Low-molecular-weight radix stemonae polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to low-molecular-weight stemona polysaccharide (STP-3-2) as well as a preparation method and application thereof, and the weight-average molecular weight (Mw) of the low-molecular-weight stemona polysaccharide (STP-3-2) is between 15 kDa and 5 kDa. The preparation method comprises the following steps: taking dry tuberous roots of stemona tuberosa, extracting to obtain non-starch total polysaccharide of stemona tuberosa, and separating and purifying to obtain the low-molecular-weight stemona tuberosa polysaccharide. The polysaccharide can obviously activate macrophages, enhance the phagocytic function of the macrophages, promote secretion of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) and up-regulate expression of inducible nitric oxide synthase (iNOS), IL-6 and TNF-alpha mRNA, shows obvious immunological enhancement activity, does not have obvious toxic or side effects, and can be used for preparing the immunopotentiator. The polypeptide can be used for preparing medicines or functional foods for treating and / or preventing diseases such as immunosuppression and the like, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a low-molecular-weight Stemona polysaccharide and its application in preparing immunomodulatory drugs or functional foods. Background Art

[0002] Immune regulation is closely linked to the development and progression of numerous diseases, and the immunomodulatory activity of polysaccharides from traditional Chinese medicines (TCMs) has garnered considerable attention. Stemona radix (Stemona sessilifolia) (Miq.) Miq., Stemona japonica (Bl.) Miq., and Stemona tuberosa (Lour.) are among the dried roots of these herbs. As a traditional Chinese medicine, Stemona radix (Stemona sessilifolia) is widely used to treat coughs, asthma, itching, and chronic lung diseases. Among these sources, Stemona tuberosa (S. tuberosa) is the most widely distributed and has become the primary source of Stemona radix on the market.

[0003] Previous studies have shown that Stemona tuberosa contains a variety of chemical components, including alkaloids, stilbenoids, and polysaccharides. However, existing polysaccharide research has mainly focused on optimizing extraction methods, conducting preliminary physicochemical analysis of crude polysaccharides, and studying the structure and anti-inflammatory activity of a low-molecular-weight component called SPS2-A (1.8 kDa). This polysaccharide is composed of D-glucose (Glc), D-galactose (Gal), L-arabinose (Ara), D-xylose (Xyl), and D-mannose (Man). Its backbone consists of α1,4-D-Glcp, α1,4-D-Galp, and β1,4,6-D-Galp residues, with α-D-Glcp as a monosaccharide side chain attached to the C-6 position of the β1,4,6-D-Galp residue (International Journal of Biological Macromolecules, 2024, 282, 136617). Despite this, research on the structural characteristics and efficacy of other major polysaccharide components in the roots of Stemona odorata is still relatively scarce. Summary of the Invention

[0004] The purpose of the present invention is to provide a low molecular weight Stemona polysaccharide (STP-3-2) with immunoenhancing activity, a preparation method thereof and an application thereof in the preparation of immunomodulatory drugs or functional foods.

[0005] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0006] In one aspect, the present invention provides a low molecular weight Stemona polysaccharide, which is a mixture of homologous polysaccharides having a structure of formula (I), wherein formula (I) is as follows:

[0007]

[0008] In formula (I):

[0009] R1 is H,

[0010] R2 is

[0011]

[0012] R3 is H or

[0013] R4 is H or -CH2CH3;

[0014] R5 is H or -CH3; the degree of methylation is (21.5±5.0)% calculated based on the peak area ratio of the anomeric hydrogen in the hydrogen spectrum;

[0015] l is an integer with a mean value of 29; m is an integer with a mean value of 8±2; and n is an integer with a mean value of 3±2.

[0016] Furthermore, the weight average molecular weight (Mw) of the low molecular weight Stemona polysaccharide ranges from 3 kDa to 20 kDa, and the polydispersity index ranges from 1.0 to 1.8. More preferably, the weight average molecular weight (Mw) of the low molecular weight Stemona polysaccharide ranges from 5 kDa to 15 kDa.

[0017] The polydispersity index (PDI) of the low molecular weight Stemona polysaccharide of the present invention refers to the ratio of the weight average molecular weight Mw to the number average molecular weight Mn of the low molecular weight Stemona polysaccharide.

