Doudan polysaccharide, and preparation method and use thereof

Abstract

This application discloses a polysaccharide derived from *Euonymus alatus*, its preparation method, and its uses, belonging to the field of polysaccharide extraction technology. The technical solution involves: drying, crushing, and sieving *Euonymus alatus* peel and / or flesh, followed by water extraction. After water extraction, the supernatant is collected by centrifugation, and then the protein in the supernatant is removed. After protein removal, the supernatant is precipitated with alcohol, and the precipitate is collected to obtain the polysaccharide. The polysaccharide provided in this application exhibits excellent antioxidant capacity, specifically in its good free radical scavenging effect and iron ion reducing power. Furthermore, when *Euonymus alatus* peel is used as the raw material for extraction, its antioxidant capacity at high concentrations is significantly higher than that extracted from *Euonymus alatus* flesh.

Description

Technical Field

[0001] This application belongs to the field of polysaccharide extraction technology, and more specifically, relates to a soybean polysaccharide, its preparation method, and its uses. Background Technology

[0002] While synthetic antioxidants exhibit good antioxidant effects, they also pose potential toxicity and safety risks. In contrast, antioxidant polysaccharides derived from natural products have advantages such as low toxicity, good biocompatibility, and strong biodegradability, which can, to some extent, avoid the bioaccumulation problems that synthetic antioxidants may cause, and therefore have attracted widespread attention.

[0003] Oxidative stress is a core pathological mechanism in many chronic diseases. Antioxidant polysaccharides can scavenge free radicals, protect cell membrane structure, and reduce oxidative damage, showing promising applications in disease prevention and functional food development. In addition to their antioxidant properties, many antioxidant polysaccharides also possess anti-inflammatory and immunomodulatory functions. This "multi-target, multi-pathway" characteristic makes them more competitive in the development of functional foods and drugs.

[0004] Bean hawk moth (Clanis bilineata) larvae are a traditional edible insect resource in some parts of my country, rich in various bioactive components such as protein, fat, chitin, and polysaccharides. In recent years, with the continuous deepening of research on the development and utilization of insect resources, insect-derived polysaccharides have received widespread attention due to their unique structure and good biological activities (such as antioxidant, immunomodulatory, and anti-inflammatory effects).

[0005] Currently, research on *Sipunculus nudus* mainly focuses on the development and utilization of its protein resources. Although some literature has touched upon preliminary research on *Sipunculus nudus* polysaccharides, related research is still very limited, especially regarding the extraction methods and bioactivity of *Sipunculus nudus* polysaccharides, which lack systematic research. Chinese patent application 201710321701.9 discloses a method for preparing *Sipunculus nudus* polysaccharide and its application. The method involves adding dried *Sipunculus nudus* powder to double-distilled water for boiling extraction, cooling, adding protease and cellulase for enzymatic hydrolysis, boiling to inactivate the enzymes, centrifuging, filtering to collect the filtrate, and freeze-drying to obtain *Sipunculus nudus* polysaccharide.

[0006] Further observation of this scheme reveals that it extracts polysaccharides from the entire body of Sipunculus nudus using a combination of water extraction and enzymatic hydrolysis. Experiments have verified that this method has effects such as lowering blood lipids, anti-oxidation, and protecting the liver.

[0007] However, existing technologies lack systematic research or related patent disclosures regarding the extraction methods and antioxidant activity of *Euonymus alatus* polysaccharides. Meanwhile, the *Euonymus alatus* industry is large-scale and has high output value, generating a large amount of *Euonymus alatus* skin during processing. As a byproduct of *Euonymus alatus* processing, this skin is usually discarded directly, resulting in low utilization rates, resource waste, and potential environmental burden. Developing a technological approach to effectively utilize *Euonymus alatus* skin resources is of great significance.

[0008] Therefore, it is still necessary in this field to develop a polysaccharide extraction method from *Euonymus alatus*, especially a polysaccharide extraction method based on *Euonymus alatus* bark, and to obtain *Euonymus alatus* polysaccharides with antioxidant activity, so as to realize the high-value utilization of *Euonymus alatus* by-product resources. Summary of the Invention

[0009] The purpose of this application is to provide a polysaccharide derived from *Eriocaulon buergerianum*, and through a series of tests on the polysaccharide, it was found that the polysaccharide has good antioxidant capacity.

