Application of sargassum polysaccharide in preparation of immune promoting product

Abstract

The application discloses application of polysaccharides in Scyphiphora hydrophyllacea in preparation of immune promoting products; the immune promoting products include products for promoting immune cells to release nitric oxide; the immune promoting products include products for promoting immune cells to express inflammatory cytokines. The application studies the polysaccharides in Scyphiphora hydrophyllacea, optimizes an extraction process of the polysaccharides in Scyphiphora hydrophyllacea, especially carries out structural analysis on uniform polysaccharides obtained after purification of the polysaccharides in Scyphiphora hydrophyllacea extracted from different parts, studies in-vitro immune promoting activity of the purified polysaccharide components in Scyphiphora hydrophyllacea, and provides a theoretical basis for further development and utilization of the polysaccharides in Scyphiphora hydrophyllacea. Experimental results show that the polysaccharides in Scyphiphora hydrophyllacea roots are the polysaccharides in Scyphiphora hydrophyllacea with the highest immune promoting activity, and the polysaccharides can significantly promote generation of mononuclear macrophage NO and inflammatory cytokine IL-6, thereby realizing a regulating effect on immune response.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of *Lepidium apetalum* polysaccharide in the preparation of immune-boosting products. Background Technology

[0002] Leicai ( Acanthopanax trifoliatus (L.) Merr Acanthopanax senticosus, also known as white thorn or three-leaf eleutherococcus, is a perennial climbing shrub belonging to the genus Acanthopanax in the family Araliaceae. It is mainly distributed in Guangdong, Guangxi, Guizhou, Sichuan, and Yunnan provinces of my country, as well as Vietnam, Thailand, India, and Japan. The entire plant can be used medicinally, with the root or root bark being the most commonly used parts. It is a traditional Chinese medicine, recorded in the quality standards of medicinal materials in Guangdong and Guizhou under the names "Sanjiapi" and "Cisanjia." It is also known as "Jiujifeng" in Yao medicine and "Baoqiluosai" in Yi medicine. It has a bitter and astringent taste, is slightly cold in nature, and its effects include dispelling wind and dampness, clearing heat and detoxifying, dispersing blood stasis and relieving pain. It is used to treat rheumatic pain, damp-heat dysentery, and carbuncles and boils.

[0003] The Chinese artichoke contains abundant terpenes and their oxygenated compounds, including monoterpenes, sesquiterpenes, diterpenes, and triterpenes. Among these, diterpenes and triterpenes are the main active ingredients of the Chinese artichoke, possessing anti-inflammatory, analgesic, hypoglycemic, and antitumor effects. Terpenes are also the main component of the volatile oil of the Chinese artichoke, with monoterpenes and sesquiterpenes being the main components, accounting for up to 92.76%.

[0004] Plant polysaccharides are compounds composed of monosaccharides produced by cell metabolism linked by α- or β-glycosidic bonds. They are mainly distributed in plant cells and participate in various metabolic activities. The relative molecular mass of plant polysaccharides ranges from tens of thousands to millions, and they are generally composed of monosaccharides such as glucose, fructose, galactose, arabinose, xylose, rhamnose, fucose, mannose, and uronic acids. The chemical structure of plant polysaccharides is very complex, which is the basis for their active effects. Plant polysaccharides are not only abundant, diverse, and widely sourced, but also possess advantages such as high safety, low toxicity, high biocompatibility, and the ability to be chemically modified. Studies have shown that there is an organic link between the different biological activities of polysaccharides and their extraction processes and structures. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes the application of *Phyllostachys edulis* polysaccharides in the preparation of immune-boosting products.

[0006] This invention also proposes an immune-boosting product.

[0007] According to a first aspect of the present invention, the application of *Lepidium apetalum* polysaccharide in the preparation of immune-boosting products is proposed.

[0008] In some embodiments of the present invention, the immune-boosting product includes a product that promotes the release of nitric oxide from immune cells.

[0009] In some embodiments of the present invention, the immune-boosting product includes a product that promotes the expression of inflammatory cytokines by immune cells.

[0010] In some embodiments of the present invention, the inflammatory cytokine includes IL-6.

[0011] In some embodiments of the present invention, the immune cells include mononuclear macrophages.

[0012] In some embodiments of the present invention, the mononuclear macrophages include RAW264.7 cells.

