Application of di-(2,2'-dipyridine) copper complex with double-cation side arm in eliminating the superoxide ion free radical
A technology of copper complexes and dications, which is applied in the directions of medical preparations, drug combinations, and pharmaceutical formulations containing active ingredients, can solve the problems of low SOD activity and achieve the effect of improving SOD activity.
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[0016] Example 1 The activity determination method of the complex (X-XO-NBT method)
[0017] The X-XO-NBT method refers to the xanthine-xanthine oxidase-chlorinated nitrotetrazolium method. Prepare a phosphate buffer solution with a pH of 7.8 and a concentration of 50mM with ultrapure water, and use the buffer as a solvent to prepare a concentration of 1.50×10 -3 M and 1.50×10 -3 M xanthine and NBT solution, and then use the buffer to prepare 1 U / mL xanthine oxidase solution, and store in a cold storage in the dark. The complex solution is prepared with phosphate buffer containing 5% DMSO. When measuring SOD activity, first measure O when no SOD or complex is added 2 - For the change in absorbance, add xanthine solution 1.0mL, NBT solution 1.0mL, phosphate buffer 0.070-0.100mL, stir and keep the temperature in a water bath at 25±0.1℃ for 10min, then add xanthine oxidase solution 0.100 under stirring -0.130mL, and start timing at the same time, after 1.5min, use Cary300 ultraviole...
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[0019] Example 2 Results of determination of SOD activity of natural enzymes and complexes
[0020] Complex and natural bovine erythrocyte superoxide dismutase (BeSOD) catalyzed O 2 - The activity of disproportionation was measured using the X-XO-NBT method.
[0021]When natural enzymes or complexes are not added and natural enzymes or complexes of different concentrations are added, the absorbance data of the samples are measured over time, and a linear regression is performed on the absorbance data of each sample to obtain a straight line. The slope of is the change in absorbance per unit time (ΔA / Δt). When no natural enzyme or complex is added, the absorbance change of the sample is A 0 Indicates that the absorbance change of the sample when the natural enzyme or complex is added is A s Expressed by the formula I(%)=(A 0 -A s )×100 / A 0 Calculate the inhibition rate I of each natural enzyme or complex to superoxide ions at various concentrations.
[0022] Then take the concentr...
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[0024] Example 3 The effect of pH change on the SOD activity of 5,5'-bis(tri-n-butylammonium methyl)-2,2'-bipyridine copper complex
[0025] IC of 5,5'-bis(tri-n-butylammonium methyl)-2,2'-bipyridine copper complex 50 The value hardly changes in the range of pH 6 to 8.5, which is consistent with the characteristics of natural enzymes. At pH 7.2, 5,5'-bis(tri-n-butylammonium methyl)-2,2'-bipyridine copper complex has the highest SOD activity (IC 50 =0.072±0.01μM). The results show that 5,5'-bis(tri-n-butylammonium methyl)-2,2'-bipyridine copper complex exhibits the highest SOD activity at human physiological pH, which exceeds that of natural bovine red blood cell superoxide dismutase ( BeSOD) activity (IC 50 =0.04μM), and compared with the reported activity of SOD from a natural plant (Wasabi, Hoseradish) (IC 50 =0.07μM) is equivalent. See the test results figure 2 .
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