[0010] To achieve the above objectives, this method provides a bean lily polysaccharide, which is obtained by drying, crushing and sieving bean lily skin and / or bean lily flesh, followed by water extraction, centrifugation to obtain the supernatant, removal of protein from the supernatant, alcohol precipitation after protein removal, collection of the precipitate, and obtaining bean lily polysaccharide.

[0011] Preferably, the bean peony bark is dried, pulverized, sieved, and then extracted with water. The supernatant is collected after extraction, and the protein in the supernatant is removed. After protein removal, the precipitate is precipitated with alcohol, and the precipitate is collected to obtain bean peony polysaccharide. In actual experiments, this application attempted to extract polysaccharides from different parts of the bean peony and compared the antioxidant capacity of polysaccharides extracted from different parts. Notably, this application unexpectedly discovered during the extraction of polysaccharides from bean peony bark that its antioxidant capacity at high concentrations is significantly superior to that extracted from bean peony flesh. The bean peony polysaccharide extracted from bean peony bark exhibits good scavenging ability against DPPH and ABTS free radicals and a strong chelating ability against iron ions. Specifically, the IC50 values ​​for DPPH and ABTS free radical scavenging and iron ion chelation are [not specified in the original text]. 50 The concentrations were 0.33 mg / mL, 1.14 mg / mL, and 0.36 mg / mL, respectively, and all indicators showed a significant positive correlation with the concentration (R²>0.99).

[0012] In addition, this application also discloses a method for preparing the above-mentioned soybean polysaccharide, comprising the following steps:

[0013] Step 1: Wash the bean skin and / or bean flesh, then dry, crush and sieve to obtain powder;

[0014] Step 2: Mix the powder with distilled water at a mass ratio of 1:25-35, and heat at 70-90 ℃ for 2-4 h to obtain an intermediate solution;

[0015] Step 3: Centrifuge the intermediate solution to obtain the supernatant, then mix the supernatant with Sevage reagent, centrifuge again and obtain the supernatant after removing the protein;

[0016] Step 4: Concentrate the supernatant after removing the protein to 0.25-0.35 of the original volume, then add ethanol for precipitation, collect the precipitate, and obtain the bean polysaccharide.

[0017] In addition, this application also discloses the use of the above-mentioned soybean polysaccharide in the preparation of antioxidant products.

[0018] Preferably, the antioxidant product is an antioxidant product with free radical scavenging ability.

[0019] Preferably, the polysaccharide in the antioxidant product is extracted from the bark of the soybean plant.

[0020] The molecular weight distribution range of the polysaccharide extracted from the bark of the dung bean is 10–250 kDa.

[0021] Its monosaccharide composition consists of glucose, galactosamine, glucuronic acid, glucosamine, galactose, and rhamnose.

[0022] Furthermore, this application compared the extraction results of polysaccharides from different extracts, such as the flesh and bark of *Euonymus alatus*, during the experiment. The results showed that at low concentrations (polysaccharide concentration less than or equal to 0.3 mg / mL), the polysaccharides extracted from the flesh of *Euonymus alatus* had good free radical scavenging ability. However, as the polysaccharide concentration increased, the polysaccharides extracted from the bark of *Euonymus alatus* showed better antioxidant properties. That is, the polysaccharides extracted from the bark of *Euonymus alatus* showed a more significant dose-dependent effect. Moreover, when the concentration was further increased, the antioxidant capacity of the two showed a significant difference. This phenomenon may be because at high concentrations, the polysaccharides extracted from the bark of *Euonymus alatus* have a large number of active molecules per unit volume, a high collision frequency, and a significantly increased reaction rate and scavenging rate.

[0023] The polysaccharides extracted from soybean flesh may contain specific, highly reactive sites. Even at low concentrations, these sites can react rapidly with DPPH, causing its free radical scavenging rate to reach a plateau more quickly.

[0024] The beneficial effects of this application are:

[0025] This application provides a polysaccharide derived from *Eupolyphaga sinensis*, and a series of tests on the polysaccharide revealed that it possesses excellent antioxidant capacity. Furthermore, our experiments showed that when *Eupolyphaga sinensis* flesh is used as the raw material for polysaccharide preparation, it exhibits better antioxidant capacity at low concentrations. However, as the polysaccharide concentration increases, the polysaccharide extracted from *Eupolyphaga sinensis* skin exhibits more significant antioxidant capacity. Attached Figure Description

[0026] Figure 1 A flowchart of the preparation process of soybean polysaccharide;

[0027] Figure 2 The ultraviolet spectrum of *Dendrobium nobile* polysaccharide;

[0028] Figure 3 This is a standard curve of protein content.

