The Study On The Determination Of Melanin Content And The Activity Of Melanocyte Lysate In Black-Bone Silky Fowl

The black-bone silky fowls（Black-bone silky fowl,BSF）,also known as black-bone chicken,Taihe chicken,etc.,was a unique dual-use resource for medicine and food in Jiangxi.It combined the three functions of medicine,supplement and food.They had special nourishing function for the human body and regulated body functions.They also improved human immunity.BSF melanin was one of the most important functional components of BSF.However,due to poor solubility（insoluble in water and organic solvents）,the chemical structure was still unclear,which resulted in the current determination of melanin in BSF had not been convenient and reliable.so it was important to find a new method that was simpler,faster and more accurate,and was not required complete dissolution of melanin.In this study,we successfully established H₂O₂ oxidation-fluorescence method for the determination of melanin content in BSF was established,which can be used to quickly and accurately analyze the content of melanin in BSF and BSF melanocytes;BSF and BSF melanin had good anti-inflammatory and anti-oxidation effects and BSF was produced by melanocytes.The BSF melanocytes were widely distributed in BSF.In this paper,Black-bone silky fowls melanocyte lysate（BMCL）was obtained by low temperature ultrasonic lysis.To investigate the anti-inflammatory effect of BMCL on LPS-induced inflammatory response in RAW264.7 cells and the protective effect of BMCL on H₂O₂-induced oxidative damage in HUVEC cells.This study provided an experimental scientific basis for further elucidating the nourishing medicinal and nutritional effects of BSF and promoting the development and utilization of active ingredients in BSF.The main research conclusions were summarized as follows:1.Establish a fluorescence analysis method for determining the melanin content in BSF meat.The results showed that the high-concentration（600μg/mL）BSF melanin standard had an excitation wavelength of 354 nm and an emission wavelength of 453 nm.The regression equation of BSF melanin in the high concentr-ation range（50-600μg/mL）was y=0.0094X+0.6926 and the value of r was 0.9986.The detection limit of the method of fluorescence analysis was 0.30μg/mL,and the detection limit of UV spectrophotometry was 3.68μg/mL.The optimal sample pretreatment conditions were as follows:NaOH concentration ranged from 0.1-0.2 M,the ultrasonic time was 40 min and the ultrasonic temperature was 70℃.The optimal oxidation condition of melanin in BSF was 55°C,the oxidation time was 2 h,and the hydrogen peroxide concentration was was in the range of 20%to 25%.The RSD of the stability test was 1.49%and the RSD value of the instrument precision test was2.03%.The average recovery rate in the spiked recovery experiment was 102.865%and the RSD value was 4.505%.It was indicated that the method was more accurate.The melanin content in the leg muscle,chest muscle and chicken skin measured by the method with the fresh weight of 0.2 g was 0.879±0.023 mg,0.681±0.012 mg and2.162±0.075 mg,respectively,which accounted for 0.439%,0.340%,1.081%of the fresh weight.The results showed that the melanin content of BSF skin was the largest,followed by the thigh meat,and the least in the breast meat.It showed that the method with high accuracy was unnecessary to extract melanin from the sample.The reaction time was greatly shortened and the analysis operation was effectively simplified.2.Establish a fluorescence analysis method for determining the melanin content in BSF melonocytes.The low-concentration（100μg/mL）BSF melanin standard had an excitation wavelength of 354 nm and an emission wavelength of 453 nm.In the low-concentration range（10-100μg/mL）,the regression equation of melanin in BSF was y=0.0147X+0.3138 and the value of r was 0.9974.The detection limit of the method is 3.04μg/mL.The optimal oxidation condition of BSF melanin was 55℃,oxidation time was 2 h,and hydrogen peroxide concentration was in the range of 24%to 26%.These results were basically consistent with the optimal oxidation reaction conditions of BSF melanin in the high-concentration range.The RSD value of the reproducibility experiment was 4.59%,and the RSD value of the precision experiment was 1.87%.Different concentrations of melanin standards（25μg/mL,40μg/mL,80μg/mL）were added to A375 cells（melanoma cells without melanin）.The relative errors between the measured values and the theoretical values were 2.78%,3.53%and 0.25%,respectively.Adding different concentrations of BSF melanin standard to BSF melanocyte samples,the average spiked recovery was 95.57%and the RSD value was 4.00%.The results showed that the fluorescence analysis