A homogeneous polysaccharide from zosterophyllus vladimirii and preparation method and use thereof

Abstract

The present application provides a kind of Cardamine macrophylla Willd. Uniform polysaccharide, it is derived from the whole grass of Brassicaceae Pteryxia plant Cardamine macrophylla Willd., and the preparation method and purpose of the Cardamine macrophylla Willd. Uniform polysaccharide are provided.The present application realizes the efficient extraction of Cardamine macrophylla Willd. Uniform polysaccharide by single factor multi-level experiment combined with Box-Behnken central combination design-response surface method optimization of high pressure auxiliary low eutectic solvent extraction Cardamine macrophylla Willd. Uniform polysaccharide optimum extraction process parameter.The Cardamine macrophylla Willd. Uniform polysaccharide prepared in the present application is rich in galacturonic acid glycan and I type rhamnose galacturonic acid glycan acidic polysaccharide, has good antioxidant activity and immune regulation activity.

Description

Technical Field

[0001] This invention relates to a homogeneous polysaccharide of *Gnaphalium affine*, its preparation method and uses, and belongs to the field of health food or pharmaceuticals. Background Technology

[0002] Cardamine macrophylla Willd., commonly known as the large-leaved stonecress, is a perennial herb belonging to the Brassicaceae family, growing 30-100 cm tall. Its rhizome is creeping and densely covered with fibrous roots. The stem is relatively stout, cylindrical, erect, sometimes prostrate at the base, unbranched or branched in the upper part, with grooved ridges on the surface. The whole plant of Cardamine macrophylla has edible and medicinal value, and is sweet, bland, and neutral in nature, possessing the effects of strengthening the spleen, promoting diuresis and reducing swelling, cooling the blood and stopping bleeding. It is harvested in spring and summer, washed, and used fresh or dried. Currently, research on Cardamine macrophylla mainly focuses on its nutritional components and physiological characteristics, while research on the structural characteristics and biological activity of its polysaccharide active substances is currently unreported in the literature.

[0003] Pectin-type acidic polysaccharides are mainly found in plant cell walls, playing a role in maintaining cell wall structure and strength. They are primarily composed of galacturonic acid polysaccharides, type I rhamnose-galacturonic acid polysaccharides, type II rhamnose-galacturonic acid polysaccharides, and xylose-galacturonic acid polysaccharides, as well as multiple branched side chains. Studies have shown that pectin-type acidic polysaccharides possess various biological activities, such as antioxidant, immunomodulatory, anti-inflammatory, anti-tumor, lipid-lowering, and gut microbiota regulation. Therefore, pectin-type acidic polysaccharides have attracted increasing attention in the development of functional foods. However, current extraction techniques for pectin-type acidic polysaccharides still need improvement. Traditionally, hot water extraction is commonly used. Since polysaccharides are highly polar and easily soluble in water, hot water extraction is frequently employed. This method requires relatively simple equipment and procedures, but its disadvantages include long extraction time, high energy consumption, and low extraction efficiency. Furthermore, hot water extraction lacks selectivity, resulting in non-homogeneous polysaccharides (low purity).

[0004] Deep eutectic solvents are mixtures composed of hydrogen bond acceptors and hydrogen bond donors that, when mixed in specific proportions, form eutectic mixtures with melting points significantly lower than those of their individual components. These solvents possess unique physicochemical properties, such as high solubility, thermal stability, biodegradability, and ease of synthesis. Deep eutectic solvents can be used as solvents for extracting polysaccharides due to their non-toxic or low-toxicity, chemical and thermal stability, non-flammability, high solubility, and low melting point. They are also cost-effective, safe, and offer higher extraction rates than traditional extraction solvents. Deep eutectic solvent extraction offers advantages such as high selectivity, high yield, and high-quality final product. Some studies also utilize ultrasound, microwave, or high-pressure extraction to assist in the extraction process. High-pressure assisted extraction offers advantages such as rapid heating, short extraction time, and high extraction efficiency. Therefore, high-pressure assisted deep eutectic solvent extraction can be used to efficiently prepare highly active homogeneous polysaccharides from *Gnaphalium affine*.

[0005] Currently, there are few reports on polysaccharides from *Symplocos buergeriana*, with only one paper published by Cai Xiaoxia in 2021 (Southwest University for Nationalities) regarding the quality research of *Symplocos buergeriana*, a Tibetan and Qiang medicinal herb, based on multi-index comprehensive analysis. This paper disclosed an optimized extraction process for *Symplocos buergeriana* polysaccharides. However, this paper used hot water extraction, which is energy-intensive, has low extraction efficiency, and lacks selectivity. Furthermore, the optimal extraction time for polysaccharides from the aerial parts of *Symplocos buergeriana* was 200 min, and for polysaccharides from the underground parts, it was 180 min. The extraction method reported in this paper requires a long time to achieve optimal extraction efficiency and did not perform structural characterization or activity evaluation of the *Symplocos buergeriana* polysaccharides. Therefore, it is necessary to provide an efficient and selective method for preparing homogeneous polysaccharides from *Symplocos buergeriana*, and to perform structural characterization and activity evaluation on these polysaccharides. Summary of the Invention

[0006] This invention provides a homogeneous polysaccharide from *Gnaphalium affine*, its preparation method, and its uses.

[0007] This invention provides a homogeneous polysaccharide derived from the whole herb of Cardamine macrophylla Willd., a plant belonging to the genus Cardamine in the Brassicaceae family. The homogeneous polysaccharide contains 90.16±1.57 mg of total polysaccharides per 100 mg.

[0008] The homogeneous polysaccharide contains 24.91±2.77 mg of total uronic acid, 1.80±0.14 mg of total protein, and 0.98±0.21 mg of total conjugated phenols per 100 mg (GAE equivalent).

[0009] The degree of esterification of the homogeneous polysaccharide from *Gnaphalium affine* was 33.01% ± 2.11%. Molecular weight Mw 4.120×10 4 Da-4.159×10 4 Da, polydispersion coefficient M w / M n The values ​​range from 2.206 to 2.233.

