Preparation of polysaccharide of tetrastigma hemsleyanum based on fermentation of phellinus sp. and use thereof

Abstract

The application discloses a kind of based on fermentation of Sanghuangjun preparation's Radix Tetrastigae polysaccharide and its use.The Radix Tetrastigae polysaccharide is composed of polysaccharide with weight percentage content more than 99%;The polysaccharide is composed of galactose, mannose, fucose and 3-O-methyl-galactose, and the molar ratio is 8.5-11.0:1.5-3:0.8-1.5:1.0.The Radix Tetrastigae polysaccharide is extracted, separated and purified from Sanghuang fermentation Radix Tetrastigae.The Radix Tetrastigae polysaccharide has significant antitumor activity and regulates intestinal flora structure efficacy, can be directly used as antitumor and / or regulate intestinal flora structure function product, and also can be used for preparing antitumor and / or regulating intestinal flora structure function product.

Description

Technical Field

[0001] This invention belongs to the field of natural products and polysaccharide technology, specifically relating to a polysaccharide of *Phellinus trifoliata* prepared by fermentation of *Phellinus linteus* and its uses. Background Technology

[0002] *Tetrastigma hemsleyanum* Diels et Gilg, commonly known as Three-Leaf Green, is a plant belonging to the genus *Tetrastigma* in the family Vitaceae. It is a traditional medicinal herb used in Zhejiang and Fujian provinces, with its underground tubers and fruits considered to have the best medicinal effects. Modern pharmacological studies have shown that *Tetrastigma hemsleyanum* possesses anti-inflammatory, analgesic, antipyretic, antitumor, antiviral, and immunomodulatory effects. Due to its various health benefits, including antipyretic, anti-proliferative, and anti-inflammatory properties, *Tetrastigma hemsleyanum* is often used as a dietary supplement.

[0003] Three-leaf clover contains active ingredients such as polysaccharides, flavonoids, and polyphenols, as well as nutrients such as starch and protein. In the traditional extraction and separation process of its active ingredients, starch, protein, and irritating components are extracted together, which increases the processing steps in the separation process, resulting in a lower content and activity of active ingredients in the final product.

[0004] Existing *Trifolium repens* polysaccharides are obtained using traditional extraction and separation methods. Chinese patent ZL201711200506.7 discloses a method for preparing *Trifolium repens* polysaccharides and its application, using *Trifolium repens* vine leaves as raw material, and implementing the following steps in sequence: (1) hot water extraction; filtration to collect the filtrate; vacuum concentration; adding ethanol to the concentrate; collecting the precipitate, i.e., crude *Trifolium repens* polysaccharides. (2) Dissolving the crude polysaccharides in water, adding Sevag reagent; stirring and centrifuging to remove the protein layer and organic layer, repeating this step multiple times until no protein appears; (3) dissolving the polysaccharides after protein removal in deionized water, and obtaining the single component of *Trifolium repens* polysaccharides by DEAE-52 ion exchange chromatography and Sephadex G-100 dextran gel chromatography. The obtained *Trifolium repens* polysaccharides can significantly reduce blood glucose and blood lipids, improve the activity level of antioxidant enzymes in the body, and have a certain repair effect on pancreatic islets, and have good medicinal and market value. Developing more valuable, safe, and environmentally friendly active substances, such as active polysaccharides, based on *Tripterygium wilfordii* would be of positive significance. Summary of the Invention

[0005] This invention reveals that over 90% of the starch in the tuberous roots of *Tripterygium wilfordii* is a viscous polymer, making it difficult to release the active polysaccharides. However, starch is a rich carbon source for the fermentation of *Sanghuang* fungus and can be fully utilized in food processing.

[0006] Based on the above findings, this invention provides a polysaccharide of *Trifolium repens* prepared by fermentation with *Sanghuang* fungus and its uses. Through research, this invention has discovered that the new *Trifolium repens* polysaccharide obtained after fermentation with *Sanghuang* fungus possesses excellent in vitro antitumor efficacy and intestinal flora regulation effects, and can be used in many fields such as food, health products, pharmaceuticals, and daily chemical products. Based on the above research results, this invention is thus completed.

[0007] The present invention also provides a method for preparing the polysaccharide of *Trifolium repens*, which has the advantages of simple operation and easy control, and is suitable for large-scale industrial production.

[0008] The present invention also provides the use of the polysaccharide of *Sanghuang* prepared by fermentation, which has significant anti-tumor activity and intestinal flora structure regulation effect. It can be used directly as an anti-tumor and / or intestinal flora structure regulating functional product, and can also be used to prepare anti-tumor and / or intestinal flora structure regulating functional products.

[0009] The fermentation of Phellinus linteus in this invention has two main functions: firstly, by introducing bio-enzymes produced by Phellinus linteus fermentation, the starchy inactive substances in Clematis chinensis are enzymatically hydrolyzed, which facilitates the release and extraction of active polysaccharides; secondly, active Clematis chinensis polysaccharides with specific structures of this invention are obtained.

[0010] A polysaccharide of *Phellinus thunbergii* prepared by fermentation is composed of polysaccharides with a weight percentage of over 99%. The polysaccharides are composed of galactose, mannose, fucose, and 3-O-methyl-galactose, wherein the molar ratio of galactose, mannose, fucose, and 3-O-methyl-galactose is 8.5-11.0:1.5-3:0.8-1.5:1.0.

[0011] To achieve better invention results, the following optimizations are made:

[0012] The galactose is α-galactose and β-galactose, more preferably α-D-galactose and β-D-galactose.

[0013] The mannose is α-mannose, and more preferably α-D-mannose.

[0014] The fucose is α-fucose, and more preferably α-L-fucose.

[0015] The 3-O-methyl-galactose is α-3-O-methyl-galactose, and more preferably α-3-O-methyl-D-galactose.

[0016] The preferred structural unit of the polysaccharide is a main chain structure consisting of (1→2) linked α-D-galactose (α-D-Galp) residues and (1→6) linked α-D-mannose (α-D-Manp) residues; at the C-6 position of one (1→2) linked α-D-galactose residue on the main chain, a β-D-galactose (β-D-Galp) end group is substituted; at the C-2 position of two consecutive (1→6) linked α-D-mannose residues on the main chain, a first branch consisting of a (1→6) linked α-3-O-methyl-D-galactose (α-3-O-Me-Galp) residue and a β-D-galactose end group is substituted, and an α-L-fucose (α-L-Fucp) end group is substituted.

[0017] Further preferably, the main chain structure consists of α-D-galactose residues linked in a (1→2) manner and α-D-mannose residues linked in a (1→6) manner connected sequentially.