[0018] Furthermore, the monosaccharide composition contained in the low molecular weight Stemona polysaccharide is a mixture of galacturonic acid (GalA), rhamnose (Rha), galactose (Gal), arabinose (Ara), xylose (Xyl), glucose (Glc), and glucuronic acid (GlcA), and the corresponding molar percentages are (41.7±5.0)%, (22.4±3.0)%, (15.1±3.0)%, (11.2±2.0)%, (4.9±1.0)%, (3.1±1.0)%, and (1.5±0.5)%, respectively.

[0019] The second aspect of the present invention provides a method for preparing the low molecular weight Stemona polysaccharide described above, comprising the following steps: taking the dried tuberous roots of Stemona radix, extracting and obtaining the non-starch Stemona total polysaccharide, and separating and purifying the non-starch Stemona total polysaccharide to obtain the low molecular weight Stemona polysaccharide.

[0020] The present invention uses Stemona tuberosa Lour. Its Latin name is Stemona tuberosa Lour. The method for extracting the non-starch total polysaccharides of Stemona tuberosa Lour. is to use conventional polysaccharide extraction technology to remove starch, protein and small molecule impurities using amylase, and then obtain the non-starch total polysaccharides of Stemona tuberosa Lour. through alcohol precipitation and drying.

[0021] Furthermore, the present invention provides a preferred method for extracting and obtaining non-starch total polysaccharides from Stemona radix, which specifically comprises: taking dried tuberous roots of Stemona radix, adding a solvent to extract 1 to 3 times, separating the extracts and combining them, adding amylase for enzymatic hydrolysis, inactivating the enzyme, ethanol precipitation, deproteinization, ethanol precipitation, and precipitation and drying to obtain non-starch total polysaccharides from Stemona radix.

[0022] Furthermore, the solvent is preferably water. The drying method is preferably freeze-drying.

[0023] Furthermore, the separation and purification of the non-starch Stemona total polysaccharide specifically includes: using the non-starch Stemona total polysaccharide as raw material, separating by anion exchange chromatography column, collecting 0.3M sodium chloride elution components, desalting and concentrating by dialysis, and further separating and purifying by gel chromatography column, collecting each fraction of the main chromatographic peak, desalting by dialysis or ultrafiltration membrane filtration, and drying to obtain the low molecular weight Stemona polysaccharide.

[0024] In the above technical solution, the filler of the anion exchange chromatography column can be a common commercially available filler, such as DEAE Sepharose Fast Flow, Amberlite FPA98Cl, etc.; the filler of the gel chromatography column can be a common commercially available filler, such as Sepharose CL-4B, Sepharose CL-6B, Sephadex G-100, Sephadex G-200, etc.

[0025] The third aspect of the present invention also provides the use of the low molecular weight Stemona polysaccharide described above in the preparation of medicines or functional foods for treating and / or preventing diseases such as immunodeficiency.

[0026] Furthermore, the use of the low-molecular-weight Stemona polysaccharide described above in the preparation of a drug or functional food has been shown to significantly activate macrophages and enhance their phagocytic function, promote the secretion of nitric oxide (NO), tumor necrosis factor-α, and interleukin-6, and upregulate the expression of inducible nitric oxide synthase, IL-6, and TNF-α mRNA. The mechanism of action may be related to the activation of the nuclear factor κB (NF-κB) signaling pathway mediated by Toll-like receptor 4 (TLR4).

[0027] The present invention has the following beneficial effects:

[0028] (1) The present invention obtains a low molecular weight Stemona polysaccharide STP-3-2 with a clear structure. This compound has significant advantages in the development of new drugs and can avoid interference from other impurities.

[0029] (2) The present invention obtained a Stemona polysaccharide STP-3-2 with unique structural characteristics, clarified that its main chain contains RG-I type and HG type structural domains of pectin polysaccharides, and revealed the specific composition and chemical modification (such as acetylation, methylation, and methyl esterification) of the side chains, providing a new paradigm for the study of the structural diversity of natural polysaccharides.

[0030] (3) The present invention confirms that the low molecular weight Stemona polysaccharide STP-3-2 can significantly enhance the phagocytic function of macrophages RAW264.7 by activating the TLR4 / NF-κB signaling pathway, upregulate the expression of iNOS, IL-6, and TNF-α mRNA, and promote the secretion of NO, TNF-α and IL-6. It has a clear immune enhancement effect and is expected to be developed as an immunomodulator. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figure is a high performance liquid gel permeation chromatogram of the molecular weight distribution of the low molecular weight Stemona polysaccharide STP-3-2 of the present invention.