[0013] In some embodiments of the present invention, the immune-boosting product comprises oleracea polysaccharide and pharmaceutically acceptable excipients.

[0014] In some embodiments of the invention, the pharmaceutically acceptable excipients include at least one of binders, disintegrants, lubricants, coating agents, suspending agents, thickeners, and surfactants.

[0015] In some embodiments of the present invention, the adhesive is selected from at least one of gum arabic, gelatin, dextrin, hydroxypropyl cellulose, methylcellulose, or polyvinylpyrrolidone.

[0016] In some embodiments of the present invention, the disintegrant is selected from at least one of corn starch, potato starch, croscarmellose, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, carboxymethyl cellulose, calcium carboxymethyl cellulose, or alginate.

[0017] In some embodiments of the present invention, the lubricant is selected from at least one of micronized silica gel, magnesium stearate, calcium stearate, stearic acid, talc, or anhydrous silica gel.

[0018] In some embodiments of the present invention, the coating agent includes at least one of hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, cellulose acetate phthalate, polyvinyl alcohol phthalate, ethyl cellulose, and cellulose acetate.

[0019] In some embodiments of the present invention, the suspending agent includes at least one of gum arabic, gelatin, methylcellulose, sodium carboxymethylcellulose, hydroxymethylcellulose, or aluminum stearate gel.

[0020] In some embodiments of the present invention, the surfactant is selected from at least one of lecithin, sorbitan monooleate, or glyceryl monostearate.

[0021] In some embodiments of the present invention, the administration methods of the anti-osteoporosis product include, but are not limited to, oral administration, enteral administration, subcutaneous injection, intramuscular injection, intravenous injection, nasal administration, transdermal administration, subconjunctival administration, intraocular administration, orbital administration, retroocular administration, retinal administration, choroidal administration, and intrathecal injection.

[0022] In some embodiments of the present invention, the dosage forms of the anti-osteoporosis products include, but are not limited to, tablets, capsules, pills, injections, inhalers, lozenges, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, suspensions, creams, gels, and sprays.

[0023] In some embodiments of the present invention, the dosage of *Phyllostachys edulis* polysaccharide in the immune-boosting product is 1-200 μg / mL.

[0024] In some embodiments of the present invention, the preparation method of the *Phyllostachys edulis* polysaccharide includes the following steps: S1: The powder of *Leymus chinensis* was defatted. The defatted powder of *Leymus chinensis* was mixed with water and subjected to ultrasonic treatment. Then it was heated for extraction. The extract was collected and concentrated. Then it was subjected to alcohol precipitation and washing to obtain crude polysaccharide of *Leymus chinensis*. S2: The crude polysaccharide of *Leymus chinensis* is subjected to protein removal and decolorization treatment, and then dialysis is performed to obtain *Leymus chinensis* polysaccharide.

[0025] In some embodiments of the present invention, the conditions for ultrasonic treatment in step S1 are 80~120 W ultrasound for 20~40 min.

[0026] In some embodiments of the present invention, the heating extraction conditions in step S1 are: extraction at 90~110℃ for 1~3 h, and extraction 2~3 times.

[0027] In some embodiments of the present invention, the degreasing process in step S1 includes degreasing by mixing anhydrous ethanol with *Gnaphalium affine* powder and heating.

[0028] In some embodiments of the present invention, the washing in step S1 includes washing and removing impurities in sequence with anhydrous ethanol, diethyl ether, and acetone.

[0029] In some embodiments of the present invention, the protein removal in step S2 is performed using Sevage reagent.

[0030] In some embodiments of the present invention, the decolorization process in step S2 is performed using a polyamide method.

[0031] In some embodiments of the present invention, the polysaccharide of *Lepidium apetalum* is extracted from at least one part of the root, stem, or leaf of *Lepidium apetalum*.

[0032] According to a second aspect of the present invention, an immune-boosting product is provided, the immune-boosting product comprising *Lepidium apetalum* polysaccharide.

[0033] In some embodiments of the invention, the immune-boosting product further includes pharmaceutically acceptable excipients.

[0034] In some embodiments of the invention, the pharmaceutically acceptable excipients include at least one of binders, disintegrants, lubricants, coating agents, suspending agents, thickeners, and surfactants.

