Research On Isolation,Structure And Bioactivies Of Melanins From Sphacelotheca Reiliana

Based on the biological functions of melanin and its wide application prospect,the research and development of melanin has become a hot topic.Natural melanin has a wide range of sources.The extraction and separation methods of melanin from different sources are also different.The structure of melanin is complex and variable,more comprehensive and accurate detection methods are still needed for accurate structural identification.Therefore,the main research topic in the future will be to find cheap,efficient and high-quality resources,establish an economic and effective extraction method,and lay a foundation for the development and application of natural melanin in production.To solve the above problems,in order to determine the optimal conditions for ultrasound-assisted extraction of melanin from sorghum Sphacelotheca reiliana,this study used sorghum S.reiliana as raw material and optimized it by response surface method,the physical and chemical properties,the ability to complex with metal ions,the structure,the physical and chemical properties,complexation ability with metal ions,structure,antioxidant,antiproliferative and hypoglycemic activity of the extracted melanin were analyzed.This paper tries to explore a new kind of natural melanin resource,and to provide theoretical basis and method for the processing,utilization and development of sorghum S.reiliana.1.Response surface method was used to optimize the optimum conditions of ultrasound-assisted alkali-soluble acid precipitation extraction of melanin from sorghum S.reiliana.Combined with single factor experiment and response surface optimization,the optimal conditions for the extraction of melanin from sorghum S.reiliana were determined as follows:the HCl hydrolysis concentration was 3 mol/L,the solid-liquid ratio(W/V)was 1:20,the water bath hydrolysis was 60?for 4 h,the p H value of Na OH leaching solution,the ultrasonic power was 240 W,the ultrasonic temperature was 50?,the ultrasonic time was 2h.2.The physical and chemical properties of melanin from sorghum S.reiliana were analyzed,the maximum absorption wavelength of melanin was 215nm;the color of the melanin has a brownish red in the p H range of 2?7 and turns to dark brown when p H>8;in addition to reducing agents,heat,light,microwave,oxidant and common food additives have no effect on it,the metal ions Cu²⁺,Mg²⁺,Ca²⁺,Zn²⁺,Al³⁺and Fe³⁺had no adverse effect on the stability of the melanin.The results showed that the melanin of sorghum S.reiliana has good stability and is a kind of natural pigment with great prospect of development and application.3.In this paper,the infrared spectral characteristics of the melanin complexes of sorghum S.reiliana and metal ions Cu²⁺,Fe³⁺were compared,and also compared with the infrared spectra of melanin of sorghum S.reiliana.It was found that the p H value of complex and solution would affect the complexation between melanin and metal ions.The effect on the two ion complexes was similar.The main interaction between metal ions and carbonyls occurs at p H 2.0 to 4.5.With the increase of p H value,the metal ions interacted with the melanin of sorghum S.reiliana.4.The structure of melanin from sorghum S.reiliana was analyzed,the melanin was systematically classified by polyamide column chromatography.A melanin component L-25-2was isolated from it and determined respectively by IR,UV,¹H NMR and UPLC-QTOF-MS.L-25-2 was identified as a member of the DOPA melanin family and was an acidic macromolecular polymer.L-25-2 contains hydroxyl and carboxyl functional groups and indoles.Accordingly,we speculated that melanin L-25-2 is a polyphenolic polymer with the structure skeleton of dopaquinone and 5,6-dihydroxyindole-2-carboxylicacid,and is synthesized by tyrosinase.5.In this study,the optimum conditions for the extraction of melanin from sorghum S.reiliana were determined,and the extracted melanin was analyzed for its physical and chemical properties,complexation ability with metal ions,structure,antioxidant activity and hypoglycemic activity,providing a theoretical basis for the development of functional foods of sorghum S.reiliana.Systematic research of the sorghum S.reiliana total extraction and L-25-2 on antioxidant capacity index(DPPH,ABTS and PSC),and intracellular antioxidant capacity assessment(CAA).The antioxidant capacity of L-25-2 and total extraction from sorghum S.reiliana were confirmed.Then determined the contribution of L-25-2 to total antioxidant capacity.The total extraction and L-25-2 were compared on the anti-proliferation ability of Hep G2 cells,and both of them had certain inhibitory effect on tumor cells.Furthermore,the protective effect of melanin on oxidative damage of Hep G2 cells induced by H₂O₂ was studied.The results showed that the melanin had good effects.It can effectively reduce the expression levels of ROS,MDA and LDH in impaired Hep G2 cells induced by hydrogen peroxide in a concentration-dependent manner.It can enhance the activity of endogenous antioxidant enzyme SOD and GSH-Px.6.In vitro?-glucosidase and PTP1B inhibitory activitives model was used to screen the inhibitory activity of melanin against them,and the melanin L-25-2 was determined to be the active substance assisting hypoglycemic activity.The lineweaver-burk double reciprocal plot method was used to determine the competitive inhibitory effect of melanin L-25-2 on?-glucosidase and PTP1B.These results provide a theoretical basis for further analysis of structure-activity relationship and computer aided simulation of docking with PTP1B to determine the action sites.Using molecular docking,the spatial binding mode of melanin and PTP1B was discussed,and Glide module in Discovery Studio software was used.The original matching small molecules were taken as the center,and it can be seen from the overall figure that the compound had a binding with the protein,but it was not completely combined.7.The hypolipidemic activity of melanins from S.reiliana was investigated by using high-fat induced C57BL/6 mouse model.The results showed that the melanins could inhibit the digestion and absorption of fat,and effectively inhibit the weight gain of mice,reduce the body fat content,and slow down the phenomenon of fatty liver without affecting the appetite.It can significantly reduce the Total Cholesterol(TC),Triglyceride(TG)and Low Density Lipoprotein(HDL-C)levels in hyperlipidemia mice,while increasing the level of High Density Lipoprotein(HDL-C).Compared with the model group,the serum enzymatic indexes of the samples were improved in both low dose and high dose groups,preliminarily proving that the melanin have a definite protective effect on liver injury and anti-inflammation caused by obesity.This study determined the best technological conditions for the extraction of melanin from sorghum S.reiliana,and analyzed the physical and chemical properties,chelating ability with metal ions,structure,antioxidant,antiproliferative and hypoglycemic activity of the extracted melanin.It also provided a theoretical basis for the development of functional food of S.reiliana.