The Preparation Identification,Stability And Formative Physiological Mechanism Of Melanins In Seed Hulls Of Coix Lacryma-jobi L

Melanins are a huge collection of irregular,heterogeneous macromolecular pigment polymers generated from the oxidative polymerization of polyphenol or indolic compounds.They are important macromolecular substances required for biological survival and widely occur in nature.Natural melanins can promote the growth and development of organisms,enhance the competitiveness of living organisms,and have many bio-activities such as photo-protection,anti-oxidation,promotion of chelation,reduction of radiation and promotion of immunity;they can be used as photo-protectants,bio-antioxidants,chelators,anti-radiation agents and immune-stimulators as well as amorphous organic semiconductors,photoconductors and electrical conductors.So they are of great prospects in medicinal,cosmetic,and functional food industries,among other materials and fields.Compared with synthetic melanins and preparations from animals or microbes,phytomelanins are safer and more widely applicable.However,due to the lack of high melanin content raw materials for development and utilization,the identification and development of natural sources of melanins from plant materials are of important theoretical significance and practical value.Coix lacryma-jobi L is an ancient food-medicine homologous and economical crop.It belongs to the genus of Coix of the family Graminea,having the largest cultivated area,the widest distribution and abundant resources in China.China is one of the important origins of Coix lacryma-jobi L in the world.In this study,the seed hull byproducts of Coix lacryma-jobi L from the processing and different populations of Coix lacryma-jobi L were used as materials.Melanins from Coix lacryma-jobi L seed hulls were extracted and prepared and the techniques for extraction and preparation,physical and chemical properties,stability as food colorant and the formative physiological mechanism during the growth of Coix lacryma-jobi L were studied.The activities and zymograms of the key enzyme polyphenol oxidase?PPO?for the formation of melanins in different plant parts and in the seed hulls of Coix lacryma-jobi L at different ripening stages,were for the first time comparatively analyzed.These results laid the theoretical and technical bases for further industrial exploitation and utilization of melanins in Coix and for the study of its formative physiological mechanism in Coix lacryma-jobi L.The main results are as follows:1.The extraction and identification of melanins from Coix lacryma-jobi L seed hullsThe natural melanins from Coix lacryma-jobi L seed hulls was extracted by chemical method and purified in order by strong acid hydrolysis,organic solvent rinsing and repeated precipitation.Based on the absorption spectra and related chemical reactions,the extracted products were identified as melanins.The results showed that the Coix lacryma-jobi L seed hull melanin extracts exhibits typical absorption spectrum of melanins:a broad absorption in short wave UV region?UVC,200-280 nm?and mid wave UV region?UVB,280-320 nm?with a maximum absorption at 229 nm.Interestingly,the melanin extracts from different Coix cultivars show identical absorption spectra.The prepared melanins are soluble in sodium hydroxide and insoluble in water,HCl and in organic solvents such as methanol,ethanol,acetone,chloroform and ethyl acetate,etc.They are bleachable in H₂O₂,NaClO,KMnO₄ and K₂Cr₂O₇,and show positive reactions in the polyphenol reaction.The optimum extraction conditions were determined to be 1.8 mol/L sodium hydroxide as the extracting reagent,with a material-to-liquid ratio of 1:10?w/v?,at 90?water-bath for3 h.?CN Patent 201710313442.5[P]?.2.The stability analysis of melanins from Coix lacryma-jobi L seed hullsThe stability of melanins from Coix lacryma-jobi L seed hulls was analyzed and assessed by absorbance at 229 nm.The results showed that the stability of melanins from Coix lacryma-jobi L seed hulls is unaffected by the factors such as temperature,sugar content,but readily discolored in the presence of oxidants,reductants,sodium chloride and in natural light.The stability of melanin is also affected by the pH,generally,the absorbance and coloration of the Coix melanin preparations increases with the increase of pH.Absorbance is relatively constant over pH3-5?acidic?,increases rapidly over pH5-7?near neutral?and increases slowly over pH7-11?alkaline?.Low temperature?4??has no significant effect on the stability.Cu²⁺increases the coloration,while Fe³⁺and Ca²⁺slightly reduce the coloration;Mg²⁺,K⁺and Zn²⁺have no significant effects on coloration.3.Establishment of the optimum in vitro assay conditions for the key enzyme polyphenol oxidase?PPO?The activity of PPO was determined via spectrophotometry.Results showed that the optimum condition in vitro PPO assay is:grinding the hulls with 0.2 mol/L phosphate buffer at pH 6.0,with a material/liquid ratio of 0.5 g:50 mL,extracting for 1 h at room temperature?25??,the substrate of 100 mmol/L o-dihydroxybenzene,reacting at40?for 10 min,and reading the absorbance at 400 nm within 20 min after the completion of the reaction.4.Comparative analyses of PPO activity and zymograms in different parts of different Coix lacryma-jobi L materialsBy comparing the PPO activity of Coix lacryma-jobi L seed hulls at different ripening stages and different parts of the plant at the grain-filling stage,it was found that the PPO activity of Coix lacryma-jobi L seed hulls showed a certain regularity at different ripening stages:lower activity in the early stage,highest activity in the mid stage and lowest in the late stage.PPO activity can be detected in seed hulls,roots,stems,leaves and kernels.There are noticeable differences between different cultivars and different plant parts of the same cultivar.The PPO activity decreases from top to bottom plant parts,with relatively higher activity in young and chlorophyll rich plant parts.The PPO isozymes were separated by polyacrylamide gel electrophoresis?PAGE?and visualized with o-dihydroxybenzene-phenylenediamine.It was found that there were 6 isozyme bands in the ripening seed hulls.The high molecular weight PPO isozyme band showed the highest activity,most likely the main PPO isozyme responsible for the formation of melanins in hulls.There are evident organ specific distribution of the PPO isozyme bands in different plant parts and the number of isozyme bands in the roots,stems and leaves was different:5 bands appeared in roots,4bands in stems and 8 bands in leaves;the most in band number and the highest PPO activity exist in leaves,and then in roots and least in band number and lowest activity,in stems.