[0029] Figure 4 Fourier transform infrared spectrum of safflower polysaccharide extracted from safflower bark in Example 1;

[0030] Figure 5 The results of differential refractive index testing of the polysaccharide extracted from the bark of *Paeonia lactiflora* in Example 1 are shown.

[0031] Figure 6 A standard diagram of monosaccharide mixing during the monosaccharide composition test of polysaccharides extracted from *Dalbergia odorifera* bark;

[0032] Figure 7 The image shows the monosaccharide composition results during the testing process of polysaccharide extracted from *Dalbergia odorifera* bark. Detailed Implementation

[0033] The present invention will now be clearly and completely described in conjunction with embodiments thereof. It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0034] The bean curd meat was purchased from Xiaobei Fresh Food Department Store in Lianyungang Economic Development Zone;

[0035] Dou Danpi was purchased from Xiaobei Fresh Food Department Store in Lianyungang Economic Development Zone;

[0036] Sevage reagent: prepared in-house by mixing chloroform and n-butanol in a volume ratio of 4:1.

[0037] Example 1

[0038] refer to Figure 1 The preparation method of soybean polysaccharide includes the following steps:

[0039] Step 1: Wash the collected bean paste sample to obtain pre-treated bean paste. Then, freeze-dry the pre-treated bean paste at -80℃ for 48 hours and pulverize it. Pass it through a 40-mesh sieve to obtain bean paste powder.

[0040] Step 2: Mix the bean powder with distilled water at a mass ratio of 1:30, and extract in a water bath at 80℃ for 3 hours to obtain the intermediate liquid;

[0041] Step 3: Cool the intermediate liquid obtained after extraction to 25°C, then centrifuge at 4000 rpm for 20 min and collect the supernatant;

[0042] The supernatant was then mixed with Sevage reagent at a volume ratio of 4:1, vortexed, and centrifuged at 4000 rpm for 15 min. The supernatant was collected, and this operation was repeated 6 times to obtain the supernatant after protein removal.

[0043] Step 4: The supernatant after protein removal was concentrated to 0.3% of its original volume using a rotary evaporator. Then, 4 times the volume of 95% ethanol (v / v) was slowly added to the concentrate, and the mixture was allowed to stand at 4°C for 12 hours for alcohol precipitation. Subsequently, the mixture was centrifuged at 4000 rpm for 15 minutes, and the precipitate was collected to obtain the bean polysaccharide.

[0044] Example 2

[0045] Step 1: Wash the collected bean paste sample to obtain pre-treated bean paste. Then, freeze-dry the pre-treated bean paste at -80℃ for 48 hours and pulverize it. Pass it through a 40-mesh sieve to obtain bean paste powder.

[0046] Step 2: Mix the bean powder with distilled water at a mass ratio of 1:25, and extract in a water bath at 90℃ for 2 hours to obtain the intermediate solution;

[0047] Step 3: Cool the intermediate liquid obtained after extraction to 25°C, then centrifuge at 4000 rpm for 20 min and collect the supernatant;

[0048] The supernatant was then mixed with Sevage reagent at a volume ratio of 4:1, vortexed, and centrifuged at 4000 rpm for 15 min. The supernatant was collected, and this operation was repeated 6 times to obtain the supernatant after protein removal.

[0049] Step 4: Concentrate the supernatant after protein removal to 0.25% of its original volume using a rotary evaporator. Then, slowly add 4 times the volume of 95% ethanol (v / v) to the concentrate and allow it to stand at 4°C for ethanol precipitation for 12 min. Subsequently, centrifuge at 4000 rpm for 15 min, collect the precipitate, and obtain the bean polysaccharide.

[0050] Example 3

[0051] Step 1: Wash the collected bean paste sample to obtain pre-treated bean paste. Then, freeze-dry the pre-treated bean paste at -80℃ for 48 hours and pulverize it. Pass it through a 40-mesh sieve to obtain bean paste powder.

