Study On Isolation,Structural Characteristics And Biological Activities Of Active Compounds From Chaenomeles Speciosa (Sweet) Nakai Seed

The fruit of Chaenomeles speciosa（Sweet）Nakai.is rich in polyphenols,polysaccharides,organic acids,superoxide dismutase,saponins and other nutritive active ingredients,and it’s medicinal and health value have been continuously explored.With the increasing demand of market,the planting scale of C.speciosa is also expanding.The seed of C.speciosa is the main industrial residue,and it contains high amounts of oil,polysaccharide,protein and other nutrients.However,this seed is still underutilized.In this paper,C.speciosa seed was used as raw material,and the quality and nutritional composition of the seed oil were evaluated.The spray drying technology was applied to prepare the C.speciosa seed oil microcapsules,meanwhile the properties of microcapsules and the effect of microencapsulation on seed oil storage stability were studied.A highly purified polysaccharide fraction was successfully isolated from defatted C.speciosa seed meal.The structural characteristics and the biological activities of the purified polysaccharide fraction were studied.The protein isolate and main protein fractions of C.speciosa seed were prepared respectively.The amino acid composition,physicochemical and functional properties of these proteins were studied.C.speciosa seed protein isolate was hydrolyzed by papain to prepare peptides with high tyrosinase inhibition activities,and the amnio acid sequence of the obtained peptides was identified.The antioxidant activity and metal ion chelation capacity of prepared peptides were evaluated.Moreover,molecular docking study was performed to investigate the possible docking model between peptides and tyrosinase.These exploratory works provided new ideas for the high-value utilization of C.speciosa seed,the detailed research contents and results are as follows:C.speciosa seed was rich in oil and it could be explored as a special woody oil resource.Based on the single factor experiment,the comparatively good technological conditions for extracting C.speciosa seed oil（CSNSO）with petroleum ether using as solvent were determined by orthogonal optimization analysis:solid-liquid ratio 1:4（g:m L）,extraction temperature 60℃,extraction time 150 min.Under the optional extraction conditions,the CSNSO yield was 28.5%.The acid value and peroxide value of CSNSO met the standard for edible oils and fats.12 fatty acids were detected in CSNSO by gas chromatography-mass spectrometry,and the major fatty acid in the oil were oleic acid（42.7%）,followed by linoleic acid（32.5%）,palmitic acid（12.9%）,stearic acid（4.8%）and arachic acid（3.3%）.The content of unsaturated fatty acids reached 77.6%.The level of CSNSO antioxidant activity was estimated by testing DPPH and hydroxyl free radicals scavenging ability,and the results were expressed in terms of IC₅₀（which were 8.51 g/L and 0.396 g/L,respectively）.C.speciosa seed oil was rich in unsaturated fatty acids,polyphenols and other readily oxidized active ingredients.In order to reduce the loss of nutrients in oil,the microcapsules of C.speciosa seed oil were prepared using spray drying technology.Based on the single-factor experiments,orthogonal experiment was carried out,and the comparatively good technical parameters for preparing C.speciosa seed oil microcapsules using spray drying technology were confirmed as follows:homogeneous pressure 30 MPa,inlet temperature 190℃,feeding rate 16m L/min.The microcapsules produced under these processing parameters were determined,and the microcapsules obtained were determined with microencapsulation efficiency 83.59%,water content 2.51%,density 0.63 g/cm^-3,hygroscopicity 9.19%,and particle size 39.44μm,respectively.The microcapsule powders observed by SEM were found to have a regular spherical shape with smooth surface and compact structure.The study of storage stability showed that the peroxide value of C.speciosa seed oil entrapped in microcapsules was 5.7 mmol/kg,whereas it was 40.2 mmol/kg found in bulk oil.The properties of microcapsule products were ideal,and the shelf life of C.speciosa seed oil was greatly extended.The defatted C.speciosa seed presented a high amount of polysaccharide.In order to better use this natural resource,the extraction process of crude polysaccharide and the physicochemical properties of purified polysaccharide were investigated.Based on the