[0010] The homogeneous polysaccharide mainly consists of the following monosaccharides: galacturonic acid, galactose, arabinose, rhamnose, xylose, glucose, glucuronic acid, and mannose. The molar percentages of each monosaccharide are as follows: (28.05%-31.66%): (28.12%-31.86%): (16.45%-18.48%): (6.46%-6.77%): (5.95%-5.96%): (5.26%-5.55%): (0.56%-0.66%): (3.20%-5.01%). Among these, galacturonic acid polysaccharide accounts for 21.59%-24.89%, and type I rhamnose galacturonic acid polysaccharide accounts for 60.14%-61.23%.

[0011] This invention also provides a method for preparing the homogeneous polysaccharide of *Gnaphalium affine*, which includes the following steps:

[0012] a. Take fresh *Gnaphalium affine*, freeze-dry it, grind it into powder, and sift it;

[0013] b. Add ethanol to the powder and remove the alcohol-soluble components by ultrasonication to obtain a precipitate;

[0014] c. High-pressure assisted eutectic solvent extraction: Take the precipitate prepared in step b, add the prepared extraction solvent, and extract under high pressure to obtain the extract;

[0015] d. Add α-amylase and saccharifying enzyme to the concentrate to remove starch;

[0016] e. Add trypsin to the concentrate to remove proteins;

[0017] f. Inactivation;

[0018] g. Take the supernatant, add ethanol, precipitate with ethanol, and let stand overnight; after centrifugation, obtain the precipitate, and then wash the precipitate with ethanol;

[0019] h. Add water to redissolve, separate by membrane, and dry to obtain homogeneous polysaccharide of *Gnaphalium affine*.

[0020] More preferably,

[0021] a. The freeze-drying conditions in this step are -30℃ to -70℃ for 40 to 70 hours; the sieve mesh size is 60 to 80 mesh.

[0022] b. The final concentration of ethanol added in the step is 20% to 90% ethanol; the weight-to-volume ratio of powder to ethanol is 1:(10-30); the ultrasonic power is 300W-800W; the ultrasonic time is 20min-60min.

[0023] c. High-pressure assisted extraction with a eutectic solvent, the extraction time is 30-70 min, and the ratio of extraction solvent to raw material is 10 mL / g-50 mL / g; wherein, the eutectic solvent is composed of choline chloride, ethylene glycol and ultrapure water, and the molar ratio of choline chloride to ethylene glycol is 1:3; the water content of the eutectic solvent is 20%-60%, the extraction pressure is 0.1 MPa, and the extraction temperature is 121℃;

[0024] d. The conditions for removing starch by α-amylase and saccharifying enzyme in the steps are as follows: α-amylase with 5 U / mL-20 U / mL, enzymatic hydrolysis temperature of 60℃-95℃, and enzymatic hydrolysis time of 3h-12h; saccharifying enzyme with 5 U / mL-20 U / mL, enzymatic hydrolysis temperature of 40℃-65℃, and enzymatic hydrolysis time of 10h-14h.

[0025] e. The conditions for trypsin to remove protein in the step are: 1 U / mL-10 U / mL trypsin, enzymatic hydrolysis temperature of 30℃-60℃, and enzymatic hydrolysis time of 3h-12h.

[0026] f. Inactivation conditions: 95℃, 30min;

[0027] g. In the step, the volume ratio of alcohol precipitation extract to ethanol is 1:(2-5), and the concentration of ethanol used is 50%-100% ethanol; the ethanol used to wash the precipitate is 50%-95% ethanol;

[0028] h. The membrane used for membrane separation in step h has a molecular weight cutoff of 1000 Da-3000 Da.

[0029] More preferably,

[0030] a. In the step, freeze-dry at -40℃ for 48 hours; sieve through an 80-mesh screen.

[0031] b. The ethanol added in this step is 80% ethanol; the weight-to-volume ratio of powder to ethanol is 1:10; the ultrasonic power is 480W; and the ultrasonic time is 30min.

[0032] c. High-pressure assisted eutectic solvent extraction, extraction time is 50 min, and the ratio of extraction solvent to raw material is 42 mL / g; wherein, the eutectic solvent is composed of choline chloride, ethylene glycol and ultrapure water, and the molar ratio of choline chloride to ethylene glycol is 1:3, the water content of the eutectic solvent is 40%, the extraction pressure is 0.1 MPa, and the extraction temperature is 121℃;

[0033] d. In this step, the amount of α-amylase added is 10 U / mL, the enzymatic hydrolysis temperature is 90℃, and the enzymatic hydrolysis time is 8 h; the amount of saccharifying enzyme added is 10 U / mL, the enzymatic hydrolysis temperature is 59℃, and the enzymatic hydrolysis time is 12 h.

[0034] e. The amount of trypsin added in this step is 5 U / mL, the enzymatic hydrolysis temperature is 40℃, and the enzymatic hydrolysis time is 8h.

[0035] f. Inactivation conditions: 95℃, 30min;

[0036] g. The volume ratio of alcohol used for precipitation in step g is 1:4; the ethanol used is 95% ethanol; the ethanol used for washing the precipitate is 76% ethanol.

[0037] h. The membrane used for membrane separation in step h has a molecular weight cutoff of 3000 Da.

[0038] This invention relates to the extraction of homogeneous polysaccharides from *Stachys macrocarpa* using a high-pressure assisted eutectic solvent method. The extraction rate of homogeneous polysaccharides from *Stachys macrocarpa* using this method was 5.70%-5.81%. The extraction rate of polysaccharides from *Stachys macrocarpa* using hot water extraction was 2.71%-2.85%. Compared to hot water extraction, the high-pressure assisted eutectic solvent method increased the extraction rate by 103.86%-110.33%. Furthermore, the optimal extraction time for the high-pressure assisted eutectic solvent method was 50 minutes, significantly shorter than the 2 hours required for hot water extraction, thus improving extraction efficiency.

[0039] The present invention also provides the use of the aforementioned homogeneous polysaccharide of *Gnaphalium affine* in the preparation of drugs or health foods with antioxidant or immunomodulatory activities.