method was accurate and reliable and its results were not affected by other proteins and lipids.3.When the concentration of IBMX was between 200-500μM,each concentr-ation significantly inhibited the growth of melanocytes（P<0.01）.The survival rate of BSF melanocytes decreased with the increase of the concentration of IBMX and the cell survival rate was range from 40.64%-80.98%.IBMX at a concentration of 100μM had no significant effect on the activity of BSF melanocytes.The IBMX concentrations of 100μM and 300μM relative to the blank group significantly promoted the synthesis of melanin in BSF melanocytes（P<0.01）.4.Observe the morphology of RAW264.7 cells,determine the cell viability by MTT assay and determine the NO secretion of RAW264.7 cells to explore the optimal LPS concentration to promote inflammation in RAW264.7 cells.It was found that when LPS was 1μg/mL,the cytotoxicity was the smallest and the NO secretion of cells was the largest.Therefore,an inflammatory model of LPS-induced RAW264.7cells was established.On this basis,we further studied the effect of diffierent concentration of BSF cell lysate on the growth of RAW264.7 cells at different concentrations（0.2-125μg/mL,the concentration of lysate of cells in the concentration of intracellular protein of BSF）and the effects on the NO secretion of RAW264.7 at different time and different modes.When the concentration of BSF cell lysate increased to 25 and 125μg/mL,the cell viability were 115.66%and 157.50%,respectively.There was a significant difference（P<0.01）compared with the blank group and BSF melanocyte lysate significantly promoted the proliferation of RAW264.7 cells.Therefore,1,5,25μg/mL were selected as low,medium and high concentrations of BSF cell lysate for subsequent experiments.when treated with different time,the NO secretion was significantly reduced after treated with BSF melanocyte lysate of high,medium and low concentration（P<0.01）.In the preventive mode,the co-action mode and the treatment mode,BSF melanocyte lysate at the concentration of 1,5,25μg/mL had different effects on the amount of NO secretion in the cells.In contrast,the BSF melanocyte lysate in the preventive mode had the highest inhibition rate on the secretion of NO,and the anti-inflammatory effect was the most obvious.5.The high（25μg/mL）,medium（5μg/mL）,low concentration（1μg/mL）BSF melanocyte lysate relative to the model group after treatment with calcium and iron significantly reduced the amount of NO secreted by the RAW264.7 cells（P<0.01）.This indicated that calcium and iron promoted the anti-inflammatory effect of BSF melanocyte lysate.The promotion of calcium chloride was stronger than that of calciun aspartate,and the promotion of Fe²⁺was stronger than that of Fe³⁺.6.Establish a model of H₂O₂ induced HUVEC cells injury.The cell morphology was observed.The cell viability was determined by MTT assay.The LDH activity,SOD activity and MDA content in cell culture medium of different concentrations of BSF melanocyte lysate were determined.The results showed that median lethal concentration（LC50）value of HUVEC cells induced by H₂O₂ was 1 mM and the LDH activity of the cells was the highest at this concentration.The model of HUVEC cells injury was established at this concentration.Based on the results of this study,the BSF melanocyte lysate with a concentration of 0.2-25μg/mL had no significant effect on the activity of HUVEC cells.When the concentration was above 25μg/mL,the promotion of HUVEC cells growth was significant（P<0.01）.In this paper,1,5,25μg/mL were selected as low,medium and high concentrations of BSF melanocyte lysate for subsequent experiments.VC of 1 and 5μg/mL had no significant effect on HUVEC cell activity,and 5μg/mL VC was used as the control group.Compare the protective effect of BSF melanocyte lysate and VC on oxidative damage.Further studies showed that compared with the model group（1 mM H₂O₂）,high and medium concentrations of BSF melanocyte lysate significantly increased the survival rate of HUVEC cells（P<0.01）,and the medium concentration of VC in the same concent-ration had no effect on the survival rate of HUVEC cells.BSF malanocyte lysate at high,medium and low concentration significantly reduced LDH activity and increase SOD activity in cell culture medium（P<0.01）.Compared with the model group（1mM H₂O₂）,the medium concentration of VC cell culture medium at the same concentration had no significant effect on LDH activity and increased SOD activity.High and low concentrations of BSF melanocyte lysate can reduce the MDA content in cells.In contrast,medium concentration of VC also significantly reduced MDA content.In summary,BSF melanocyte lysate had stronger protective effect on oxidatively damaged HUVEC cells than VC.