[0052] Step 2: Mix the bean powder with distilled water at a mass ratio of 1:35, and extract in a water bath at 70℃ for 4 hours to obtain the intermediate solution;

[0053] Step 3: Cool the intermediate liquid obtained after extraction to 25°C, then centrifuge at 4000 rpm for 20 min and collect the supernatant;

[0054] The supernatant was then mixed with Sevage reagent at a volume ratio of 4:1, vortexed, and centrifuged at 4000 rpm for 15 min. The supernatant was collected, and this operation was repeated 6 times to obtain the supernatant after protein removal.

[0055] Step 4: Concentrate the supernatant after protein removal to 0.35% of its original volume using a rotary evaporator. Then, slowly add 4 times the volume of 95% ethanol (v / v) to the concentrate and allow it to stand at 4°C for ethanol precipitation for 12 min. Subsequently, centrifuge at 4000 rpm for 15 min, collect the precipitate, and obtain the bean polysaccharide.

[0056] Example 4

[0057] It is basically the same as Example 1, except that the flesh of the bean is used instead of the skin of the bean.

[0058] Performance testing:

[0059] 1. Polysaccharide content determination (anthrone sulfuric acid method):

[0060] 72% concentrated sulfuric acid: Add 72 mL of concentrated sulfuric acid to 28 mL of distilled water and store at 4 °C.

[0061] Anthrone reagent: Dissolve 0.2g of anthrone in 100mL of 72% H2SO4. Prepare fresh before use.

[0062] Preparation of glucose standard solution: Weigh 10 mg of D-anhydrous glucose standard, dissolve it in a small amount of ultrapure water, transfer it to a 100 mL volumetric flask, dilute to volume with ultrapure water, shake well, and you will get a 0.1 mg / mL glucose standard solution. Store at low temperature for later use.

[0063] Sample solution: Weigh 10 mg of sample, dissolve in ultrapure water, and prepare a 0.1 mg / mL sample solution.

[0064] Plotting the glucose standard curve:

[0065] Take 7 centrifuge tubes and prepare a series of glucose solutions of different concentrations according to the data in Table 1:

[0066] Table 1. Glucose Solution Preparation Table

[0067] <![CDATA[H2O (mL)]]> 1.0 0.9 0.8 0.6 0.4 0.2 0 Concentration (μg / mL) 0 10 20 40 60 80 100

[0068] Add 4.0 mL of anthrone reagent to each tube. After adding all the reagent, immerse all tubes in a boiling water bath, capping the tubes to prevent evaporation. Start timing from the moment the water bath boils, and boil accurately for 10 minutes. Remove the tubes and cool them to room temperature in an ice-water bath. Using the first tube as a blank, quickly measure the absorbance of the remaining tubes at a wavelength of 620 nm. Plot a standard curve with the standard glucose content (µg) on ​​the x-axis and the absorbance on the y-axis.

[0069] Determination: Pipette 1 mL of sample solution into a test tube, add 4.0 mL of anthrone reagent, and repeat in triplicate; replace the blank tube with an equal volume of distilled water. The following procedures are the same as for the standard curve preparation. According to A... 620 The polysaccharide content is calculated from the average value on the standard curve.

[0070] 2. DPPH free radical scavenging ability:

[0071] Take 100 μL of sample solution (dissolution method as per polysaccharide content test, using ultrapure water as solvent), mix thoroughly with 100 μL of 0.1 mM DPPH ethanol solution, react in the dark at room temperature for 30 min, and measure the absorbance value at 517 nm. The calculation formula is as follows:

[0072] Formula 1

[0073] In Formula 1: S2—DPPH free radical scavenging rate (%);

[0074] A3—Absorbance value of the sample after reaction with DPPH;

[0075] A4—Absorbance value of the sample after reaction with ethanol;

[0076] A0—Control absorbance value.

[0077] Furthermore, it should be noted that the polysaccharide concentrations in the above 100 μL sample solutions were 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, and 0.9 mg / mL, respectively. The detection results are shown in Table 2.

[0078] Table 2. In vitro antioxidant activity test of *Dendrobium nobile* polysaccharide

[0079] Polysaccharide content / % 14.04% 5.74% 0.1 mg / mL DPPH free radical scavenging rate / % 18.12% 45.52% 0.2 mg / mL DPPH free radical scavenging rate / % 34.09% 47.73% 0.3 mg / mL DPPH free radical scavenging rate / % 47.04% 50.68% 0.4 mg / mL DPPH free radical scavenging rate / % 59.99% 52.67% 0.5 mg / mL DPPH free radical scavenging rate / % 70.38% 54.54% 0.9 mg / mL DPPH free radical scavenging rate / % 70.56% 61.14% <![CDATA[IC 50 ]]> 0.33 mg / mL 0.29 mg / mL

[0080] Results analysis:

[0081] As can be seen from Table 2, the only difference between Example 1 and Example 4 is that the polysaccharide in the former is extracted from the skin of the bean peony, while that in the latter it is extracted from the flesh of the bean peony.