[0040] The present invention also provides a health food that helps with anti-oxidation or helps enhance immunity, which contains the homogeneous polysaccharide of the large-leaved stonewort.

[0041] The homogeneous polysaccharide of *Gnaphalium affine* described in this invention is an acidic polysaccharide with a single symmetrical chromatographic peak, mainly composed of galacturonic acid polysaccharide and type I rhamnose galacturonic acid polysaccharide.

[0042] The beneficial effects of this invention are:

[0043] 1. This invention employs a eutectic solvent for extraction. Eutectic solvents generally have lower toxicity and are more suitable for food and pharmaceutical extraction compared to traditional organic solvents. Furthermore, the composition and ratio of the eutectic solvent can be adjusted to optimize the extraction conditions for specific polysaccharides. Polysaccharides extracted using eutectic solvents may exhibit higher bioactivity, while water extraction may not maximize the preservation of polysaccharide bioactivity.

[0044] 2. This invention uses high-pressure assisted eutectic solvent extraction to extract homogeneous polysaccharides from *Gnaphalium affine*. Compared with traditional extraction methods, the high-pressure extraction process is shorter, which can save extraction time and improve work efficiency.

[0045] 3. The molecular weight chromatogram of the homogeneous polysaccharide of the large-leaved stonegrass of the present invention shows a single symmetrical chromatographic peak, which means that the polysaccharide in the sample has high purity, that is, there is mainly a specific polysaccharide molecule, without interference from polysaccharides or impurities of other molecular weights. This helps to ensure its consistency in biological function and application.

[0046] The polysaccharide extract of this invention is a homogeneous polysaccharide with high purity. Furthermore, starch-like substances have been removed. Starch-like substances in polysaccharides can cross-react with other polysaccharides or biomolecules, interfering with the analysis and evaluation of the polysaccharides. Moreover, polysaccharides possess specific biological activities, such as immunomodulatory effects, and the presence of starch may reduce these activities or alter the bioavailability of the polysaccharides. Therefore, removing starch from polysaccharides yields a purer polysaccharide product with more specific functions. Attached Figure Description

[0047] Figure 1 Process flow diagram of high-pressure assisted eutectic solvent extraction;

[0048] Figure 2 Effects of extraction time (A), solid-liquid ratio (B), and water content of eutectic solvent (C) on the extraction rate of homogeneous polysaccharides from *Gnaphalium affine* using high-pressure assisted eutectic solvent extraction;

[0049] Figure 3 Three-dimensional stereographic plots (A, B, C) and two-dimensional contour plots (D, E, F) of the response surface to various factors on the homogeneous polysaccharide extraction rate of *Gnaphalium affine*.

[0050] Figure 4 Size exclusion chromatogram (A) of DYP polysaccharide extracted by high pressure-assisted eutectic solvent extraction, high performance liquid chromatogram (B) and Fourier transform infrared spectrum (C) of the constituent monosaccharides;

[0051] Figure 5 High-pressure assisted eutectic solvent extraction of DYP (dichloro ... 1 H nuclear magnetic resonance spectroscopy and 13 C nuclear magnetic resonance spectrum;

[0052] Figure 6 The scavenging rate (A) and total reducing power (B) of ABTS by homogeneous polysaccharide (DYP) extracted from *Gnaphalium affine* under high pressure-assisted eutectic solvent extraction;

[0053] Figure 7Effects of high-pressure assisted eutectic solvent extraction of homogeneous polysaccharide (DYP) from *Gnaphalium affine* on cytotoxicity (A), nitric oxide (NO) production (B), interleukin-6 (IL-6) production (C), and tumor necrosis factor-α (TNF-α) production (D) in RAW 264.7 macrophages.

[0054] Figure 8 Effects of high-pressure assisted eutectic solvent extraction of homogeneous polysaccharide (DYP) from *Gnaphalium affine* on the secretion of NO (A), TNF-α (B), and IL-6 (C) in RAW264.7 macrophages treated with TAK-242. Detailed Implementation

[0055] Example 1: This invention uses a traditional hot water extraction method to prepare polysaccharides from *Sedum macrocephalum*.

[0056] (1) Ultrasonic degreasing: Fresh *Gnaphalium affine* was freeze-dried, ground into powder, and passed through an 80-mesh sieve; fat-soluble compounds were removed by ultrasonication at a ratio of *Gnaphalium affine* powder to 80% ethanol = 1:10, with an ultrasonic power of 480W and an ultrasonic time of 30min. After ultrasonication, the precipitate was retained after centrifugation at 5000g for 10min.

[0057] (2) Extraction: The material-to-liquid ratio was 1:30 (w / v), the extraction temperature was 95℃, and the extraction time was 2h. After extraction, the sample was centrifuged at 4500g for 15min and the supernatant was retained.

[0058] (3) In the steps, the amount of α-amylase added was 10 U / mL, the enzymatic hydrolysis temperature was 90℃, and the enzymatic hydrolysis time was 8h; the amount of saccharifying enzyme added was 10 U / mL, the enzymatic hydrolysis temperature was 59℃, and the enzymatic hydrolysis time was 12h.

[0059] (4) The amount of trypsin added in the step is 5 U / mL, the enzymatic hydrolysis temperature is 40℃, and the enzymatic hydrolysis time is 8h;

[0060] (5) Inactivation: 95℃ for 30 min; centrifuge at 4500g for 15 min and retain the supernatant.

[0061] (6) Alcohol precipitation: Add 4 times the volume of 95% ethanol to the extract, and let it stand overnight after alcohol precipitation.

[0062] (7) Reconstitution: Remove the supernatant, retain the precipitate, centrifuge at 5000g for 10min to obtain the precipitate, then wash the precipitate with 76% ethanol; then reconstitute with ultrapure water. Take the supernatant and remove small molecules (molecular weight cutoff, 3000Da) by membrane separation. Freeze-dry to obtain the polysaccharide of *Gnaphalium affine*.

[0063] The extraction rate of *Gnaphalium affine* using traditional hot water extraction is 2.71%-2.85%.