[0032] Figure 2 This is a high performance liquid chromatogram of the monosaccharide composition analysis of the low molecular weight Stemona polysaccharide STP-3-2 of the present invention.

[0033] Figure 3 This is the total ion current diagram of the methylated sugar alcohol acetyl derivative of the low molecular weight Stemona polysaccharide STP-3-2 reduced derivative STP-3-2Re of the present invention.

[0034] Figure 4 This is the hydrogen spectrum of the low molecular weight Stemona polysaccharide STP-3-2 of the present invention.

[0035] Figure 5 This is the carbon spectrum of the low molecular weight Stemona polysaccharide STP-3-2 of the present invention.

[0036] Figure 6 The low molecular weight Stemona polysaccharide STP-3-2 of the present invention 1 H- 13 C HSQC-TOCSY spectrum.

[0037] Figure 7 The low molecular weight Stemona polysaccharide STP-3-2 of the present invention 1 H- 1 H ROESY spectrum.

[0038] Figure 8The low molecular weight Stemona polysaccharide STP-3-2 of the present invention 1 H- 13 C HMBC spectrum.

[0039] Figure 9 This is the effect of the low molecular weight Stemona polysaccharide STP-3-2 of the present invention on the proliferation ability of macrophage RAW264.7 cells.

[0040] Figure 10 The present invention shows the effect of the low molecular weight Stemona polysaccharide STP-3-2 on the phagocytic ability of macrophage RAW264.7 cells.

[0041] Figure 11 This is the effect of the low molecular weight Stemona polysaccharide STP-3-2 of the present invention on the release of NO in macrophage RAW264.7 cells.

[0042] FIG12 shows the effect of the low molecular weight Stemona polysaccharide STP-3-2 of the present invention on the release of IL-6 (A) and TNF-α (B) from macrophage RAW264.7 cells.

[0043] FIG13 shows the effects of the low molecular weight Stemona polysaccharide STP-3-2 of the present invention on the expression of iNOS mRNA (A), IL-6 mRNA (B), and TNF-α mRNA (C) in macrophage RAW264.7 cells.

[0044] FIG14 shows the effects of specific receptor inhibitors on the secretion of NO (A), TNF-α (B) and IL-6 (C) by macrophage RAW264.7 cells stimulated by STP-3-2.

[0045] Figure 15 The effect of the low-molecular-weight Stemona polysaccharide STP-3-2 on the expression of NF-κB pathway proteins in RAW264.7 macrophage cells. (AB): Western blot expression bands; (C): Relative expression intensity of p-NF-κB p65 protein; (D): Relative expression intensity of p-IκB-α protein. DETAILED DESCRIPTION

[0046] In order to more clearly illustrate the purpose, features and advantages of the present invention, the following will be combined with specific examples and drawings to describe the embodiments of the present invention in detail. In the following description, we provide many specific details so that the core content of the present invention can be more fully understood. However, it should be noted that the embodiments of the present invention are not limited to the specific forms described herein. Those skilled in the art can make various similar improvements and expansions without departing from the core idea of ​​the present invention. Therefore, the scope of the present invention should not be limited by the specific examples disclosed below.

[0047] Example 1 Preparation and structural analysis of a low molecular weight Stemona polysaccharide STP-3-2

[0048] 1.1 Materials

[0049] The dried tubers of Stemona radix were collected from the medicinal material market in Yichang City, Hubei Province. The raw materials were dug in spring and autumn according to traditional harvesting specifications, and the main root was retained after removing the fibrous roots.

[0050] All reagents used, including ethanol, acetonitrile, sodium chloride, sodium borohydride, deuterium oxide, iodomethane, glacial acetic acid, and monosaccharide standards, were commercially available analytical grade reagents.

[0051] Anion exchange resin DEAE Sepharose Fast Flow and cross-linked agarose gel Sepharose CL-6B were from GE Healthcare, USA.

