Method for extracting acidic pinus sylvestris var. mongolica polysaccharide by bionic-microwave-assisted alkali extraction and ultrasonic extraction

Abstract

This invention discloses a biomimetic-microwave-assisted alkaline extraction combined with ultrasonic extraction method for extracting acidic pine polysaccharides, belonging to the field of polysaccharide technology. The method includes the following steps: Step 1, enzymatic hydrolysis; Step 2, microwave-assisted alkaline extraction; Step 3, ultrasonic-assisted extraction; Step 4, combining the extracts to obtain a concentrated solution, precipitating with alcohol overnight, centrifuging, discarding the supernatant, redissolving the precipitate in deionized water, adding trichloroacetic acid (TCA), stirring, incubating overnight at 0-4°C, centrifuging, dialyzing the supernatant, and then freeze-drying to obtain acidic pine polysaccharides. This biomimetic-microwave-assisted alkaline extraction combined with ultrasonic extraction method for extracting acidic pine polysaccharides significantly improves the polysaccharide yield, is environmentally friendly, enhances the immunomodulatory activity of the polysaccharides, and solves various problems in traditional extraction methods.

Description

Technical Field

[0001] This invention relates to a method for preparing acidic pine polysaccharide, and more particularly to a biomimetic-microwave-assisted alkaline extraction method combined with ultrasonic extraction of acidic pine polysaccharide, belonging to the field of polysaccharide technology. Background Technology

[0002] Due to its specific climate and altitude, the seeds of the dwarf pine mature only once every three years, making it one of the tree species with the longest seed-bearing period in the world. Trees over 200 years old can still bear fruit. The dwarf pine is a typical native tree species in the Huzhong District of the Greater Khingan Mountains, with a distribution area exceeding 70,000 hectares, ranking first among forest areas in China. In the Natural Forest Protection Project, the dwarf pine is listed as a soil and water conservation forest and is a key tree species in ecological public welfare forests. In addition, dwarf pine kernels contain various nutrients and unsaturated fatty acids needed by the human body, and can be used for food, medicine, and oil extraction. Therefore, the dwarf pine is also an important economic tree species. Dwarf pine kernels are a gift from nature, with a very considerable harvest, generating hundreds of millions of yuan in economic output annually. Due to advancements in international molecular research, research on polysaccharides is receiving increasing attention both domestically and internationally, and the 21st century is considered the century of polysaccharides. As humanity's fight against disease deepens, traditional treatments have side effects on normal cells and can damage the immune system. Polysaccharides are essential macromolecules found in almost all life forms, possessing important biological functions. Their broad range of biological and pharmacological activities, such as antitumor, immunomodulatory, antioxidant, and hepatoprotective activities, coupled with the relatively few side effects of novel drugs or vaccines prepared based on their bioactivity, make them promising candidates in the biomedical and pharmaceutical fields. However, research on polysaccharides from *Pinus sylvestris* is currently lacking, hindering the high-value development and utilization of this specialty nut from Northeast China. Furthermore, existing extraction technologies suffer from complex processes, low yields, and solvent residues and environmental pollution during processing. Therefore, providing an extraction method that improves polysaccharide yield, is environmentally friendly, and exhibits high activity is of significant importance. Summary of the Invention

[0003] The purpose of this invention is to address the problems existing in the prior art by providing a biomimetic-microwave-assisted alkali extraction method combined with ultrasonic extraction for the extraction of acidic pine polysaccharides.

[0004] Another objective of this invention is to provide an acidic pine polysaccharide with good immunomodulatory activity.

[0005] Another object of the present invention is to provide the use of acidic pine polysaccharide in the preparation of drugs that promote immune activity.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A biomimetic-microwave-assisted alkaline extraction combined with ultrasonic extraction method for extracting acidic pine polysaccharides includes the following steps:

[0008] Step 1, enzymatic hydrolysis: Add defatted pine nut powder to deionized water at a ratio of 1:(20-40)g / mL, then add papain at a ratio equivalent to 0.5-3.0wt% of deionized water. Extract for 30-80 minutes at pH 6-7 and 25-75℃. Inactivate the enzyme, centrifuge, concentrate the supernatant to obtain material 1 and pine nut oil meal 1.

[0009] Step 2, microwave-assisted alkaline extraction: Add 0.2-1 mol / L NaOH to the pine kernel oil meal 1 at a material-to-liquid ratio of 1:(5-160) g / mL, then microwave extract at 100-700W for 10-110S, centrifuge, concentrate the supernatant to obtain material 2 and pine kernel oil meal 2.

[0010] Step 3, Ultrasonic-assisted extraction: Add 0.4-0.6 mol / L NaOH to the pine kernel oil meal 2 at a material-to-liquid ratio of 1:(20-40) g / mL, and extract for 10-60 min at 30-80℃ and ultrasonic power of 50-100W. Centrifuge, concentrate the supernatant to obtain material 3 and pine kernel oil meal 3.

[0011] Step 4: Combine materials 1, 2, and 3 and concentrate to 3-5 times the weight of defatted pine nut powder to obtain a concentrated solution. Add 3-5 times the volume of 90-95% ethanol to precipitate overnight at 0-4°C. Centrifuge, discard the supernatant, and redissolve the precipitate in 5-10 times the weight of defatted pine nut powder in deionized water. Add 10-20% trichloroacetic acid (TCA) in an equal volume of the redissolved deionized water, stir for at least 30 minutes, and let stand overnight at 0-4°C. Centrifuge, dialyze the supernatant, and freeze-dry to obtain acidic pine polysaccharide.

[0012] Specifically, in step one, the enzyme activity of papain is 200 u / mg.

[0013] In step one, the enzyme inactivation method is boiling water bath for 8-12 minutes, centrifugation at 6000-9000 rpm for 5-15 minutes, and concentration temperature is 40-60℃; in steps two and three, the concentration temperature is 40-60℃.

[0014] In step four, centrifugation is performed at 3000-6000 rpm for 10-20 min; dialysis time is at least 48 h, with tap water and deionized water each dialyzed for at least 24 h; the sample is then frozen in a freezer at at least -20°C for at least 12 h; after freezing, the sample is placed in a vacuum freeze dryer and dried under vacuum for at least 36 h.

[0015] The monosaccharides of the acidic dansonia polysaccharide obtained by this method exist in the form of pyranose; the glycosidic bonds of the acidic dansonia polysaccharide are α-glycosidic bonds.

[0016] The application of acidic pine polysaccharide obtained by this method in the preparation of drugs that promote immune activity.