[0064] Example 2: Extraction of homogeneous polysaccharide (hereinafter referred to as DYP) from *Stachys macrocarpa* using the high-pressure assisted eutectic solvent extraction method of the present invention.

[0065] (1) Ultrasonic degreasing: Fresh *Gnaphalium affine* was freeze-dried, ground into powder, and passed through an 80-mesh sieve; fat-soluble compounds were removed by ultrasonication at a ratio of *Gnaphalium affine* powder to 80% ethanol = 1:10, with an ultrasonic power of 480W and an ultrasonic time of 30min. After ultrasonication, the precipitate was retained after centrifugation at 5000g for 10min.

[0066] (2) Extraction: The homogeneous polysaccharide from *Gnaphalium affine* was extracted using a high-pressure assisted eutectic solvent at a material-to-liquid ratio of 1:42 (w / v) for 50 min. The eutectic solvent consisted of choline chloride, ethylene glycol, and ultrapure water. The molar ratio of choline chloride to ethylene glycol was 1:3, the water content of the eutectic solvent was 40%, the extraction pressure was 0.1 MPa, and the extraction temperature was 121℃. After extraction, the sample was centrifuged at 4500g for 15 min, and the supernatant was retained.

[0067] (3) In the steps, the amount of α-amylase added was 10 U / mL, the enzymatic hydrolysis temperature was 90℃, and the enzymatic hydrolysis time was 8h; the amount of saccharifying enzyme added was 10 U / mL, the enzymatic hydrolysis temperature was 59℃, and the enzymatic hydrolysis time was 12h.

[0068] (4) The amount of trypsin added in the step is 5 U / mL, the enzymatic hydrolysis temperature is 40℃, and the enzymatic hydrolysis time is 8h;

[0069] (5) Inactivation: 95℃ for 30 min; centrifuge at 4500g for 15 min and retain the supernatant.

[0070] (6) Alcohol precipitation: Add 4 times the volume of 95% ethanol to the extract, and let it stand overnight after alcohol precipitation.

[0071] (7) Reconstitution: Remove the supernatant, retain the precipitate, centrifuge at 5000g for 10min to obtain the precipitate, then wash the precipitate with 76% ethanol; then reconstitute with ultrapure water. Take the supernatant and remove small molecules (molecular weight cutoff, 3000Da) by membrane separation. Freeze-dry to obtain homogeneous polysaccharide of *Gnaphalium affine*. (See the preparation process flow chart) Figure 1 )

[0072] Example 3: Screening experiment of condition parameters for high-pressure assisted eutectic solvent extraction method:

[0073] The prepared extraction solvent (choline chloride: ethylene glycol = 1:3) was added to the precipitate after ultrasonic defatting, and the mixture was extracted in a laboratory autoclave. A single-factor experimental design was used to optimize the extraction method, and the conditions are shown in Table 1. In summary, the solid-liquid ratio and the water content of the eutectic solvent were determined first, and then the extraction time was optimized. (See the single-factor optimization diagram below.) Figure 2 )

[0074] Table 1 Single-factor experimental design table

[0075]

[0076] Secondly, a three-factor response surface methodology was employed to further optimize the effects of various extraction parameters on the homogeneous polysaccharide extraction rate of *Gnaphalium affine*. Independent variables included extraction time (X1, 40, 50, 60 min), solid-liquid ratio (X2, 30, 40, 50 mL / g), and eutectic solvent water content (X3, 30, 40, 50%). Seventeen experiments were designed, and the response surface factor codes are shown in Table 2. The experimental data were then analyzed using Design-Expert. The obtained data were fitted using a second-order polynomial model, as shown in Table 2.

[0077] Table 2. Response Surface Experimental Design and Results

[0078]

[0079]

[0080] Compared with traditional extraction methods, high-pressure assisted extraction (HPLC) of polysaccharides offers advantages such as high efficiency and energy saving. Therefore, this study utilized HPLC and a eutectic solvent (DYP) as extraction solvents to extract homogeneous polysaccharides from *Gnaphalium affine*. Extraction time, solid-liquid ratio, and the water content of the eutectic solvent significantly affected the extraction rate of homogeneous polysaccharides from *Gnaphalium affine* using HPLC-assisted eutectic solvent extraction. When the extraction time increased from 30 min to 50 min, the DYP extraction rate increased. This is because as the HPLC extraction time increased, the polysaccharides were gradually extracted from the sample. However, after 50 min, the extraction rate gradually decreased with increasing time, possibly due to polysaccharide degradation caused by prolonged HPLC extraction. When the solid-liquid ratio was too high, excessive solvent could lead to over-dilutement of the active ingredients in the raw material, thus reducing the actual extraction efficiency. Therefore, the optimal conditions optimized in this experiment were 1:42. High water content in the eutectic solvent affected the interaction between the solvent and the polysaccharides, while low water content resulted in an overly viscous solvent. Therefore, the final optimized conditions were 40%.

[0081] Based on the results of the single-factor experiment, a Box-Behnken experimental design was further used to optimize the extraction rate of DYP. The final BBD experimental data are shown in Table 3. The derived second-order polynomial equation is as follows:

[0082] Y=-37.94688+1.03466X1+0.106933X2+0.753120X3+0.001445X1X2-0.00060

[0083] 2X1X3-0.001072X2X3-0.010592X1 2 -0.001631X2 2 -0.008329X3 2

[0084] Where Y represents the extraction rate; X1, X2, and X3 are the extraction time (min), solid-liquid ratio (mL / g), and eutectic solvent water content (%), respectively.

[0085] Table 3. Analysis of variance of the regression model for high-pressure assisted eutectic solvent extraction.

[0086]

[0087] Note: X1: Extraction time (min); X2: Solid-liquid ratio (mL / g); X3: Water content of eutectic solvent (%)

[0088] R 2 =0.9988, R 2 adj =0.9972, coefficient of variation (CV) =0.83%, adeq.precision =72.1287.

[0089] *Significant difference (p < 0.05), **Highly significant difference (p < 0.01).