single-factor experiments,orthogonal experiment was performed,and the comparatively good parameters using water extraction and alcohol precipitation for preparing C.speciosa seed crude polysaccharide were presented as follows:extraction temperature 70℃,extraction time 3 h,solid-liquid ratio 1:40（g:m L）.The yield of crude polysaccharide reached 7.46%under the optional extraction conditions.The crude polysaccharide was sequentially purified with Cellulose DEAE-52 and Toyopearl HW-65F gel-filtration chromatography,and a highly purified polysaccharide fraction（F3）was obtained.The physicochemical properties of F3 purified from crude C.speciosa seed polysaccharide were determined using high-performance gel permeation chromatography（HPGPC）,high performance liquid chromatography（HPLC）,ultraviolet-visible（UV）,Fourier transform infrared（FT-IR）spectrum.The results indicated that F3 was a homogeneous heteropolysaccharide fraction with a molecular weight of 8.65×10⁶ u,and it was composed of Rha（6.34%）,Glc A（5.73）,Gal（47.14%）,and Ara（40.13%）.The primary structure of F3 purified from crude C.speciosa seed polysaccharide was characterized,and it’s bioactivities were also evaluated in this section.Nuclear magnetic resonance（NMR）spectrum,together with methylation,periodate oxidation and Smith degradation analysis,scanning electron microscopy（SEM）,atomic force microscope（AFM）,and Congo-red test analysis were hired for structural analysis.The backbone of F3 was consisted of→3,6)-Galp-(1→,and the side chains of F3 were composed of Araf-（1→,→4）-Glcp A-（1→,→4）-Galp-（1→and→3）-Rhap-(1→.The repeating unit of F3 was identified.In the antioxidant activity evaluations,F3 presented DPPH,hydroxyl and superoxide anion free radicals scavenging ability and metal ion chelation ability,and they all showed an obvious dose-dependent manner.Moreover,F3 demonstrated good inhibition activities onα-amylase andα-glucosidase,and their IC₅₀ values were 6.24 g/L and 4.59 g/L respectively.C.speciosa seed was rich in protein,and it could be applied as a high-quality protein resource.The protein isolate and different protein fractions were prepared respectively by alkali dissolving acid precipitation method and classical Osborne fraction method.The amino acid compositions of protein isolate,and main protein fractions（albumin and glutelin）were studied.The result showed that all these three protein samples exhibited appropriate essential amino acid compositions,and they basically met the minimum recommendations（World Health Organization/Food and Agriculture Organization,WHO/FAO）of essential amino acids for adults diet.Among these three kinds of protein samples,glutelin showed the highest denaturation temperature（T_d108.36℃）and the best heat coagulability（25.39%）.Albumin exhibited better solubility than protein isolate and glutelin.In addition,albumin showed the strongest emulsifying ability and foaming capacity.Whereas,protein isolate performed best in fat adsorption capacity（10.77 g/g）and glutelin showed the highest capacity in holding water（5.44 g/g）.The viscosity measurement showed that all these three protein solutions were pseudoplastic fluids with typical characteristics of shear thinning.In order to enhance the utilization value of C.speciosa seed protein isolate,it was hydrolyzed by papain to produce active peptides.The tyrosinase inhibition activity was set as the index,and two peptides were successfully prepared after ultrafiltration,gel filtration chromatography,and reversed phase high performance liquid chromatography purifications.MALDI-TOF-TOF was applied to identified the amino acid sequences of these two purified peptides,and they were Asparagine-Tyrosine-Arginine-Arginine-Glutamicacid（NYRRE,737.059 u）and Arginine-Histidine-Alanine-Lysine-Phenylalanine（RHAKF,658.010 u）,respectively.In the antioxidant activity evaluation tests,RHAKF showed better performance in scavenging DPPH,superoxide anion free radicals and lipid peroxidation inhibition capacity.RHAKF also exhibited better copper chelating activity than NYRRE,as it’s IC₅₀ value was 0.93g/L and it was lower than that of NYRRE（2.11 g/L）.Moreover,RHAKF exhibited excellent tyrosinase inhibition activity with a low IC₅₀ value（1.15 g/L）,and it was more efficient than that of citric acid(IC₅₀9.77g/L).The molecular docking simulation was used to reveal the interactions between tyrosinase and peptides.The results revealed that more docking sites were existed between RHAKF and tyrosinase.