[0052] 1.2 Methods

[0053] 1.2.1 Extraction of Stemona polysaccharides

[0054] Dry the dried root tubers of Stemona radix to constant weight in a 60°C constant-temperature drying oven and then crush them. Accurately weigh 800g of powder and place it in a reactor. Add 16L of pure water at a material-liquid ratio of 1:20 (w / v), then heat to 90°C and stir for 3 hours. After heating, cool the solution to room temperature, centrifuge at 4700 rpm for 15 minutes, collect the supernatant, and filter. The centrifuged residue is returned to the reactor, and 8L of pure water is added. The solution is heated to 90°C and stirred for 3 hours for a second extraction. After extraction, cool and centrifuge at 4700 rpm for 15 minutes. Filter the supernatant from the second extraction and combine it with the filtrate from the first extraction. Add 4g of α-amylase to the combined filtrate, adjust the temperature to 50°C, and allow enzymatic hydrolysis overnight. After enzymatic hydrolysis, adjust the pH of the extract to neutral and boil for 5 minutes to inactivate the enzyme. Ethanol precipitation is then performed, stirring well, allowing the extract to stand for 24 hours, and then centrifuging at 4700 rpm for 15 minutes to collect the precipitate. The precipitate was dissolved in an appropriate amount of pure water, deproteinized four times using the Sevage method, ethanol precipitated for 24 hours, and finally centrifuged at 4700 rpm for 15 minutes to collect the precipitate. The precipitate was dissolved in pure water and freeze-dried to obtain crude Stemona tuberosa polysaccharide (C-STP).

[0055] 1.2.2 Isolation and purification of Stemona polysaccharides

[0056] 500 mg of the total non-starch polysaccharide C-STP from Stemona paraphylla was weighed and dissolved in 20 mL of pure water. After centrifugation, the supernatant was loaded onto a DEAE FF chromatography column (2.6 cm × 40 cm) equilibrated with pure water. Gradient elution was performed using different concentrations of NaCl (0 M, 0.1 M, and 0.3 M) as the mobile phase at a flow rate of 2 mL / min. The eluted fractions were collected using an automatic fraction collector. The polysaccharide content of each eluate was determined using the phenol-sulfuric acid method, and the corresponding elution curve was plotted. Fractions of the same type were combined according to the elution curve, concentrated, and desalted using a dialysis bag with a molecular weight cutoff of 1000 Da. The fractions were then concentrated and lyophilized to obtain STP-3. 150 mg of STP-3 was weighed and dissolved in 5 mL of 0.2 M NaCl solution. After centrifugation at 12000 rpm for 5 min, the supernatant was loaded onto a Sepharose CL-6B gel chromatography column (1.5 m × 1.5 cm). According to the phenol-sulfuric acid method and high performance liquid chromatography (HPLC) results, the fractions of each elution peak were combined, dialyzed and desalted using a dialysis bag with a molecular weight cutoff of 1000 Da, and lyophilized to obtain STP-3-2.

[0057] 1.2.3 Determination of molecular weight and distribution of STP-3-2

[0058] The molecular weight and distribution of STP-3-2 were determined using high-performance liquid gel permeation chromatography (HPGPC). A standard curve was generated using a series of commercially available dextran standards of known molecular weight. Chromatographic conditions were as follows: a Shimadzu HPLC (LC-2030C 3D Plus) using a Shodex SB-804HQ (8.0 mm × 300 mm) gel column, a differential refractive index detector (RID), a flow rate of 0.5 mL / min, and a 0.1 M NaCl mobile phase.

[0059] 1.2.4 Analysis of Monosaccharide Composition of STP-3-2

[0060] The monosaccharide composition of STP-3-2 was determined using PMP pre-column derivatization coupled with high-performance liquid chromatography (HPLC). Chromatographic conditions were as follows: a diode array detector (DAD) at a wavelength of 245 nm, an Eclipse Plus C18 column (4.6 × 250 mm, 5 μm), a column temperature of 30°C, and a mobile phase consisting of a mixture of phosphate buffer (0.1 M, pH 6.7) and acetonitrile (83:17 by volume) at a flow rate of 1.0 mL / min. The injection volume was 20 μL.