[0090] As shown in Table 3, one-way ANOVA was used to evaluate the influence of extraction parameters on the extraction rate of *Gnaphalium affine* and the validity of the fitted model. Based on the p-value (<0.0001) and F-value (625.01), the fitted model was highly significant. Furthermore, the values ​​of the lack-of-fit term, coefficient of determination, and corrected coefficient of determination demonstrated that the model had a high good fit. Additionally, the values ​​of the coefficient of variation and moderate precision also demonstrated that the fitted model had good repeatability and reliability. Moreover, the p-values ​​of the linear coefficients, interaction coefficients, and quadratic coefficients of all extraction factors were less than 0.05, indicating that all extraction parameters significantly affected the extraction rate of homogeneous polysaccharides from *Gnaphalium affine*. In fact, the three-dimensional response surface... Figure 3 A, B, C and two-dimensional contour lines Figure 3Figures D, E, and F further indicate significant interactions between extraction time and the solid-liquid ratio, extraction time and the eutectic solvent water content, and the solid-liquid ratio and the eutectic solvent water content. All these data suggest that extraction time, solid-liquid ratio, and eutectic solvent water content are important parameters affecting the extraction of homogeneous polysaccharides from *Gnaphalium affine*. Based on the analysis of the experimental results, the predicted optimal extraction conditions were determined as follows: extraction time of 50.536 min, solid-liquid ratio of 1:41.766 mL / g, and eutectic solvent water content of 40.691%. Considering the operability of the actual process, the verification experiment was conducted under the conditions of an extraction time of 50 min, a solid-liquid ratio of 1:42 mL / g, and a eutectic solvent water content of 40%. Under these conditions, the actual extraction rate was 5.765% ± 0.12%, very close to the predicted value of 5.754%.

[0091] Example 4: Chemical composition of homogeneous polysaccharide from *Gnaphalium affine* of the present invention.

[0092] Total polysaccharides, total uronic acids, total bound phenols, and total protein in homogeneous polysaccharides from *Gnaphalium affine* were determined using colorimetric methods. The molecular weight and dispersibility of diethylstilbestrol (DYP) were determined using SEC-MALLS-RID (Wyatt Technology Co., Santa Barbara, CA, USA). The monosaccharide composition of the polysaccharide samples was determined using high-performance liquid chromatography (L-20A, Shimadzu, Japan) combined with pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP). The functional groups and degree of esterification of the samples were analyzed by Fourier transform infrared spectroscopy (PerkinElmer, Waltham, MA, USA). The glycosidic bond types of the polysaccharides were analyzed using nuclear magnetic resonance (NMR) analysis (Bruker, Rheinstetten, Germany). For specific methodological references: Authors: Ding-Tao Wu, Meng-Xi Fu, Huan Guo, Yi-Chen Hu, Xiao-Qin Zheng, Ren-You Gan and LiangZou; Title: Microwave-assisted deep eutectic solvent extraction, structural characteristics, and biological functions of polysaccharides from sweet tea (Lithocarpus litseifolius) leaves; Journal: ANTIOXIDANTS, Volume 11, Issue 8; DOI: 10.3390 / antiox11081578.

[0093] Table 4. Chemical composition, molecular weight, and monosaccharide composition of homogeneous polysaccharides from *Gnaphalium affine* extracted by high-pressure assisted eutectic solvent extraction.

[0094]

[0095] HG, galacturonic acid polysaccharide, HG (%) = GalA (%) - Rha (%);

[0096] RG-I, type I rhamnose galacturonic acid polysaccharide, RG-I (%) = 2 * Rha (%) + Gal (%) + Ara (%)

[0097] The homogeneous polysaccharide DYP from *Gnaphalium affine* was obtained by high-pressure assisted eutectic solvent extraction, and its chemical composition was determined. DYP contained 90.16±1.57 mg of total polysaccharides, 24.91±2.77 mg of total uronic acids, 1.80±0.14 mg of total protein, and 0.98±0.21 mg of total bound phenols (GAE).

[0098] To further understand the structural characteristics of DYP, we investigated its molecular weight, functional groups, monosaccharide composition, and glycosidic bonds using high-performance size exclusion chromatography, high-performance liquid chromatography, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy.

[0099] like Figure 4 As shown in Figure A, the molecular weight of DYP was determined by high-performance size exclusion chromatography (HPLC). The DYP molecular weight chromatogram shows a single symmetrical peak, indicating that the polysaccharide in the sample has high purity, meaning that it mainly contains a specific polysaccharide molecule without interference from other polysaccharides or impurities. Molecular weight M w 4.120×10 4 Da-4.159×10 4 Da, polydispersion coefficient M w / M n The values ​​range from 2.206 to 2.233.

[0100] like Figure 4As shown in B, the main sugar components of DYP are the following monosaccharides: galacturonic acid, galactose, arabinose, rhamnose, xylose, glucose, glucuronic acid, and mannose. The molar percentages of each monosaccharide are: (28.05%-31.66%): (28.12%-31.86%): (16.45%-18.48%): (6.46%-6.77%): (5.95%-5.96%): (5.26%-5.96%). 55%): (0.56%-0.66%): (3.20%-5.01%); among which, galacturonic acid polysaccharide accounted for 21.59%-24.89% and type I rhamnose galacturonic acid polysaccharide accounted for 60.14%-61.23%. The experimental results showed that the sample obtained by high pressure assisted eutectic solvent extraction was mainly composed of polysaccharides, and was a pectin-type acidic polysaccharide rich in galacturonic acid polysaccharide and type I rhamnose galacturonic acid polysaccharide.

