects were enhanced because of the coordination of the central metal ion to parent compounds,which was beneficial to the increment of antioxidant activity. |
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