[0017] Preferably, a biomimetic-microwave-assisted alkaline extraction combined with ultrasonic extraction method for acidic pine polysaccharide extraction includes the following steps: Step 1, enzymatic hydrolysis: 5g of defatted pine nut powder is added to deionized water at a material-to-liquid ratio of 1:20g / mL, and papain is added at a ratio equivalent to 2.29wt% of deionized water. Extraction is carried out at pH 6.2 and 45℃ for 43.04min, enzyme is inactivated, centrifuged, and the supernatant is concentrated to obtain material 1 and pine nut oil meal 1;

[0018] Step 2, microwave-assisted alkaline extraction: Add 0.6mol / L NaOH to the pine kernel oil meal 1 at a material-liquid ratio of 1:32g / mL, extract by microwave at 400W for 50s, centrifuge, concentrate the supernatant to obtain material 2 and pine kernel oil meal 2;

[0019] Step 3, Ultrasonic-assisted extraction: Add 0.6mol / L NaOH to the pine kernel oil meal 2 at a material-to-liquid ratio of 1:20g / mL, extract at 71℃ and 81W for 45min, centrifuge, concentrate the supernatant to obtain material 3 and pine kernel oil meal 3;

[0020] Step 4: Combine materials 1, 2, and 3 and concentrate to 40 mL to obtain a concentrated solution. Add 95% ethanol (equivalent to 4 times the volume of the concentrated solution) and precipitate overnight at 4°C. Centrifuge, discard the supernatant, redissolve the precipitate in 80 mL of deionized water, add 80 mL of 15% trichloroacetic acid (TCA), stir for 30 min, place at 4°C overnight, centrifuge, dialyze the supernatant, and freeze-dry to obtain acidic pine polysaccharide.

[0021] Specifically, in step one, the enzyme activity of papain is 200 u / mg.

[0022] In step one, the enzyme inactivation method is boiling water bath for 10 minutes, centrifugation at 7500 rpm for 10 minutes, and concentration temperature is 50℃; in steps two and three, the concentration temperature is 50℃.

[0023] In step four, the centrifugation was performed at 6000 rpm for 15 min; the dialysis time was 48 h, with tap water and deionized water each dialyzed for 24 h; the samples were then frozen at -20 ℃ for 12 h; and the frozen samples were placed in a vacuum freeze dryer for 36 h of vacuum drying.

[0024] The monosaccharides of the acidic dansonia polysaccharide obtained by this method exist in the form of pyranose; the glycosidic bonds of the acidic dansonia polysaccharide are α-glycosidic bonds.

[0025] In acidic pine polysaccharide, the mass ratio of polysaccharide:protein:tannin is 52.17:1.26:0.61.

[0026] Application of acidic pine polysaccharide in the preparation of immunomodulatory drugs.

[0027] The present invention has the following beneficial effects:

[0028] Currently, there are no reported studies on pine polysaccharides, which greatly hinders the utilization and development of Northeast China's unique nut resources. Furthermore, existing extraction technologies face problems such as complex extraction processes, low yields, poor activity, and solvent residues and environmental pollution during processing. Compared to traditional extraction methods, this invention provides a biomimetic-microwave-assisted alkaline extraction combined with ultrasonic extraction of acidic pine polysaccharides, which significantly improves polysaccharide yield. The biomimetic (enzymatic) polysaccharide yield is 27.34%, the biomimetic-microwave-assisted alkaline extraction yield is 31.06%, and the final yield of the biomimetic-microwave-assisted alkaline extraction combined with ultrasonic extraction is 32.94% (based on the optimal results of single-factor response surface optimization, the optimal yield of 27.34% was obtained first through enzymatic hydrolysis, then increased to 31.06% under optimal microwave conditions, and further increased to 32.94% under optimal ultrasonic conditions). Moreover, this method is green and environmentally friendly, improves the immunomodulatory activity of polysaccharides, and solves various problems in the aforementioned traditional extraction methods. Attached Figure Description

[0029] Figure 1 Bar chart showing the yield of nut polysaccharides extracted with ultrasound-assisted solvents;

[0030] Figure 2 A bar chart showing the proliferation rate of immune cells in RAW264.7 mice;

[0031] Figure 3 The graph shows the determination of total reducing power;

[0032] Figure 4 The graph shows the determination of ABTS scavenging ability;

[0033] Figure 5 The graph shows the determination of hydroxyl radical scavenging ability;

[0034] Figure 6 Bar chart showing the yield of pine polysaccharides extracted with different enzymes;

[0035] Figure 7 Single-factor line graph for biomimetic extraction of pine polysaccharides;

[0036] Figure 8 3D response surface model for biomimetic extraction of pine polysaccharides;

[0037] Figure 9 Single-factor line graph for microwave-assisted alkaline extraction of pine polysaccharides;

[0038] Figure 10 3D response surface model for microwave-assisted alkaline extraction of pine polysaccharides;

[0039] Figure 11 Single-factor line graph for ultrasound-assisted extraction of pine polysaccharides;

[0040] Figure 12 3D response surface model for ultrasound-assisted extraction of pine polysaccharides;

[0041] Figure 13 This is a standard curve for polysaccharide content.

[0042] Figure 14 This is a standard curve for protein content.

[0043] Figure 15 This is a standard curve for tannin content;

[0044] Figure 16 Infrared spectrum of pine polysaccharide;

[0045] Figure 17 Bar chart showing the effect of polysaccharides extracted by different methods on the proliferation rate of immune cells in RAW264.7 mice;

[0046] Figure 18 Bar chart showing the effect of polysaccharides extracted by different methods on the phagocytic rate of immune cells in RAW264.7 mice. Detailed Implementation

[0047] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0048] Example 1

[0049] 1. Screening and identification of raw materials and extraction methods

[0050] 1.1 Ultrasonic-assisted extraction of polysaccharides

[0051] 10g of defatted pine nut powder (containing hickory, hazelnut, red pine, and dwarf pine nuts respectively) was taken separately and added to deionized water, 75% ethanol, 0.2mol / L HCl, and 0.2mol / L NaOH at a material-to-liquid ratio of 1:20. Extraction was carried out simultaneously at 60℃ for 30 min and 80W. The extracts were filtered and combined. Each extract was concentrated to 40mL, and precipitated overnight at 4℃ with 4 times its volume of 95% ethanol. The mixture was centrifuged at 6000rpm / min for 15 min, the supernatant was discarded, and the precipitate was redissolved in 80mL of water. An equal volume of 15% TCA was added, the mixture was stirred for 30 min, incubated overnight at 4℃, centrifuged, and the supernatant was dialyzed for 48 h and lyophilized. The polysaccharide content was determined using the phenol-sulfuric acid method, and the polysaccharide yield was calculated. Figure 1 The results show that: alcohol extraction yields low polysaccharide yields for hazelnut and dwarf pine, while alcohol-soluble polysaccharides from red pine and hickory are almost zero and negligible; alkali extraction yields higher yields for red pine and dwarf pine; acid extraction yields higher yields for red pine, dwarf pine, and hazelnut polysaccharides; and water extraction yields higher yields for dwarf pine and red pine polysaccharides. Based on subsequent research on immunomodulatory activity, a literature search revealed numerous studies on the immunomodulatory activity of red pine polysaccharides, and polysaccharide extraction experiments also showed higher yields for red pine and dwarf pine. Therefore, to further screen and determine the optimal method for polysaccharide extraction, the extracted red pine and dwarf pine polysaccharides were selected as the main research subjects for in vitro immunomodulatory activity tracking.