[0018] The three branches on the main chain of the *Trifolium repens* polysaccharide of this invention have various combinations. The β-D-galactose end group can replace any (1→2) linked α-D-galactose residue at the C-6 position on the main chain. The first branch and an α-L-fucp end group can be arranged in any order at the C-2 position of two consecutive (1→6) linked α-D-mannose residues, for example, they can have the following configurations: Figure 5a The structural unit shown, Figure 5a The structural unit shown is only an example of one arrangement of the two end bases and the first branch, and is not intended to restrict the arrangement of the two end bases and the first branch.

[0019] Figure 5a In this context, Galp is pyranose, Manp is pyranose-mannose, and Fucp is pyranose-fucose. Figure 5a The structure shown is a repeating unit, and the specific number of repeating units is determined based on the molecular weight of the *Trifolium repens* polysaccharide.

[0020] Optionally, the weight-average molecular weight of the polysaccharide prepared based on *Sanghuang* fermentation is preferably 10 kDa-15 kDa, further preferably 12 kDa-13 kDa, and most preferably 12.3 kDa-12.8 kDa.

[0021] The polysaccharide of *Trifolium repens* prepared based on *Sanghuang* fermentation can be extracted, separated, and purified from fermented *Trifolium repens* using a method for preparing water-soluble polysaccharides, preferably obtained by fermentation of *Trifolium repens* with *Sanghuang* followed by hot water extraction and purification. This includes: obtaining crude polysaccharide from fermented *Trifolium repens* with *Sanghuang* followed by water extraction and alcohol precipitation, and deproteinization with Sevage reagent; purifying this crude polysaccharide by anion exchange chromatography and gel filtration chromatography; and freeze-drying to obtain the polysaccharide prepared based on *Sanghuang* fermentation. The specific technical solution is as follows:

[0022] A method for preparing *Trifolium repens* polysaccharide based on *Sanghuang* fermentation includes the following steps:

[0023] (1) Preparation of fermented crude polysaccharide of *Trifolium repens*: *Trifolium repens* tubers were fermented with *Sanghuang* fungus. The fermentation product was extracted with water, precipitated with alcohol, and the protein was removed and the supernatant was freeze-dried to obtain crude polysaccharide of *Trifolium repens* fermented with *Sanghuang* fungus (numbered F-THDP).

[0024] (2) Purification: The aqueous solution of the crude polysaccharide of *Sanghuang* obtained in step (1) after fermentation by *Sanghuang* (F-THDP) was subjected to column chromatography packed with diethylaminoethyl cellulose (DEAE cellulose) ion exchange resin. The eluent eluted with 0.15 mol / L-0.3 mol / L NaCl aqueous solution was collected and then subjected to gel filtration chromatography. The eluent containing polysaccharide was collected, and then dialyzed and freeze-dried to obtain the polysaccharide of *Sanghuang* after fermentation by *Sanghuang* (numbered F-THDP2).

[0025] To achieve better invention results, the preferred method is:

[0026] The fungus used in this invention is Sanghuangporus sanghuang, a commercially available product.

[0027] Step (1) includes: cutting the tuber of *Trifolium repens* into thin slices, sterilizing, cooling and inoculating with *Sanghuang* fungus, fermenting and culturing, extracting the fermentation product with water at 90℃-100℃ to obtain an aqueous extract, concentrating to obtain a concentrated solution, removing the protein from the precipitate obtained by alcohol precipitation with Sevage reagent, and freeze-drying the supernatant to obtain crude polysaccharide of *Trifolium repens* fermented with *Sanghuang* fungus.

[0028] The fermentation culture conditions can be any suitable conditions for Sanghuang fungus. The preferred fermentation culture conditions are: temperature 24℃-28℃, humidity 50%-65%, and culture time 18-25 days.

[0029] In the step of extracting the fermentation product with water, water is used as the extraction solvent, and the amount used is not strictly limited; water can be used in quantities of 2 to 6 times the weight of the fermentation product. The extraction time of the fermentation product with water at 90℃-100℃ should preferably be at least 2 hours.

[0030] The alcohol precipitation step uses commonly used alcohol precipitation reagents in the art, preferably ethanol or an aqueous ethanol solution. The aqueous ethanol solution is selected with a volume percentage concentration of ≥90%.

[0031] The Sevage reagent used is a commonly used Sevage reagent in the art, preferably chloroform and n-butanol, wherein the volume ratio of chloroform to n-butanol is preferably 4:1.

[0032] In step (2), the preferred conditions for column chromatography filled with DEAE cellulose ion exchanger are: elution with NaCl aqueous solution in a gradient increasing from 0 to 0.7 mol / L.

[0033] The DEAE cellulose ion exchanger can be a commercially available product, such as DEAE cellulose-52 ion exchanger.

[0034] The gel filtration chromatography uses polyacrylamide dextran gel column filtration chromatography, and commercially available products such as Sephacryl S series (Sephacryl S-100) can be used.

[0035] The preferred conditions for gel filtration chromatography are: elution with 0.05 mol / L phosphate buffer and 0.15 mol / L NaCl aqueous solution, wherein the volume ratio of phosphate buffer to NaCl aqueous solution is 2-3:1.

[0036] The phosphate buffer solution is prepared according to methods commonly used in the art, such as referring to the Chinese Pharmacopoeia.

[0037] The dialysis uses dialysis bags with a pore size of 5000Da-8000Da to remove small molecules.

[0038] This invention relates to F-THDP2, a polysaccharide from *Sanghuang* fungus prepared by fermentation, which exhibits good antitumor activity. For example, at a concentration of 0.6 mg / mL, F-THDP2 showed an inhibition rate of 51.63%-57.51% against three types of tumor cells: MCF-7, A549, and HeLa, which was significantly enhanced compared to the inhibition rate of unfermented *Sanghuang* polysaccharide THDP2 against these three tumor cells.

[0039] This invention relates to F-THDP2, a polysaccharide from *Sanghuang* fungus prepared through fermentation, which, when applied to mice, significantly increases the number of beneficial intestinal bacteria such as *Lactobacillus* and *Bifidobacterium*, and decreases the number of pathogenic intestinal bacteria such as *Escherichia coli*, compared to unfermented F-THDP2 and the control group. This regulates the intestinal flora structure and enhances intestinal motility. F-THDP2, prepared by fermentation of *Sanghuang* fungus, exhibits activity in regulating intestinal flora structure (promoting the proliferation of beneficial intestinal bacteria, such as *Lactobacillus* and *Bifidobacterium*, and inhibiting the proliferation of pathogenic intestinal bacteria, such as *Escherichia coli*) and enhancing intestinal motility.

