Low molecular weight astragalus polysaccharide, and preparation method and application thereof

Abstract

The application discloses a low-molecular-weight astragalus polysaccharide, which is obtained by the following steps: pretreating raw materials, once alcohol precipitation, drying, impurity removal and concentration, and finally adopting medium barrier discharge low-temperature plasma degradation. The application does not use strong acid, strong base, peroxide or metal catalyst, reduces the risk of residues and environmental protection pressure, and is green, safe and environmentally-friendly. The DBD treatment time is short, the energy utilization efficiency is high, the amplification can be realized through a continuous flow mode, and the method is fast and efficient.

Description

Technical Field

[0001] This invention relates to the fields of traditional Chinese medicine and natural product processing, bioengineering and functional food technology, specifically to a low molecular weight astragalus polysaccharide and its preparation method and application. Background Technology

[0002] Astragalus polysaccharides are important active components of Astragalus membranaceus, possessing immunomodulatory, antioxidant, and metabolic regulatory effects. However, natural Astragalus polysaccharides typically have high molecular weights, limiting their solubility and bioavailability, resulting in insufficient effective exposure in vivo and restricting their application and development in metabolic diseases such as hyperlipidemia and NAFLD.

[0003] To improve the solubility, bioavailability, and functional activity of polysaccharides, existing technologies often employ degradation methods such as acid hydrolysis, oxidation (hydrogen peroxide / Fenton), enzymatic hydrolysis (cellulase, pectinase, etc.), or ultrasound / microwave-assisted degradation. These methods often suffer from relatively harsh reaction conditions, excessive damage to the polysaccharide backbone and substituents, large batch-to-batch fluctuations in molecular weight distribution, and difficulty in achieving precise and controllable degradation. Furthermore, some chemical degradation routes may introduce byproducts or residues, affecting food / pharmaceutical safety and regulatory compliance.

[0004] Existing technologies lack systematic research on the relationship between the structure, quality properties, and efficacy of degraded Astragalus polysaccharides: most publications only report the decrease in molecular weight or a single characterization index, and lack systematic elucidation of the monosaccharide composition, uronic acid content, and retention of key functional groups of different molecular weight segments (such as ultrafiltration fractionation products), which limits the application potential of degraded polysaccharides.

[0005] Low-temperature plasma (especially deep-dip ionization) can generate a large number of reactive oxygen species (ROS / NOx) at near-room temperature, enabling rapid modification and degradation of macromolecular biopolymers, and possessing the potential for "additive-free, low-energy consumption, and online application." In recent years, patents have been published for the use of DBD low-temperature plasma to reduce the molecular weight of polysaccharides such as pectin. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a low molecular weight Astragalus polysaccharide, its preparation method, and its applications. By using low-temperature plasma (especially DBD), a large number of active oxygen / nitrogen species can be generated at near-room temperature, enabling rapid modification and degradation of Astragalus polysaccharide. This method has the potential to be "additive-free, low-energy, and online-processable."

[0007] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0008] A low molecular weight Astragalus polysaccharide is obtained by pretreating the raw material, followed by alcohol precipitation, drying, impurity removal and concentration, and finally low-temperature plasma degradation by dielectric barrier discharge (DBD).

[0009] The preparation method of low molecular weight Astragalus polysaccharide as described above includes the following steps:

[0010] (1) Raw material pretreatment: After pulverizing the Astragalus membranaceus, add water and extract with hot water 1-3 times, and combine the extracts;

[0011] (2) Alcohol precipitation and drying: Add ethanol to the filtrate obtained in step (1), let stand, centrifuge to collect the precipitate, redissolve the precipitate with deionized water, remove the ethanol by rotary evaporation, and obtain a polysaccharide aqueous solution.

[0012] (3) Removal and concentration: The polysaccharide aqueous solution obtained in step (2) was precipitated by TCA method to remove protein. The supernatant was dialyzed, concentrated by rotary evaporation, and freeze-dried to obtain Astragalus polysaccharide (APS).