[0035] In some embodiments of the present invention, the adhesive is selected from at least one of gum arabic, gelatin, dextrin, hydroxypropyl cellulose, methylcellulose, or polyvinylpyrrolidone.

[0036] In some embodiments of the present invention, the disintegrant is selected from at least one of corn starch, potato starch, croscarmellose, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, carboxymethyl cellulose, calcium carboxymethyl cellulose, or alginate.

[0037] In some embodiments of the present invention, the lubricant is selected from at least one of micronized silica gel, magnesium stearate, calcium stearate, stearic acid, talc, or anhydrous silica gel.

[0038] In some embodiments of the present invention, the coating agent includes at least one of hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, cellulose acetate phthalate, polyvinyl alcohol phthalate, ethyl cellulose, and cellulose acetate.

[0039] In some embodiments of the present invention, the suspending agent includes at least one of gum arabic, gelatin, methylcellulose, sodium carboxymethylcellulose, hydroxymethylcellulose, or aluminum stearate gel.

[0040] In some embodiments of the present invention, the surfactant is selected from at least one of lecithin, sorbitan monooleate, or glyceryl monostearate.

[0041] In some embodiments of the present invention, the administration methods of the anti-osteoporosis product include, but are not limited to, oral administration, enteral administration, subcutaneous injection, intramuscular injection, intravenous injection, nasal administration, transdermal administration, subconjunctival administration, intraocular administration, orbital administration, retroocular administration, retinal administration, choroidal administration, and intrathecal injection.

[0042] In some embodiments of the present invention, the dosage forms of the anti-osteoporosis products include, but are not limited to, tablets, capsules, pills, injections, inhalers, lozenges, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, suspensions, creams, gels, and sprays.

[0043] In some embodiments of the present invention, the dosage of *Phyllostachys edulis* polysaccharide in the immune-boosting product is 1-200 μg / mL.

[0044] In some embodiments of the present invention, the immune-boosting product includes a product that promotes the release of nitric oxide from immune cells.

[0045] In some embodiments of the present invention, the immune-boosting product includes a product that promotes the expression of inflammatory cytokines by immune cells.

[0046] In some embodiments of the present invention, the inflammatory cytokine includes IL-6.

[0047] In some embodiments of the present invention, the immune cells include mononuclear macrophages.

[0048] In some embodiments of the present invention, the mononuclear macrophages include RAW264.7 cells.

[0049] The present invention has at least the following beneficial effects: This invention studies the polysaccharides of *Leymus chinensis*, optimizes the extraction process, and, in particular, analyzes the structure of homogeneous polysaccharides obtained from different parts of *Leymus chinensis* after purification. The in vitro immunomodulatory activity of the purified *Leymus chinensis* polysaccharide fraction is investigated, providing a theoretical basis for the further development and utilization of *Leymus chinensis* polysaccharides. Experimental results show that *Leymus chinensis* root polysaccharides have the highest immunomodulatory activity, significantly promoting the production of NO and the inflammatory cytokine IL-6 by monocytes and macrophages, thereby regulating the immune response. Attached Figure Description

[0050] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a glucose standard curve diagram from Example 2 of the present invention; Figure 2 This is a protein standard curve diagram from Example 2 of the present invention; Figure 3 The infrared spectrum of the polysaccharide from the root of *Lepidium apetalum* in Example 2 of this invention; Figure 4 The infrared spectrum of the polysaccharide from the stem of *Gnaphalium affine* in Example 2 of this invention; Figure 5 The infrared spectrum of the polysaccharide from the leaves of *Gnaphalium affine* in Example 2 of this invention; Figure 6 This is a graph showing the cell viability detection results after treatment with *Leymus chinensis* root polysaccharide in Example 3 of the present invention; Figure 7 This is a graph showing the detection results of NO release from cells after treatment with *Leymus chinensis* root polysaccharide in Example 3 of the present invention. Figure 8 This is a graph showing the detection results of IL-6 release from cells after treatment with polysaccharides from different parts of *Leymus chinensis* in Example 3 of the present invention. Detailed Implementation

