Extraction And Degradation Of Melanin From Black-bone Silky Fowl And Isolation And Analysis Of Degradation Products

Black-Bone Silky Fowl is a local resource in Jiangxi province. Compared with other chicken, the most special feature of Black-bone Silky Fowl is rich in melanin. Researches showed that Melanin has a wide range of biochemical activities, including antioxidation, immunomodulatory, antimutagenesis, freeradical scavenging, delay senility effect and so on. Because of the solubility properties of melanin, insolubility in water, acid and almost all solvents, only dissolved in alkali solution, the activity mechanism and chemical structure of melanin hasn’t been explained clearly, thus there are many difficulties to study the structure of melanin in terms of methods and techniques.Oxidative controlling degradation method was adopted in this paper to degradated melanin for breaking its polymeric structure, the soluble degradation products were obtained. Various separation technology of natural products, modern spectroscopic techniques and research methods were used synthetically to separate, purify and identify components in degradation products, and reveal the chemical structure composition of melanin. Theses results could not only provide an important theoretical foundations for studying of animal melanin, also could provide a theoretical basis for further research on function mechanism and structure-activity relationship of melanin, and had outstanding contributions to promote the development of silky industry in Jiangxi Province and exploit the unique resources in China. The contents and conclusions were summarized as follows:1、Method of "defating-enzymolysis-acidolysis" was established for extraction of BSFM. The conditions of defating were selected and the optimum conditon were refluxing4times, refluxing time1h, liquid-solid ratio1:2, and under this condition the removal rate was7.5%. The hydrochloric acid hydrolysis conditions were selected and the optimum conditions were hydrochloric concentration2M, refluxing time3h, liquid-solid ratio1:10, refluxing3times.2、The morphological features of BSFM prepared by double enzyme-alkali method, acid hydrolysis method and "defating-enzymolysis-acidolysis" method were observed through transmission electron microscope. Results showed that melanin was ellipsoidal and shape integrity particle, with clear and smooth borderline and without destruct. The melanin extracted by "defating-enzymolysis-acidolysis" method was more pure than which extracted by enzyme-alkali method, and had similar morphological features with melanin extraxted by acid hydrolysis method. The infrared spectrum of melanin suggested there were amide and aliphatic structure in melanin. Element analysis of melanin showed that mass percentage of C (59.18%), H (3.28%), N (9.09%) element was similar to synthetic eumelanin, but mass percentage of S(1.79%) element was much more than that in eumelanin and much lower than that in pheomelanin. This suggested that melanin from Black-Bone Silky Fowl may be a specail melanin which was different with eumelanin and phomelanin.3、A method with rate-controlling H2O2for degradation of melanin from Black-bone Silky Fowl has been established, which made the degradation reaction more moderate and controllble. The NaOH-H2O2degradation method for degradation of melanin has been founded compared with K2CO3-H2O2method, the content of inorganic salt had been decreased in NaOH-H2O2method, and this method made the separation and purificcation more convinient.4、Dissolution characteristices of degradation products were studied. Results showed that degradation products have good solubility in water, methanol, acetic acid, pyridine, dimethyl sulfoxide, N, N-dimethylformamide and tetrahydrofuran, slightly soluble in acetidin, acetone and acetonitrile, insoluble in chloroform, dimethyl benzene and ligroin.5、BSFM was degradated with NaOH-H2O2method, the degradation products of different degradation time, pH and H2O2dosage were analyzed with UV-visible absorption spectroscopy and infrared spectrum. The UV-vis absorption spectroscopy of different degradation products had no significant differences with each other, and maximum absorption wavelength was210nm nearby. Compared with other products, degradation products at pH13and pH14had strong absorption in the range of1300-1500cm-1, other products at different condition had no prominent difference in