[0052] 1.2. RAW264.7 mouse immune cell MTT assay for cell proliferation

[0053] 1.2.1 Preparation of polysaccharide solution and experimental grouping

[0054] Take the water-soluble *Pinus koraiensis* polysaccharide, water-soluble *Pinus koraiensis* polysaccharide, acidic *Pinus koraiensis* polysaccharide, acidic *Pinus koraiensis* polysaccharide, alkaline *Pinus koraiensis* polysaccharide, and alkaline *Pinus koraiensis* polysaccharide obtained by the above-mentioned water extraction method; prepare five different concentration groups with concentrations of 0.1, 1, 10, 100, and 1000 μg / mL, respectively. First, weigh 10 mg of each polysaccharide and place it in a sterilized EP tube, and prepare a 1 mg / mL polysaccharide solution under aseptic conditions. Then, dilute the polysaccharide samples several times with culture medium. Seal the prepared polysaccharide sample solutions with sealing film and store them in a refrigerator at 4°C for later use.

[0055] 1.2.2 Culture of mouse macrophages RAW264.7

[0056] Frozen mouse macrophages were removed from the liquid nitrogen container and immediately placed in a 37°C water bath, then transferred to sterile centrifuge tubes. 10 mL of DMEM complete medium was added to the tubes, and the cells were gently mixed by pipetting. The cells were centrifuged at 1000 rpm for 5 min. After centrifugation, the supernatant was removed, and fresh medium was added. The cells were gently mixed by pipetting, then transferred to cell culture dishes. Finally, the dishes were placed in an incubator at 37°C and 5% CO2. Cell growth should be monitored daily, and the medium should be replaced regularly with fresh medium.

[0057] 1.2.3 Effect of polysaccharides on the survival rate of RAW264.7 cells

[0058] The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-bromodiphenyltetrazolium) method was used. Cells were placed in 96-well plates and treated with polysaccharide solutions of different concentrations (0.1, 1, 10, 100, and 1000 μg / mL) dissolved in DMEM. After 24 h, 10 mL / well of 5 mg / mL MTT solution was added. After 4 h of culture, the cell supernatant was removed, and 50 μL of dimethyl sulfoxide (DMSO) was added for dissolution and crystallization. The absorbance was then recorded at 490 nm. Figure 2 The results show that the effects of acidic pine polysaccharide, water-soluble pine polysaccharide, basic pine polysaccharide, and acidic red pine polysaccharide on the proliferation rate of immune cells in RAW 264.7 mice are dose-dependent, with acidic pine polysaccharide showing the strongest proliferative effect.

[0059] 1.3 Determination of the antioxidant capacity of three polysaccharides (the three polysaccharides were acidic ginseng polysaccharide, basic ginseng polysaccharide, and water-soluble ginseng polysaccharide).

[0060] 1.3.1 Reducing power

[0061] Preparation of phosphate buffer: Prepare a 0.2 mol / L phosphate buffer solution with a pH of 7.4. 1% potassium ferricyanide solution: Accurately weigh 0.5 g of potassium ferricyanide and dilute to 50 mL with distilled water; 10% trichloroacetic acid solution: Accurately weigh 5 g of trichloroacetic acid and dilute to 50 mL with distilled water; 0.1% ferric chloride solution: Accurately weigh 50 mg of ferric chloride and dilute to 50 mL with distilled water. Operating Procedure: Take 1 mL of sample solution (sample solution concentrations are set at 1, 2, 4, 6, 8, 10, and 12 mg / mL; the preparation method is to first prepare a 10 mg / mL sample and then dilute it to a lower concentration using multiples; the 12 mg / mL sample is prepared directly by weighing a certain sample mass). Add 2.5 mL of phosphate buffer and 2.5 mL of 1% potassium ferricyanide solution, mix well, and incubate in a 50°C water bath for 20 min. Remove and cool rapidly. Add 2.5 mL of 10% trichloroacetic acid solution, mix well, centrifuge at 3000 rpm for 10 min, and transfer 2.5 mL of the supernatant to another test tube. Add 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride solution, mix well, and let stand for 10 min. Measure the absorbance at 700 nm.

[0062] according to Figure 3 The results show that the total reducing power increases with increasing polysaccharide concentration in the reaction system, indicating a dose-dependent relationship. When the polysaccharide concentration is in the range of 1.0–8.0 mg / mL, the dose-response relationship of the total reducing power increases significantly, and the reducing power is ranked from strongest to weakest as follows: acidic pine polysaccharide > alkaline pine polysaccharide > water-soluble pine polysaccharide.

[0063] 1.3.2, ABTS

[0064] Preparation of ABTS solution: Accurately weigh 0.19 g of ABTS into a beaker, add 50 mL of distilled water, and mix to dissolve; then weigh 0.095 g of potassium persulfate and add 50 mL of distilled water, and mix to dissolve; then mix the two solutions thoroughly, and let stand in the dark for 12–16 h to form a mother liquor; dilute the mother liquor with phosphate buffer at pH 4 to OD. 734 =0.7±0.02.

[0065] Procedure: Add 0.1 mL of sample solution to 1.9 mL of ABTS detection solution and vortex to mix. Incubate at room temperature in the dark for 6 min, and measure the absorbance at 734 nm. Perform the experiment in triplicate and calculate the clearance rate using the following formula:

[0066] In the formula: 0.1 mL of distilled water is used instead of the sample solution to measure the absorbance value A0; A1 is the absorbance value after the sample solution of different concentrations is mixed and reacted with ABTS solution; and 1.9 mL of distilled water is used instead of ABTS solution as the background to measure the absorbance value A2.

[0067] ABTS is a decolorization test used to screen the antioxidant activity of lipophilic and hydrophilic antioxidants. The reaction directly generates blue and green chromophores of ABTS through the reaction of ABTS with potassium persulfate. Then, an antioxidant is added to scavenge free radicals, leading to a decrease in absorbance. This decrease is used to indicate the strength of the antioxidant activity. ABTS free radical scavenging is an electron migration process; the antioxidant reacts with an ABTS free radical stock solution, exhibiting a maximum absorbance at 734 nm, thus determining the ABTS free radical scavenging ability. Figure 4 The results show that the ABTS scavenging activities of the three *Pinus sylvestris* polysaccharides were weaker than those of vitamin C. In the reaction system, the ABTS scavenging ability increased with increasing polysaccharide concentration, indicating a dose-dependent relationship. The dose-effect relationship stabilized at 8.0 mg / mL, but even at a polysaccharide concentration of 12.0 mg / mL, the ABTS scavenging rate remained between 30% and 40%. Acidic *Pinus sylvestris* polysaccharide exhibited the best scavenging ability.

[0068] 1.3.3 Hydroxyl radicals

[0069] Preparation of reagents: Prepare 9 mmol / L salicylic acid (accurately weigh 0.1249 g of salicylic acid and dilute to 100 mL with 70% ethanol), 9 mmol / L FeSO4 (accurately weigh 0.2508 g of FeSO4·7H2O and dilute to 100 mL with distilled water) and 6 mmol / L H2O2 (accurately measure 61 μL of H2O2 and dilute to 100 mL with distilled water), and prepare fresh each time you need to use them.