[0101] FT-IR spectroscopy is often used to compare the preliminary structural properties of polysaccharides. For example... Figure 4 As shown in Figure C, DYP exhibits typical signals of pectin polysaccharides. In short, the FT-IR spectrum of DYP is in the range of 4000-400 cm⁻¹. -1 The wavelength range showed typical absorption peak characteristics of polysaccharides. At 3410.6 cm⁻¹... -1 and 2949.9cm -1 The broad peaks at 1745.1 cm⁻¹ are caused by the stretching vibrations of the hydroxyl group and the asymmetric stretching vibrations of the CH bond, respectively. -1 and 1623.4cm -1 The absorption band at 1447.3 cm⁻¹ is caused by the stretching vibrations of the esterified groups C=O and -COOR, confirming that DYP is mainly composed of acidic polysaccharides. Meanwhile, at 1447.3 cm⁻¹... -1 and 1245.5cm -1 The absorption peaks at 1101.4 cm⁻¹ are attributed to CH / OH and -OCH₃, respectively. -1 and 1018.1cm -1 The absorption peak at 1745.1 cm⁻¹ is attributed to the absorption of pyranoside. Furthermore, based on the... -1 and 1623.4cm -1 Based on the signal intensity, the degree of esterification of DYP was calculated to be 33.01% ± 2.11%.

[0102] To better understand the structure of DYP, 1D NMR analysis was performed. Figure 5As shown, both α and β configurations exist in DYP. Signals at 1.24 ppm, 5.25 ppm, and 16.61 ppm are attributed to H-6, H-1, and C-6 of 1,2,4-α-L-Rhap. Signals at 4.97 ppm, 100.31 ppm, and 170.65 ppm are attributed to H-1, C-1, and C-6 of 1,4-α-D-GalAMep. Signals at 5.03 ppm and 99.39 ppm are attributed to H-1 and C-1 of 1,4-α-D-GalAp. Signals at 5.09 ppm and 107.31 ppm are associated with H-1 and C-1 of 1,5-α-L-Araf. Signals at 5.15 ppm and 109.13 ppm are associated with H-1 and C-1 of T-α-L-Araf, respectively. The signals at 3.81 ppm and 52.75 ppm originate from the methyl ester of 6-O GalA (GalA-OCH3). The signal at 2.07 ppm represents the O-acetyl group of GalAp. Additionally, the signal at 20.08 ppm also represents the O-acetyl group. The signal at 4.46 ppm belongs to the H-1 group of 1,3,6-β-D-Galp. The signal at 4.53 ppm belongs to the H-1 group of 1,3-β-D-Galp. An H-1 signal of 1,4-β-D-Galp was observed at 4.62 ppm. In summary, these results confirm that DYP is mainly composed of galacturonic acid polysaccharides and type I rhamnose galacturonic acid polysaccharides, with galactan or arabinogalactan serving as side chains of type I rhamnose galacturonic acid polysaccharides.

[0103] The following efficacy tests demonstrate the beneficial effects of the present invention.

[0104] Experimental Example 1: Antioxidant Activity Test of the Homogeneous Polysaccharide DYP of the Invention from *Sedum macrocephalum*

[0105] 1. ABTS free radical scavenging ability:

[0106] Reagents: 7mM ABTS free radical cation solution, 2.45mM potassium persulfate solution.

[0107] Methods: The ABTS radical cation solution was prepared by mixing 7 mM ABTS solution and 2.45 mM potassium persulfate aqueous solution at a 1:1 volume ratio and reacting in the dark for at least 16 hours at room temperature. The ABTS radical cation solution was diluted with phosphate buffer (0.05 M, pH 6.6) to achieve an absorbance of 0.750 ± 0.02 at 734 nm. Then, in a 96-well microplate, 200 μL of the ABTS radical cation working solution was mixed with 20 μL of samples of different concentrations and shaken in the dark for 6 min, and the absorbance was measured at 734 nm. Vitamin C (Vc) was used as a positive control. The ABTS radical scavenging activity was calculated as follows:

[0108] ABTS free radical scavenging activity (%) = (1 - (A1 - A2) / A2) × 100%

[0109] Where A1 is the absorbance value of the sample group and A2 is the absorbance value of the blank group.

[0110] Experiments were conducted using different concentrations of DYP, and the IC50 values ​​were finally determined. 50 Value. And vitamin C was used as a positive control. For example... Figure 6 As shown in Figure A, DYP exhibits certain antioxidant activity. Specifically, the IC50 value of DYP's ABTS free radical scavenging ability... 50 The value was 5.02 mg / mL.

[0111] 2. Overall restoration capability:

[0112] Reagents: 1% potassium ferricyanide (prepared with 0.2M PBS solution, pH 6.6), 10% trichloroacetic acid, 0.1% ferric chloride

[0113] Method: Mix 100 μL of sample with 100 μL of 1% potassium ferricyanide and shake well. Incubate in a water bath at 50 °C for 20 min. Then, add 100 μL of 10% trichloroacetic acid, 300 μL of distilled water, and 60 μL of 0.1% ferric chloride sequentially. Incubate in a water bath at 50 °C for 30 min. Finally, take 200 μL of the reaction solution into a 96-well microplate and measure the absorbance at 700 nm. Use vitamin C (Vc) as a positive control.

[0114] This experiment reflects the reducing power by measuring the degree of color change; the darker the color and the larger the OD value, the stronger the reducing power. Figure 6 As shown in Figure B, the total reducing power of DYP increases with increasing concentration.

[0115] Experimental Example 2: Immunomodulatory Effect of the Homogeneous Polysaccharide DYP from *Gnaphalium affine* of this Invention

[0116] Reagents: DMEM medium, thiazolyl blue (MTT), dimethyl sulfoxide (DMSO), lipopolysaccharide (LPS), TAK-242, Griess kit, ELISA kit

[0117] 1. Cytotoxicity assay:

[0118] Methods: RAW 264.7 macrophages were cultured at a concentration of 5 × 10⁻⁶ cells / mL. 3Cells were cultured overnight in 96-well microplates at a concentration of [number] cells / well. The supernatant was aspirated, and 100 μL of DYP polysaccharide (25, 50, 100, 200, 400 μg / mL) was added to each well. Incubation was performed for 24 h. A culture medium blank served as a blank control, and LPS (1 μg / mL) served as a positive control. The supernatant was aspirated, and 100 μL of MTT solution (1 mg / mL) was added to each well. Incubation was performed for 4 h. The supernatant was aspirated, and 100 μL of DMSO was added to each well. OD was measured at 570 nm. Cell proliferation rate was calculated using the following formula:

[0119] Cell proliferation rate (%) = 1 - (A1 - A2) / A1 × 100%

[0120] Where A1 is the absorbance value of the blank group and A2 is the absorbance value of the sample group.