infrared spetrum.6、Effects of different factors on the degradation rate, molecular weight distribution and yields of degradation products had been studied. No significant difference had been found in degradation products of different degradation time, pH and H2O2dosage on molecular weight distribution. With increase of degradation time, pH, and H2O2dosage, the degradation rate inreased, the effect order of this three factos was pH> degradation time>H2O2dosage. With increase of degradation time and H2O2dasage, the yields of degradation products increased at first, and then decreased. When pH and H2O2dasage were fixed and degradation time was12h, the yield of degradation product was maximum, which was56%. When degradation time and pH were fixed and H2O2dosage was3.4%(calculated in melanin weight, m:v), the yield of degradation product was maximum, which was57.07%.7、Degradation products were dissolved in acetonitrile by ultrasound, and the dissolved samples was derived by BSTFA. Then the derivant of degradation products has been isolated and identified by GC-MS. The degradation products were proved to contain short chain organic acid, such as succinic acid, adipic acid, hexadecanoic acid, margaric acid and so on.8、Adopt solvent separation method, the degradation products were separated with methanol, ethyl alconol, n-butanol, chloroform, acetone, acetic ether and so on, seven main components (B-1、B-2、B-3、B-4、D、F、G) were obtained. The seven components were analyzed by chemical components pretestin system, results showed that except composition D, other components contained organic acid, indole derivatives or aromatic amine substabce; composition B-2、B-3and B-4contained amino acids or carbohydrate subtance; composition B-1and F contained higher alcohol, phenol, steriod and essential oil compounds.9、Components B-1, B-2, B-3, B-4, F and G were analyzed with Q-TOF/MS. Results showed that the molecular weight of all components were under900Da. Through comparation of molecular weight with amino acids and known melanin structure unit, we found that more than90%of the degradation products was nitrogen-containning compounds, but not amino acids. And structure similar to melanin structural units, like PDCA, PDCA, and TTCA, hadn’t been found in degradation products.10、The Separation effecct of degradation products in different chromatographic colums and mobile phases had been examined. Results showed that the separation was effectual with strongly basic anion column and KH2PO4-H3PO4buffer, and with C18colum and0.1%formic acid/water mobile phase. With the crystal separation method, the crystallization D-1had been gained from component D, when the solvent was60%ethanol and the temperature was4℃. The crystallization L was obtained from derivative samples with methanol at room temperature.11、Elemental analysis, chemical testing method, nfrared spectroscopy and mass spectrometry were adopted to analyze chemical composition of crystallization D-1. The elemental analysis results showed that D-1contained H (3.73%), S (25.45%) elements, excluding C, N elements. Chemical test results showed that D-1could react with BaCl2and produce white precipitate whih could dissolve in hydrochloric acid. These results suggested that D-1contained SO42-. IR spectroscopy showed that D-1had a strong absorption at1116cm-1which was consistent with the infrared spectra of anhydrous Na2SO4in this range. Mass spectrum showed m/z of D-1was164.9244[M+Na]+, and molecular weight of D-1was141.9346closed to the molecular weight (141.9333) of Na2SO4. Based on the above informations, crystallization D-1was determined tobe Na2SO4. Because S-containing reagents didn’t be used in experimental procedure, the S in Na2SO4derived from melanin structure. Researches showed that H2O2could oxidize and catalytic thiol ether into sulfoxide and sulfone, and then sulfoxide and sulfone could easily be oxidized into sulfate in high temperature, this suggested that the S element of D-1was the form of sulfide in BSFM12、Crystallization L was analyzed with Q-TOF/MS and NMR, the MS results showed that the molecular weight of crystallization L250.09.1H NMR (δ, ppm):8.428,8.320,8.150,7.630,7.617,7.596,7.242(aromatic protons).13C NMR8, ppm:151.85(C-1),139.69(C-2),129.79(C-3),129.57(C-4),129.25(C-5),127.64(C-6),126.90(C-7),123.33(C-8),119.82(C-9),115.08(C-10),44.73(C-11).Based on the above informations, crystallization L was determined to be dansyl amide.