[0040] Therefore, the *Trifolium repens* polysaccharide F-THDP2 prepared by fermentation of *Sanghuang* fungus in this invention can be directly used as an anti-tumor functional product, and can also be used to prepare anti-tumor functional products. The *Trifolium repens* polysaccharide F-THDP2 prepared by fermentation of *Sanghuang* fungus in this invention can also be directly used as a functional product to enhance intestinal microbial motility, such as a functional product to regulate intestinal flora structure, and can also be used to prepare functional products to enhance intestinal microbial motility, such as functional products to regulate intestinal flora structure. The functional products are pharmaceuticals, etc. For example, the *Trifolium repens* polysaccharide F-THDP2 prepared by fermentation of *Sanghuang* fungus can be used to prepare anti-tumor drugs, drugs to enhance intestinal motility, etc.

[0041] The raw materials, reagents, consumables, instruments, etc. used in this invention can all be commercially available products.

[0042] Compared with the prior art, the present invention has the following advantages:

[0043] This invention, based on the nutritional and active ingredient characteristics of *Tripterygium wilfordii*, addresses the shortcomings of current *Tripterygium wilfordii* products, such as high content of inactive starch components. It utilizes bioenzymes produced by *Sanghuang* fungus fermentation to enzymatically hydrolyze the inactive starch components in *Tripterygium wilfordii*. The resulting fermented polysaccharide exhibits excellent bioactivity in anti-tumor activity and enhancing intestinal motility, thus fully utilizing *Tripterygium wilfordii* resources. This preparation process effectively biodegrades and utilizes the nutritional and active ingredients in *Tripterygium wilfordii*, contributing to improved activity of the polysaccharides in the final product. The raw materials used in this invention are natural, of controllable quality, environmentally friendly, and suitable for industrial production.

[0044] The higher-order structure of the *Trifolium repens* polysaccharide F-THDP2 produced by fermentation of *Sanghuang* fungus in this invention is completely different from that of the original, unfermented *Trifolium repens* polysaccharide, and its biological activity also varies. The newly produced *Trifolium repens* polysaccharide after fermentation by *Sanghuang* fungus has higher medicinal value. The *Trifolium repens* polysaccharide F-THDP2 produced by fermentation of *Sanghuang* fungus in this invention has good biological activity, such as good anti-tumor activity and good activity in enhancing intestinal microbial motility, and has broad practical application value. The method for obtaining the *Trifolium repens* polysaccharide F-THDP2 produced by fermentation of *Sanghuang* fungus in this invention is convenient to operate and can produce a macromolecule with high orderliness and a well-defined structure, which can be industrially promoted for production.

[0045] This invention investigated the structural and activity changes of F-THDP2, a polysaccharide derived from fermented *Phellinus thunbergii* (mulberry tree). Compared to unfermented *Phellinus thunbergii* polysaccharide THDP2, the main monosaccharide composition and molecular weight of F-THDP2 from fermented *Phellinus thunbergii* were significantly altered. The molecular weight of F-THDP2 ranged from 12.3 kDa to 12.8 kDa. Furthermore, the glycosidic chains of F-THDP2 differed significantly from those of THDP2. F-THDP2 established a 1,2-linked α-D-Galp and 1,6-linked α-D-Manp backbone, which is distinctly different from the 1,4-linked α-D-Glcp and 1,4-linked β-D-Galp backbone of THDP2. In addition, F-THDP2 exhibited a more flexible chain conformation in aqueous solution than THDP2. Notably, compared to the original polysaccharide, F-THDP2 exhibited better inhibitory effects on HeLa cells via the Fas / fasl-mediated Caspase-3 signaling pathway. These structural and bioactivity changes suggest that fermentation modification of *Sanghuang* fungus is a promising new method for effectively converting starch and other polysaccharides from *Trifolium repens* into highly bioactive biomacromolecules, with potential industrial applications. Attached Figure Description

[0046] Figure 1 The absorbance curve at 490 nm (denoted as A490) of the eluent collected by DEAE cellulose-52 ion exchange chromatography of the aqueous solution of F-THDP crude polysaccharide from *Sanghuang* fermented with *Phellinus sylvestris* in Example 1. Tube numbers represent the number of tubes.

[0047] Figure 2 The image shows the absorbance at 490 nm of the polysaccharide-containing F-THDP2 eluent purified by polyacrylamide dextran gel (Sephacryl S-100) in Example 1. Tube numbers represent the number of tubes.

[0048] Figure 3 The images show the GC-MS spectra of polysaccharide acetylation products; A is the standard reference spectrum; B is the spectrum of *Trifolium repens* polysaccharide THDP2 sample before fermentation; C is the spectrum of *Trifolium repens* polysaccharide F-THDP2 sample after fermentation. The ordinate represents Relative Abundance, and the abscissa represents retention time in minutes (min). Rham represents rhamnose, Rib represents ribose, Ara represents arabinose, Fuc represents fucose, Xyl represents xylose, Man represents mannose, Glc represents glucose, and Gal represents galactose.

[0049] Figure 4aFor F-THDP2 1 H-NMR spectrum, Figure 4b For F-THDP2 13 C-NMR spectrum, Figure 4c The HSQC spectrum of F-THDP2, Figure 4d The COSY spectrum of F-THDP2, Figure 4e The TCOSY spectrum of F-THDP2 Figure 4f For F-THDP2 1 H- 13 C HMBC map, Figure 4g The NOESY spectrum of F-THDP2.

[0050] Figure 5a The structural formula for F-THDP2; Figure 5b The structural formula for THDP2.

[0051] Figure 6a For THDP2 1 H-NMR spectrum, Figure 6b For THDP2 13 C-NMR spectrum, Figure 6c The HSQC spectrum of THDP2, Figure 6d The COSY spectrum of THDP2, Figure 6e The TCOSY plot of THDP2 Figure 6f For THDP2 1 H- 13 C HMBC map, Figure 6g The NOESY spectrum of THDP2.

[0052] Figure 7a The laser light scattering diagram of THDP2; Figure 7b The laser light scattering diagram of F-THDP2; Figure 7c The graph shows the molecular weight Mw of F-THDP2 versus the logarithm of viscosity [η]; where the vertical axis represents the relative scale and the horizontal axis represents time (minutes).

[0053] Figure 8a Two-dimensional atomic force microscopy (AFM) spectra of THDP2; Figure 8b for Figure 8a Curve of straight-line height measurements in the topography; Figure 8c for Figure 8a Linear energy spectrum curves in morphology; Figure 8d Two-dimensional atomic force microscopy pattern of F-THDP2; Figure 8e for Figure 8d Curve of straight-line height measurements in the topography; Figure 8f for Figure 8d Linear energy spectrum curve in morphology.