[0051] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0052] Example 1: Extraction and purification of polysaccharides from *Lepidium apetalum* This embodiment provides a method for preparing *Phyllostachys edulis* polysaccharide, and the preparation process parameters have been screened and optimized, as detailed below: 1. Extraction of polysaccharides from *Lepidium apetalum* Dried roots, stems, and leaves of *Lepidium apetalum* were separately pulverized and passed through a 20-mesh sieve. 100 g of the sieved powder from different parts (roots, stems, and leaves) was collected. The powder was mixed with anhydrous ethanol at a volume ratio of 1:10 and added to a beaker. The mixture was refluxed twice in a water bath at 90°C for 2 hours each time to defatted the powder. After evaporating the ethanol, the powder was weighed to obtain defatted white *Lepidium apetalum* stem powder. The defatted white *Lepidium apetalum* stem powder was then mixed with distilled water at a material-to-liquid ratio of 1 g:20 mL and sonicated at 100 W for 30 min. The mixture was then extracted twice in a 95°C water bath for 2 hours each time. The extracts were combined and concentrated under reduced pressure to 20% of the total volume. Four times the volume of anhydrous ethanol was added to the supernatant, and the mixture was precipitated at -4°C for 7 hours (6-8 hours is acceptable). The precipitate was then filtered, and the precipitate was collected. The precipitate was then washed successively with anhydrous ethanol, ether, and acetone to remove impurities, yielding crude polysaccharides from different parts of *Lepidium apetalum*.

[0053] 2. Purification of *Lepidium apetalum* polysaccharides Crude polysaccharides from *Leymus chinensis* were dissolved in water and deproteinized using the Sevage method. Sevage reagent (n-butanol:chloroform = 1:4) was prepared, with a polysaccharide-to-Sevage reagent volume ratio of 5:1. The mixture was stirred with a rotor until a water flow sound was heard, for 30 min, followed by centrifugation at 8000 rpm for 10 min. The supernatant was collected, and the deproteinization process was repeated 4-6 times. The deproteinized polysaccharide solution was then rotary evaporated to remove the organic reagents, followed by decolorization. Decolorization was performed using polyamide; 25% polyamide powder (by volume of the solution) was added, and the mixture was heated and stirred at 50℃ for 30 min, centrifuged, and the supernatant was collected. The supernatant was dialyzed for 48 h, filtered through a membrane, centrifuged at 8000 rpm for 10 min, and the supernatant was freeze-dried to obtain total polysaccharides from different parts of *Leymus chinensis*.

[0054] Example 2 Characterization of polysaccharides from *Lepidium apetalum* This embodiment characterizes the polysaccharides extracted from different parts of *Gnaphalium affine* obtained in Example 1 from various aspects. The specific experimental methods and results are as follows: 1. The polysaccharide content of the crude polysaccharide from *Gnaphalium affine* obtained in step 1 of Example 1 was determined using the phenol-sulfuric acid spectrophotometric method. 1) Glucose standard curve preparation: Accurately weigh 10 mg of glucose standard into a 100 mL volumetric flask, and dilute to 100 mL with distilled water to prepare a 0.1 mg / mL glucose standard solution; transfer the prepared glucose standard solution and dilute to 5 mL with distilled water to prepare 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, 0.10 mg / mL, 0.12 mg / mL, and 0.14 mg / mL solutions respectively. Take 200 μL of each glucose dilution and place it in a 2 mL test tube. Add 100 μL of 6% phenol and 500 μL of concentrated sulfuric acid to each test tube, let stand for 10 min, shake well, and incubate at room temperature for 20 min. Dispense 200 μL of each solution into three wells of a 96-well plate. Measure the absorbance at 490 nm using a microplate reader, with distilled water as a blank control. Plot a standard curve with glucose concentration on the x-axis and absorbance on the y-axis. The resulting standard curve is shown below. Figure 1 As shown. Its regression equation is y = 8.6042x + 0.0166; the goodness of fit of the equation is Rfit. 2 = 0.9992. Within the range of 0–0.14 mg / mL, glucose concentration showed a good linear relationship with absorbance.

[0055] 2) Determination of polysaccharide content: Crude polysaccharides extracted from different parts of *Lepidium apetalum* were prepared into a 0.1 mg / mL solution using distilled water. 200 μL of the crude polysaccharide solution was added to each of three test tubes, along with phenol and sulfuric acid in the specified proportions. The mixture was allowed to stand for 5 minutes, then shaken well and incubated at room temperature for 20 minutes. The absorbance was measured at 490 nm using a microplate reader. The polysaccharide content of the crude polysaccharide was calculated using the following formula: Polysaccharide content (%) = ; In the formula, C is the sugar content (mg) calculated from the standard curve; V2 is the total volume of the extract (mL); V1 is the volume used for determination (mL); D is the dilution factor; and W is the sample weight (g).