[0013] (4) Dielectric barrier discharge (DBD) low-temperature plasma degradation: The astragalus polysaccharide (APS) obtained in step (3) is dissolved in water to obtain an astragalus polysaccharide solution;

[0014] The obtained Astragalus polysaccharide solution was subjected to dielectric barrier discharge low-temperature plasma degradation with a liquid layer thickness of 0.5-10 mm. The DBD electrode had a plate-plate structure, and the dielectric layer was selected from quartz, ceramic, or glass with an air gap of 1-10 mm. Air was used as the discharge medium, and an AC power supply was applied for a treatment time of 600-3600 s. After treatment, the solution was immediately cooled, filtered, and small molecule byproducts and salts were removed by dialysis and ultrafiltration. The solution was then freeze-dried to obtain low molecular weight Astragalus polysaccharide dAPS (degradedAPS).

[0015] Preferably, in step (1), after the Astragalus membranaceus is pulverized, deionized water is added at a material-to-liquid ratio of 1:30 (g / mL), and the mixture is extracted with hot water at 85℃ 1-3 times, each time for 1-3 hours. The extracts are then combined and the insoluble matter is filtered out.

[0016] Preferably, in step (2), anhydrous ethanol is added to the filtrate obtained in step (1) to a final concentration of 80% (v / v), the mixture is allowed to stand for 8 h, the precipitate is collected by centrifugation, the precipitate is re-dissolved with deionized water, and the ethanol is removed by rotary evaporation at 45°C.

[0017] Preferably, in step (3), the TCA precipitation to remove protein involves adding 80% trichloroacetic acid (TCA) to the polysaccharide aqueous solution obtained in step (2) to achieve a final TCA concentration of 4%. After mixing evenly, the solution is allowed to stand overnight at 4°C, and the protein precipitate is removed by centrifugation to obtain the supernatant. In step (3), the dialysis is performed with a molecular weight cutoff of 3.5 kDa for 36 hours, with 6 water changes.

[0018] Preferably, the freeze-drying in steps (3) and (4) involves pre-freezing at -80°C for 12 hours, then placing the freeze dryer in a freeze dryer and freeze-drying for 24-48 hours under conditions of cold trap temperature of approximately -50°C and vacuum degree ≤20 Pa.

[0019] Preferably, in step (4), the obtained Astragalus polysaccharide (APS) is dissolved in deionized water to obtain an Astragalus polysaccharide solution with a mass concentration of 2 mg / mL; in step (4), insoluble matter is removed by 0.45 μm filtration; the dialysis is performed with a molecular weight cutoff of 0.5 kDa for 12-36 h, with water changed 3-6 times; the ultrafiltration is performed with a molecular weight cutoff of 1 / 3 / 10 kDa.

[0020] Preferably, the air gap spacing in step (4) is 2-5 mm; the applied AC power supply has an input voltage of 90 V, a peak voltage of 120 kV, and a frequency of 100 Hz; and the system temperature is maintained at no higher than 45℃ during the process.

[0021] As mentioned above, low molecular weight Astragalus polysaccharides are used in the preparation of products related to regulating abnormal lipid metabolism and alleviating hyperlipidemia or non-alcoholic fatty liver disease.

[0022] Preferably, the product is a drug or a functional food.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1) This invention does not use strong acids or bases, peroxides or metal catalysts, reducing residual risks and environmental pressure, making it green, safe and environmentally friendly; it adopts DBD treatment with short processing time and high energy utilization efficiency, and can be scaled up through continuous flow, making it fast and efficient;

[0025] 2) Linking degradation processes with lipid metabolism improvement efficacy evaluation forms a closed loop of "process-structure-efficacy" to enhance the targeting of product development; it can be used in functional foods and health foods, as well as in the development of drug compositions or special medical foods. Attached Figure Description

[0026] Figure 1 This involves the determination of the molecular weight of APS and dAPS.