[0054] Figure 9 The absorbance curve at 490 nm (denoted as A490) of the eluent collected by DEAE cellulose-52 ion exchange chromatography of the aqueous solution of crude polysaccharide THDP from *San Ye Qing* without fermentation by *Sanghuang* in Comparative Example 1 is shown. Tubenumbers represent the number of tubes.

[0055] Figure 10 The image shows the absorbance at 490 nm of the polysaccharide-containing THDP2 eluent purified by polyacrylamide dextran gel (Sephacryl S-100) in Comparative Example 1. Tube numbers represent the number of tubes. Detailed Implementation

[0056] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but it is not intended to limit the scope of implementation of the technical solutions of the present invention.

[0057] The fungus used is Sanghuang from the mulberry tree (Sanghuangporus sanghuang).

[0058] Example 1

[0059] (1) Preparation of fermented crude polysaccharide of *Trifolium repens*: 500g of *Trifolium repens* tubers dried by vacuum freeze-drying were cut into thin slices of 3mm±2mm, sterilized at 121℃ for 25min, cooled to room temperature and inoculated with *Sanghuang* fungus; in a sealed Erlenmeyer flask, the humidity was maintained at 58%±2% and fermented at 26℃ for 20 days. The fermentation product was extracted with 3 times the weight of the fermentation product of distilled water at 95℃±5℃ for 2.5h. The water extract was concentrated to obtain a concentrate. Ethanol was added to the concentrate until the final concentration of the concentrate was 80% (v / v) for alcohol precipitation. The precipitate was treated with Sevage reagent (chloroform and n-butanol in a volume ratio of 4:1) to remove protein. The supernatant was freeze-dried to obtain crude polysaccharide of *Trifolium repens* fermented with *Sanghuang* fungus (numbered F-THDP).

[0060] (2) Purification: The crude polysaccharide (F-THDP) of *Sanghuang* obtained in step (1) after fermentation by *Sanghuang* fungus was separated and purified. 10 mL of the crude polysaccharide aqueous solution (15 mg / mL) was injected into a column (6.5 cm × 40 cm) packed with DEAE cellulose-52 ion exchange resin. Elution was performed with a gradient of NaCl aqueous solution (0-0.7 mol / L) at a flow rate of 2.5 mL / min. 6 mL of the eluent was collected from each tube, and the absorbance of each tube of eluent at 490 nm was measured using the phenol-sulfuric acid method. The results are shown in the figure. Figure 1The polysaccharides in the eluent collected from 0.05 mol / L to 0.1 mol / L NaCl aqueous solutions were designated F-THDP1, those in the eluent collected from 0.15 mol / L to 0.3 mol / L NaCl aqueous solutions were designated F-THDP2, and those in the eluent collected from 0.35 mol / L to 0.5 mol / L NaCl aqueous solutions were designated F-THDP3. Figure 1 The eluents containing polysaccharide F-THDP1, F-THDP2, and F-THDP3 were dialyzed separately (dialysis bags with pore sizes of 5000 Da-8000 Da). Each dialysate was then chromatographyd on a polyacrylamide dextran gel (Sephacryl S-100) column (1.2 cm × 90 cm) with a loading volume of 5 mL. Elution was performed using 0.05 mol / L phosphate buffer (pH 7.0) and 0.15 mol / L NaCl aqueous solution (phosphate buffer to NaCl aqueous solution volume ratio 2:1) at a flow rate of 0.5 mL / min. 3 mL of eluent was collected from each tube, and the absorbance of each eluent at 490 nm was measured using the phenol-sulfuric acid method. The eluent rich in polysaccharide F-THDP2 was collected after chromatography. The results are shown in the figure below. Figure 2 The eluent rich in polysaccharide F-THDP1 and F-THDP3 was collected after chromatography. The eluent rich in polysaccharide F-THDP2 was dialyzed (using a dialysis bag with a pore size of 5000 Da-8000 Da), and the dialysate was freeze-dried to obtain the F-THDP2 polysaccharide fraction of *Trifolium repens* fermented with *Sanghuang* fungus. The eluent rich in polysaccharide F-THDP1 was dialyzed (using a dialysis bag with a pore size of 5000 Da-8000 Da), and the dialysate was freeze-dried to obtain the F-THDP1 polysaccharide fraction of *Trifolium repens* fermented with *Sanghuang* fungus. The eluent rich in polysaccharide F-THDP3 was dialyzed (using a dialysis bag with a pore size of 5000 Da-8000 Da), and the dialysate was freeze-dried to obtain the F-THDP3 polysaccharide fraction of *Trifolium repens* fermented with *Sanghuang* fungus.

[0061] Comparative Example 1

[0062] (1) Preparation of crude polysaccharide of Trifoliate orange: 500g of Trifoliate orange tuber obtained by vacuum freeze-drying was extracted with distilled water at 95℃±5℃ for 2.5h. The water extract was concentrated to obtain a concentrate. Ethanol was added to the concentrate until the final concentration of the concentrate was 80% (v / v) for alcohol precipitation. The precipitate was treated with Sevage reagent (chloroform and n-butanol in a volume ratio of 4:1) to remove protein. The supernatant was freeze-dried to obtain crude polysaccharide of Trifoliate orange before fermentation (numbered THDP).

[0063] (2) Purification: The crude polysaccharide (THDP) obtained in step (1) was separated and purified according to the purification steps in Example 1. The polysaccharide in the eluent of 0.05 mol / L-0.1 mol / L NaCl aqueous solution was designated as THDP1, the polysaccharide in the eluent of 0.15 mol / L-0.3 mol / L NaCl aqueous solution was designated as THDP2, and the polysaccharide in the eluent of 0.35 mol / L-0.5 mol / L NaCl aqueous solution was designated as THDP3. Finally, the unfermented polysaccharide component THDP2 was obtained.

[0064] Example 2

[0065] (1) Preparation of fermented crude polysaccharide of *Trifolium repens*: 500g of *Trifolium repens* tubers dried by vacuum freeze-drying were cut into thin slices of 3mm ± 2mm, sterilized at 120℃ for 30min, cooled to 24℃ and inoculated with *Sanghuang* fungus; in a sealed Erlenmeyer flask, the humidity was maintained at 55% ± 5% and fermented at 24℃ for 25 days. The fermentation product was extracted with 6 times the weight of the fermentation product of distilled water at 95℃ ± 5℃ for 4h. The water extract was concentrated to obtain a concentrate. 90% ethanol aqueous solution was added to the concentrate until the final concentration of the concentrate was 80% (v / v) for alcohol precipitation. The precipitate was treated with Sevage reagent (chloroform and n-butanol in a volume ratio of 4:1) to remove protein. The supernatant was freeze-dried to obtain crude polysaccharide of *Trifolium repens* fermented with *Sanghuang* fungus (numbered F-THDP).

