Study On The Determination And Physicochemical Properties Of Melanin From Black-bone Silky Fowl

Black-bone silky fowl(Gallus gellus demesticus brissen, BSF), a unique breed of chicken belonging to phasianidae, grows primarily in South China, Taihe, Jiangxi Province. As a traditional Chinese medicine, it can be used alone or in compound, BSF has been proved to have the effect of nourishing liver and kidney, profiting vitality, reducing asthenia heat, curing abdominal pain and so on. BSF contains a large amount of melanin which is one of the most important active compounds responsible for its unique functions. It has been approved that BSF melanin has many pharmacological effects including anti-mutation, enhancing immunity, anti-aging, eliminating free radicals and anti-oxidation activities. Thus it can be seen there is a close correlation between the pharmacological action and BSF melanin.So far, there have been some research achievements on BSF melanin, however, the systematic deep research was still lacked and the action mechanism was also unclear. Therefore there will be great practical significance for structural analysis and product development in studying melanin deeply. Through investigating the determination method, solubility, spectrum characteristics and amino acid residues of BSF melanin, this paper further clarified the properties, formation mechanism, structural characteristics and deposition distribution of BSF melanin in the purpose of providing the reliable scientific foundation for melanin extraction and product development, furthermore, there were also high economic value and practical significance in increasing the medicinal value and exploring the best species resources in our country. The main results in the paper are mentioned as follows:1. A spectrophotometry method was established for the determination of BSF melanin. The method was on the basis of BSF melanin dissolved better in the presence of 0.1 mol/L sodium hydroxide solution by investigating its solubility in different solvents. The developed method has good precision, high recovery and reproducibility. Determination of different parts of BSF melanin were showed that: there are great differences in different parts of BSF melanin, in which periosteum is the highest reaching 3.749 mg/g and heart is the lowest only had 0.138 mg/g. 2. A HPLC method was established for the determination of BSF melanin. The method was developed by the determination of pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA) which were oxidative products of melanin. PDCA and PTCA standard were self-made and then structures were identified by using LC-MS and NMR. The chromatographic conditions were Kromasil C₁₈ column (4.6 mm×150 mm, 5μm) using gradient elution with a mobile phase of 0.1% formic acid and methanol; PDCA and PTCA were monitored by the diode array detector at 282.8 nm and 269.8 nm, respectively. The developed method has good precision, high recovery and reproducibility. Determination of different parts of BSF melanin showed that: periosteum > trachea > crista galli > Claw > intestine >muscle of thigh > skin > gizzard > muscles of thorax > heart. A good correlation is showed as this method compared with the spectrophotometry method. The correlation coefficient is 0.978.3. Differences in spectroscopy of BSF melanin in different parts was studied through the ultraviolet-visible spectrum and infrared spectrum. The results showed that the Uv-Vis spectrum of BSF melanin presents no significant characteristic absorption peak compared with the synthetic melanin, the peak of 290-300 nm is attributed to protein absorption in conjunction with melanin; The infrared spectrum revealed that the characteristics of BSF melanin absorption concentrates in 3600-3000 cm^-1, 2850-2950 cm^-1, 1610-1690 cm^-1, 1520-1540 cm^-1, 1380-1360 cm^-1, 1240-1200 cm^-1, of which the strongest absorption peak symboles the indole ring, therefore it is speculated that BSF melanin represents in the form of indole eumelanin. In addition, the infrared spectrum shows that BSF melanin contains certain amount of amino acids or protein.4. A HPLC method was established to determine amino acids of BSF melanin by pre-column derivation with 2,4-dinitrofluorobenzene. The method was developed by investigating several factors including column temperature, the quantity of derivative reagent, the mobile phase system and the pH of mobile phase. The experimental results demonstrated that the separation of amino acids were performed on a kromasil C₁₈ column (4.6 mm×250 mm, 5μm); the mobile phase were sodium acetate buffer(pH6.8) and acetonitrile-water with gradient elution; the column temperature was 27℃; the detection wavelength was 360 nm; the flow rate was 1.0 ml/min. The amino acid analysis were showed that there are great differences in different parts of BSF melanin, in which trachea, crista galli, intestine are lower, for example, the amino acids of crista galli is only 13.70μg/100μg, and gizzard is the highest reaching 35.54μg/100μg.