[0027] Figure 2 These are scanning electron microscope (SEM) images of APS and dAPS.

[0028] Figure 3 It is the Fourier transform infrared spectroscopy determination of APS and dAPS.

[0029] Figure 4 The effects of APS and dAPS intervention on serum biochemical and lipid parameters in mice on a high-fat diet.

[0030] Figure 5 These are Oil Red O and HE staining images of liver tissue from each group of mice.

[0031] Figure 6 This is the quantitative detection result of the content of 3-succinyl cholic acid (3-sucCA) in the feces of mice in each group. Detailed Implementation

[0032] The following detailed description, in conjunction with the accompanying drawings, outlines specific embodiments. However, it should be understood that the scope of protection of this invention is not limited to these specific embodiments. Unless otherwise specified, all raw materials and reagents used in the examples are commercially available.

[0033] Example 1

[0034] A method for preparing low molecular weight Astragalus polysaccharide, the operation steps are as follows:

[0035] (1) Raw material pretreatment: After pulverizing Astragalus membranaceus, add deionized water at a material-to-liquid ratio of 1:30 (g / mL), extract with hot water at 85℃ 1-3 times, 1-3 h each time, combine the extracts, and filter out insoluble matter;

[0036] (2) Alcohol precipitation and drying: Add anhydrous ethanol to the filtrate obtained after filtering out insoluble matter in step (1) to a final concentration of 80% (v / v), let stand for 8 h, centrifuge to collect the precipitate, redissolve the precipitate with deionized water, and remove the ethanol by rotary evaporation at 45℃ to obtain a polysaccharide aqueous solution.

[0037] (3) Impurity removal and concentration: Add 80% TCA to the polysaccharide aqueous solution obtained in step (2) to make the final TCA concentration reach 4%. After mixing evenly, let stand at 4℃ overnight, centrifuge to remove protein precipitate, and use a dialysis bag (molecular weight cutoff 3.5 kDa, 36 h, water changed 6 times) to remove small molecule impurities, inorganic salts and residual reagents. Concentrate by rotary evaporation, pre-freeze the concentrated solution at -80℃ for 12 h, and then place it in a freeze dryer. Freeze dry for 24-48 h under the conditions of cold trap temperature of about -50℃ and vacuum degree ≤20Pa to obtain Astragalus polysaccharide (APS).

[0038] (4) Dielectric barrier discharge low-temperature plasma degradation: The APS obtained in step (3) was dissolved in deionized water to obtain an Astragalus polysaccharide solution with a mass concentration of 2 mg / mL;

[0039] The obtained Astragalus polysaccharide solution was placed in a glass reaction vessel for dielectric barrier discharge (DBD) low-temperature plasma degradation, with a liquid layer thickness of 0.5-10 mm. The DBD electrode was a plate-to-plate structure, and the dielectric layer was made of glass with an air gap of 1-10 mm (preferably 2-5 mm, more preferably 3 mm; in specific implementation, the air gap was pre-adjusted and fixed according to the structure of the reaction device, and no dynamic adjustment was made during the treatment). Air was used as the discharge medium, and an AC power supply was applied with an input voltage of 90 V (peak high voltage 120 kV), a frequency of 100 Hz, and a treatment time of 600-3600 s. During the treatment, the system temperature was maintained at no higher than 45℃. After the treatment, the solution was immediately cooled and filtered through a 0.45 μm microporous membrane to remove insoluble matter. Dialysis (molecular weight cutoff 0.5 kDa, 12-36 h, water change 3-6 times) and ultrafiltration (1 / 3 / 10 kDa) were used to remove small molecule byproducts and salts. The substances obtained after dialysis and ultrafiltration were pre-frozen at -80℃ for 12 days. After h, it was placed in a freeze dryer and freeze-dried for 24-48 h under the conditions of cold trap temperature of about -50℃ and vacuum degree ≤20 Pa to obtain low molecular weight astragalus polysaccharide (dAPS).