[0070] Operating procedures: Add 9×10 to the reaction system sequentially. -3 1 mL of mol / L FeSO4 solution, 9 × 10 -3 1 mL of mol / L salicylic acid-ethanol solution and 1 mL of pine nut polysaccharide sample solutions of different concentrations were added, and finally 6 × 10⁻⁶ mol / L was added. -3 1 mL of mol / L H₂O₂ was added to start the reaction, and the reaction was carried out in a water bath at 37℃ for 0.5 h. The absorbance of each concentration of sample was measured at 510 nm. The blank group was prepared by replacing the sample solution with the same volume of distilled water, and by replacing 6 × 10⁻⁶ mol / L H₂O₂ with distilled water. -3 Using mol / L H2O2 as the sample control group and ascorbic acid (VC) solution of the same concentration as the positive control, the experiment was repeated three times in parallel. The clearance rate was calculated using the following formula:

[0071]

[0072] In the formula: A0 is the absorbance of the blank control solution; A1 is the absorbance after adding the pine polysaccharide sample solution; A2 is the background absorbance of the pine polysaccharide solution without adding H2O2.

[0073] Hydroxyl radicals are the most reactive free radicals in the human body and the precursors of most oxygen free radicals. They can generate various free radicals and are also the most toxic free radicals. They directly damage various biological macromolecules, biological membranes, cell membranes, and cellular DNA, leading to the occurrence of various diseases and aging. Figure 5 The results show that the scavenging capacity of the three pine polysaccharides for hydroxyl radicals increased with increasing concentration in the reaction system, indicating a dose-dependent relationship. The dose-response relationship for hydroxyl radical scavenging increased significantly within the concentration range of 0.5–5.0 mg / mL, and then stabilized at 5.0 mg / mL. The basic pine polysaccharide exhibited the best scavenging capacity. This may be due to the activity of the polysaccharides being related to their coexisting proteins, pigments, and other substances.

[0074] 1.4 Enzymatic hydrolysis

[0075] Deionized water was added at a material-to-liquid ratio of 1:20. The pH was adjusted using 0.5 mol / L hydrochloric acid and 0.5 mol / L sodium hydroxide. Papain, neutral protease, acidic protease, and alkaline protease were added at 2% each, and extracted for 50 min at their optimal pH and temperature (specifically, papain 200 u / mg: pH 6.5, 55℃; neutral protease 50 u / mg: pH 7.0, 50℃; acidic protease 50 u / mg: pH 3.0, 45℃; alkaline protease 200 u / mg: pH 10, 50℃). The enzymes were inactivated by boiling in a water bath for 10 min, followed by centrifugation at 7500 rpm for 10 min. The supernatant was collected and concentrated to 40 mL at 50℃, then precipitated overnight at 4℃ with four times its volume of 95% ethanol. The polysaccharide content was determined using the phenol-sulfuric acid method, and the polysaccharide yield was calculated. Figure 6 The results show that papain yielded the highest amount of pine polysaccharide through enzymatic hydrolysis, followed by neutral protease > alkaline protease > acidic protease.

[0076] 2. Extraction of Pineapple Polysaccharides

[0077] 2.1 Enzymatic hydrolysis

[0078] 5g of defatted pine nut powder was added to deionized water at a material-to-liquid ratio of 1:20 (g / mL), followed by 2% papain. Extraction was performed at pH 6 and 45℃ for 50 min, followed by enzyme inactivation in a boiling water bath for 10 min, and centrifugation at 7500 rpm for 10 min. The collected supernatant was concentrated at 50℃, and the polysaccharide content was determined using the phenol-sulfuric acid method, and the polysaccharide yield was calculated. The single-factor setup consisted of four factors at six levels: enzyme dosage (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%), hydrolysis temperature (25, 35, 45, 55, 65, 75℃), pH (5, 5.5, 6, 6.5, 7, 7.5), and hydrolysis time (30, 40, 50, 60, 70, 80 min).

[0079] according to Figure 7 The results show that the polysaccharide yield initially increased and then slowly decreased. The extraction rate increased with increasing enzyme dosage, reaching its maximum at 2.0% enzyme concentration. Afterward, the extraction rate decreased with further increases in enzyme dosage. This may be because the polysaccharides were largely extracted, and other impurities entered the extract, increasing viscosity and slowing diffusion, thus hindering polysaccharide dissolution. Alternatively, excessive enzyme dosage could saturate the enzyme molecules in the solution, inhibiting polysaccharide dissolution. Considering all factors, an enzyme dosage of 2.0% is optimal. The polysaccharide yield reached its maximum at pH 6.0. Afterward, the yield decreased with increasing pH, as pH affects the affinity between the enzyme and substrate, inhibiting enzyme activity and thus reducing polysaccharide yield. Considering all factors, pH 6 is optimal. Insufficient hydrolysis time leads to incomplete hydrolysis, while excessively long hydrolysis times do not significantly increase polysaccharide yield. Considering all factors, a hydrolysis time of 50 minutes is optimal. As the enzymatic hydrolysis temperature increased, the polysaccharide extraction rate decreased. This may be because excessively high temperatures reduce enzyme activity, decreasing the effectiveness of cell wall hydrolysis and thus lowering the polysaccharide yield. Considering all factors, an optimal enzymatic hydrolysis temperature of 45℃ was determined. Response surface methodology was used to optimize the enzyme dosage, pH, and time.

[0080] By employing the central composite experimental design principle of Box-Behnken and using response surface methodology, response surface analysis (RSM) was conducted. Based on the analysis scheme and results, the DeSign-Expert software was used to generate RSM plots for different factors, and the optimal extraction process parameters for polysaccharide extraction were obtained through analysis and optimization. Figure 8 As shown, the optimal parameters for enzymatic hydrolysis were finally determined to be: enzyme dosage 2.29%, pH 6.02, time 43.04 min, and temperature 45℃.

[0081] 2.2 Microwave-assisted alkaline extraction

[0082] 5g of defatted pine nut powder was added to 0.6mol / L NaOH at a material-to-liquid ratio of 1:40 (g / mL), and extracted by microwave extraction at 400W for 30s. After centrifugation, the collected supernatant was concentrated at 50℃. The polysaccharide content was then determined using the phenol-sulfuric acid method, and the polysaccharide yield was calculated. The single-factor setup was a four-factor, five-level system with material-to-liquid ratios of 1:5, 1:10, 1:20, 1:40, 1:80, and 1:160, NaOH concentrations of 0.2, 0.4, 0.6, 0.8, and 1 mol / L, microwave power of 100, 250, 400, 550, and 700W, and microwave time of 10, 30, 50, 70, 90, and 110s.