[0056] The polysaccharide contents of different parts of the root, stem, and leaves of *Lepidium apetalum* were calculated using the standard curve to be 68.48%, 79.6%, and 60.6%, respectively.

[0057] 2. The protein content of *Leonurus japonicus* polysaccharides was determined using the Coomassie Brilliant Blue G250 method. Accurately weigh 100 mg of bovine serum albumin (BSA) and add 0.15 mol / L NaCl solution to the mark in a 100 mL volumetric flask. Aliquot the solution and set aside. Weigh 100 mg of Coomassie Brilliant Blue powder and dissolve it first in 50 mL of 95% ethanol solution. Then add 100 mL of 85% phosphoric acid solution and finally add ultrapure water to bring the volume to 1 L. After the Coomassie Brilliant Blue is fully dissolved, filter the solution through a 0.22 or 0.45 μm syringe filter. Accurately pipette 0, 20, 40, 60, 80, and 100 μL of 1.0 mg / mL bovine serum albumin standard solution, respectively, and add 0.15 mol / L NaCl solution to bring the volume to 100 μL. Then add 5 mL of Coomassie Brilliant Blue solution to each tube and vortex thoroughly to mix. React at room temperature for 5 min and measure the absorbance at 595 nm. Repeat each sample three times and take the average value. A standard curve was plotted with standard protein concentration (mg / mL) on the x-axis and absorbance on the y-axis, and the regression equation was calculated. The resulting standard curve is shown below. Figure 2 As shown in the figure. The regression equation is y = 688.89x - 280596; the goodness of fit is R² = 0.994. Within the range of 0–2.5 mg / mL, bovine serum albumin concentration and absorbance show a good linear relationship.

[0058] Weigh the crude polysaccharide and total polysaccharide samples obtained in Example 1, and prepare 1.00 mg / mL solutions with distilled water. Take 0.10 mL of each solution, and then add 5 mL of Coomassie Brilliant Blue solution, vortexing thoroughly to mix. React at room temperature for 5 min, and measure the absorbance of each tube at a wavelength of 595 nm. Repeat each sample three times, and take the average. Calculate the protein removal rate using the following formula: Protein Removal Rate (%) ; Wherein, ρ1 is the mass concentration of protein in the solution prepared before protein removal from the sample, and ρ2 is the mass concentration of protein in the solution prepared after protein removal from the sample.

[0059] The absorbance values ​​of crude polysaccharides from *Leymus chinensis* before protein removal were measured and substituted into the regression equation, yielding protein concentrations of 51.19%, 57.8%, and 46.2% for the roots, stems, and leaves, respectively. The absorbance values ​​of total polysaccharides from *Leymus chinensis* after protein removal were measured and substituted into the regression equation, yielding protein concentrations of 11.7%, 13.6%, and 4.9% for the roots, stems, and leaves, respectively.

[0060] 3. Infrared spectral analysis of polysaccharides from *Lepidium apetalum* The total polysaccharides from *Gnaphalium affine* obtained in Example 1 were further purified by column chromatography and then analyzed by infrared spectroscopy.

[0061] 1) DEAE cellulose ion chromatography purification: ①Pretreatment of DEAE-52 packing: The DEAE-52 packing was activated by adding approximately two liters of pure water to half the column volume of DEAE-52 packing and soaking it overnight at 4°C to remove the upper layer of flocculation. The next day, the packing was soaked in a 0.5 mol / L NaOH solution for 1 hour, followed by repeated washing with distilled water until neutral. Then, it was soaked in a 0.5 mol / L HCl solution for 1 hour, followed by repeated washing with distilled water until neutral. Finally, the packing was soaked in a 0.5 mol / L NaOH solution for 1 hour, followed by repeated washing with distilled water until neutral. An appropriate amount of distilled water was added, and the packing was soaked at 4°C for later use. Before packing the packing material into the column, the packing material was stirred with a magnetic stirrer for 30 min and then degassed by ultrasound for 1 h. The degassed packing material was then slowly and continuously guided into the cylindrical chromatography column (4×60 cm) using a glass rod. The constant flow pump was turned on, and an automatic pressurizing pump was used, along with gentle tapping of the chromatography column to compact the packing material until the liquid level remained unchanged. The constant flow pump was adjusted to maintain a flow rate of 1.5 mL / min, and the volumes of the three columns were balanced.