[0082] Furthermore, in Example 1, the polysaccharide content of the bean peony bark extracted reached 14.04%, while that of the bean peony flesh was only 5.74%.

[0083] The concentrations of *Eupolysaccharide* extracted from *Eupolysaccharide* bark in Example 1 and from *Eupolysaccharide* flesh in Example 4 were both significantly positively correlated with DPPH free radical scavenging rate, and the half-inhibition rate (IC50) concentration was also significantly higher. 50 The concentrations were 0.33 mg / mL and 0.29 mg / mL, respectively, with no significant difference; however, at higher concentrations (≥0.4 mg / mL), the polysaccharide extracted from the bark of the dung bean showed a higher DPPH free radical scavenging rate.

[0084] 3. The purity of the two polysaccharides was further characterized by ultraviolet scanning spectroscopy and protein content determination:

[0085] 3.1 Ultraviolet (UV) spectroscopy determination: Prepare a 0.1 mg / mL aqueous solution of Sophora flavescens polysaccharide and perform a full UV scan in the range of 200–500 nm.

[0086] Figure 2 The results showed that both types of *Eupolyphaga sinensis* polysaccharides exhibited significant strong absorption in the 210–230 nm region. This is attributed to the σ→σ* or n→σ* electronic transitions in the glycosidic bonds, the hydroxyl (-OH) related structure, and a small number of carbonyl (C=O) or carboxyl groups. This indicates that the main component of the sample is a polysaccharide, and this is a typical polysaccharide absorption region. However, the overall absorption of the *Eupolyphaga sinensis* polysaccharide extracted from the flesh of the *Eupolyphaga sinensis* was higher than that extracted from the bark, especially in the 260–290 nm region. This suggests that the *Eupolyphaga sinensis* polysaccharide extracted from the flesh of the *Eupolyphaga sinensis* contained more protein residue, resulting in lower sample purity. Different raw materials lead to different protein removal efficiencies.

[0087] 3.2 Determination of protein content in polysaccharides: The protein content in crude polysaccharides from *Solanum tuberosum* was determined using the Coomassie Brilliant Blue G-250 method. Figure 3 The standard curve is y = 3.7706x + 0.4519, R0 2 =0.9959, substituting the values, the protein concentration in the polysaccharide extracted from the bark of *Euonymus alatus* was found to be 0.0835±0.0006 mg / mL, with a content of 16.69±0.12%; the protein concentration in the polysaccharide extracted from the flesh of *Euonymus alatus* was found to be 0.1122±0.0016 mg / mL, with a content of 22.45±0.33%. Similarly, the protein content results also indicate that the polysaccharide extracted from the flesh of *Euonymus alatus* has a higher content of protein impurities.

[0088] In summary, the polysaccharide content and purity of the polysaccharide extracted from the bark of *Euonymus alatus* in Example 1 are superior to those extracted from the flesh of *Euonymus alatus*, and it also has better free radical scavenging ability, especially in terms of DPPH free radical scavenging ability.

[0089] 4. ABTS Free Radical Inhibition Rate Test

[0090] Mix equal volumes of 7 mM ABTS and 5 mM potassium persulfate and store in the dark for 12 hours. Before use, dilute to approximately 0.7 to obtain the working solution. Take 20 μL of sample (dissolve using the same method as the polysaccharide content test, using ultrapure water as the solvent) and mix thoroughly with 180 μL of the working solution. React in the dark at room temperature for 10 minutes, and measure the absorbance at 734 nm. The calculation formula is as follows:

[0091] Formula 2

[0092] Where: S3—ABTS free radical scavenging rate (%); A5—absorbance value after the sample reacts with the working solution; A6—absorbance value after the sample reacts with distilled water; A0—control absorbance value.

[0093] The results are shown in Table 3:

[0094] Table 3. Results of ABTS free radical inhibition rate (%) of polysaccharides extracted from *Paeonia lactiflora* bark.