[0061] 1.2.5 Uronate reduction treatment

[0062] 50mg sample was dissolved in 2-morpholineethanesulfonic acid (MES, 0.05M) solution, followed by addition of methyl paratoluenesulfonate (CMC, 500mg / mL). After reacting for 2h at room temperature, 4M imidazole (pH 7.0) was added to the solution and cooled in an ice bath. Subsequently, freshly prepared sodium borohydride solution (60mg / mL) was slowly added dropwise and stirred for 3h at room temperature. After completion of the reaction, the reaction was terminated with glacial acetic acid to destroy excess sodium borohydride. Then, the reaction solution was dialyzed, concentrated and lyophilized. This process was repeated four times to ensure that most of the uronic acid was reduced. The reduced polysaccharide sample STP-3-2Re was finally obtained.

[0063] The analysis was performed according to the methylation method reported by Ciucanu et al. (Journal of the American Chemical Society, 2003, 125(52):16213–16219).

[0064] 1.2.6 Nuclear magnetic resonance spectroscopy analysis

[0065] Weigh 50 mg of STP-3-2 dried polysaccharide sample, dissolve in 0.5 mL of D2O, centrifuge to remove the supernatant, freeze-dry, repeat three times. The freeze-dried sample is dissolved in D2O containing internal standard. The polysaccharide is measured on a 600 MHz nuclear magnetic resonance spectrometer. 1 H. 13 C. 1 H- 1 HCOSY / TOCSY / ROESY and 1 H- 13 C HSQC / HMBC spectrum.

[0066] 1.3 Results

[0067] 1.3.1 Molecular weight and its distribution

[0068] The HPLC gel permeation chromatogram of STP-3-2 is as follows Figure 1 As shown. The purity of STP-3-2 was calculated to be >98% by area normalization. According to the standard curve equation, the weight-average molecular weight of STP-3-2 was approximately 12.01 kDa, and the polydispersity index (PDI) was approximately 1.6.

[0069] 1.3.2 Monosaccharide composition

[0070] The monosaccharide composition of STP-3-2 was determined by PMP pre-column derivatization combined with high performance liquid chromatography. Figure 2The peak elution time was compared with that of standard monosaccharides and the molar percentage of monosaccharide composition was calculated by peak area. The results showed that STP-3-2 was composed of GalA, Rha, Gal, Ara, Xyl, Glc, and GlcA, with molar percentages of 41.74%, 22.36%, 15.14%, 11.25%, 4.91%, 3.12%, and 1.49%, respectively.

[0071] 1.3.3 Methylation analysis results

[0072] Since STP-3-2 contains a large amount of GalA, its glycosidic bond is difficult to hydrolyze, and some methylated derivatives have low ionization efficiency and weak signal response in GC-MS analysis, which to a certain extent limits the structural analysis of the sample. Therefore, the uronic acid carboxyl part was reduced before structural analysis. The PMAAs total ion current chromatogram of the reduced STP-3-2Re is shown in the attached figure. Figure 3 As shown. STP-3-2Re contains 12 sugar residues. Among them, the main connection modes of GalA and Gal are →4)-Galp-(1→(50.5) and Galp-(1→(12.3), followed by →3,4)-Galp-(1→(3.8) and →2,4)-Galp-(1→(2.7). The main connection modes of Rha are →2)-Rhap-(1→(9.4), Rhap-(1→(4.0) and →2,4)-Rhap-(1→(3.8). Ara residues exist in the form of Araf-(1→(4.9), →5)-Araf-(1→(2.4) and →3,5)-Araf-(1→(1.7). Glc residues are Glcp-(1→(1.1). Xyl residues are Xylp-(1→(3.3).

[0073] 1.3.4 Nuclear Magnetic Resonance Spectrum Analysis

[0074] The structure of STP-3-2 was further analyzed by nuclear magnetic resonance spectroscopy (1D NMR and 2D NMR) to determine its chemical structure. 1 H. 13 C NMR, 1 H- 13 C HSQC-TOCSY, ROESY, HMBC and other spectra and their attribution Figures 4 to 8 As shown, STP-3-2 1 H and 13 The assignments of C NMR signals are shown in Table 1.

[0075] Table 1. STP-3-2 1 H and 13 C NMR signal assignment results

[0076]

[0077]

[0078]

[0079] Note: a Bold values ​​indicate glycosylation positions, and t indicates the terminal end.

