Method for improving extraction rate and antioxidant activity of angelica polysaccharide

Abstract

The invention discloses a method for improving the extraction rate and antioxidant activity of angelica polysaccharide. The method comprises the steps of raw material pretreatment and degreasing, electron beam irradiation, polysaccharide extraction and preliminary purification and polysaccharide refining. The radix angelicae sinensis powder is pretreated by using the ethanol and the surfactant, so that the permeability of cell walls is preliminarily changed while lipid and protein are removed; then, a polymer degradation accelerant is introduced, so that the polymer degradation accelerant and irradiation energy generate a synergistic effect under electron beam irradiation, hydrogen bonds and glucosidic bonds are efficiently broken, and the cell structure becomes loose and porous, so that the polysaccharide extraction rate is increased by 8.64%-62.13%, the extraction efficiency is remarkably improved, and the molecular weight is reduced by 46.61%; the molecular weight is reduced, the solubility is improved, and meanwhile, the core functional group and triple helix conformation of the polysaccharide are maintained, so that the antioxidant activity of the polysaccharide is remarkably enhanced instead of activity loss caused by simple degradation. According to the invention, the integration of extraction, modification and purification of angelica sinensis is realized, and the product purity and biological activity are synchronously improved.

Description

Technical Field

[0001] This invention belongs to the field of natural drug extraction technology, specifically relating to a method for improving the extraction rate and antioxidant activity of Angelica polysaccharides. Background Technology

[0002] Angelica sinensis, a traditional Chinese medicine, is the dried root of the Angelica sinensis plant (family Apiaceae). As a widely used traditional Chinese medicine, Angelica sinensis was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and has a medicinal history of over 2000 years. It is mainly cultivated in Gansu, Yunnan, Sichuan, and Shaanxi provinces of my country, with the highest quality product coming from Minxian County, Gansu Province. Angelica sinensis has a sweet and pungent taste and is warm in nature, possessing the effects of nourishing blood, promoting blood circulation, regulating menstruation, relieving pain, and moistening the intestines to relieve constipation. Furthermore, as a typical representative of medicinal and edible plants, Angelica sinensis is widely used in health foods, cosmetics, and dietary supplements in Asia, Europe, and the Americas. With increasing health awareness, products containing active ingredients from medicinal and edible plants are gradually becoming a preferred choice in the health and wellness consumer market.

[0003] The main active components of Angelica sinensis include volatile oils, organic acids, and polysaccharides. Among these components, polysaccharides are one of the most effective bioactive components, possessing good safety and bioactivity. Angelica sinensis polysaccharides have become a new option for natural antioxidant and anti-aging drugs by reducing oxidative damage to cells, increasing the activity of antioxidant enzymes, and inhibiting the production of reactive oxygen species. The wide molecular weight distribution and high proportion of high molecular weight components of Angelica sinensis polysaccharides not only result in poor water solubility but also, in conjunction with the intestinal absorption barrier, limit their efficiency in transmembrane transport and intracellular function. The bioactivity of polysaccharides is closely related to their structural characteristics; therefore, to enhance their function, the structure of natural polysaccharides must be modified. Polysaccharide modification methods mainly include biological (enzymatic) degradation, physical degradation, and chemical modification. Biological degradation reagents are expensive and inefficient, making them unsuitable for large-scale production. Chemical methods bring environmental challenges and the potential problem of chemical residues. Therefore, there is an urgent need for an environmentally friendly, simple, economical, and rapid method for modifying Angelica sinensis polysaccharides. Summary of the Invention

[0004] The purpose of this invention is to provide a method for improving the extraction rate and antioxidant activity of Angelica polysaccharides.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for improving the extraction rate and antioxidant activity of Angelica sinensis polysaccharides, comprising the following steps: (1) Raw material pretreatment and degreasing: The dried Angelica sinensis slices were crushed and sieved. The resulting Angelica sinensis powder was added to an ethanol solution and a surfactant for heating and reflux treatment, filtration and drying to obtain pretreated Angelica sinensis powder. (2) Electron beam irradiation: Add a polymer degradation promoter to the pretreated Angelica sinensis powder, mix evenly, put it into a sealed bag, and perform electron beam irradiation pretreatment to obtain irradiated Angelica sinensis powder; (3) Polysaccharide extraction and preliminary purification: Irradiated Angelica sinensis powder was mixed with water, and the pH was adjusted to 3-5 with acid. After ultrasonic extraction or heating extraction, Angelica sinensis polysaccharide extract was obtained. After the Angelica sinensis polysaccharide extract was concentrated by vacuum distillation, anhydrous ethanol was added for alcohol precipitation, centrifugation, collection of precipitate and freeze-drying to obtain crude Angelica sinensis polysaccharide. (4) Polysaccharide refining: Dissolve the crude polysaccharide of Angelica sinensis in water, and then obtain refined Angelica sinensis polysaccharide by dialysis, vacuum concentration and freeze drying.