[0066] (2) Purification: 10 mL of the crude polysaccharide F-THDP (20 mg / mL) aqueous solution was injected into a DEAE cellulose-52 ion exchange column (6.5 cm × 40 cm). During the Sephacryl S-100 column (1.2 cm × 90 cm) chromatography, elution was performed with 0.05 mol / L phosphate buffer (pH 7.0) and 0.15 mol / L NaCl aqueous solution (the volume ratio of phosphate buffer to NaCl aqueous solution was 3:1) at a flow rate of 0.5 mL / min. The remaining operations were the same as in Example 1 to obtain the F-THDP2 polysaccharide component of F. trifoliate polysaccharide after fermentation by Phellinus linteus.

[0067] Example 3

[0068] (1) Preparation of fermented crude polysaccharide of *Trifolium repens*: 500g of *Trifolium repens* tubers dried by vacuum freeze-drying were cut into thin slices of 3mm±2mm, sterilized at 125℃ for 20min, cooled to 25℃ and inoculated with *Sanghuang* fungus; in a sealed Erlenmeyer flask, the humidity was maintained at 60%±5% and fermented at 28℃ for 18 days. The fermentation product was extracted with twice the weight of the fermentation product of distilled water at 95℃±5℃ for 2h. The water extract was concentrated to obtain a concentrate. 95% ethanol aqueous solution was added to the concentrate until the final concentration of the concentrate was 80% (v / v) for alcohol precipitation. The precipitate was treated with Sevage reagent (chloroform and n-butanol in a volume ratio of 4:1) to remove protein. The supernatant was freeze-dried to obtain crude polysaccharide of *Trifolium repens* fermented with *Sanghuang* fungus (numbered F-THDP).

[0069] (2) Purification: 10 mL of the aqueous solution (25 mg / mL) of crude polysaccharide of *Trifolium repens* (F-THDP) was injected into a column (6.5 cm × 40 cm) filled with DEAE cellulose-52 ion exchange resin. The remaining operations were the same as in Example 1 to obtain the polysaccharide component F-THDP2 of *Trifolium repens* after fermentation by *Sanghuang* fungus.

[0070] Identification and performance analysis of polysaccharides from *Trifolium repens* before and after fermentation:

[0071] I. Monosaccharide Composition

[0072] Take 3 mg of polysaccharide and place it in a thin-walled long test tube. Add 4.0 mL of 2.0 mol / L trifluoroacetic acid, seal the tube, and hydrolyze at 110 °C for 2 h. After hydrolysis, evaporate the solution in the test tube to dryness under reduced pressure below 40 °C, then add methanol and evaporate to dryness. Repeat the "add methanol and evaporate to dryness" step 4-5 times to completely remove trifluoroacetic acid and obtain a completely acid-hydrolyzed sample.

[0073] Various monosaccharide standards and fully acid-hydrolyzed samples were dissolved separately in 3 mL of distilled water. 30 mg of sodium borohydride was added, the mixture was sealed, and reduction was carried out at room temperature for 3 h. Excess sodium borohydride was then neutralized with glacial acetic acid, a small amount of methanol was added, and the mixture was concentrated under reduced pressure and evaporated to dryness. This process of adding a small amount of methanol and concentrating under reduced pressure was repeated 4-5 times. Then, 4 mL of acetic anhydride was added, and the mixture was reacted at 100 °C for 1 h. Finally, 3 mL of toluene was added, and the mixture was concentrated under reduced pressure and evaporated to dryness to obtain the acetylated products. Each acetylated product was dissolved separately in 3 mL of chloroform and transferred to a separatory funnel. An equal volume of distilled water was added, and the mixture was thoroughly mixed. After standing, the supernatant was removed. This process was repeated 3-4 times. The chloroform layer was dried with an appropriate amount of anhydrous sodium sulfate, filtered, and then diluted to 10 mL with chloroform to obtain the acetylated products, which were then analyzed by GC-MS.

[0074] GC-MS conditions: DB-5 capillary column (30m×0.25mm×0.25μm) was used, with programmed temperature ramp (initial column temperature 120℃, ramped to 240℃ at 10℃ / min, held for 6.5min), interface temperature 250℃, ion source temperature 250℃, injection volume 2.0μL, helium as carrier gas, and flow rate 1.0mL / min.

[0075] GC-MS spectrum of the acetylated products of polysaccharide F-THDP2 hydrolysate after fermentation is shown below. Figure 3 C, GC-MS spectrum of the acetylated products of the pre-fermentation polysaccharide THDP2 hydrolysate as shown in Figure 1. Figure 3 B, corresponding monosaccharide standard spectrum ( Figure 3 A) Display:

[0076] In Example 1, the polysaccharide F-THDP2 has a monosaccharide composition of D-galactose, D-mannose, and L-fucose, with a molar ratio of 12.6:2.3:1.0. F-THDP2 does not contain glucose, but the content of galactose increases dramatically. Galactose is the main monosaccharide of F-THDP2, with a molar percentage of 79.25%.

[0077] In Example 2, the polysaccharide F-THDP2 has a monosaccharide composition of D-galactose, D-mannose, and L-fucose, with a molar ratio of 11.8:2.6:1.0. F-THDP2 does not contain glucose, but the content of galactose increases sharply. Galactose is the main monosaccharide of F-THDP2, with a molar percentage of 76.62%.

[0078] In Example 3, the polysaccharide F-THDP2 has a monosaccharide composition of D-galactose, D-mannose, and L-fucose, with a molar ratio of 12.0:2.0:1.0. F-THDP2 does not contain glucose, but the content of galactose increases sharply. Galactose is the main monosaccharide of F-THDP2, with a molar percentage of 80.0%.

[0079] The monosaccharide composition of polysaccharide THDP2 in Comparative Example 1 consists of D-glucose, D-galactose and D-mannose, with a molar ratio of 9.5:4.1:1.1, indicating that THDP2 does not contain fucose and glucose is the main monosaccharide of THDP2, with a molar percentage of 64.63%.

[0080] The above results indicate that glucose in *Trifolium repens* is metabolized during fermentation and mainly converted into galactose. Furthermore, compared to THDP2, F-THDP2 contains a novel monosaccharide: fucose, suggesting that F-THDP2 is a new polysaccharide biosynthesized during fermentation, rather than a degradation product of the original *Trifolium repens* polysaccharide.