[0095] 0 9.71 0.2 15.40 0.4 24.83 0.6 30.52 0.8 38.18 1 42.20 2 81.88

[0096] Table 3 shows that the concentration of polysaccharides extracted from *Paeonia suffruticosa* bark was significantly positively correlated with its ability to inhibit ABTS free radicals. 2 The half-maximum inhibitory concentration (IC50) was 0.9965. 50 It has a concentration of 1.14 mg / mL and a good ability to inhibit ABTS free radicals.

[0097] 5. Iron ion suppression ability test (%)

[0098] Take 3 mL of sample (dissolution method as per polysaccharide content test, using ultrapure water as solvent), mix thoroughly with 3 mL of potassium ferricyanide solution (1%, w / v), and react in a 50℃ water bath for 20 min. After the reaction, cool the solution to room temperature, add 3 mL of trichloroacetic acid solution (10%, w / v), mix well, let stand for 10 min, and centrifuge at 4000 rpm for 10 min. Take 150 μL of the supernatant, mix with 150 μL of distilled water and 30 μL of ferric chloride solution (0.1%, w / v), and react in a 37℃ water bath for 30 min. After the reaction, measure the absorbance at 700 nm. The higher the absorbance value, the stronger the total reducing power of the sample. The highest value in the group is A. maxThe changes in total reducing power of *Dendrobium nobile* polysaccharide at different concentration gradients were compared.

[0099] Formula 3

[0100] Where: S1—total reducing power (%); A1—absorbance value after the sample reacts with the working solution; A2—absorbance value after the sample reacts with distilled water.

[0101] The results are shown in Table 4:

[0102] Table 4. Iron ion inhibition capacity (%)

[0103] 0.1 26.35 0.2 34.10 0.3 46.79 0.4 55.15 0.6 71.31 0.8 84.72 1 100

[0104] Table 4 shows that the concentration of polysaccharides extracted from *Paeonia suffruticosa* bark was significantly positively correlated with its ability to inhibit iron ions, R. 2 The half-maximum inhibitory concentration (IC50) was 0.9935. 50 It has a concentration of 0.36 mg / mL and a good ability to inhibit iron ions.

[0105] 6. Further infrared spectral analysis of the polysaccharide extracted from the bark of *Paeonia lactiflora* in Example 1.

[0106] Infrared spectroscopy was performed using the potassium bromide (KBr) pellet method. Appropriate amounts of dried *Dendrobium nobile* polysaccharide samples were mixed with KBr powder in agate. The mixture was then pressed into granules using the KBr disc method, and the granules were analyzed using an IRAffinity-1S infrared spectrometer at 400–4000 cm⁻¹. -1 Infrared spectral scanning analysis was performed within the specified range.

[0107] The infrared spectral results of the polysaccharides extracted from the bark of *Paeonia lactiflora* are as follows: Figure 4 As shown, the polysaccharide is at 3292.085 cm⁻¹ -1 The absorption peak at 2962.836 cm⁻¹ is the stretching vibration peak of the OH group of the polysaccharide, indicating the presence of hydroxyl groups; -1 The narrower absorption peaks at 1660 cm⁻¹ are due to the bending and stretching vibrations of CH, both of which are characteristic absorption peaks of polysaccharides; -1 The absorption peaks around the left and right are either due to the stretching vibration of C=O or caused by bound water; 1cm -1 The vibrational peak of CH is 1239.058 cm⁻¹. −1 The absorption peak at 1250 cm⁻¹ is the stretching vibration peak of the C=O bond; -1 -1000cm -1 The stronger peaks in between should be caused by the stretching vibration of glycosidic bonds.

[0108] Therefore, the antioxidant activity of polysaccharides extracted from peony bark is likely closely related to the abundance of functional groups such as hydroxyl, carboxyl, and amino groups in its molecule. These functional groups exert their antioxidant effects synergistically through mechanisms such as hydrogen atom donation, electron transfer, and metal ion complexation.

[0109] 7. Further molecular weight determination of the polysaccharide extracted from the bark of *Paeonia lactiflora* in Example 1.

[0110] High-performance liquid chromatography-size exclusion chromatography coupled with laser light scattering and differential detector (HPSEC-MALLS-RI) in series can directly determine the absolute molecular weight, dispersion index, root mean square radius of rotation, and conformation in solution of polysaccharides without the need for reference standards.

[0111] (1) Sample solution preparation

[0112] Accurately weigh 10.0 mg of polysaccharide sample and dilute to 1.0 mL to prepare a 10.0 mg / mL sample solution. Filter all solutions through a 0.22 μm microporous membrane and then perform high-performance liquid chromatography (HPLC) for size exclusion analysis.