[0006] Further, in step (1), the sieving involves passing the pulverized Angelica powder through a 40-60 mesh sieve.

[0007] Further, in step (1), the volume fraction of the ethanol solution is 85% to 95%.

[0008] Further, in step (1), the mass-to-volume ratio of the Angelica sinensis powder and the ethanol solution is 1g:8-16mL.

[0009] Further, in step (1), the surfactant is at least one of sodium dodecylbenzenesulfonate, sodium fatty alcohol polyoxyethylene ether sulfate, cocamidopropyl betaine, alkyl glycoside, polyoxyethylene fatty alcohol ether, methyl castor oil ethoxylate, and fatty alcohol polyoxyethylene ether.

[0010] Further, in step (1), the mass ratio of the Angelica sinensis powder to the surfactant is 1:(0.01~0.08).

[0011] Further, in step (1), the temperature of the heating reflux treatment is 60-80°C, each time for 2 hours, and repeated 2-3 times.

[0012] Furthermore, in step (1), the drying temperature is 45-55℃.

[0013] Further, in step (2), the polymer degradation promoter is a 0.5-2 wt% urea or sodium persulfate solution, and the amount of polymer degradation promoter is 5-10% of the mass of the pretreated Angelica sinensis powder.

[0014] Furthermore, in step (2), the irradiation energy of the electron beam irradiation pretreatment is 5 to 10 MeV. When irradiating one side, the thickness of the Angelica sinensis powder does not exceed 4 cm, and when irradiating both sides, the thickness of the Angelica sinensis powder does not exceed 8 cm.

[0015] Furthermore, in step (2), the irradiation dose of the electron beam irradiation pretreatment is 10 to 80 kGy.

[0016] Furthermore, in step (3), the mass-to-volume ratio of irradiated Angelica sinensis powder to water is 1g:10-20mL.

[0017] Furthermore, in step (3), the acid is one or more of hydrochloric acid, citric acid, and acetic acid.

[0018] Furthermore, in step (3), the ultrasonic extraction power is 300-500W, the temperature is 60-80℃, and the extraction time is 30-60min.

[0019] Furthermore, in step (3), the heating extraction temperature is 70-80℃ and the extraction time is 2-3h.

[0020] Furthermore, in step (4), dialysis is performed using a dialysis bag with a capacity of 3500-10000 Da.

[0021] Furthermore, in step (4), the freeze-drying conditions are: temperature -80 to -60°C, vacuum degree 0.75 to 1.2 mbar.

[0022] Compared with the prior art, the beneficial technical effects of the present invention include: 1. This invention utilizes ethanol and surfactants to pretreat Angelica sinensis powder, removing lipids and proteins while initially altering cell wall permeability. Subsequently, a high-molecular-weight degradation promoter is introduced, which, under electron beam irradiation, produces a synergistic effect with the irradiation energy, efficiently breaking hydrogen bonds and glycosidic bonds, making the cell structure loose and porous. This increases the polysaccharide extraction rate by 8.64%-62.13%, significantly improving extraction efficiency and reducing molecular weight by 46.61%. While reducing molecular weight and improving solubility, the core functional groups and triple helix conformation of the polysaccharides are maintained, thereby significantly enhancing their antioxidant activity rather than the loss of activity caused by simple degradation.

[0023] 2. This invention integrates the extraction, modification, and purification of Angelica sinensis, simultaneously improving product purity and bioactivity, ultimately yielding a high-purity product with a polysaccharide content exceeding 70%. The entire process avoids the use of toxic chemical reagents, relying on physical processes and biodegradable additives, making it environmentally friendly. The electron beam irradiation treatment is rapid, uniform, and easy to control, providing a reliable industrialization path for developing high-value-added Angelica sinensis polysaccharide products. Attached Figure Description

[0024] Figure 1 The results of scanning electron microscopy analysis of Angelica sinensis powder from Examples 1-5 and the comparative examples are shown. Figure 2 The results show the detection of protein content in the Angelica polysaccharides prepared in Examples 1-5 and the comparative examples. Figure 3The infrared spectral analysis results are for the Angelica polysaccharides prepared in Examples 1-5 and the comparative examples. Figure 4 The results of the triple helix structure analysis of the Angelica polysaccharides prepared in Examples 1-5 and the comparative examples are shown. Figure 5 The results show the solubility of Angelica polysaccharides prepared in Examples 1-5 and the comparative examples. Figure 6 The results show the antioxidant activity of Angelica polysaccharides prepared in Examples 1-5 and the comparative examples. Detailed Implementation