[0081] II. Physicochemical Properties, Components, and Molecular Weight Detection

[0082] The total polysaccharide F-THDP2 from *Sanghuang* fungus fermentation in Example 1 was determined to have a total polysaccharide content of 99.76% by weight using the phenol-sulfuric acid method. Its laser light scattering pattern (...) Figure 7b The results showed that the 90° light scattering (LS) signal, the differential detection (RI) signal, and the viscosity detector (VIS) signal peaks had similar peak shapes and almost completely overlapped, indicating that the delay between the two detectors had been accurately corrected. The RI signal of sample F-THDP2 showed a single symmetrical peak shape for the polysaccharide, indicating that F-THDP2 is a polysaccharide with a uniform molecular weight distribution, with a molecular weight Mw = 1.23 × 10⁻⁶. 4 Da.

[0083] In Example 2, the total polysaccharide F-THDP2 from *Sanghuang* fungus fermentation was found to contain 99.13% polysaccharide by weight, as determined by the phenol-sulfuric acid method. Its RI signal showed a single, symmetrical peak, indicating that F-THDP2 is a polysaccharide with a uniform molecular weight distribution (Mw = 1.26 × 10⁻⁶). 4 Da.

[0084] In Example 3, the total polysaccharide F-THDP2 from *Sanghuang* fungus fermentation was found to have a total polysaccharide weight percentage of 99.37% using the phenol-sulfuric acid method. Its RI signal showed a single symmetrical peak shape, indicating that F-THDP2 is a polysaccharide with a uniform molecular weight distribution, and its molecular weight Mw = 1.28 × 10⁻⁶. 4 Da.

[0085] The total polysaccharide content (THDP2) of *Trifolium repens* polysaccharide fraction before fermentation (unfermented) in Comparative Example 1 was determined to be 99.71% by weight using the phenol-sulfuric acid method. Its laser light scattering pattern (...) Figure 7a The results showed that the peaks detected by the 90° light scattering (LS), differential detection (RI), and viscosity detector (VIS) signals had similar shapes and almost completely overlapped, indicating that the delay between the two detectors had been accurately corrected. The RI signal of sample THDP2 showed a single symmetrical peak shape, indicating that THDP2 is a polysaccharide with a uniform molecular weight distribution, with a molecular weight Mw = 3.58 × 10⁻⁶. 4 Da.

[0086] The molecular weights of THDP2 and F-THDP2 are 3.58 × 10⁻⁶ and 3.58 × 10⁻⁶, respectively.4 g / mol and 1.23×10 4 g / mol - 1.28 × 10 4 The g / mol indicates that the molecular weight of F-THDP2, a polysaccharide from fermented Tripterygium wilfordii, is significantly lower than that of THDP2, a polysaccharide from unfermented Tripterygium wilfordii.

[0087] III. Methylation Analysis

[0088] 2 mg of polysaccharide sample was dissolved in 1 mL of dimethyl sulfoxide (DMSO), sealed with nitrogen gas, and sonicated for a short time to aid dissolution. Then, methylation was performed according to the method of Ciucanu, et al. (Ciucanu, L., & Kerek, F.. A simple and repid method for permethylation of carbohydrates. Carbohydrate Research, 131, 209-217).

[0089] F-THDP2 underwent three methylation processes, followed by acid hydrolysis, reduction, and acetylation to prepare a partially methylated alkaloid acetate derivative, which was then analyzed by GC-MS (see Table 1). Table 1 shows that the polysaccharide F-THDP2 after fermentation of *Trifolium repens* contains six residues: 2-Galp-1→ (26.72 mol%), 6-Galp-1→ (6.43 mol%), 2,6-Galp-1→ (21.15 mol%), 2,6-manp-1→ (13.35 mol%), T-Fucp (6.32 mol%), and T-Galp (26.03 mol%).

[0090] After three methylation processes, THDP2 was further hydrolyzed, reduced, and acetylated to prepare a partially methylated ardiol acetate derivative, which was then analyzed by GC-MS (see Table 2). Table 2 shows that the glycosidic bond linkages of the unfermented *Tripterygium wilfordii* polysaccharide THDP2 mainly consist of T-Manp (7.11 mol%), T-Glcp (21.44 mol%), 4-Galp-1→ (28.49 mol%), 4,6-Glcp-1→ (28.67 mol%), and 4-Glcp-1→ (14.29 mol%). The glycosidic bond linkages of the polysaccharide changed significantly before and after fermentation.

[0091] Table 1. F-THDP2 Methylation Analysis

[0092]

[0093] Table 2 THDP2 methylation analysis

[0094]

[0095] IV. Nuclear Magnetic Resonance

[0096] 60 mg of polysaccharide was dissolved in 0.5 mL of deuterium water and NMR scanned at 600 MHz using a Bruker-AVIII500M (Switzerland).

[0097] According to F-THDP2 1 H-NMR (see H-NMR) Figure 4a ), 13 C-NMR (see C-NMR) Figure 4b Combined with HSQC spectrum (see) Figure 4c Six peaks were detected that were relatively significant and could be used for analysis. The main chain of the polysaccharide F-THDP2 after fermentation of Trifoliate L. consists of the main chain of →2)-α-D-Galp-(1→ and →2,6)-α-D-Manp-(1→), and the side chains consist of terminal residues β-D-Galp, →6)-α-3-O-Me-D-Galp-(1→ and terminal residue α-L-Fucp.

[0098] According to THDP2 1 H-NMR (see H-NMR) Figure 6a ), 13 C-NMR (see C-NMR) Figure 6b Combined with HSQC spectrum (see) Figure 6c The main chain of the unfermented polysaccharide THDP2 of *Trifolium repens* consists of →4)-α-D-Glcp-(1→ and →4)-β-D-Galp-(1→), and the side chains consist of α-D-Manp and α-D-Glcp.

[0099] Table 3. All chemical shifts of F-THDP2 sugar residues.

[0100]

[0101] Table 4. Chemical shift assignments of THDP2 sugar residues

[0102]

[0103] In summary, it was confirmed that F-THDP2 is composed of polysaccharides with a weight percentage of over 99%. The monosaccharide composition consists of D-galactose, D-mannose, L-fucose, and 3-O-methyl-D-galactose, with a molar ratio of 8.5-11.0:1.5-3:0.8-1.5:1.0 for D-galactose, D-mannose, L-fucose, and 3-O-methyl-D-galactose. Laser light scattering analysis confirmed that it is a single-component product with a weight-average molecular weight of 12.3 kDa-12.8 kDa. Nuclear magnetic resonance (NMR) spectra determined the glycosidic bond linkages. The main chain structure of the polysaccharide unit consisted of (1→2) linked α-D-galactose residues and (1→6) linked α-D-mannose residues. At the C-6 position of one (1→2) linked α-D-galactose residue on the main chain, a β-D-galactose terminal group was substituted. At the C-2 position of two consecutive (1→6) linked α-D-mannose residues on the main chain, a first branch consisting of a (1→6) linked α-3-O-methyl-D-galactose residue and a β-D-galactose terminal group, and an α-L-fucose terminal group, were substituted, respectively. Specific structural units could be… Figure 5a The structural unit shown can also be a structural unit with three branches arranged in other orders.