[0113] (2) Chromatographic analysis conditions

[0114] Instrument: Agilent 1260 high performance liquid chromatography;

[0115] Chromatographic column: The chromatographic column was Shodex Ohpak 806HQ (13μm, 8.0mM×300mM) and Shodex Ohpak...

[0116] 804HQ (10μm, 8.0mM × 300mM) used in series;

[0117] Mobile phase: 0.2M NaCl;

[0118] Flow rate: 1.0 mL / min;

[0119] Detectors: (RID-G7162A) Differential detector and DAWN detector;

[0120] Use specialized software to collect and calculate data;

[0121] Results analysis:

[0122] refer to Figure 5(The dRI curve in the figure is the result of differential refractive index detection.) The molecular weight distribution range of the bean polysaccharide prepared in Example 1 is 10.35-14.27 kDa (accounting for 10% of the total), 14.27-70.51 kDa (accounting for 80% of the total), and 70.51-238.31 kDa (accounting for 10% of the total). It belongs to the medium molecular weight polysaccharide (10-100 kDa range), with a large number of hydroxyl groups, moderate molecular flexibility, easy formation of free radical stable structure, and good electron transfer ability.

[0123] 8. Testing of the monosaccharide composition of the *Dendrobium nobile* polysaccharide prepared in Example 1

[0124] Information on the instruments, reagents, and standards used in the experiment is shown in Table 5-7:

[0125] Table 5 Instrument Information Sheet

[0126] Ion Chromatograph Thermo Fisher ICS5000 Electric constant temperature drying oven Lichen Scientific Instruments (Zhejiang) Co., Ltd. 101-1BS Nitrogen blowing device Lichen Scientific Instruments (Zhejiang) Co., Ltd. UGC-24M Electronic balance Sartorius BS 210 S centrifuge Thermo Fisher D-37520 pipette Dalong Xingchuang Experimental Instruments (Beijing) Co., Ltd. 19050983

[0127] Table 6 Reagent Information Sheet

[0128] Trifluoroacetic acid (TFA) ACROS A0356762 139725000 AR 50% sodium hydroxide solution (50% NaOH) Alfa Aesar Z21E036 33382 GR Sodium acetate (NaOAc) Thermo Fisher 191126 059326 GR

[0129] Table 7 Standard Product Information Table

[0130]

[0131] Preparation and calculation methods of standard solutions

[0132] A standard stock solution was prepared by acid hydrolysis of 15 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosyl hydrochloride, glucosamine hydrochloride, guluronic acid, and mannuronic acid).

[0133] Precisely prepare concentration standards from the standard solutions of each monosaccharide as a mixed standard. Determine the mass of different monosaccharides using an absolute quantification method, and calculate the molar ratio according to Equation 2 (molar mass of monosaccharides).

[0134] Formula 4

[0135] In Equation 4, C (standard) is the concentration of the standard, A (standard) is the peak area of ​​the standard, C (sample) is the concentration of the sample, and A (sample) is the peak area of ​​the sample.

[0136] Sample preparation

[0137] Accurately weigh 5 mg of sample into an ampoule, add 2 mL of 3M TFA, and hydrolyze at 120 °C for 3 h. Accurately pipette the acid hydrolysis solution into a tube and blow it dry under nitrogen. Add 5 mL of water and vortex to mix. Pipe 50 μL of the sample into 950 μL of deionized water, centrifuge at 12000 rpm for 5 min, and filter the supernatant through a 0.22 μm microporous membrane for HPAEC analysis.

[0138] Chromatographic methods

[0139] Column: Dionex Carbopac™ PA20 (3mM*150mM);

[0140] Mobile phase: A: H2O; B: 15mM NaOH; C: 15mM NaOH & 100mM NaOAc;

[0141] Flow rate: 0.3 mL / min;

[0142] Injection volume: 25 μL;

[0143] Column temperature: 30 ℃;

[0144] The elution gradients are as follows: 0 min A / B / C phase (98.8:1.2:0, V / V), 18 min A / B / C phase (98.8:1.2:0, V / V), 20 min A / B / C phase (50:50:0, V / V), 30 min A / B / C phase (50:50:0, V / V), 30.1 min A / B / C phase (0:0:100, V / V), 46 min A / B / C phase (0:0:100, V / V), 46.1 min A / B / C phase (0:100:0, V / V), 50 min A / B / C phase (0:100:0, V / V), 50.1 min A / B / C phase (98.8:1.2:0, V / V), 80 min Phase A / Phase B / Phase C (98.8:1.2:0, V / V).