[0062] ② Weigh 1 g of total polysaccharides from *Leymus chinensis*, dissolve in 30 mL of distilled water, centrifuge, filter through a 0.22 mm PES membrane, and add to a chromatography column. After all the liquid has flowed into the column, turn off the constant flow pump and allow it to stand for 1 h to ensure sufficient contact between the sample solution and the packing material. Turn on the constant flow pump and elute first with deionized water, then sequentially with NaCl solutions of different concentration gradients: 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L. Collect approximately 100 tubes of each fraction using an automatic collector at a rate of one tube every 7 minutes. Concentrate the collected polysaccharide solutions of different fractions under reduced pressure. Mix and concentrate the same fraction to 30 mL, transfer to a dialysis bag with a molecular weight cutoff of 8000 Da, and dialyze with distilled water. Freeze-dry the dialyzed polysaccharide solutions to obtain purified polysaccharides of different fractions.

[0063] 2) G-100 dextran gel column chromatography: ① Pretreatment of G-100 dextran gel filler: Soak G-100 dextran gel filler in distilled water for 2 hours until it expands, remove the upper layer of floating flocculation, and then place it at 4℃ for later use.

[0064] The DEAE-52 packing material was degassed according to the degassing method described above. The degassing was then directed into a cylindrical chromatography column (1.5 × 60 cm). The constant flow pump was adjusted to equilibrium, and the column was gently tapped to compact the packing. The polysaccharide sample purified by DEAE cellulose ion chromatography was reconstituted with distilled water to a 10 mg / mL polysaccharide solution. This solution was then spread evenly on the packing material using a dropper. Elution was performed using an automatic collector, collecting one tube every 15 minutes, to obtain the final fractions of *Leymus chinensis* polysaccharides from different parts.

[0065] Two mg of total polysaccharides from different parts of *Leymus chinensis* extracted in Example 1 were weighed out and placed in a mortar. Dry KBr was added at a ratio of 1:100, and the mixture was thoroughly ground and mixed. After tableting, the tablets were scanned using a Fourier transform infrared spectroscopy (FTIR) spectrometer, with a scanning range of 4000–400 cm⁻¹. -1 The results obtained are as follows: Figures 3-5 As shown.

[0066] Depend on Figure 3-5 It can be seen that 3600-3200 cm -1 The absorption band within this range corresponds to the stretching vibration of -OH, a characteristic peak of polysaccharides. Specifically, 3407.1 cm⁻¹ -1 The absorption peak at 2931.2 cm⁻¹ indicates the stretching vibration of the OH groups in the sugar. -1 The band observed at 1739.40 cm is attributed to CH stretching vibration. -1 The absorption peak at 1610.20 cm⁻¹ and 1610.20 cm⁻¹ -1 The absorption peak at 1243.07 cm⁻¹ corresponds to the stretching vibration of C=O, indicating the presence of a carbonyl group. Furthermore, the absorption peak at 1243.07 cm⁻¹... -1 The peak at that location, and 1099.40 cm. -1 and 1019.40 cm -1 The absorption at this site may be related to the stretching vibrations of CO and COC, indicating the presence of glycosidic bonds and pyranose ring structures in the polysaccharide. These features collectively confirm the typical polysaccharide structure of ATP.

[0067] Example 3: Immunostimulatory activity of *Lepidium apetalum* polysaccharides This embodiment tested the cytotoxicity and immunopromoting activity of total polysaccharides from different parts of *Leymus chinensis* from Example 1 using the RAW264.7 mononuclear macrophage cell line. The specific experimental methods and results are as follows: 1. Assay for cytotoxicity RAW264.7 macrophages in the logarithmic growth phase were resuspended in DMEM medium to a concentration of 5 × 10⁻⁶ cells / mL. 5 Cells were counted at a density of 100 μL / mL. The cell suspension was then seeded into 96-well plates at 200 μL per well and cultured at 37°C and 5% carbon dioxide for 24 h. Cell morphology and number were observed under a microscope.