[0121] like Figure 7 As shown in Figure A, DYP showed no toxicity to RAW 264.7 macrophages at concentrations ranging from 25 to 400 μg / mL.

[0122] 2. Measurement of NO and cytokine levels:

[0123] RAW264.7 cells 1×10 5 Cells were cultured overnight in 24-well plates at a concentration of [number] cells / well. The supernatant was aspirated, and 1 mL of DYP polysaccharide (100, 200, or 400 μg / mL) was added to each well. The plates were incubated for 48 hours. A culture medium blank served as a control, and LPS (1 μg / mL) served as a positive control. Subsequently, the supernatant from each well was collected, and the levels of NO and cytokines (TNF-α and IL-6) were measured according to the kit instructions.

[0124] like Figure 7 As shown in B, C, and D, DYP stimulated the release of NO, TNF-α, and IL-6 from RAW 264.7 macrophages at concentrations ranging from 100 μg / mL to 400 μg / mL, with the increase occurring in a concentration-dependent manner.

[0125] 3. Potential Mechanism of Cellular Immune Function of DYP

[0126] Cells were spaced at 1×10⁶ cells per well. 5 Cells were cultured overnight in 24-well plates. After removing the culture medium, the wells were incubated with medium containing or without TAK-242 (1 μM) for 4 h. Then, the cells were treated with DYP (400 μg / mL) or LPS (1 μg / mL) for 48 h. The total NO content was determined by the Griess method, and the levels of TNF-α and IL-6 were determined by an ELISA kit.

[0127] TAK-242 is a small-molecule specific inhibitor of TLR4 that selectively binds to TLR4 and interferes with the interaction between TLR4 and its linker molecules. Previous studies have shown that a 1 μM concentration of TAK-242 has no toxic effect on RAW264.7 macrophages. Therefore, cells were treated with 1 μM TAK-242 for 4 h, and then the release of NO, TNF-α, and IL-6 from RAW 264.7 macrophages was measured after 48 h of treatment. Figure 8 As shown in A, B, and C, the addition of TAK-242 significantly inhibited the release of NO, TNF-α, and IL-6 from both DYP (400 μg / mL) and positive LPS (1 μg / mL). These results indicate that DYP can immunomodulate RAW264.7 macrophages through the TLR4 receptor.

[0128] In summary, this invention optimizes the method for extracting homogeneous polysaccharides from *Gnaphalium affine*. The DYP extracted using this method exhibits good antioxidant and immunomodulatory activities, providing a theoretical basis for its development into functional foods.

Claims

1. A homogeneous polysaccharide from *Gnaphalium affine*, characterized in that: It comes from the plant *Capsella macrantha*, belonging to the genus *Capsella* in the Brassicaceae family. Cardamine macrophylla The homogeneous polysaccharides of the whole plant of Willd.; among them, the total polysaccharides in the homogeneous polysaccharides of *Gnaphalium affine* are 90.16 ± 1.57 mg per 100 mg; The degree of esterification of the homogeneous polysaccharide was 33.01% ± 2.11%, and the molecular weight was... M w 4.120 × 10 4 Da - 4.159 × 10 4 Da, polydispersion coefficient M w / M n The values ​​range from 2.206 to 2.

233. The homogeneous polysaccharide mainly comprises the following monosaccharides: galacturonic acid, galactose, arabinose, rhamnose, xylose, glucose, glucuronic acid, and mannose. The molar percentages of each monosaccharide are as follows: (28.05%-31.66%): (28.12%-31.86%): (16.45%-18.48%): (6.46%-6.77%): (5.95%-5.96%): (5.26%-5.55%): (0.56%-0.66%): (3.20%-5.01%). Among these, galacturonic acid polysaccharide accounts for 21.59%-24.89%, and type I rhamnose galacturonic acid polysaccharide accounts for 60.14%-61.23%. The preparation method of the homogeneous polysaccharide of *Gnaphalium affine* includes the following steps: a. Take fresh *Gnaphalium affine*, freeze-dry it, grind it into powder, and sift it; b. Add ethanol to the powder and remove the alcohol-soluble components by ultrasonication to obtain a precipitate; c. High-pressure assisted eutectic solvent extraction: Take the precipitate prepared in step b, add the prepared extraction solvent, and extract under high pressure to obtain the extract; d. Add α-amylase and saccharifying enzyme to the concentrate to remove starch; e. Add trypsin to the concentrate to remove proteins; f. Inactivation; g. Take the supernatant, add ethanol, precipitate with ethanol, and let stand overnight; after centrifugation, obtain the precipitate, and then wash the precipitate with ethanol; h. Add water to reconstitute, separate by membrane, and dry to obtain homogeneous polysaccharide of *Gnaphalium affine*. in, In step a, the freeze-drying conditions are -30 ℃ to -70 ℃ for 40 to 70 hours; the sieve mesh size is 60 to 80 mesh. In step b, the final concentration of ethanol added is 20% to 90% ethanol; the weight-to-volume ratio of powder to ethanol is 1:(10-30); the ultrasonic power is 300 W to 800 W; and the ultrasonic time is 20 min to 60 min. In step c, high-pressure assisted eutectic solvent extraction is performed for 30-70 minutes, with the solvent-to-raw material ratio being 10 mL / g-50 mL / g. The eutectic solvent is composed of choline chloride, ethylene glycol, and ultrapure water. The molar ratio of choline chloride to ethylene glycol is 1:3, the water content of the eutectic solvent is 20%-60%, the extraction pressure is 0.1 MPa, and the extraction temperature is 121 °C. In step d, the conditions for starch removal by α-amylase and saccharifying enzyme were as follows: α-amylase with a concentration of 5 U / mL - 20 U / mL, hydrolysis temperature of 60 ℃ - 95 ℃, and hydrolysis time of 3 h - 12 h; saccharifying enzyme with a concentration of 5 U / mL - 20 U / mL, hydrolysis temperature of 40 ℃ - 65 ℃, and hydrolysis time of 10 h - 14 h. In step e, the conditions for trypsin to remove proteins are: 1 U / mL - 10 U / mL of trypsin, a hydrolysis temperature of 30 ℃ - 60 ℃, and a hydrolysis time of 3 h - 12 h. In step f, the inactivation conditions are 95 °C for 30 min. In step g, the volume ratio of alcohol precipitation extract to ethanol is 1:(2-5), and the concentration of ethanol used is 50%-100% ethanol; the ethanol used to wash the precipitate is 50%-95% ethanol; The membrane used for membrane separation in step h has a molecular weight cutoff of 1000 Da - 3000 Da.