[0040] Example 2

[0041] Characterization of APS and dAPS

[0042] (1) Molecular weight: The weight-average molecular weight (Mw) was determined by high-performance gel permeation chromatography (HPGPC), and the results are as follows: Figure 1 As shown.

[0043] (2) Physicochemical indicators: total sugar (phenol-sulfuric acid method), uronic acid (m-hydroxydiphenyl method), protein (Qubit protein detection), the results are shown in Table 1.

[0044] (3) Structural characterization: SEM results are as follows Figure 2 As shown, the FT-IR results are as follows: Figure 3 As shown.

[0045] Table 1

[0046] APS dAPS Total sugar content (%) 78.41±0.43 92.35±1.30 Glucuronic acid content (%) 3.23±0.12 1.82±0.24 Protein content (%) 5.32±0.64 0.92±0.06

[0047] Depend on Figure 1 It can be seen that after DBD low-temperature plasma treatment, the molecular weight of dAPS is significantly lower than that of APS, indicating that this method can effectively break the molecular chains of Astragalus polysaccharides and obtain low molecular weight products. Figure 2 It can be seen that the surface morphology of APS is relatively dense, while that of dAPS is more loose, fragmented, or has increased porosity, indicating that the degradation treatment alters the aggregation state of Astragalus polysaccharides, which is beneficial to improving their dispersibility and application suitability. Figure 3It can be seen that both APS and dAPS retain the typical infrared absorption peaks of polysaccharides, and no obvious new characteristic peaks appear, indicating that DBD treatment mainly reduces the molecular weight without destroying the basic sugar chain backbone structure of Astragalus polysaccharides. Table 1 shows that the total sugar content of dAPS increased from 78.41±0.43% to 92.35±1.30%, while the protein content decreased from 5.32±0.64% to 0.92±0.06%, suggesting that degradation and subsequent purification treatment help improve the purity of the polysaccharides; the uronic acid content decreased from 3.23±0.12% to 1.82±0.24%, but still retained certain acidic group characteristics.

[0048] Example 3

[0049] Animal experiments verified

[0050] (1) Model construction: Male C57BL / 6J mice (8 weeks old) were selected and randomly divided into groups (n=8) after 1 week of acclimatization. The model group was fed a high-fat diet (Beijing Huafukang) for 12 weeks to induce the NAFLD phenotype; the control group was fed a normal diet.

[0051] (2) Grouping and administration:

[0052] a) Control group (NC, normal feed): administered physiological saline by gavage daily;

[0053] b) Model group (MD, high-fat diet): daily gavage with physiological saline;

[0054] c) APS group (high-fat diet): Astragalus polysaccharide solution was administered by gavage daily, 200 mg / kg / day;

[0055] d) dAPS group (high-fat diet): dAPS, the ultrafiltration fraction after DBD degradation, was administered by gavage daily at 200 mg / kg / day.

[0056] (3) Evaluation indicators:

[0057] a) Blood lipids and liver function: ALT, AST, TC, TG, HDL-C, and LDL-C were measured using a Nanjing Jiancheng reagent kit. The results are as follows: Figure 4 As shown, ALT: alanine aminotransferase, AST: aspartate aminotransferase, HDL-C: high-density lipoprotein cholesterol, TC: total cholesterol, TG: triglycerides, and LDL-C: low-density lipoprotein cholesterol.

[0058] b) Oil Red O and HE staining of liver tissue: to evaluate hepatocyte morphology, lipid accumulation, and inflammatory infiltration (results as follows). Figure 5 (As shown).