[0080] According to the monosaccharide composition, methylation analysis and nuclear magnetic resonance spectrum analysis results of STP-3-2, the structural formula of STP-3-2 is:

[0081]

[0082] In formula (I): R1, R2, and R3 are side chains;

[0083] R1 is H,

[0084] R2 is

[0085]

[0086] R3 is H or

[0087] R4 is a hydrogen atom (H) or an ethyl group (-CH2CH3); R5 is a hydrogen atom (H) or a methyl group (-CH3). The degree of methylation is 21.5% calculated based on the peak area ratio of the anomeric hydrogen in the H spectrum. The average value of l is 29; the average value of m is 8; and the average value of n is 3.

[0088] Example 2 Immunomodulatory Activity of Stemona polysaccharide STP-3-2

[0089] 2.1 Materials

[0090] Cells and reagents: Mouse macrophage RAW264.7 cells were provided by the Scientific Experimental Center of Guangxi University of Chinese Medicine. Mouse IL-6 and TNF-α ELISA kits were purchased from Xinbosheng Biotechnology Co., Ltd. Thiazolyl blue (MTT) was purchased from MacLean Reagent Co., Ltd. HiScript III RT SuperMix for qPCR (with gDNA wiper) and ChamQ Universal SYBR qPCR Master Mix were purchased from Novozymes Biotech Co., Ltd. Penicillin-streptomycin double antibody, Australian imported fetal bovine serum, and TRIzol were purchased from Thermo Fisher Scientific. FITC-Dextran, TLR4 inhibitor (TAK-242), and TLR2 inhibitor (C29) were purchased from MedChemExpress, USA. Antibodies to NF-κB p65, p-NF-κB p65, IκBα, p-IκBα, and β-actin were purchased from Cell Signaling Technology, USA. PBS, BCA protein concentration assay kit, 5× loading buffer, and the protease inhibitor PMSF were purchased from Beijing Solebao Technology Co., Ltd. Protein markers, a rapid PAGE gel preparation kit, and an ECL chemiluminescence detection kit were purchased from Shanghai Yazyme Biotechnology Co., Ltd. Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich. Other reagents (such as sulfonamide, dimethyl sulfoxide, and isopropanol) were commercially available and of analytical grade.

[0091] Cell culture medium and additives: DMEM high glucose medium, penicillin / streptomycin double antibody, fetal bovine serum, etc. were purchased from Thermo Fisher Scientific.

[0092] 2.2 Instruments

[0093] Cell culture and operation instruments: The biological safety cabinet (HR1500-ⅡB2) was purchased from Qingdao Haier Biomedical Co., Ltd. The carbon dioxide incubator (MCO-170AICDL-PC) and high-pressure steam sterilizer (MLS-3781L-PC) were purchased from Puhexi Co., Ltd.

[0094] Detection and analysis instruments: A real-time fluorescence quantitative PCR instrument (LightCycler 480) was purchased from Roche Applied Science. A full-wavelength multifunctional microplate reader (VICTOR Nivo) was purchased from PerkinElmer. An inverted phase-contrast microscope (ICX41) was purchased from Ningbo Sunny Optical Instrument Co., Ltd. A micro-volume nucleic acid protein analyzer (NanoDrop One) was purchased from Thermo Fisher Scientific. A PCR amplifier (C1000 Touch) was purchased from Bio-Rad Laboratories. A transfer and decolorization shaker (NYC-80) was purchased from Taizhou Nomi Medical Technology Co., Ltd. A vertical electrophoresis system (552BR) was purchased from Bio-Rad, USA. A flow cytometer (CNL18-C0462) was purchased from Becton Dickinson Medical Devices, Inc., USA.

[0095] 2.3 Methods

[0096] 2.3.1 Cell culture

[0097] Cell culture, including cell recovery, passage, and cryopreservation, was performed according to conventional experimental methods. RAW264.7 cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum in a 5% CO2, 37°C incubator.

[0098] 2.3.2 Effect of STP-3-2 on the proliferation of RAW264.7 cells

[0099] Referring to the method of Sun et al. (International Journal of Biological Macromolecules, 2018, 108: 314–323), the MTT assay was used to determine the effect of different concentrations (400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL) of STP-3-2 on the proliferation ability of RAW264.7 cells, and a blank control and a positive control (1 μg / mL LPS) were set up. The cells were seeded in a 96-well plate at a density of 1×104 cells / well. After culturing for 12 hours, different concentrations of STP-3-2 were added to treat the cells for 24 hours. After adding MTT solution (5 mg / mL), the cells were incubated for another 4 hours. The supernatant was discarded, dimethyl sulfoxide was added to dissolve the crystals, and the absorbance (OD) of each well was measured using a microplate reader. 490 ), and cell viability was calculated.

