Study On Antioxidant Activity And Stability Of Melanin From Black-Bone Silky Fowl

Black-bone Silky Fowl were one of the local food resources of Jiangxi province and traditional Chinese medicine,and its health-promoting values have been well known for more than one thousand years.The most special feature of Black-bone Silky Fowl is rich in melanin.The melanin is an important functional component of Black-bone Silky Fowl.In this paper,the antioxidative activity of melanin from Black-bone Silky Fowl(SM_BSF)was studied in terms of scavenging free radicals,inhibiting lipid peroxidation,and protecting human umbilical vein endothelial cells（HUVEC）from oxidative damage.At the same time,the stability of SM_BSFwas studied.These can provide a certain theoretical basis and promote the development of Black-bone Silky Fowl resource.The main findings were as follows:1、The DPPH radical scavenging rate,superoxide radical scavenging rate,hydroxyl radical scavenging rate,reducing power,and ability to sequester Fe²⁺were used as indicators to study the in vitro antioxidant capacity of SMBSF.The results showed that:（1）The IC₅₀values of DPPH free radical scavenging by positive control ascorbic acid,SM_BSF1and SM_BSF2were 6μg/m L,182μg/m L,and 178μg/m L.The positive control ascorbic acid had a DPPH free radical scavenging activity about 30times that of SM_BSF.（2）The IC₅₀values of the positive control ascorbic acid,SM_BSF1,and SM_BSF2for scavenging superoxide anion radicals were 85μg/m L,398μg/m Land400μg/m L,respectively.Positive control ascorbic acid scavenging superoxide anion radicals was approximately 4.7 times as effective as SM_BSF.（3）The IC₅₀values of the positive control ascorbic acid,SM_BSF1,and SM_BSF2for scavenging hydroxyl radicals were 1315μg/m L,2374μg/m L and 2119μg/m L.The positive control ascorbic acid hydroxyl radical capacity was approximately 1.7 times that of SM_BSF.（4）In the experimentally set concentration range,the reducing power of SM_BSF1and SM_BSF2increased with the increase of their concentrations.At a concentration of 1000μg/m L,SM_BSF1and SM_BSF2showed the greatest reduction power,both was 0.32.（5）The chelating ability to Fe²⁺of SM_BSF1and SM_BSF2increased as the concentration increased.When the concentrations of SM_BSF1and SM_BSF2were 786μg/m L and 797μg/m L,the chelation ability of Fe²⁺reached 50%.2、The effect of SM_BSFon oxidative hemolysis of erythrocytes was studied by using auto-oxidation,oxidative of red blood cells induced by H₂O₂and ultraviolet irradiation in vitro.The results showed that:（1）The IC₅₀values of the positive control of ascorbic acid,SM_BSF1and SM_BSF2on erythrocyte autolysis and hemolysis were 150μg/m L,874μg/m Land 912μg/m L.The positive control ascorbic acid inhibited the Erythrocyte autooxidation hemolysis,and it is 6 times that of SM_BSF.（2）The IC₅₀values of positive control group of ascorbic acid,SM_BSF1and SM_BSF2on the H₂O₂-induced red blood cell oxidative hemolysis half inhibition was 217μg/m L,1121μg/m L and 1129μg/m L,positive control ascorbic acid on H₂O₂induced red blood cell oxidative hemolysis inhibition rate was about 5 times that of SM_BSF.（3）The IC₅₀values of the positive control group of ascorbic acid,SM_BSF1and SM_BSF2on the UV-induced red blood cell oxidative hemolysis half inhibition were 435μg/m L,1160μg/m L and1168μg/m L.The positive control ascorbic acid inhibited the erythrocyte oxidative hemolysis induced by UV.It is 2.7 times that of SM_BSF.3、The effect of SM_BSFon liver homogenate and mitochondrial lipid peroxidation was studied by CCl₄-induced lipid peroxidation of liver homogenates in vitro.The results showed that the IC₅₀of the positive control of ascorbic acid,SM_BSF1and SM_BSF2by CCl₄-induced liver lipid peroxidation were 1164μg/m L,1392μg/m L,and1387μg/m L,and SM_BSFinduced CCl₄-induced hepatic lipid metabolism.Oxidative inhibition was comparable to positive control