[0083] according to Figure 9 The results show that the yield of *Pinus sylvestris* polysaccharides increases with increasing material-to-liquid ratio, showing a significant increase at a ratio of 1:20. However, the extraction rate plateaus after a ratio of 1:40, possibly because further increases in NaOH volume become insufficient to disrupt the cross-linking between *Pinus sylvestris* polysaccharides and cellulose polysaccharides once a certain volume is reached. Excessive NaOH addition prolongs the solution concentration time, therefore, a material-to-liquid ratio of 1:40 is optimal. The polysaccharide yield exhibits a near-linear increase in NaOH concentration from 0.2 to 0.6 mol / L, with a turning point at 0.6 mol / L, followed by a plateau. This may be because higher alkaline concentrations cause the chemical bonds between cellulose polysaccharides to break, converting insoluble polysaccharides like cellulose into soluble polysaccharides, while also increasing non-polysaccharide impurities. Excessively high alkaline concentrations result in a strong alkaline odor and other undesirable flavors. Therefore, a NaOH concentration of 0.6 mol / L is recommended. The polysaccharide yield increased with increasing microwave time, with 50 seconds showing a significant promoting effect on polysaccharide extraction. However, prolonged microwave treatment caused the solution to boil and overflow. Therefore, a time of 50 seconds is preferable. Within the range of 100–400 W, the polysaccharide yield increased with increasing power; further increases in power slightly decreased the yield. This may be because higher radiation power results in greater absorption of microwave energy by the system, accelerating molecular motion and solid-liquid mass transfer, thus promoting polysaccharide dissolution. However, when the microwave power increases to a certain level, microwaves may promote polysaccharide decomposition, leading to a decrease in the polysaccharide extraction rate. Therefore, a power of 400 W is preferable. Response surface methodology experiments were conducted to optimize the material-to-liquid ratio, microwave power, and microwave time.

[0084] By employing the central composite experimental design principle of Box-Behnken and using response surface methodology, response surface analysis (RSM) was conducted. Based on the analysis scheme and results, the DeSign-Expert software was used to generate RSM plots for different factors, and the optimal extraction process parameters for polysaccharide extraction were obtained through analysis and optimization. Figure 10As shown, the optimal parameters for enzymatic hydrolysis were finally determined to be: a material-to-liquid ratio of 1:32, a NaOH concentration of 0.6 mol / L, a microwave power of 400 W, and a microwave time of 50 s.

[0085] 2.3 Ultrasonic-assisted extraction

[0086] 5g of defatted pine nut powder was added to 0.6mol / L NaOH at a material-to-liquid ratio of 1:20 (g / mL), and extracted at 50℃ and 70W for 30min. After centrifugation, the collected supernatant was concentrated at 50℃. The polysaccharide content was then determined using the phenol-sulfuric acid method, and the polysaccharide yield was calculated. The single-factor setup consisted of three factors at six levels: ultrasonic time (10, 20, 30, 40, 50, 60 min), ultrasonic power (50, 60, 70, 80, 90, 100 W), and ultrasonic temperature (30, 40, 50, 60, 70, 80℃).

[0087] according to Figure 11 The results show that increased temperature facilitates solvent penetration into plant cell membranes, leading to increased polysaccharide extraction. Initially, the polysaccharide yield increases steadily, but the increase slows down after reaching approximately 70℃. Therefore, the ultrasonic temperature was controlled at 55℃. Extended time helps the alkali solution penetrate the cells, making chemical components easier to dissolve and thus increasing the polysaccharide yield; however, excessive time may promote degradation, leading to a decrease in yield. Therefore, an ultrasonic time of 40 minutes was chosen. When the ultrasonic power is between 50 and 80 W, the disruption of the cell wall by ultrasound intensifies with increasing power, increasing the intracellular polysaccharide extraction rate and gradually improving the extraction rate. However, above 80 W, the polysaccharide yield decreases with increasing power. This may be because excessively high-frequency ultrasonic radiation degrades the polysaccharides, leading to their destruction. Therefore, an ultrasonic power of 80 W was chosen as the optimal experimental power. Response surface methodology was used to optimize the ultrasonic time, temperature, and power.

[0088] By employing the central composite experimental design principle of Box-Behnken and using response surface methodology, response surface analysis (RSM) was conducted. Based on the analysis scheme and results, the DeSign-Expert software was used to generate RSM plots for different factors, and the optimal extraction process parameters for polysaccharide extraction were obtained through analysis and optimization. Figure 12 As shown, the optimal parameters for enzymatic hydrolysis were finally determined to be: ultrasonic time 45 min, ultrasonic power 81 W, and ultrasonic temperature 71 ℃.

[0089] 3. Component analysis

[0090] 3.1 Determination of polysaccharide content

[0091] ① Preparation of glucose standard solution

[0092] After drying 1g of glucose standard to constant weight, accurately weigh 0.15g and dissolve it in deionized water. Dilute to 100mL using a volumetric flask to obtain a 1.5mg / mL glucose standard solution.

[0093] ② Preparation of phenol solution

[0094] Weigh 5g of phenol quickly, dissolve it in deionized water, and bring the volume up to 100mL using a brown volumetric flask. Protect from light, sonicate for a few seconds, and mix thoroughly.

[0095] ③ Preparation of glucose standard curve

[0096] Prepare a glucose standard curve. Add reagents (such as...) to each tube... Figure 13 As shown, the concentrations of glucose standard solutions in each tube were 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, and 0.10 mg / mL, respectively. After shaking well, the solutions were placed at room temperature for 15 minutes. After cooling to room temperature, the absorbance of each solution at a wavelength of 490 nm was measured using a spectrophotometer.

[0097] ④ Determination of polysaccharide content in samples

[0098] Accurately pipette 1.0 mL of the diluted polysaccharide solution, add 1 mL of phenol solution and 5 mL of concentrated sulfuric acid, measure the absorbance values, and calculate the polysaccharide content in the solution based on the glucose standard curve.

[0099] The polysaccharide contents determined by the phenol-sulfuric acid method were 41.62%, 42.63%, 48.5%, and 52.17% respectively after ultrasonic-assisted water extraction (i.e., 1.1, ultrasonic-assisted polysaccharide extraction using deionized water), ultrasonic-assisted acid extraction (i.e., 1.1, ultrasonic-assisted polysaccharide extraction using 0.2 mol / L HCl), ultrasonic-assisted alkali extraction (i.e., 1.1, ultrasonic-assisted polysaccharide extraction using 0.2 mol / L NaOH), and biomimetic-microwave-assisted alkali extraction combined with ultrasonication. (Standard curve y = 5.24571x + 0.08821, R² = 0.9991, see...) Figure 13 ).

[0100] The specific method of biomimetic-microwave-assisted alkali extraction synergistic ultrasound is as follows:

[0101] Step 1, enzymatic hydrolysis: Add 5g of defatted pine nut powder to deionized water at a material-to-liquid ratio of 1:20 (g / mL), then add papain at a ratio of 2.29%, extract at pH 6.2 and 45℃ for 43.04 min, inactivate the enzyme by boiling in a water bath for 10 min, centrifuge at 7500 rpm for 10 min, and concentrate the collected supernatant at 50℃ to obtain material 1 and pine nut oil meal 1;

[0102] Step 2, microwave-assisted alkaline extraction: Add 0.6 mol / L NaOH to the pine kernel oil meal 1 at a material-to-liquid ratio of 1:32 (g / mL), extract with microwave at 400W for 50 seconds, centrifuge, and concentrate the collected supernatant at 50℃ to obtain material 2 and pine kernel oil meal 2.