[0025] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings: Example 1 (1) Raw material pretreatment and degreasing Weigh dried Angelica sinensis slices, pulverize them, and pass them through a 60-mesh sieve. Add the obtained Angelica sinensis powder to a 90% ethanol solution at a material-to-liquid ratio of 1g:10mL, and add sodium dodecylbenzenesulfonate, with a mass ratio of 1:0.02 to the Angelica sinensis powder. Heat and reflux at 80℃ for 2 hours, repeating the process twice to fully remove fat-soluble components and some proteins. Then filter, collect the filter residue, and dry it in a 50℃ oven for 24 hours to obtain defatted and deproteinized pretreated Angelica sinensis powder.

[0026] (2) Electron beam irradiation Add 5% (by weight) of 0.5wt% urea solution to the pretreated Angelica sinensis powder sample as a polymer degradation promoter, mix well, pack the treated powder into a sealed packaging bag, spread it evenly, and control the thickness to 4 cm; irradiate one side under normal temperature and pressure using a high-energy electron accelerator with an energy of 10 MeV and a power of 20kW, and set the irradiated absorbed dose to 10.0 kGy to obtain irradiated Angelica sinensis powder.

[0027] (3) Polysaccharide extraction and preliminary purification Irradiated Angelica sinensis powder was mixed with ultrapure water at a material-to-liquid ratio of 1g:10mL, and the pH of the solution was adjusted to 3.5 with citric acid. Then, ultrasonic-assisted extraction was performed at a power of 400W and a frequency of 40kHz, with the temperature controlled at 75℃ and the extraction time at 40min. After extraction, the extract was collected and concentrated to about one-third of the original volume by vacuum distillation. Four times the volume of anhydrous ethanol was added to the concentrate for alcohol precipitation. The concentrate was allowed to stand at 4℃ for 12h, and the precipitate was collected by centrifugation. The precipitate was then freeze-dried at -80℃ and a vacuum of 0.9 mbar to obtain crude Angelica sinensis polysaccharide.

[0028] (4) Polysaccharide refining The crude polysaccharide of Angelica sinensis was redissolved in ultrapure water and injected into a dialysis bag with a molecular weight cutoff of 3500 Da. The solution was dialyzed in deionized water for 48 hours. After dialysis, the retentate in the dialysis bag was collected, concentrated under reduced pressure, and then freeze-dried again at a temperature of -80℃ and a vacuum degree of 0.9 mbar to finally obtain the refined Angelica sinensis polysaccharide product, named EAP-10.

[0029] According to the test results, the extraction rate of Angelica polysaccharide EAP-10 prepared in this embodiment was 10.90%, the polysaccharide content was 72.74%, and the weight-average molecular weight of the polysaccharide was 680.10 kDa.

[0030] Example 2 (1) Raw material pretreatment and degreasing Weigh dried Angelica sinensis slices, pulverize them, and pass them through a 50-mesh sieve. Take this Angelica sinensis powder and add a 95% ethanol solution at a material-to-liquid ratio of 1g:10mL, and add sodium dodecylbenzenesulfonate, with a mass ratio of 1:0.04 to the Angelica sinensis powder. Heat and reflux at 80℃ for 2.0h, and repeat the process twice. Then filter, collect the filter residue, and dry it in a 50℃ oven for 24h to obtain defatted and deproteinized pretreated Angelica sinensis powder.

[0031] (2) Electron beam irradiation pretreatment: Add 6% (by weight) of 0.8 wt% sodium persulfate solution to the pretreated Angelica sinensis powder sample and mix thoroughly. Pack the treated powder into a sealed packaging bag, spread it evenly, control the thickness to 4 cm, and irradiate it on one side using a high-energy electron accelerator with an energy of 10 MeV and a power of 20 kW at room temperature and pressure. Set the irradiated absorbed dose to 20.0 kGy to obtain irradiated Angelica sinensis powder.

[0032] (3) Polysaccharide extraction and preliminary purification Irradiated Angelica sinensis powder was mixed with ultrapure water at a material-to-liquid ratio of 1g:20mL, and the pH of the solution was adjusted to 3.5 using citric acid. Subsequently, ultrasonic-assisted extraction was performed at a power of 450W and a frequency of 40kHz, with the temperature controlled at 75℃ and the extraction time at 40min. After extraction, the extract was collected and concentrated to about one-third of the original volume by vacuum distillation. Four times the volume of anhydrous ethanol was added to the concentrate for alcohol precipitation, and the mixture was allowed to stand at 4℃ for 12h. The precipitate was collected by centrifugation and freeze-dried at -80℃ and a vacuum of 0.9mbar to obtain crude Angelica sinensis polysaccharide.