ascorbic acid.4、Mitochondrial lipid peroxidation was induced by Fe²⁺-H₂O₂in vitro to study the effect of SM_BSF1on mitochondrial lipid peroxidation.The results showed that:SM_BSF1can inhibit mitochondrial swelling.When SM_BSF1concentration was 1200μg/m L,the inhibitory rate of Fe²⁺-H₂O₂induced mitochondrial lipid peroxidation reached 43.51%.5、HUVEC cells were induced by hydrogen peroxide to establish the oxidative damage model of HUVEC cells.The protective effect of SM_BSF1on oxidative damage HUVEC cells was studied.The results showed that:（1）When SM_BSF1at concentrations of 12.5,25,50,100,and 200 ug/m L acted on HUVEC cells for 24 h,SM_BSF1had no effect on the survival rate of HUVEC cells,indicating that SM_BSF1had no toxic effects on HUVEC cells.（2）H₂O₂of different concentrations acted on HUVEC cells for 2 h.Compared with the group without H₂O₂,H₂O₂at a concentration of 1.00 mmol/L decreased the survival rate of HUVEC cells to 50%.Therefore,the H₂O₂of 1.00mmol/L was selected as the injury model.（3）SM_BSF1can significantly increase the survival rate of HUVEC cells after H₂O₂-induced injury,and improve the H₂O₂damage morphology of HUVEC cells in a dose-effect relationship.（4）Compared with the blank group administered with H₂O₂,SM_BSF1significantly inhibited the increase of LDH activity caused by oxidative injury,showing a dose-effect relationship.（5）Compared with blank H₂O₂injury group,SM_BSF1significantly increased SOD activity attenuated by oxidative damage and showed a dose-effect relationship.（6）Compared with blank H₂O₂injury group,SM_BSF1can significantly inhibit the MDA content caused by oxidative injury,showing a dose-effect relationship.6、Taking activity retention rate of SM_BSFto scavenge DPPH radicals as an index,the effects of temperature and time on the stability of SM_BSFwere investigated.The results showed that:under different temperature conditions,the DPPH free radical scavenging rate of the melanin soluble parts showed a downward trend with increasing temperature,but the change was not significant.When stored at 4℃,SM_BSF1had the highest DPPH radical scavenging rate.Under normal temperature conditions,as the storage time prolonged,SM_BSF1and SM_BSF2had no significant effects on the DPPH radical scavenging rate.7、Taking activity retention rate of SM_BSFto scavenge DPPH radicals as an index,the effects of food ingredients on the stability of SM_BSFwere investigated.The results showed that:after addition of 2%～10%sodium chloride solution for 1 hour,the DPPH radical scavenging rate of SM_BSF1and SM_BSF2remained unchanged remained unchanged,but after 24 hour,the clearance rate decreased slightly.Addition of different mass fractions of sucrose and citric acid,regardless of 1hour or 24 hour after addition,the DPPH radical scavenging rates of SM_BSF1and SM_BSF2remained basically unchanged.Therefore,the commonly used ingredients of food ingredients had no effect on the stability of silky melanin pigments.8、Taking activity retention rate of SM_BSFto scavenge DPPH radicals as an index,the effects of metal ions on the stability of SM_BSFwere investigated.The results showed that:the common metal ions K⁺,Ca²⁺,Zn²⁺,and Fe³⁺had different effects on the stability of SM_BSF1and SM_BSF2.In the range of 0～250μg/m L,K⁺and Ca²⁺had little effects on the DPPH free radical scavenging activity.The effects of Zn²⁺was small,but Fe³⁺had a significant effect on the melanin.With the increase of Fe³⁺concentration,the DPPH free radical scavenging rates of both SM_BSF1and SM_BSF2decreased.9、Taking activity retention rate of SM_BSFto scavenge DPPH radicals as an index,the effects of preservative on the stability of SM_BSFwere investigated.The results showed that:potassium sorbate and sodium benzoate,which were commonly used as food preservatives,had no obvious effect on SM_BSF1and SM_BSF2,and could be used as a preservative in melanin products.