[0103] Step 3, Ultrasonic-assisted extraction: Add 0.6 mol / L NaOH to the pine kernel oil meal 2 at a material-to-liquid ratio of 1:20 (g / mL), extract at 71℃ and 81W for 45 min, centrifuge, and concentrate the collected supernatant at 50℃ to obtain material 3 and pine kernel oil meal 3.

[0104] Step 4: Combine materials 1, 2, and 3 and concentrate to 40 mL. Add 4 times the volume of 95% ethanol and precipitate overnight at 4°C. Centrifuge at 6000 rpm / min for 15 min, discard the supernatant, and redissolve the precipitate in 80 mL of deionized water. Add an equal volume of 15% trichloroacetic acid (TCA), stir for 30 min, and incubate overnight at 4°C. Centrifuge and dialyze the supernatant for 48 h (24 h each with tap water and deionized water). Freeze at -20°C for 12 h. Place the frozen sample in a vacuum freeze dryer and vacuum dry for 36 h to obtain acidic pine polysaccharide.

[0105] 3.2 Protein content determination

[0106] ① Preparation of Coomassie Brilliant Blue G-250 solution

[0107] Accurately weigh 50 mg of Coomassie Brilliant Blue, dissolve it in 25 mL of ethanol solution (50%), then add an equal volume of phosphoric acid (85%), and dilute to 500 mL with deionized water. Store in the dark for later use.

[0108] ② Preparation of protein standard solutions

[0109] Accurately weigh 10 mg of bovine serum albumin, dissolve it in deionized water, and dilute to 100 mL using a volumetric flask to obtain a protein standard solution with a concentration of 0.1 mg / mL.

[0110] ③ Preparation of protein standard curve

[0111] Prepare a protein standard curve. Add reagents (such as...) to each tube... Figure 14 As shown, the protein standard solutions in each tube had concentrations of 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, and 0.10 mg / mL, respectively. After shaking, the solutions were allowed to stand at room temperature for 10 minutes, and the absorbance of each solution at a wavelength of 590 nm was measured using a spectrophotometer.

[0112] ④ Determination of protein content in samples

[0113] Accurately pipette 1.0 mL of polysaccharide solution and add 5 mL of Coomassie Brilliant Blue solution. Measure the absorbance of each tube using the method described above. Calculate the protein content in the solution based on the protein standard curve.

[0114] The protein contents determined by the Coomassie Brilliant Blue method after ultrasound-assisted water extraction, ultrasound-assisted acid extraction, ultrasound-assisted alkali extraction, and biomimetic-microwave-assisted alkali extraction combined with ultrasound were 0.79%, 0.54%, 1.75%, and 1.26%, respectively (standard curve y = 8.07714x + 0.01014, R² = 0.9994, see...). Figure 14 ).

[0115] 3.3 Determination of Tannin Content

[0116] ① Preparation of tannic acid standard solution

[0117] Dissolve and dilute 1g of tannic acid and bring the volume to 100mL in a volumetric flask to prepare a 10mg / mL standard stock solution. Accurately pipette 1mL of the stock solution and bring the volume to 100mL to prepare a 0.1mg / mL standard solution.

[0118] ② Preparation of saturated sodium carbonate solution

[0119] Quickly weigh out a sufficient amount of sodium carbonate powder, dissolve it in a certain amount of distilled water, and prepare a saturated sodium carbonate solution for later use.

[0120] ③ Preparation of tannic acid standard curve

[0121] Prepare a standard curve for tannic acid. (Each tube, e.g., ...) Figure 15 As shown, the tannic acid masses in each tube were 20 μg, 40 μg, 60 μg, 80 μg, and 100 μg, respectively. 6 mL of distilled water, 0.2 mL of tannic acid standard solution, 0.6 mL of 0.6 mL of 0.6 mL of 1 mL of distilled water, and 0.5 mL of Folin-Ciocalteu were added. After shaking each tube well, it was allowed to stand at room temperature for 6 min. Then, 2 mL of saturated sodium carbonate was added, and finally, the volume was adjusted to 10 mL with distilled water. The solution was then allowed to stand at room temperature in the dark for 30 min. The absorbance of each solution at 760 nm was measured using a spectrophotometer.

[0122] ④ Determination of tannin content in samples

[0123] Accurately pipette 1.0 mL of the diluted polysaccharide solution, add Folin-Ciocalteu solution and saturated sodium carbonate solution, measure the absorbance values, and calculate the tannin content in the solution based on the tannin standard curve.

[0124] The tannin content, determined by the Folin-Ciocalteu method, was 0.28%, 0.31%, 0.33%, and 0.61% after ultrasonic-assisted water extraction, ultrasonic-assisted acid extraction, ultrasonic-assisted acid extraction, and biomimetic-microwave-assisted alkali extraction combined with ultrasound, respectively (standard curve y = 0.0082x + 0.005, R² = 0.9994, see...). Figure 15 ).

[0125] 4. Infrared spectroscopy analysis

[0126] Take 2 mg of acidic pine polysaccharide obtained from the synergistic method (i.e., biomimetic-microwave-assisted alkali extraction synergistic ultrasound), mix it thoroughly with 100 mg of KBr powder, and press it into a thin sheet. Separately, weigh 100 mg of KBr powder, press it into a thin sheet as a blank control, and scan it in the range of 4000-400 cm⁻¹.

[0127] To investigate the effect of this extraction method on important functional groups of *Pinus thunbergii* polysaccharides, infrared spectroscopy analysis was performed on the extracted polysaccharides. Figure 16 The conclusion can be drawn from this: at 3384.4cm -1 There is a broad and strong absorption peak, which is the -OH stretching vibration peak; at 2927 cm⁻¹ -1 There is a relatively weak absorption peak at 1650 cm⁻¹, which is the absorption peak of the -CH stretching vibration; at 1650 cm⁻¹... -1 There is a sharp and strong absorption peak in between, which is the stretching vibration absorption peak of uronic acid; at 1546 cm⁻¹ -1 There is an absorption peak at 1408 cm⁻¹, which is the absorption peak of -C=C⁻; -1 The vibrations at these locations are CH-angled; 1153.2, 1079.9, and 1024.9 cm. -1 The absorption peak at 835.5 cm⁻¹ is caused by two CO stretching vibrations, one belonging to COH and the other to COC of the sugar ring. This characteristic suggests that the monosaccharide of pine polysaccharide exists in the form of pyranose. -1 The absorption peak at 792.6 cm⁻¹ is due to the α-glycosidic bond. -1 The absorption peak at that point is caused by the symmetric stretching vibration of the α-pyran ring.

[0128] 5. Tracking of in vitro immunomodulatory activity of polysaccharide fractions

[0129] (1) Culture of RAW264.7 cells

[0130] Frozen mouse macrophages were removed from the liquid nitrogen container and immediately placed in a 37°C water bath, then transferred to sterile centrifuge tubes. 10 mL of DMEM complete medium was added to the tubes, and the cells were gently mixed by pipetting. The cells were centrifuged at 1000 rpm for 5 min. After centrifugation, the supernatant was removed, and fresh medium was added. The cells were gently mixed by pipetting, then transferred to cell culture dishes. Finally, the dishes were placed in an incubator at 37°C and 5% CO2. Cell growth should be monitored daily, and the medium should be replaced regularly with fresh medium.