[0033] (4) Polysaccharide refining The crude Angelica polysaccharide was redissolved in ultrapure water and injected into a dialysis bag with a molecular weight cutoff of 5000 Da. Dialysis was performed in flowing deionized water for 48 hours. After dialysis, the retentate in the dialysis bag was collected, concentrated under reduced pressure, and then freeze-dried again at a temperature of -80℃ and a vacuum of 0.9 mbar to finally obtain the refined Angelica polysaccharide product, named EAP-20.

[0034] The extraction rate of Angelica sinensis polysaccharide EAP-20 prepared in this embodiment was 12.13%, the polysaccharide content was 76.88%, and the weight-average molecular weight of the polysaccharide was 640.80 kDa. Compared with Example 1, under a higher irradiation dose, the extraction rate and content of polysaccharides were further improved, and the molecular weight was further reduced, demonstrating the controllable regulation effect of irradiation dose on the yield and structure of polysaccharides.

[0035] Example 3 (1) Raw material pretreatment and degreasing Weigh dried Angelica sinensis slices, pulverize them, and pass them through a 50-mesh sieve. Take this Angelica sinensis powder and add a 95% ethanol solution at a material-to-liquid ratio of 1g:15mL. Add sodium polyoxyethylene ether sulfate as a surfactant, with a mass ratio of 1:0.03 to the Angelica sinensis powder. Heat and reflux at 80℃ for 2.0h, and repeat the process twice. Then filter, collect the filter residue, and dry it in a 50℃ oven for 24h to obtain defatted and deproteinized pretreated Angelica sinensis powder.

[0036] (2) Electron beam irradiation pretreatment Add 7% by weight of 1wt% urea solution as a polymer degradation promoter to the pretreated Angelica sinensis powder sample obtained in step (1), and mix evenly; put the treated powder into a sealed packaging bag, spread it evenly, and control the thickness to 6cm; under normal temperature and pressure, use a high-energy electron accelerator with an energy of 10MeV and a power of 20 kW to perform double-sided irradiation treatment, and set the irradiation absorbed dose to 40.0kGy to obtain irradiated Angelica sinensis powder.

[0037] (3) Polysaccharide extraction and preliminary purification Irradiated Angelica sinensis powder was mixed with ultrapure water at a material-to-liquid ratio of 1g:20mL, and the pH of the solution was adjusted to 3.5 with acetic acid. Then, ultrasonic-assisted extraction was performed at a power of 350W and a frequency of 40kHz, with the temperature controlled at 75℃ and the extraction time at 40min. After extraction, the extract was collected and concentrated to about one-third of the original volume by vacuum distillation. Four times the volume of anhydrous ethanol was added to the concentrate for alcohol precipitation, and the mixture was allowed to stand at 4℃ for 12h. The precipitate was collected by centrifugation and freeze-dried at -80℃ and a vacuum of 0.9mbar to obtain crude Angelica sinensis polysaccharide.

[0038] (4) Polysaccharide refining The crude Angelica polysaccharide was redissolved in ultrapure water and injected into a dialysis bag with a molecular weight cutoff of 3500 Da. The solution was dialyzed in flowing deionized water for 48 hours. After dialysis, the retentate in the dialysis bag was collected, concentrated under reduced pressure, and then freeze-dried again at a temperature of -80℃ and a vacuum degree of 0.9 mbar to finally obtain the refined Angelica polysaccharide product, named EAP-40.

[0039] The extraction rate of Angelica sinensis polysaccharide EAP-40 prepared in this embodiment was 13.20%, the polysaccharide content was 77.34%, and the weight-average molecular weight of the polysaccharide was 496.10 kDa. The results indicate that under moderate-high dose irradiation of 40 kGy, the molecular weight of the polysaccharide significantly decreased, and the extraction rate was further improved.

[0040] Example 4 (1) Raw material pretreatment and degreasing Weigh dried Angelica sinensis slices, pulverize them, and pass them through a 50-mesh sieve. Take this Angelica sinensis powder and add an 85% ethanol solution at a material-to-liquid ratio of 1g:10mL. Add cocamidopropyl betaine as a surfactant, with a mass ratio of 1:0.05 between the surfactant and the Angelica sinensis powder. Heat and reflux at 80℃ for 2.0h, and repeat the process twice. Then filter, collect the filter residue, and dry it in a 50℃ oven for 24h to obtain defatted and deproteinized pretreated Angelica sinensis powder.