[0068] Discard the culture medium and prepare 20 mg / mL *Leymus chinensis* root polysaccharide solutions with PBS. Filter the solutions through a 33 nm syringe filter to remove impurities, obtaining *Leymus chinensis* root polysaccharide stock solutions. Dilute the *Leymus chinensis* root polysaccharide stock solutions to 200 μg / mL, 100 μg / mL, 50 μg / mL, 10 μg / mL, and 1 μg / mL using fresh DMEM complete medium containing 10% FBS and 1% antibiotics. Add the prepared medium along the wall of the plate, repeating five wells for each concentration. After culturing for 24 h, add 0.01 mL of MTT (5 mg / mL) to each well and incubate for another 2 h in a constant temperature incubator. After culturing, remove the 96-well plate and aspirate all the culture medium (be careful not to aspirate the bottom cells). Add 100 μL of dimethyl sulfoxide to each well to dissolve any crystals formed. Shake for 5 min. Then, use a SYNERGY NEO2 microplate reader to measure the absorbance of each well at 550 nm. The results are shown below. Figure 6 As shown.

[0069] Depend on Figure 6 It can be seen that the polysaccharide of *Leymus chinensis* root has a slight effect on promoting the proliferation of RAW264.7 cells. However, the growth-promoting effect on cells weakens with increasing treatment concentration, but no cytotoxic effect has been observed. This indicates that the polysaccharide of *Leymus chinensis* root has no cytotoxic effect on RAW264.7 cells at concentrations of 1-200 μg / mL.

[0070] 2. Determination of NO release To evaluate the effect of *Leymus chinensis* root polysaccharide on NO release from macrophages, a blank control group, a positive control group, and an experimental group were set up. The blank control group received no LPS treatment and was cultured in DMEM complete medium for 24 h, followed by a 24 h culture with fresh DMEM complete medium. The positive control group was cultured in 1 µg / mL LPS solution (prepared in DMEM complete medium) for 24 h. The experimental groups were cultured in different concentrations of *Leymus chinensis* root polysaccharide (1, 2, 5, 10, and 25 µg / mL) for 24 h. After culture, the NO release from macrophages in each group was measured using the Griess method. The results are shown below. Figure 7 As shown.

[0071] Depend on Figure 7 It was found that the effect of *Leymus chinensis* root polysaccharide on NO release increased with increasing drug concentration. At a drug concentration of 1 μg / mL, the effect of *Leymus chinensis* root polysaccharide on NO release from RAW264.7 cells was significantly increased. When the drug concentration reached 25 μg / mL, NO release further increased, indicating that the promoting effect of *Leymus chinensis* root polysaccharide on NO release from macrophages was concentration-dependent.

[0072] 3. Determination of IL-6 production IL-6 can stimulate the proliferation and differentiation of T cells and B cells, promote antibody production and the formation of immune memory. Furthermore, IL-6 can participate in the initiation and maintenance of the immune response by regulating the migration and activation of immune cells. In this embodiment, the mRNA expression of the inflammatory factor IL-6 in RAW264.7 cells after treatment with polysaccharides from the roots, stems, and leaves of *Lespedeza bicolor* at different concentrations was detected by qRT-PCR. The experimental methods and results are as follows: RAW264.7 cells in good condition and in the logarithmic growth phase were selected. The supernatant in the culture dish was discarded, and the cells were washed once or twice with preheated PBS. Next, 2 mL of DMEM medium was added to the cell culture dish, and the cells were gently pipetted to detach them from the surface of the culture dish. Then, 10 μL of the cell suspension was mixed with 10 μL of trypan blue solution, and the cells were counted. Subsequently, the cells were seeded at a density of 500,000 per well in 6-well plates, with 2 mL of cell suspension seeded in each well. After culturing for 24 h, the supernatant was discarded, and DMEM medium containing different concentrations and fractions of *Leymus chinensis* polysaccharide was added, and the cells were cultured for another 24 h. The concentrations of *Leymus chinensis* polysaccharide were set at 1, 5, 10, and 100 μg / mL, and 1 μg / mL LPS was used as a positive control.