[0131] (2) RAW264.7 mouse macrophage MTT proliferation assay

[0132] Adjust cells to 1×10 5 Cells were seeded per well in a 96-well plate with 100 μL of cell suspension per well and cultured for 24 h. After complete cell adhesion, the cells were treated with a solution of each component polysaccharide (200 μg / mL) dissolved in DMEM at the same concentration. After 24 h, 10 mL of 5 mg / mL MTT solution was added to each well. After 4 h of culture, the cell supernatant was removed, and 50 μL of dimethyl sulfoxide (DMSO) was added for dissolution and crystal dissolution. The absorbance was then recorded at 490 nm.

[0133] (3) Neutral red phagocytosis experiment

[0134] Adjust cells to 1×10 5 Cells were seeded per well in a 96-well plate with 100 μL of cell suspension per well and cultured for 24 h. Once the cells were fully adhered, 200 μg / mL of each polysaccharide solution was added to the 96-well plate. After 20 h of culture, the supernatant was discarded, and the cells were washed three times with phosphate-buffered saline (PBS). 100 μL of 0.1% neutral red solution was added, and after 4 h, the supernatant was discarded, the cells were washed with PBS, and 200 μL of lysis buffer (ethanol:acetic acid = 1:1) was added. The mixture was stirred for 10 min, and the absorbance was measured at 540 nm.

[0135] Currently, lentinan is the most common acidic polysaccharide on the market. Therefore, this study used lentinan (LN) as a positive control and used ultrasound-assisted water extraction (WP), ultrasound-assisted acid extraction (CP), ultrasound-assisted alkali extraction (LP), and biomimetic-microwave assisted alkali extraction combined with ultrasound extraction (PSP) as experimental groups, all with the same concentration of 200 μg / mL. The results were verified by MTT and neutral red phagocytosis experiments (see [link to study]. Figures 17-18The immunomodulatory activity of acidic ginseng polysaccharide extracted by a biomimetic-microwave-assisted alkaline extraction combined with ultrasound method was significantly higher than that of the positive control, and its phagocytic capacity was comparable to that of lentinan. Based on the determination of polysaccharide, protein, and tannin content, the polysaccharide:protein:tannin ratio was found to be 41.62:0.79:0.28 in WP, 42.63:0.54:0.31 in CP, 48.5:1.75:0.33 in LP, and 52.17:1.26:0.61 in PSP. Therefore, it is inferred that this ratio is more effective in promoting immunomodulatory activity in PSP.

[0136] The application of the acidic pine polysaccharide PSP obtained in this embodiment in the preparation of drugs that promote immune activity.

[0137] Example 2

[0138] The only difference between this embodiment and Embodiment 1 is that:

[0139] Step 1, enzymatic hydrolysis: Add defatted pine nut powder to deionized water at a ratio of 1:30 g / mL, then add papain at a ratio equivalent to 0.5 wt% of deionized water, extract at pH 6 and 25℃ for 30 min, inactivate the enzyme, centrifuge, concentrate the supernatant to obtain material 1 and pine nut oil meal 1.

[0140] Step 2, microwave-assisted alkaline extraction: Add 0.2mol / L NaOH to the pine kernel oil meal 1 at a material-liquid ratio of 1:5g / mL, extract by microwave at 100W for 10s, centrifuge, concentrate the supernatant to obtain material 2 and pine kernel oil meal 2;

[0141] Step 3, Ultrasonic-assisted extraction: Add 0.4mol / L NaOH to the pine kernel oil meal 2 at a material-to-liquid ratio of 1:30g / mL, extract for 10min at 30℃ and ultrasonic power of 50W, centrifuge, concentrate the supernatant to obtain material 3 and pine kernel oil meal 3;

[0142] Step 4: Combine materials 1, 2, and 3 and concentrate to 3 times the weight of defatted pine nut powder to obtain a concentrated solution. Add 3 times the volume of 90% ethanol to precipitate overnight at 0°C. Centrifuge, discard the supernatant, and redissolve the precipitate in 5 times the weight of defatted pine nut powder in deionized water. Add an equal volume of 10% trichloroacetic acid (TCA) to the redissolved deionized water, stir for 40 minutes, place at 0°C overnight, centrifuge, dialyze the supernatant, and freeze-dry to obtain acidic pine polysaccharide.

[0143] In step one, the enzyme inactivation method is boiling water bath for 8 minutes, centrifugation at 6000 rpm for 5 minutes, and concentration temperature at 40℃; in steps two and three, the concentration temperature is 40℃. In step four, centrifugation is performed at 3000 rpm for 10 minutes; dialysis time is 60 hours, with dialysis using tap water and deionized water for 30 hours each, followed by freezing at -40℃ for 20 hours. After freezing, the sample is placed in a vacuum freeze dryer and dried under vacuum for 48 hours.

[0144] In this embodiment, the ratio of polysaccharide:protein:tannin in the acidic pine polysaccharide PSP is slightly different from that in Example 1.

[0145] Example 3

[0146] The only difference between this embodiment and Embodiment 1 is that:

[0147] Step 1, enzymatic hydrolysis: After adding defatted pine nut powder to deionized water at a material-to-liquid ratio of 1:40 g / mL, papain was added at a ratio equivalent to 3.0 wt% of deionized water. The mixture was extracted at pH 7 and 75℃ for 80 min, the enzyme was inactivated, the mixture was centrifuged, and the supernatant was concentrated to obtain material 1 and pine nut oil meal 1.

[0148] Step 2, microwave-assisted alkaline extraction: Add 1 mol / L NaOH to the pine kernel oil meal 1 at a material-liquid ratio of 1:160g / mL, extract by microwave at 700W for 110s, centrifuge, concentrate the supernatant to obtain material 2 and pine kernel oil meal 2.

[0149] Step 3, Ultrasonic-assisted extraction: Add 0.5mol / L NaOH to the pine kernel oil meal 2 at a material-to-liquid ratio of 1:40g / mL, extract at 80℃ and ultrasonic power of 100W for 60min, centrifuge, concentrate the supernatant to obtain material 3 and pine kernel oil meal 3;

[0150] Step 4: Combine materials 1, 2, and 3 and concentrate to 5 times the weight of defatted pine nut powder to obtain a concentrated solution. Add 5 times the volume of 95% ethanol to precipitate the solution overnight at 4°C. Centrifuge, discard the supernatant, and redissolve the precipitate in 10 times the weight of deionized water. Add an equal volume of 20% trichloroacetic acid (TCA) to the redissolved deionized water, stir for 60 minutes, and let stand overnight at 4°C. Centrifuge, dialyze the supernatant, and freeze-dry to obtain acidic pine polysaccharide.

[0151] The enzyme inactivation method was a boiling water bath for 12 minutes, centrifugation at 9000 rpm for 15 minutes, and concentration at 60℃; in steps two and three, the concentration temperature was 60℃. In step four, centrifugation was performed at 4500 rpm for 20 minutes; dialysis time was 50 hours, with dialysis using tap water and deionized water for 25 hours each; the sample was then frozen at -30℃ for 20 hours, and finally placed in a vacuum freeze dryer for 50 hours of vacuum drying.