[0041] (2) Electron beam irradiation pretreatment Add 10% of the pretreated Angelica sinensis powder sample obtained in step (1) to a 2wt% sodium persulfate solution as a polymer degradation promoter and mix evenly. Pack the treated powder into a sealed packaging bag, spread it evenly, control the thickness to 4cm, and irradiate it on one side at room temperature and pressure using a high-energy electron accelerator with an energy of 10MeV and a power of 20kW. Set the irradiation absorbed dose to 60.0kGy to obtain irradiated Angelica sinensis powder.

[0042] (3) Polysaccharide extraction and preliminary purification Irradiated Angelica sinensis powder was mixed with ultrapure water at a material-to-liquid ratio of 1g:20mL, and the pH of the solution was adjusted to 3.5 with citric acid. Then, ultrasonic-assisted extraction was performed at a power of 400W and a frequency of 40kHz, with the temperature controlled at 80℃ and the extraction time being 40min. After extraction, the extract was collected and concentrated to about one-third of the original volume by vacuum distillation. Four times the volume of anhydrous ethanol was added to the concentrate for alcohol precipitation, and the mixture was allowed to stand at 4℃ for 12h. The precipitate was collected by centrifugation and freeze-dried at a temperature of -80℃ and a vacuum degree of 0.9mbar to obtain crude Angelica sinensis polysaccharide.

[0043] (4) Polysaccharide refining The crude Angelica polysaccharide was redissolved in ultrapure water and injected into a dialysis bag with a molecular weight cutoff of 6000 Da. Dialysis was performed in flowing deionized water for 48 hours. After dialysis, the retentate in the dialysis bag was collected, concentrated under reduced pressure, and then freeze-dried again at a temperature of -80℃ and a vacuum degree of 0.9 mbar to finally obtain the refined Angelica polysaccharide product, named EAP-60.

[0044] The extraction rate of Angelica sinensis polysaccharide EAP-60 prepared in this embodiment was 15.76%, the polysaccharide content was 78.31%, and the weight-average molecular weight of the polysaccharide was 475.60 kDa. The results indicate that high-dose irradiation significantly increases the extraction rate and further reduces the molecular weight, demonstrating the advantages of this method in efficient extraction and molecular weight control.

[0045] Example 5 (1) Raw material pretreatment and degreasing Weigh dried Angelica sinensis slices, pulverize them, and pass them through a 50-mesh sieve. Take this Angelica sinensis powder and add an 85% ethanol solution at a material-to-liquid ratio of 1g:12mL. Add polyoxyethylene fatty alcohol ether as a surfactant, with a mass ratio of 1:0.06 to the Angelica sinensis powder. Heat and reflux at 80℃ for 2.0h, and repeat the process twice. Then filter, collect the filter residue, and dry it in an oven at 45℃ for 24h to obtain defatted and deproteinized pretreated Angelica sinensis powder.

[0046] (2) Electron beam irradiation pretreatment Add 8% of the mass of 1.5wt% urea solution as a polymer degradation promoter to the pretreated Angelica sinensis powder sample obtained in step (1), and mix evenly; put the treated powder into a sealed packaging bag, spread it evenly, and control the thickness to 4cm; under normal temperature and pressure, use a high-energy electron accelerator with an energy of 10MeV and a power of 20kW to perform single-sided irradiation treatment, and set the irradiation absorbed dose to 80.0kGy to obtain irradiated Angelica sinensis powder.

[0047] (3) Polysaccharide extraction and preliminary purification Irradiated Angelica sinensis powder was mixed with ultrapure water at a material-to-liquid ratio of 1g:15mL, and the pH of the solution was adjusted to 3.0 with citric acid. Then, ultrasonic-assisted extraction was performed at a power of 400W and a frequency of 40kHz, with the temperature controlled at 80℃ and the extraction time being 40min. After extraction, the extract was collected and concentrated to about one-third of the original volume by vacuum distillation. Four times the volume of anhydrous ethanol was added to the concentrate for alcohol precipitation, and the mixture was allowed to stand at 4℃ for 12h. The precipitate was collected by centrifugation and freeze-dried at a temperature of -80℃ and a vacuum degree of 0.9mbar to obtain crude Angelica sinensis polysaccharide.

[0048] (4) Polysaccharide refining The crude Angelica polysaccharide was redissolved in ultrapure water and injected into a dialysis bag with a molecular weight cutoff of 4000 Da. Dialysis was performed in flowing deionized water for 48 hours. After dialysis, the retentate in the dialysis bag was collected, concentrated under reduced pressure, and then freeze-dried again at a temperature of -80℃ and a vacuum of 0.9 mbar to finally obtain the refined Angelica polysaccharide product, named EAP-80.