[0059] Depend on Figure 4It was found that, compared with the control group (NC), the model group (MD) mice had increased serum ALT, AST, TC, TG, and LDL-C levels, and decreased HDL-C levels, indicating that a high-fat diet induced liver function damage and lipid metabolism disorders in the mice. Compared with the model group, both the APS group and the dAPS group reduced ALT, AST, TC, TG, and LDL-C levels to varying degrees and increased HDL-C levels, with the dAPS group showing a more significant improvement trend, indicating that low molecular weight Astragalus polysaccharides after DBD degradation have a better effect on regulating lipid metabolism and improving liver function damage. Figure 5 It can be seen that Oil Red O staining of liver tissue in the model group showed a large amount of lipid deposition, and HE staining showed obvious fat vacuoles and disordered hepatocyte structure. After APS intervention, lipid deposition and fat vacuoles were reduced. After dAPS intervention, lipid deposition in liver tissue was significantly reduced, and the morphology and tissue structure of hepatocytes were closer to those of the control group, further proving that dAPS can alleviate hepatic steatosis induced by high-fat diet.

[0060] Example 4

[0061] The regulatory effect of low molecular weight Astragalus polysaccharides on bile acid-related metabolites, and the quantitative detection of 3-sucCA in feces.

[0062] The content of 3-succinyl cholic acid (3-sucCA) in the fecal samples of mice in each group in Example 3 was quantitatively detected by liquid chromatography-mass spectrometry. 3-sucCA is a bile acid derivative formed by the participation of intestinal flora in bile acid conversion and can be used as one of the indicators for evaluating changes in intestinal flora-bile acid metabolism.

[0063] (1) Sample collection and pretreatment: Fresh feces from mice in each group were collected before the end of the experiment and immediately stored at -80℃. Before detection, the fecal samples were freeze-dried and ground evenly. An appropriate amount of fecal powder was weighed and extracted with pre-cooled methanol solution. After vortexing and ultrasonic extraction, the supernatant was collected by centrifugation at 12000 rpm for 15 min at 4℃ and filtered through a 0.22 μm microporous membrane for liquid chromatography-mass spectrometry analysis.

[0064] (2) Establishment of standard curve: Prepare a series of standard solutions with different concentrations using 3-sucCA standard, and analyze them under the same detection conditions as the samples. Establish a standard curve using the peak area of ​​3-sucCA and the concentration of the standard, and calculate the content of 3-sucCA in the samples based on the standard curve. The detection results are normalized according to the dry weight of feces.

[0065] (3) Liquid chromatography-mass spectrometry conditions: Liquid chromatography-mass spectrometry system was used for detection. The retention time and characteristic ion signals were determined using 3-sucCA standard. The negative ion electrospray ionization mode was used for detection, and 3-sucCA was quantitatively analyzed based on multiple reaction monitoring (MRM).

[0066] The results are as follows Figure 6 As shown in the figure, compared with the control group, the fecal 3-sucCA content in the model group mice was significantly reduced, suggesting that a high-fat diet can disrupt gut microbiota-mediated bile acid metabolism. Compared with the model group, the fecal 3-sucCA content in the APS group mice was increased, and the increase was more significant in the dAPS group, indicating that the low molecular weight Astragalus polysaccharide prepared in this invention can more effectively regulate the level of intestinal bile acid derivatives. Combined with the blood lipid, liver function and histological results in Example 3, it is suggested that dAPS may participate in improving high-fat diet-induced lipid metabolism abnormalities and hepatic lipid deposition by regulating the gut microbiota-bile acid metabolism axis.

[0067] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A low molecular weight astragalus polysaccharide, characterized in that: After pretreatment of the raw materials, they undergo alcohol precipitation, drying, impurity removal, and concentration. Finally, they are degraded by dielectric barrier discharge low-temperature plasma to obtain the final product.