[0152] In this embodiment, the ratio of polysaccharide:protein:tannin in the acidic pine polysaccharide PSP is slightly different from that in Example 1.

[0153] It should be understood that, in order to simplify this disclosure and aid in understanding one or more of the various aspects of the invention, features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof in the above description of exemplary embodiments of the invention. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than expressly recited in each claim. Rather, as reflected in the claims, inventive aspects lie in fewer than all the features of the foregoingly disclosed embodiments. Therefore, the claims, following the detailed description, are hereby expressly incorporated into that detailed description, wherein each claim itself is a separate embodiment of the invention.

[0154] Although the invention has been described with reference to a limited number of embodiments, those skilled in the art will understand from the foregoing description that other embodiments are conceivable within the scope of the invention described herein. Furthermore, it should be noted that the language used in this specification has been chosen primarily for readability and instructional purposes, and not for the purpose of interpreting or limiting the subject matter of the invention. Therefore, many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the appended claims. The disclosure of the invention is illustrative and not restrictive, and the scope of the invention is defined by the appended claims.

[0155] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for biomimetic-microwave-assisted alkaline extraction combined with ultrasonic extraction of acidic P. mongolicus polysaccharides, characterized in that, The method comprises the following steps: Step one, enzymolysis: defatted pine nut powder is added into deionized water at a ratio of 1:(20-40) g / mL, and papain is added at a ratio of 0.5-3.0 wt% of the deionized water, and then the mixture is extracted at 6-7 of pH and 25-75 DEG C for 30-80 min, and then the enzyme is inactivated, and then the mixture is centrifuged, and then the supernatant is concentrated to obtain material 1 and pine nut oil meal 1; Step two, microwave-assisted alkali extraction: the pine nut oil meal 1 is added into 0.2-1 mol / L NaOH at a ratio of 1:(5-160) g / mL, and then the mixture is subjected to microwave extraction at 100-700 W for 10-110 s, and then the mixture is centrifuged, and then the supernatant is concentrated to obtain material 2 and pine nut oil meal 2; Step three, ultrasonic-assisted extraction: the pine nut oil meal 2 is added into 0.4-0.6 mol / L NaOH at a ratio of 1:(20-40) g / mL, and then the mixture is extracted at 30-80 DEG C and under the condition of 50-100 W ultrasonic power for 10-60 min, and then the mixture is centrifuged, and then the supernatant is concentrated to obtain material 3 and pine nut oil meal 3; Step four, the material 1, the material 2 and the material 3 are combined and concentrated to 3-5 times the weight of the defatted pine nut powder to obtain a concentrated solution, and then 90-95% ethanol is added to the concentrated solution at a volume ratio of 3-5 to precipitate the acid-resistant pine polysaccharide at 0-4 DEG C overnight, and then the mixture is centrifuged, and then the supernatant is discarded, and then the precipitate is redissolved in deionized water at a weight ratio of 5-10 times the defatted pine nut powder, and then 10-20% trichloroacetic acid TCA is added in an amount equal to the volume of the redissolved deionized water, and then the mixture is stirred for at least 30 min, and then the mixture is placed at 0-4 DEG C overnight, and then the mixture is centrifuged, and then the supernatant is dialyzed and freeze-dried to obtain the acid-resistant pine polysaccharide.

2. The method of claim 1, wherein, The method comprises the following steps: Step one, enzymolysis: 5 g of defatted pine nut powder is added into deionized water at a ratio of 1:20 g / mL, and then papain is added at a ratio of 2.29 wt% of the deionized water, and then the mixture is extracted at 6.2 of pH and 45 DEG C for 43.04 min, and then the enzyme is inactivated, and then the mixture is centrifuged, and then the supernatant is concentrated to obtain material 1 and pine nut oil meal 1; Step two, microwave-assisted alkali extraction: the pine nut oil meal 1 is added into 0.6 mol / L NaOH at a ratio of 1:32 g / mL, and then the mixture is subjected to microwave extraction at 400 W for 50 s, and then the mixture is centrifuged, and then the supernatant is concentrated to obtain material 2 and pine nut oil meal 2; Step three, ultrasonic-assisted extraction: the pine nut oil meal 2 is added into 0.6 mol / L NaOH at a ratio of 1:20 g / mL, and then the mixture is extracted at 71 DEG C and under the condition of 81 W for 45 min, and then the mixture is centrifuged, and then the supernatant is concentrated to obtain material 3 and pine nut oil meal 3; Step four, the material 1, the material 2 and the material 3 are combined and concentrated to 40 mL to obtain a concentrated solution, and then 95% ethanol is added to the concentrated solution at a volume ratio of 4 to precipitate the acid-resistant pine polysaccharide at 4 DEG C overnight, and then the mixture is centrifuged, and then the supernatant is discarded, and then the precipitate is redissolved in 80 mL of deionized water, and then 80 mL of 15% trichloroacetic acid TCA is added, and then the mixture is stirred for 30 min, and then the mixture is placed at 4 DEG C overnight, and then the mixture is centrifuged, and then the supernatant is dialyzed and freeze-dried to obtain the acid-resistant pine polysaccharide.

3. The method according to claim 1 or 2, characterized in that, In step one, the enzyme activity of the papain is 200 u / mg.

4. The method of claim 1, wherein, In step one, the method of enzyme inactivation is boiling water bath for 8-12 min, centrifugation is 6000-9000 rpm for 5-15 min, and the concentration temperature is 40-60℃; in step two and step three, the concentration temperature is 40-60℃.

5. The method of claim 2, wherein, In step one, the method of enzyme inactivation is boiling water bath for 10 min, centrifugation is 7500 rpm for 10 min, and the concentration temperature is 50℃; in step two and step three, the concentration temperature is 50℃.

6. The method of claim 1, wherein, In step four, the centrifugation is 3000-6000 rpm for 10-20 min; the dialysis time is at least 48 h, tap water and deionized water are each dialyzed for at least 24 h, are placed in a refrigerator at least -20℃ for freezing for at least 12 h, the frozen sample is placed in a vacuum freeze dryer, and is vacuum dried for at least 36 h.

7. The method of claim 2, wherein, In step four, the centrifugation is 6000 rpm for 15 min; the dialysis time is 48 h, tap water and deionized water are each dialyzed for 24 h, are placed in a refrigerator at -20℃ for freezing for 12 h, the frozen sample is placed in a vacuum freeze dryer, and is vacuum dried for 36 h.

8. Acidic Pinus sylvestris polysaccharides obtained according to the method of claim 1, characterized in that, The monosaccharide of the acid P. mongolicum polysaccharide is in the form of pyranose; the glycosidic bond of the acid P. mongolicum polysaccharide is an α-glycosidic bond.

9. Acidic Pinus sylvestris polysaccharides obtained according to the method of claim 2, characterized in that, In the acid P. mongolicum polysaccharide, the mass ratio of polysaccharide: protein: tannin is 52.17: 1.26: 0.

61.

10. Use of the acid P. mongolicum polysaccharide according to claim 9 in the preparation of an immune activity promoting drug.

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