[0049] Testing revealed that the extraction rate of Angelica sinensis polysaccharide EAP-80 prepared in this embodiment was 16.27%, the polysaccharide content was 77.34%, and the weight-average molecular weight of the polysaccharide was 374.90 kDa. Under the highest irradiation dose in this series of embodiments, the highest extraction rate and lowest molecular weight were obtained, fully demonstrating the superior effect of electron beam irradiation in improving yield and precisely controlling the molecular weight of polysaccharides.

[0050] Comparative Example Step (2) is excluded, and the rest is the same as in Example 1, to obtain refined Angelica polysaccharide ASP.

[0051] The extraction rate of Angelica polysaccharides was 10.03%, the polysaccharide content was 72.05%, and the molecular weight of the polysaccharides was 702.20 kDa.

[0052] Compared with the comparative examples, Examples 1-5 used electron beams with doses of 10kGy-80kGy to irradiate Angelica sinensis powder, which could precisely control and reduce the molecular weight of Angelica sinensis polysaccharides. Moreover, with the increase of irradiation dose, the extraction rate of Angelica sinensis polysaccharides increased by 8.64-62.13%.

[0053] Experimental Example The Angelica polysaccharides prepared in Examples 1-5 and the comparative examples were subjected to scanning electron microscopy, Congo red assay, infrared spectroscopy, and immunomodulation analysis. The relevant methods are as follows: (1) Scanning electron microscopy analysis The Angelica powder treated by step (2) was placed on a sample holder and fixed to an aluminum plate with conductive tape. It was then plated with gold under high vacuum conditions. The microstructure of the sample was observed using a scanning electron microscope (Nano-SEM 450, FEI Company, Hillsboro, OR, USA), and the image was magnified 5000X for imaging.

[0054] Scanning electron microscopy analysis results are as follows Figure 1 As shown, the unirradiated Angelica sinensis powder in the comparative example (0 kGy) exhibits a polyhedral irregular morphology with a relatively smooth and flat surface. In Examples 1-5 (10 kGy → 80 kGy), the irradiated Angelica sinensis powder showed a significantly increased surface roughness and the appearance of obvious pores and cracks. This structural change indicates that EBI disrupts cell wall integrity, reduces particle size, and increases porosity and solvent permeability.

[0055] (2) Protein content detection Accurately weigh 25 mg of Coomassie Brilliant Blue and dissolve it in 25 mL of 95% ethanol, stirring until completely dissolved. Then, add 25.0 mL of 85% phosphoric acid dropwise while stirring to form an acidic colorimetric system. Transfer the solution to a 250 mL volumetric flask and dilute to volume with ultrapure water. Store the Coomassie Brilliant Blue solution at 4°C protected from light.

[0056] Weigh 2 mg of bovine serum albumin and dissolve it in ultrapure water in a 10 mL volumetric flask to obtain a BSA standard solution with a concentration of 0.2 mg / mL. Transfer 0, 200, 400, 600, 800, and 1000 μL of the stock solution to 1.5 mL centrifuge tubes, respectively, and add ultrapure water to a final volume of 1 mL to obtain a six-concentration gradient system solution of 0-200 μg / mL.

[0057] Add 200 μL of gradient standard solution to a 1.5 mL centrifuge tube, and finally add 1.0 mL of Coomassie Brilliant Blue solution. Mix well, centrifuge, and incubate in the dark for 15 min. Add 200 µL of the reaction solution to a 96-well plate and measure the absorbance using a microplate reader (540 nm). Plot a standard curve with BSA concentration X (mg / mL) on the x-axis and absorbance Y on the y-axis.

[0058] Protein content test results as follows Figure 2 As shown, the protein content gradually decreases with increasing irradiation dose, demonstrating the advantages of this method in efficiently extracting and reducing protein content.

[0059] (3) Infrared spectroscopy analysis 2.0 mg of polysaccharide sample was mixed with 300 mg of potassium bromide and pressed into thin sheets. The mixture was analyzed using an FT-IR spectrometer (Nicolet™ 470, Thermo Fisher Scientific, USA) with a scanning range of 4000 cm⁻¹. -1 -400cm -1 .

[0060] Infrared spectroscopy analysis results as follows Figure 3 As shown, the spectra of Angelica polysaccharides prepared in Examples 1-5 and Example 1 show a high degree of similarity in the positions of the main characteristic absorption peaks, confirming that the types of core functional groups of Angelica polysaccharides prepared in this invention have not changed.