2. The homogeneous polysaccharide of *Gnaphalium affine* according to claim 1, characterized in that: The homogeneous polysaccharide contains, per 100 mg, total uronic acid: 24.91 ± 2.77 mg; total protein: 1.80 ± 0.14 mg; and total conjugated phenols: 0.98 ± 0.21 mg GAE (gallic acid equivalent).

3. A method for preparing a homogeneous polysaccharide of *Gnaphalium affine* as described in claim 1 or 2, characterized in that: It includes the following steps: a. Take fresh *Gnaphalium affine*, freeze-dry it, grind it into powder, and sift it; b. Add ethanol to the powder and remove the alcohol-soluble components by ultrasonication to obtain a precipitate; c. High-pressure assisted eutectic solvent extraction: Take the precipitate prepared in step b, add the prepared extraction solvent, and extract under high pressure to obtain the extract; d. Add α-amylase and saccharifying enzyme to the concentrate to remove starch; e. Add trypsin to the concentrate to remove proteins; f. Inactivation; g. Take the supernatant, add ethanol, precipitate with ethanol, and let stand overnight; after centrifugation, obtain the precipitate, and then wash the precipitate with ethanol; h. Add water to redissolve, separate by membrane, and dry to obtain homogeneous polysaccharide of *Gnaphalium affine*.

4. The method for preparing homogeneous polysaccharide from *Gnaphalium affine* according to claim 3, characterized in that: a. The freeze-drying conditions in this step are -30 ℃ to -70 ℃ for 40 to 70 hours; the sieve mesh size is 60 to 80 mesh. b. The final concentration of ethanol added in this step is 20% to 90% ethanol; the weight-to-volume ratio of powder to ethanol is 1:(10-30); the ultrasonic power is 300 W to 800 W; the ultrasonic time is 20 min to 60 min; c. High-pressure assisted eutectic solvent extraction, with an extraction time of 30 min - 70 min and a solvent-to-raw material ratio of 10 mL / g - 50 mL / g; wherein the eutectic solvent is composed of choline chloride, ethylene glycol, and ultrapure water, with a molar ratio of choline chloride to ethylene glycol of 1:3, a water content of 20% - 60%, an extraction pressure of 0.1 MPa, and an extraction temperature of 121 ℃; d. The conditions for removing starch by α-amylase and saccharifying enzyme in the steps are as follows: α-amylase with 5 U / mL - 20 U / mL, enzymatic hydrolysis temperature of 60 ℃ - 95 ℃, and enzymatic hydrolysis time of 3 h - 12 h; saccharifying enzyme with 5 U / mL - 20 U / mL, enzymatic hydrolysis temperature of 40 ℃ - 65 ℃, and enzymatic hydrolysis time of 10 h - 14 h. e. The conditions for trypsin to remove protein in the step are: 1 U / mL - 10 U / mL of trypsin, enzymatic hydrolysis temperature of 30 ℃ - 60 ℃, and enzymatic hydrolysis time of 3 h - 12 h. f. Inactivation conditions: 95 ℃, 30 min; g. In the step, the volume ratio of alcohol precipitation extract to ethanol is 1:(2-5), and the concentration of ethanol used is 50%-100% ethanol; the ethanol used to wash the precipitate is 50%-95% ethanol; h. The membrane used for membrane separation in step h has a molecular weight cutoff of 1000 Da - 3000 Da.

5. The method for preparing homogeneous polysaccharide from *Gnaphalium affine* according to claim 4, characterized in that: a. In the step, freeze-dry at -40 ℃ for 48 h; sieve through an 80-mesh screen. b. The ethanol added in this step is 80% ethanol; the weight-to-volume ratio of powder to ethanol is 1:10; the ultrasonic power is 480 W; the ultrasonic time is 30 min. c. High-pressure assisted eutectic solvent extraction, with an extraction time of 50 min and a solvent-to-raw material ratio of 42 mL / g; wherein the eutectic solvent is composed of choline chloride, ethylene glycol, and ultrapure water, with a molar ratio of choline chloride to ethylene glycol of 1:3, a water content of 40%, an extraction pressure of 0.1 MPa, and an extraction temperature of 121 ℃; d. In this step, the amount of α-amylase added is 10 U / mL, the enzymatic hydrolysis temperature is 90 ℃, and the enzymatic hydrolysis time is 8 h; the amount of saccharifying enzyme added is 10 U / mL, the enzymatic hydrolysis temperature is 59 ℃, and the enzymatic hydrolysis time is 12 h. e. The amount of trypsin added in this step is 5 U / mL, the enzymatic hydrolysis temperature is 40 ℃, and the enzymatic hydrolysis time is 8 h; f. Inactivation conditions: 95 ℃, 30 min; g. The volume ratio of alcohol used for precipitation in step g is 1:4; the ethanol used is 95% ethanol; the ethanol used for washing the precipitate is 76% ethanol. h. The membrane used for membrane separation in step h has a molecular weight cutoff of 3000 Da.

6. The use of the homogeneous polysaccharide of *Gnaphalium affine* according to claim 1 or 2 in the preparation of drugs or health foods with antioxidant or immunomodulatory activities.

7. A health food product that helps with antioxidation or boosts immunity, characterized in that: It contains the homogeneous polysaccharide of *Gnaphalium affine* as described in claim 1 or 2.

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