Study On Comprehensive Utilization Of Acer Truncatum Bunge Seed Kernel In Northeast China

Acer truncatum Bunge is a unique tree species with high application value in China,which integrates economic,ecological,oil and medical values.However,it is still regarded as an ornamental tree in most parts of China,and its seed kernels are used for oil production in only a few places,which produces a large amount of residue that is often discarded and wasted.Therefore,the comprehensive utilization of A.truncatum Bunge seed kernels has important ecological significance,economic significance and social significance.In this study,a simple,rapid,efficient and green enzyme-assisted three-phase partitioning method was developed for the one-step extraction and separation of seed oil,peptides and polysaccharides from seed kernels of Northeastern A.truncatum Bunge,and their applications were studied,respectively.As follows:（1）Extraction and isolation of seed oil,peptides and polysaccharides from Northeast A.truncatum seedsThe enzyme-assisted three-phase partitioning was used to extract and separate the seed oil,polypeptides and polysaccharides from the seed kernel of A.truncatum.Firstly,a complex enzyme of cellulase and alkaline protease was identified as the experimental enzyme through enzyme reagent screening.The yield of A.truncatum seed oil was used as the response value.The effects of enzyme dosage,enzymolysis time,solid-liquid ratio,extraction temperature,ratio of tert-butanol to water,ammonium sulfate mass fraction,p H value and extraction time on the yield of A.truncatum seed oil were investigated by single factor and response surface experiments.Under the optimal extraction conditions（solid/liquid ratio of 1:15,enzyme dosage of 4%,enzymatic hydrolysis time of 4.3 h,mass fraction of ammonium sulfate of 40%,p H of 4,ratio of tert-butanol to water of 1.4:1,extraction temperature of 40℃and extraction time of 1h）,the seed oil yield was 48.72±0.51%.The mixed fatty acids of A.truncatum seed oil were prepared by saponification method,and then the molecular distillation-low temperature crystallization method was used to purify the nervonic acid and the purity of nervonic acid was determined by gas chromatography.The results showed that the concentration of nervonic acid increased from 6.25%to 37.30%,which was 5.97-fold increase.（2）Anti-Alzheimer’s disease study of Northeast A.truncatum neuronic acidThe purified nervonic acid was used as raw material to prepare the water-soluble micronized powder of A.truncatum Bunge neuronic acid.Then the mouse model of Alzheimer’s disease（AD）was established by subcutaneous injection of D-gal and intragastric administration of Al Cl₃,and the intervention treatment was performed by nasal administration.The water maze experiment results showed that the water-soluble powder of A.truncatum nervonic acid could significantly shorten the time of AD mice to explore the hidden platform and enhance their learning and memory ability.Compared with the model group,after nasal administration of high-dose A.truncatum nervonic acid water-soluble powder,the levels of Aβ₄₀ and Aβ₄₂ in the hippocampus of mice decreased by 32.16%and 49.80%,respectively;the levels of MDA decreased by 84.19%and the levels of antioxidant enzymes（SOD,CAT and GSH-Px）increased by 130.79%,124.95%and 80.03%,respectively.And the corresponding levels of ALT and AST decreased by 43.33%and 41.40%,respectively,the levels of serum aminotransferases decreased by 43.33%and41.40%,respectively,among the central cholinergic indicators of brain tissue,their Ach E levels decreased by 50.92%,and Ach and Ch AT levels increased by 207.90%and 181.84%,respectively.Among the ATPase activities of brain tissue,Na⁺K⁺-ATPase and Ca²⁺Mg²⁺-ATPase activities increased by 170.68%and 38.01%,respectively.At the same time,the nasal administration group also showed far better therapeutic effect than the equal dose oral administration and close to the positive drug donepezil group.In addition,through the pathological observation of mouse brain tissue and liver tissue,it was further found that A.truncatum nervonic acid powder had a certain protective and improving effect on the organs of AD mice.The above results indicate that the water-soluble powder of A.truncatum nervonic acid has a good anti-Alzheimer’s disease effect,which also provides data support for neuronic acid to treat Alzheimer’s disease clinically.（3）Physicochemical properties,functional characterization and antioxidant activity of peptides from seed kernels of Northeast A.truncatumThe physicochemical properties and functional properties of the seed kernel peptides obtained using enzyme-assisted three-phase partitioning were analyzed.The results showed that the yield was 22.84±2.52%,and the contents of moisture,fat,total sugar,protein and ash were5.23%,0.25%,2.79%,83.64%and 4.54%,respectively.According to the analysis of amino acid composition,the kernel polypeptide contains all the amino acids necessary for the human body.Compared with the seed kernel powder,the content of eight essential amino acids in the kernel polypeptide can reach 18.83%,which is much higher than 8.73%in the kernel powder,which is about 2.16 times,and also much higher than the amount of essential amino acids required for adults（10.75%）recommended by the International Food and Agriculture Organization（FAO）.The total amount of hydrophobic amino acids in the kernel polypeptide was 26.95%,which was about 2.18 times higher than that of seed kernel powder（12.37%）.Compared with the kernel powder,the kernel polypeptide had higher degree of hydrolysis（15.72%）,free sulfhydryl content（15.48μmol/g）,and lower disulfide bond content（15.31μmol/g）.In addition,the infrared spectrum analysis of A.truncatum kernel polypeptide showed that it had five typical amide bands.The