[0061] (4) Analysis of Congo Red Experiment Polysaccharide sample solutions with a mass concentration of 0.5 mg / mL, Congo red solutions with a mass concentration of 80 μg / mL, and NaOH solutions of different concentrations (0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol·L⁻¹) were prepared using ultrapure water. -1Then, the polysaccharide solution, Congo red solution, and NaOH solution were mixed in a volume ratio of 1:1:1. The mixture was thoroughly mixed and allowed to stand at room temperature for 10 minutes. The maximum absorption wavelength in the range of 400-600 nm was measured using a UV spectrophotometer.

[0062] The results of the Congo Red test analysis are as follows: Figure 4 As shown, the complex formed by Angelica polysaccharide and Congo red has a λ max All values ​​were significantly higher than the Congo red background value, confirming that the Angelica polysaccharides prepared in this invention can maintain the triple helix conformation.

[0063] (5) Solubility test Accurately weigh 120.0 mg of sample into a 2 mL centrifuge tube, add 1.0 mL of ultrapure water, and dissolve intermittently at room temperature, vortexing for 10 s every 5 min for 2 h. Stir at 12000 r·min⁻¹. -1 Centrifuge for 10 min, accurately transfer 0.5 mL of the supernatant and freeze-dry at -50℃ for 6 h, weigh the dried residue, and characterize the solubility as the mass of polysaccharide dissolved in each mL of distilled water.

[0064] Solubility test results as follows Figure 5 As shown, the solubility of irradiated Angelica polysaccharides in Examples 1-5 showed a significant dose-dependent increase (P<0.05), with a solubility increase of 6.62%-20.61% compared to unirradiated Angelica polysaccharides in the comparative examples.

[0065] (6) Antioxidant activity DPPH free radical scavenging activity: Precisely prepare DPPH ethanol solution (0.25 mg / mL). -1 Store at 4°C, protected from light, for later use. The initial concentration was 0.5 mg / mL. -1 Angelica polysaccharide solutions were prepared in the range of 0.5-2.5 mg / mL through a series of dilutions. -1 Concentration gradient. Take 2.0 mL of sample solution and mix with an equal volume of DPPH solution, shake well, and allow to react at room temperature in the dark for 30 min. Measure the absorbance value A1 at 517 nm. The DPPH free radical scavenging rate is calculated as follows, where A0, A1, and A2 are the absorbances of the control group (sample without DPPH solution), the sample, and the sample without DPPH solution, respectively: ABTS radical scavenging activity was detected using a kit (BC4775, Beijing Solarbio Science & Technology Co., Ltd.). 10 μL of sample extract and ABTS working solution were added to each well of a 96-well plate, mixed thoroughly, and reacted at room temperature in the dark for 6 min. The absorbance (A1) was measured at 405 nm. The ABTS radical scavenging activity was calculated using the following formula, where A0, A1, and A2 are the absorbances of the control group (ABTS solution without sample), the sample, and the sample without ABTS solution, respectively: Hydroxyl radical scavenging activity: This was performed using a kit (BC1325, Beijing Solarbio Science & Technology Co., Ltd.). 300 μL of working solution was added to a microcentrifuge tube, followed by 50 μL of the sample solution to be tested, and the mixture was thoroughly mixed. The mixture was then incubated in a 37℃ water bath for 60 min. After the reaction, 200 μL of the supernatant was transferred to a 96-well plate, and the absorbance (A1) was measured at 536 nm using a microplate reader. The hydroxyl radical scavenging activity was calculated using the following formula, where A0 and A2 represent the blank group (ultrapure water instead of the sample solution) and the control group (ultrapure water instead of reagent 4), respectively.

[0066] Total antioxidant capacity (T-AOC): This was determined using a kit (BC1315, Beijing Solarbio Science & Technology Co., Ltd.). 180 μL of working solution, 6 μL of sample extraction buffer, and 18 μL of deionized water were added to a 96-well plate. After thorough mixing, the mixture was reacted at room temperature for 10 min, and the absorbance was measured at 593 nm. (The last sentence appears to be incomplete and possibly refers to a different topic.) 2+ Final concentration of standard solution (X, μmol·mL) -1 Plot a standard curve with absorbance (Y) as the x-axis and absorbance (Y) as the y-axis, and calculate the sample concentration.

[0067] Total antioxidant activity is based on the formula T-AOC (μmol·g) -1 ) Calculate the total antioxidant capacity of the sample (μmol·g) -1 ), where X is the sample concentration (μmol·mL). -1 W represents the sample mass (g).

[0068] Antioxidant activity test results are as follows Figure 6 As shown, the antioxidant activity of the Angelica polysaccharide prepared by the method of the present invention was significantly increased compared with the unirradiated group (0 kGy) (P<0.05).