2. The method for preparing low molecular weight astragalus polysaccharide according to claim 1, characterized in that, The operation includes the following steps: (1) Raw material pretreatment: After pulverizing the Astragalus membranaceus, add water and extract with hot water 1-3 times, and combine the extracts; (2) Alcohol precipitation and drying: Add ethanol to the filtrate obtained in step (1), let stand, centrifuge to collect the precipitate, redissolve the precipitate with water, and evaporate by rotary evaporation to obtain a polysaccharide aqueous solution; (3) Removal and concentration: The polysaccharide aqueous solution obtained in step (2) was precipitated by TCA method to remove protein. The supernatant was dialyzed, concentrated by rotary evaporation, and freeze-dried to obtain Astragalus polysaccharide. (4) Dielectric barrier discharge low-temperature plasma degradation: Dissolve the Astragalus polysaccharide obtained in step (3) in water to obtain an Astragalus polysaccharide solution; The obtained Astragalus polysaccharide solution was subjected to dielectric barrier discharge low-temperature plasma degradation with a liquid layer thickness of 0.5-10 mm. The DBD electrode had a plate-plate structure, and the dielectric layer was selected from quartz, ceramic, or glass with an air gap of 1-10 mm. Air was used as the discharge medium, and an AC power supply was applied for a treatment time of 600-3600 s. After the treatment, the solution was immediately cooled, filtered, dialyzed, and ultrafiltered, and then freeze-dried to obtain low molecular weight Astragalus polysaccharide dAPS.

3. The method for preparing low molecular weight Astragalus polysaccharide according to claim 1, characterized in that: In step (1), after the Astragalus membranaceus is pulverized, water is added at a material-to-liquid ratio of 1:30 (g / mL), and the mixture is extracted with hot water at 85℃ 1-3 times, each time for 1-3 hours, and the extracts are combined.

4. The method for preparing low molecular weight astragalus polysaccharide according to claim 1, characterized in that: In step (2), ethanol is added to the filtrate obtained in step (1) to a final concentration of 80%, and the mixture is allowed to stand for 8 hours. The precipitate is collected by centrifugation, and the precipitate is reconstituted with water and evaporated at 45°C.

5. The method for preparing low molecular weight astragalus polysaccharide according to claim 1, characterized in that: The TCA precipitation method for protein removal in step (3) involves adding 80% trichloroacetic acid to the polysaccharide aqueous solution obtained in step (2) to achieve a final TCA concentration of 4%. After mixing, the solution is allowed to stand overnight at 4°C, centrifuged, and the supernatant is obtained. The dialysis method in step (3) involves a molecular weight cutoff of 3.5 kDa, 36 h, and 6 water changes.

6. The method for preparing low molecular weight astragalus polysaccharide according to claim 1, characterized in that: The freeze-drying described in steps (3) and (4) involves pre-freezing at -80℃ for 12 h, followed by freeze-drying at a cold trap temperature of approximately -50℃ and a vacuum degree of ≤20 Pa for 24-48 h.

7. The method for preparing low molecular weight astragalus polysaccharide according to claim 1, characterized in that: In step (4), the obtained Astragalus polysaccharide is dissolved in water to obtain an Astragalus polysaccharide solution with a mass concentration of 2 mg / mL; in step (4), the solution is filtered through 0.45 μm; the dialysis is performed with a molecular weight cutoff of 0.5 kDa for 12-36 h, with water changed 3-6 times; the ultrafiltration is performed with a molecular weight cutoff of 1 / 3 / 10 kDa.

8. The method for preparing low molecular weight Astragalus polysaccharide according to claim 1, characterized in that: In step (4), the air gap spacing is 2-5 mm; the AC power supply is applied with an input voltage of 90 V, a peak voltage of 120 kV, and a frequency of 100 Hz; and the system temperature is maintained at no higher than 45℃ during the process.

9. The use of the low molecular weight astragalus polysaccharide as described in claim 1 or the low molecular weight astragalus polysaccharide prepared by any of the methods in claims 2-8 in the preparation of products related to regulating abnormal lipid metabolism and alleviating hyperlipidemia or non-alcoholic fatty liver disease.

10. The application according to claim 9, characterized in that: The product in question is a medicine or a functional food.

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