[0100] 2.3.3 Effect of STP-3-2 on the phagocytic ability of RAW264.7 cells

[0101] FITC-labeled dextran (1 mg / mL) was used as a tracer for phagocytic activity. Cells were seeded in 24-well plates at a density of 1×105 cells / well and incubated overnight. The cells were treated with different concentrations of STP-3-2 (50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL, respectively). Blank culture medium and LPS (1 μg / mL) were set as control groups and cultured for 24 hours. FITC-labeled dextran (1 mg / mL) was used as a tracer for phagocytic activity and co-cultured with the cells. After 1 hour, the cells were washed with PBS and the fluorescence intensity was detected using a flow cytometer to assess the phagocytic ability of the cells.

[0102] 2.3.4 Effect of STP-3-2 on NO secretion in RAW264.7 cells

[0103] The effect of STP-3-2 on NO secretion in RAW264.7 cells was determined by Griess method according to conventional methods. 4 Cells were seeded at a density of 100 μg / well in a 96-well plate. After culturing for 12 h, different concentrations of STP-3-2 (50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL) were added to treat the cells for 24 h. A blank control and a positive control (1 μg / mL LPS) were set up. The supernatant was collected, Griess reagent was added, and the absorbance (OD) was measured by spectrophotometer. 540 ), calculate the NO content.

[0104] 2.3.5 Effect of STP-3-2 on TNF-α and IL-6 secretion in RAW264.7 cells

[0105] ELISA kits were used to detect the effect of STP-3-2 on the secretion of TNF-α and IL-6 in RAW264.7 cells. 4 Cells were seeded at a density of 100 μg / well in a 96-well plate. After 12 hours of culture, different concentrations of STP-3-2 (50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL) were added and treated for 24 hours. A blank control and a positive control (1 μg / mL PLs) were also set up. The supernatant was collected and the ELISA kit instructions were followed. The absorbance was measured using a microplate reader, and the cytokine concentration was calculated.

[0106] 2.3.6 Effects of STP-3-2 on the mRNA expressions of iNOS, IL-6, and TNF-α in RAW264.7 cells

[0107] qRT-PCR was used to detect the effect of STP-3-2 on the expression of iNOS, IL-6 and TNF-α mRNA in RAW264.7 cells. 5 Cells were seeded at a density of 100 μg / well in a 6-well plate. After 12 hours of culture, different concentrations of STP-3-2 (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL) and 1 μg / mL LPS were added to treat the cells for 6 hours. The supernatant was discarded, the cells were washed with PBS, and TRIzol reagent was added to lyse the cells and extract total RNA. Total RNA was reverse transcribed into cDNA according to the instructions of the reverse transcription kit, and qPCR amplification was performed using cDNA as a template. GAPDH was used as an internal reference, and 2- △△Ct The target gene was quantitatively analyzed.

[0108] 2.3.7 Effects of TLR2 / TLR4 inhibitors on the secretion of NO, TNF-α, and IL-6 by RAW264.7 cells stimulated by STP-3-2

[0109] Before stimulation with 400 μg / mL of STP-3-2 polysaccharide, cells were pretreated with the TLR4 inhibitor TAK-242 (1 μM) and / or the TLR2 inhibitor C29 (100 μM) for 1 h. LPS (1 μg / mL) was used as a positive control. NO and cytokine (TNF-α and IL-6) release were measured using the Griess assay and ELISA, respectively.