[0104] V. Higher Conformations of Polysaccharide Chains

[0105] Using the SEC-MALLS-Vis laser light scattering system, the molecular weights of THDP2 and F-THDP2 were determined to be 3.58 × 10⁻⁶. 4 g / mol and 1.23×10 4 The g / mol indicates that the molecular weight of the polysaccharide F-THDP2 after fermentation of *Trifolium repens* is lower than that of the unfermented polysaccharide THDP2.

[0106] Calculations using the Mark-Houwink-Sakurada equation showed that the α value of polysaccharide THDP2 before fermentation was 0.32, while the α value of F-THDP2 after fermentation was 0.76. This indicates that polysaccharide THDP2 had a compact chain structure in solution before fermentation, and that polysaccharide F-THDP2 underwent a transformation from a compact chain structure to a freely extended chain conformation in solution after fermentation.

[0107] The F-THDP2 aqueous solutions from Examples 1, 2, and 3 (5%-10% by mass) were respectively coated onto mica sheets for testing. Two-dimensional atomic force morphology of the polysaccharides was obtained. The results showed that the polysaccharide chains of THDP2 intertwined to form aggregates, creating tightly packed coils with a height of 1.18 nm ± 0.09 nm (Example 1, see below). Figure 8a Conversely, many extended molecular chains can be observed on F-THDP2 (as in Example 1). Figure 8dWith a chain height of 0.51nm±0.07nm, F-THDP2 exhibits a more extended flexible conformation.

[0108] VI. Comparison of the bioactivity of *Trifolium repens* polysaccharides before and after fermentation

[0109] 1. In vitro antitumor therapy (MTT method)

[0110] A549, HeLa, and MCF-7 cells in logarithmic growth phase were collected. Adherent cells were digested with 0.25% (w / v) trypsin and the cell concentration was adjusted to 1-10 × 10⁻⁶ cells / mL using RPMI 1640 complete culture medium. 4 Cell suspensions of 500 cells / mL were seeded into 96-well plates at a rate of 100 μL per well after accurate cell counting. The plates were then incubated at 37°C in a 5% (v / v) CO2 saturated humidity incubator for 24 h. The samples were then diluted to different concentrations by adding 10 μL of complete culture medium. The blank control group was prepared by adding an equal volume of RPM11640 complete culture medium. Each group had 5 replicates. The cell culture plates were then transferred to a CO2 incubator and incubated at 37°C in a 5% CO2 saturated humidity incubator for 24 h and 48 h. After culture, add 50 μL of freshly prepared MTT solution (5 mg / mL) to each well and incubate for 4 hours to reduce MTT. When filamentous purple crystals are observed around the cells in the wells under an inverted microscope, discard the supernatant. Add 200 μL of dimethyl sulfoxide (DMSO) to each well, mix well on a plate shaker, and measure the optical density (OD) at 570 nm using a microplate reader. Set up a control group and calculate the cell inhibition rate using the formula: Inhibition rate (%) = (OD value of control group - OD value of drug group) / OD value of control group × 100%.

[0111] Table 5. Comparison of antitumor activities of *Trifolium repens* polysaccharides before and after fermentation.

[0112]

[0113] Note: When comparing F-THDP2 with F-THDP1, F-THDP3, and the blank control group, * indicates P<0.05, and ** indicates p<0.01. When comparing F-THDP2 with Comparative Example 1 THDP2, # indicates P<0.05, and ## indicates p<0.01.

[0114] Table 6. Comparison of antitumor activities of *Trifolium repens* polysaccharides before and after fermentation.

[0115]

[0116] Note: When comparing F-THDP2 with F-THDP1, F-THDP3, and the blank control group, * indicates P<0.05, and ** indicates p<0.01. When comparing F-THDP2 with Comparative Example 1 THDP2, # indicates P<0.05, and ## indicates p<0.01.

[0117] Table 7 Comparison of antitumor activities of *Trifolium repens* polysaccharides before and after fermentation

[0118]

[0119] Note: When comparing F-THDP2 with F-THDP1, F-THDP3, and the blank control group, * indicates P<0.05, and ** indicates p<0.01. When comparing F-THDP2 with Comparative Example 1 THDP2, # indicates P<0.05, and ## indicates p<0.01.

[0120] The in vitro antitumor activity of three components, F-THDP2, F-THDP1, and F-THDP3, obtained by separating crude polysaccharide from *Trifolium repens* after fermentation using DEAE cellulose-52 was compared. Table 5-7 shows that, compared with the blank control group, F-THDP1 and F-THDP3 showed no significant difference in inhibition rates against the three cell lines (A549, HeLa, and MCF-7), indicating that F-THDP1 and F-THDP3 did not have the ability to inhibit tumor cells. Compared with THDP2, at the same concentration, F-THDP2 showed significantly better anticancer activity against all three cell lines (P<0.01), indicating that the anticancer activity of polysaccharide F-THDP2 was significantly enhanced after fermentation of *Trifolium repens*.

[0121] 2. Effects on gut microbiota

[0122] One hundred healthy mice were randomly divided into two groups of 10 each. Mice in the treatment group were administered the polysaccharide solution once daily by gavage at a concentration of 0.1 mg / mL, at a dose of 50 mg polysaccharide / kg body weight. After 14 days of continuous administration, the mice were sacrificed, and tissues were collected. Mice in the control group were administered the same volume of physiological saline as the treatment group by gavage once daily for 14 days, after which they were sacrificed, and tissues were collected. The contents of the mouse cecum were placed in centrifuge tubes containing 4.5 mL of sterile physiological saline and shaken well in a micro-shaker. This was 10... -1 Diluted solution, 10 -1 Centrifuge the diluted solution, take 0.5 mL of the supernatant, add it to a centrifuge tube containing 4.5 mL of sterile physiological saline, and mix well in a micro-shaker to obtain 10... -2 Dilute the solution by 10 times, following the steps above until diluted to 10. -7 Times. 10 -7Bacterial cultures were performed using diluted solutions. Lactobacillus was cultured anaerobically at 37°C for 48 hours on LBS agar medium; Escherichia coli was cultured aerobically at 37°C for 24 hours on EMB agar medium; and Bifidobacterium was cultured anaerobically at 37°C for 48 hours on BS agar medium. Each bacterial culture experiment was performed in triplicate. After the bacterial culture experiments, plate counts were performed to determine the number of bacteria in each of the three bacterial groups (Lactobacillus, Bifidobacterium, and Escherichia coli).