amino acid sequence analysis of A.truncatum kernel polypeptide by LC-MS/MS secondary mass spectrometry showed that in the secondary mass spectrometry of the 18 best-matched peptides,the proportion of hydrophobic amino acids to residues was more than 71.43%,and many were almost 100%,which indicates that A.truncatum kernel polypeptide is an excellent antioxidant peptide.In addition,among the 18 peptides,we also found that there are superimposed hydrophobic amino acids,such as V-V（Val-Val）,E-E（Glu-Glu）,P-P（Pro-Pro）,etc.,and most of the peptides also contain aromatic amino acids,which further indicates that the A.truncatum kernel polypeptide has good antioxidant potential and can be further developed into antioxidant peptides for medical use in the future.The antioxidant activity of A.truncatum kernel polypeptide was evaluated by chemical model method and cell biological method.The results showed that A.truncatum kernel polypeptide had good DPPH and hydroxyl radical scavenging ability.A.truncatum Bunge kernel polypeptide can also play a good antioxidant role by regulating MDA,antioxidant enzymes（SOD,CAT and GSH-px）and ROS levels in RAW264.7 cells induced by H₂O₂.These results indicate that the polypeptides can effectively improve the oxidative stress response of Raw264.7 cells treated with H₂O₂,and can be used as potential antioxidant peptides in the fields of food and medicine.（4）Structural analysis and hypoglycemic activity of polysaccharides in seed kernels of Northeast A.truncatumThe A.truncatum seed kernel polysaccharide in the combined enzyme-assisted t three-phase partitioning and the residue water extract was precipitated by alcohol,and then separated and purified by deproteinization,DEAE-52 cellulose and dextran gel G-100 chromatography column to obtain a main component PATM-3,and its chemical composition and structure were analyzed.The results showed that the contents of polysaccharide,protein and uronic acid in PATM-3 were97.17±1.62%,1.14±0.45%and 68.88±2.65%,respectively,indicating that the A.truncatum seed polysaccharide was an acidic polysaccharide.The monosaccharide composition of PATM-3 was galacturonic acid,arabinose,galactose,rhamnose,glucose,xylose,mannose,glucuronic acid,fucose,ribose and D-glucosamine with a molar ratio of12.40:12.81:5.08:3.86:2.69:2.31:1:0.66:0.32:0.06:0.02.Finally,the results of IR spectra and NMR spectra showed that PATM-3 is a pyranose withβ-configuration and consists mainly ofβ-D-galacturonic acid.Type II diabetic model mice were established by high-fat diet combined with streptozotocin（STZ）and the hypoglycemic effect of A.truncatum Bunge seed kernel polysaccharide was evaluated.Firstly,the results showed that seed kernel polysaccharide（PATM）could significantly improve weight loss,high food intake and water intake in T2DM model mice,as well as reduce fasting blood glucose in diabetic mice.Secondly,PATM could reduce insulin resistance and enhanceβ-cell function index and insulin sensitivity in diabetic mice by lowering blood glucose and insulin levels,which in turn promoted insulin secretion and glucose uptake in peripheral tissues to achieve hypoglycemic effects.PATM also significantly reduced GSP levels in mouse serum,improved serum and liver lipid levels（including free fatty acids（FFA）,total cholesterol（TC）,total triglycerides（TG）,high-density lipoprotein（HDL-C）and low-density lipoprotein（LDL-C）,etc.）,regulated liver glycogen levels,improved liver oxidative stress levels（MDA,SOD,CAT and GSH-Px）and protection of liver and pancreatic tissues in mice.（5）Regulation of intestinal microbial community in diabetic mice by polysaccharide from seed kernels of Northeast A.truncatumThe changes of fecal short-chain fatty acids（SCFAs）content and intestinal flora in mice treated with PATM were analyzed by gas chromatography and 16S r RNA sequencing techniques and corresponding bioinformatics analysis,respectively.The results showed that PATM-H produced SCFAs through intestinal enzymatic digestion,thus effectively regulating the function of adipose and liver tissues in mice,thereby improving glucose homeostasis and insulin sensitivity,and playing a role in lowering blood glucose.Meanwhile,PATM-H can effectively increase the abundance of intestinal flora and improve the structure of intestinal flora in diabetic mice.In addition,PATM-H could also regulate the increase of the ratio of Firmicutes to Bacteroidetes and the decrease of the abundance of Muribaculaceae and Lachnospiraceae＿NK4A136＿group caused by diabetes to the normal level in mice.Overall,PATM-H can protect and regulate the gut microbial community structure in diabetic mice in terms of phylum and genus,which in turn affects the host health.In summary,this thesis aims to develop a simple,rapid,efficient,low-cost and green enzyme-assisted three phase partitioning for the extraction and separation of seed oil,peptides and polysaccharides from the seed kernels of Northeast A.truncatum with the aim of their utilization.Then,neuronic acid was purified from the seed oil and prepared as a water-soluble micronized powder for anti-Alzheimer’s disease studies.The seed kernel peptides were analyzed for physicochemical properties and functional properties,and then their protective effects against H₂O₂-induced oxidative damage in RAW264.7 cells were evaluated.The seed kernel polysaccharides were isolated and purified and structurally characterized,and their hypoglycemic effects on type II diabetes and the regulation of intestinal flora were examined.This stu provides a scientific basis for the high-value comprehensive utilization of northeast A.truncatum seed kernels and the development of northeast A.truncatum resources processing industry,and contributes to the theoretical basis and technical support for the common development of green,health and economy.