[0069] The above description is a preferred embodiment of the present invention, used to explain the technical solution of the present invention, and is not intended to limit the present invention. Those skilled in the art can make conventional modifications, equivalent substitutions and improvements within the spirit and principles of the present invention, all of which are still included within the protection scope of the present invention.

Claims

1. A method for improving the extraction rate and antioxidant activity of Angelica sinensis polysaccharides, characterized in that, Includes the following steps: (1) Raw material pretreatment and degreasing: The dried Angelica sinensis slices were crushed and sieved. The resulting Angelica sinensis powder was added to an ethanol solution and a surfactant for heating and reflux treatment, filtration and drying to obtain pretreated Angelica sinensis powder. (2) Electron beam irradiation: Add a polymer degradation promoter to the pretreated Angelica sinensis powder, mix evenly, put it into a sealed bag, and perform electron beam irradiation pretreatment to obtain irradiated Angelica sinensis powder; (3) Polysaccharide extraction and preliminary purification: Irradiated Angelica sinensis powder was mixed with water, and the pH was adjusted to 3-5 with acid. After ultrasonic extraction or heating extraction, Angelica sinensis polysaccharide extract was obtained. After the Angelica sinensis polysaccharide extract was concentrated by vacuum distillation, anhydrous ethanol was added for alcohol precipitation, centrifugation, collection of precipitate and freeze-drying to obtain crude Angelica sinensis polysaccharide. (4) Polysaccharide refining: Dissolve the crude polysaccharide of Angelica sinensis in water, and then obtain refined Angelica sinensis polysaccharide by dialysis, vacuum concentration and freeze drying.

2. The method for improving the extraction rate and antioxidant activity of Angelica sinensis polysaccharides according to claim 1, characterized in that... In step (1), the sieving involves passing the pulverized Angelica powder through a 40-60 mesh sieve, the volume fraction of the ethanol solution is 85%-95%, and the mass-volume ratio of the Angelica powder to the ethanol solution is 1g:8-16mL.

3. The method for improving the extraction rate and antioxidant activity of Angelica sinensis polysaccharides according to claim 1, characterized in that... In step (1), the surfactant is at least one of sodium dodecylbenzenesulfonate, sodium fatty alcohol polyoxyethylene ether sulfate, cocamidopropyl betaine, alkyl glycoside, polyoxyethylene fatty alcohol ether, methyl ricinoleate ethoxylate, and fatty alcohol polyoxyethylene ether; the mass ratio of the angelica powder to the surfactant is 1:(0.01-0.08).

4. The method for improving the extraction rate and antioxidant activity of Angelica sinensis polysaccharides according to claim 1, characterized in that... In step (1), the temperature of the heating reflux treatment is 60-80℃, each time for 2 hours, and repeated 2-3 times; the drying temperature is 45-55℃.

5. The method for improving the extraction rate and antioxidant activity of Angelica sinensis polysaccharides according to claim 1, characterized in that... In step (2), the polymer degradation promoter is a 0.5-2 wt% urea or sodium persulfate solution, and the amount of polymer degradation promoter is 5-10% of the mass of the pretreated Angelica sinensis powder.

6. The method for improving the extraction rate and antioxidant activity of Angelica sinensis polysaccharides according to claim 1, characterized in that... In step (2), the irradiation energy of the electron beam irradiation pretreatment is 5-10 MeV, the irradiation dose is 10-80 kGy, the thickness of Angelica sinensis powder does not exceed 4 cm when irradiating one side, and does not exceed 8 cm when irradiating both sides; the mass-volume ratio of irradiated Angelica sinensis powder to water is 1 g: 10-20 mL.

7. The method for improving the extraction rate and antioxidant activity of Angelica sinensis polysaccharides according to claim 1, characterized in that... In step (3), the acid is one or more of hydrochloric acid, citric acid, and acetic acid.

8. The method for improving the extraction rate and antioxidant activity of Angelica sinensis polysaccharides according to claim 1, characterized in that... In step (3), the ultrasonic extraction power is 300-500W, the temperature is 60-80℃, and the extraction time is 30-60min.

9. The method for improving the extraction rate and antioxidant activity of Angelica sinensis polysaccharides according to claim 1, characterized in that... In step (3), the temperature for heating and extraction is 70-80℃, and the extraction time is 2-3h.

10. The method for improving the extraction rate and antioxidant activity of Angelica sinensis polysaccharides according to claim 1, characterized in that... In step (4), dialysis is performed using dialysis bags with a capacity of 3500-10000 Da; the conditions for freeze drying are: temperature -80 to -60℃, vacuum degree 0.75 to 1.2 mbar.

Images