[0145] Detector: Electrochemical detector.

[0146] The standard product sequence is shown in Table 8.

[0147] Table 8 Standard Product Sequence

[0148]

[0149] Results analysis:

[0150] Standard diagram of monosaccharide mixtures as follows Figure 6As shown, the signal peak at 2.0 min is the solvent peak of sodium hydroxide, and the chromatographic peak at 40 min is the signal peak of sodium acetate.

[0151] The monosaccharide composition of the sample is as follows Figure 7 As shown in Table 9, the proportions of each monosaccharide are as follows.

[0152] Table 9. Results of Monosaccharide Composition Test

[0153] galactosamine 7.179 10.134 0.221 1.389 Rhamnose 0.344 10.642 0.087 0.418 Glucosamine 3.018 12.95 0.117 0.736 Galactose 0.625 15.059 0.117 0.613 glucose 4.391 17.209 0.333 1.753 Glucuronic acid 0.998 45.275 0.125 0.710

[0154] Monosaccharide composition analysis showed that the polysaccharides extracted from *Paeonia suffruticosa* bark mainly consisted of glucose, galactosamine, glucuronic acid, glucosamine, galactose, and rhamnose. Glucose was the primary monosaccharide component, while also containing a certain proportion of amino sugars and uronic acid monosaccharides, classifying it as a heteropolysaccharide containing both amino sugars and uronic acids.

[0155] Structurally, this polysaccharide molecule contains a large number of hydroxyl groups. Hydroxyl groups can participate in free radical scavenging processes and, to some extent, provide a source of hydrogen atoms, forming an important structural basis for the polysaccharide's antioxidant activity. The amino sugar residues in the molecule help enhance electron transfer capabilities and improve its reducing properties. Simultaneously, the carboxyl group of glucuronic acid can coordinate with metal ions, playing a regulatory role in oxidation reactions involving metal ions.

[0156] Based on the above structural characteristics, the polysaccharide extracted from the bark of *Paeonia lactiflora* exhibits significant DPPH free radical scavenging ability, ABTS cationic free radical scavenging ability, and iron ion reducing ability, demonstrating good antioxidant effects.

Claims

1. A kind of bean polysaccharide, characterized in that, After drying, crushing and sieving the skin and / or flesh of the bean peony, water extraction was performed. After water extraction, the supernatant was collected by centrifugation. Then, the protein in the supernatant was removed. After removing the protein, alcohol precipitation was performed, and the precipitate was collected to obtain bean peony polysaccharide.

2. The soybean polysaccharide according to claim 1, characterized in that, After drying, crushing and sieving, the bean bark is extracted with water. After extraction, the supernatant is collected by centrifugation. Then, the protein in the supernatant is removed. After removing the protein, the precipitate is precipitated with alcohol and collected to obtain bean polysaccharide.

3. A method for preparing the soybean polysaccharide according to any one of claims 1-2, characterized in that, Includes the following steps: Step 1: Wash the bean skin and / or bean flesh, then dry, crush and sieve to obtain powder; Step 2: Mix the powder with distilled water at a mass ratio of 1:25-35, and heat at 70-90℃ for 2-4 hours to obtain an intermediate solution; Step 3: Centrifuge the intermediate solution to obtain the supernatant, then mix the supernatant with Sevage reagent, centrifuge again and obtain the supernatant after removing the protein; Step 4: Concentrate the supernatant after removing the protein to 0.25-0.35 of the original volume, then add ethanol for precipitation, collect the precipitate, and obtain the bean polysaccharide.

4. Use of the soybean polysaccharide as described in any one of claims 1-2 in the preparation of antioxidant products.

5. The use according to claim 4, characterized in that, The antioxidant product is an antioxidant product with free radical scavenging ability.

6. The use according to claim 4, characterized in that, The polysaccharide in the antioxidant product is extracted from the bark of the soybean plant. The molecular weight distribution range of the polysaccharide extracted from the bark of the dung bean is 10–250 kDa. Its monosaccharide composition consists of glucose, galactosamine, glucuronic acid, glucosamine, galactose, and rhamnose.

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