[0073] After culturing, RNA was extracted from each group of cells using the following methods: (1) Discard the supernatant and wash 2-3 times with pre-cooled PBS, and blot dry as much as possible on the last wash; (2) On ice, add 1 mL of Trizol reagent to each well and gently pipette the cells in the well repeatedly to ensure that the cells are fully lysed. Then transfer the lysate to a 1.5 mL centrifuge tube and let it stand at room temperature for 5 min. (3) Take 200 μL of chloroform and add it to each of the 1.5 mL centrifuge tubes containing the lysis buffer. Invert the tubes to mix thoroughly, then place them on a vortex mixer and vortex for 15 s. Let them stand at room temperature for 3 min to separate into layers. (4) Centrifuge at 4℃ and 11000 rpm for 15 min (after centrifugation, the liquid surface is divided into 3 layers, of which RNA is in the upper layer, protein is in the middle layer, and chloroform is in the lower layer). Carefully collect the upper aqueous phase and transfer it to a clean enzyme-free centrifuge tube. (5) Add the same volume of isopropanol solution as the aqueous phase, invert and mix well, let stand at room temperature for 10 min; centrifuge at 11000 rpm for 10 min at 4℃; discard the supernatant, and aspirate the liquid remaining on the inner wall of the centrifuge tube, being careful not to aspirate the precipitate; add 1 mL of anhydrous ethanol to the centrifuge tube and wash the precipitated RNA twice, inverting the tube to suspend the RNA and wash thoroughly, being careful not to blow away the RNA, otherwise the RNA content will be lost; centrifuge at 8500 rpm for 5 min at 4℃; pour out the supernatant after centrifugation, open the cap, and then place the centrifuge tube on the table to allow the ethanol to evaporate; (6) Add 20 μL of DEPC water to each tube and let it stand on ice for 10 min to allow the RNA to dissolve completely. You can gently tap the tube wall to dissolve the RNA. (7) After the RNA purity is detected by the nucleic acid protein analyzer, the RNA is stored in a -80℃ freezer; (8) RNA was reverse transcribed into cDNA, and qRT-PCR was performed using cDNA as a template. The reaction procedure is shown in Table 1, and the results are as follows. Figure 8 As shown.

[0074] The primer sequence used for IL-6 is as follows: F: 5'-GAGAGGAGACTTCACAGAGGTACC-3' (SEQ ID NO: 1); R: 5'-TCATTTCCCGATTTCCCAGAGAAC-3' (SEQ ID NO: 2).

[0075] Table 1 qRT-PCR reaction procedure

[0076] Depend on Figure 8 It can be seen that, compared with other parts of the plant, the root polysaccharide of the plant can significantly increase the expression level of IL-6 inflammatory factor mRNA in a dose-dependent manner.

[0077] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Application of *Lepidium apetalum* polysaccharides in the preparation of immune-boosting products.

2. The application according to claim 1, characterized in that, The immune-boosting products include those that promote the release of nitric oxide from immune cells.

3. The application according to claim 1, characterized in that, The immune-boosting products include those that promote the expression of inflammatory cytokines by immune cells.

4. The application according to claim 1, characterized in that, The immune-boosting product includes oleracea polysaccharide and pharmaceutically acceptable excipients.

5. The application according to claim 1, characterized in that, The preparation method of the *Phyllostachys edulis* polysaccharide includes the following steps: S1: The powder of *Leymus chinensis* was defatted. The defatted powder of *Leymus chinensis* was mixed with water and subjected to ultrasonic treatment. Then it was heated for extraction. The extract was collected and concentrated. Then it was subjected to alcohol precipitation and washing to obtain crude polysaccharide of *Leymus chinensis*. S2: The crude polysaccharide of *Leymus chinensis* is subjected to protein removal and decolorization treatment, and then dialysis is performed to obtain *Leymus chinensis* polysaccharide.

6. The application according to claim 5, characterized in that, The conditions for ultrasonic treatment in step S1 are 80~120 W ultrasound for 20~40 min.

7. The application according to claim 5, characterized in that, The heating extraction conditions described in step S1 are: extraction at 90~110℃ for 1~3 h, and extraction 2~3 times.

8. The application according to claim 1, characterized in that, The polysaccharide from *Lepidium apetalum* is extracted from at least one part of the root, stem, or leaf of *Lepidium apetalum*.

9. An immune-boosting product, characterized in that, The immune-boosting product includes *Lepidium apetalum* polysaccharide.

10. The immune-boosting product according to claim 9, characterized in that, The immune-boosting products also include pharmaceutically acceptable excipients.

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