[0123] Table 8. Effects on the microbial community (unit: lg CFU / g, 10⁻⁶) 1 CFU / g)

[0124]

[0125] Note: F-THDP2 was compared with F-THDP1, F-THDP3, and the control group, respectively. * indicates P<0.05, and ** indicates p<0.01. F-THDP2 was compared with Comparative Example 1 THDP2, # indicates P<0.05, and ## indicates p<0.01.

[0126] The effects of three components (F-THDP2, F-THDP1, and F-THDP3) obtained from fermented *Trifolium repens* crude polysaccharide isolated via DEAE cellulose-52 on the intestinal microbiota of mice were compared. Table 8 shows that, compared with the control group, F-THDP1 and F-THDP3 showed no significant differences in the number of *Lactobacillus*, *Bifidobacterium*, and *Escherichia coli*, indicating that F-THDP1 and F-THDP3 components did not have a regulatory effect on intestinal flora. Compared with THDP2, F-THDP2 significantly increased the number of *Lactobacillus* and *Bifidobacterium* (p<0.01) and significantly decreased the number of *Escherichia coli* (p<0.01). *Lactobacillus* and *Bifidobacterium* are common probiotics; their abundant proliferation can inhibit the proliferation of pathogenic microorganisms and is of great significance for ensuring intestinal health, indicating that F-THDP2 can regulate the intestinal flora structure.

[0127] The polysaccharide of *Trifolium repens* fermented with *Sanghuang* fungus in this invention is composed of polysaccharides with a weight percentage of over 99%. This polysaccharide consists of galactose, mannose, fucose, and 3-O-methyl-galactose, with a molar ratio of 8.5-11.0:1.5-3:0.8-1.5:1.0. The weight-average molecular weight of the *Trifolium repens* polysaccharide fermented with *Sanghuang* fungus is 10 kDa-15 kDa. This polysaccharide exhibits strong biological activity, such as enhanced antitumor activity and enhanced intestinal microbial motility. Variations in the parameters of the preparation method of this invention do not affect the preparation of this polysaccharide; therefore, any combination of parameters in the preparation method of this invention can achieve the preparation of this polysaccharide. Further details are omitted here.

Claims

1. A polysaccharide of *Trifolium repens* prepared by fermentation of *Sanghuang* fungus, characterized in that, It is composed of polysaccharides with a weight percentage of more than 99%; the polysaccharides are composed of galactose, mannose, fucose and 3-O-methyl-galactose, wherein the molar ratio of galactose, mannose, fucose and 3-O-methyl-galactose is 8.5-11.0:1.5-3:0.8-1.5:1.

0.

2. The *Trifolium repens* polysaccharide according to claim 1, characterized in that, The galactose is α-galactose and β-galactose, the mannose is α-mannose, the fucose is α-fucose, and the 3-O-methyl-galactose is α-3-O-methyl-galactose.

3. The *Trifolium repens* polysaccharide according to claim 1 or 2, characterized in that, The galactose is α-D-galactose and β-D-galactose, the mannose is α-D-mannose, the fucose is α-L-fucose, and the 3-O-methyl-galactose is α-3-O-methyl-D-galactose.

4. The *Trifolium repens* polysaccharide according to claim 3, characterized in that, The main chain structure of the polysaccharide's structural unit consists of (1→2) linked α-D-galactose residues and (1→6) linked α-D-mannose residues. At the C-6 position of one (1→2) linked α-D-galactose residue on the main chain, a β-D-galactose end group is substituted. At the C-2 position of two consecutive (1→6) linked α-D-mannose residues on the main chain, a first branch consisting of a (1→6) linked α-3-O-methyl-D-galactose residue and a β-D-galactose end group, and an α-L-fucose end group are substituted, respectively.

5. The *Trifolium repens* polysaccharide according to claim 4, characterized in that, The β-D-galactose end group replaces the C-6 position of any (1→2) linked α-D-galactose residue on the main chain, and the first branch and an α-L-fucose end group are arranged in any order at the C-2 position of two consecutive (1→6) linked α-D-mannose residues.

6. The *Trifolium repens* polysaccharide according to claim 1, 2, 4, or 5, characterized in that, The weight-average molecular weight of the polysaccharide from *Trifolium repens* is 10 kDa-15 kDa.

7. The method for preparing *Trifolium repens* polysaccharide according to any one of claims 1-6, characterized in that, Including the following steps: (1) Preparation of fermented crude polysaccharide of *Trifolium repens*: *Trifolium repens* tubers were fermented with *Sanghuang* fungus. The fermentation product was extracted with water, precipitated with alcohol, and the protein was removed and the supernatant was freeze-dried to obtain crude polysaccharide of *Trifolium repens* fermented with *Sanghuang* fungus. (2) Purification: The aqueous solution of the crude polysaccharide of *San Ye Qing* obtained in step (1) after fermentation by *Sanghuang* was subjected to column chromatography packed with DEAE cellulose ion exchange resin. The eluent eluted with 0.15 mol / L-0.3 mol / L NaCl aqueous solution was collected and then subjected to gel filtration chromatography. The eluent containing polysaccharide was collected and then dialyzed and freeze-dried to obtain the polysaccharide of *San Ye Qing* after fermentation by *Sanghuang*.

8. The preparation method according to claim 7, characterized in that, Step (1) includes: cutting the tuber of *Trifolium repens* into thin slices, sterilizing, cooling and inoculating with *Sanghuang* fungus, fermenting and culturing, extracting the fermentation product with water at 90℃-100℃ to obtain an aqueous extract, concentrating to obtain a concentrated solution, removing the protein from the precipitate obtained by alcohol precipitation with Sevage reagent, and freeze-drying the supernatant to obtain crude polysaccharide of *Trifolium repens* fermented with *Sanghuang* fungus.

9. The preparation method according to claim 7 or 8, characterized in that, The fermentation conditions are: temperature 24℃-28℃, humidity 50%-65%, and fermentation time 18-25 days.

10. The use of the polysaccharide of *Trifolium repens* according to any one of claims 1-6 in the preparation of functional products for antitumor and / or enhancing intestinal motility, wherein the functional product is a pharmaceutical product.

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