[0110] 2.3.8 Effects of STP-3-2 on the expression of key proteins in the NF-κB pathway in RAW264.7 cells

[0111] Western blot analysis was used to examine the activation of the NF-κB signaling pathway by STP-3-2 in RAW264.7 cells. Different concentrations of STP-3-2 (50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL) and 1 μg / mL LPS were added to 12-well plates of RAW264.7 cells cultured for 12 hours. The cells were incubated in a humidified incubator for 2 hours. The supernatant was discarded, and the cells were washed with pre-chilled PBS. RIPA lysis buffer (200 μL / well) containing PMSF and phosphatase inhibitors was added and lysed at 4°C for 10 minutes. The lysate was collected and centrifuged, and the supernatant was collected. The protein concentration was determined using the BCA assay and adjusted to a consistent level. The protein was mixed with loading buffer and denatured at 95°C. A 12.5% ​​SDS-PAGE gel was prepared, and 20 μg of protein was loaded for electrophoresis and transferred to a PVDF membrane. After blocking, primary antibodies (NF-κB p65, p-NF-κB p65, IκBα, p-IκBα, and β-actin) were added and incubated overnight at 4°C. After washing with TBST, secondary antibodies were added and incubated for 1 hour, and the membranes were analyzed by ECL imaging.

[0112] 2.4 Results

[0113] 2.4.1 Effect of STP-3-2 on the proliferation of RAW264.7 cells

[0114] like Figure 9 As shown, STP-3-2 significantly promoted the proliferation of RAW264.7 cells within the tested concentration range (50-400 μg / mL) (P<0.05). Compared with the blank control group, STP-3-2 exhibited significant cell proliferation promotion at all tested concentrations, indicating that it has good cell growth stimulating activity.

[0115] 2.4.2 Effect of STP-3-2 on the phagocytic ability of RAW264.7 cells

[0116] like Figure 10 As shown, STP-3-2 significantly enhanced the phagocytic capacity of RAW264.7 cells (p < 0.05). Within the concentration range of 50-400 μg / mL, STP-3-2 increased the phagocytic activity of FITC-dextran in a dose-dependent manner. Compared with the blank control group, cells treated with STP-3-2 exhibited enhanced phagocytic activity, demonstrating that it can effectively enhance the phagocytic function of macrophages.

[0117] 2.4.3 Effect of STP-3-2 on NO secretion in RAW264.7 cells

[0118] like Figure 11 As shown, STP-3-2 significantly promoted NO secretion in RAW264.7 cells within a concentration range of 50-400 μg / mL (p < 0.05), exhibiting a clear concentration-dependent effect. At a concentration of 400 μg / mL, NO secretion reached 30.18 μmol / L, exceeding the LPS-positive control (28.5 μmol / L). This suggests that STP-3-2 has a strong NO-inducing ability, likely through activation of relevant intracellular signaling pathways.

[0119] 2.4.4 Effects of STP-3-2 on cytokine secretion in RAW264.7 cells

[0120] As shown in Figure 12, STP-3-2 significantly increased the secretion of TNF-α and IL-6 by RAW264.7 cells (p < 0.05). At a concentration of 400 μg / mL, TNF-α secretion reached a maximum of 86,447 pg / mL, significantly higher than at other tested concentrations. IL-6 secretion reached a maximum of 1,410 pg / mL at 400 μg / mL, showing a clear dose-dependent effect. This suggests that STP-3-2 can effectively activate the inflammatory response of macrophages and promote cytokine secretion.

[0121] 2.4.5 Effects of STP-3-2 on the mRNA Expression of iNOS, IL-6, and TNF-α in RAW264.7 Cells

[0122] As shown in Figure 13, STP-3-2 significantly increased the expression levels of iNOS, IL-6, and TNF-α mRNA in RAW264.7 cells (p < 0.05). iNOS mRNA expression reached its highest value at a concentration of 400 μg / mL, showing a concentration-dependent pattern. IL-6 mRNA expression was significantly enhanced within the 100-400 μg / mL concentration range. TNF-α mRNA expression approached the level of the LPS-treated group at a concentration of 400 μg / mL. These results indicate that STP-3-2 promotes cytokine secretion by upregulating the expression of related genes.

[0123] 2.4.6 Effects of TLR2 / TLR4 inhibitors on the secretion of NO, TNF-α, and IL-6 by RAW264.7 cells stimulated by STP-3-2

[0124] As shown in Figure 14 (the positive sign under each data set indicates the treatment method of that group), compared with treatment with STP-3-2 alone, the TLR4 inhibitor TAK-242 almost completely inhibited the secretion of NO, IL-6, and TNF-α (p<0.05), indicating that TLR4 plays a key role in the STP-3-2-induced inflammatory response. Although the TLR2 inhibitor C29 had a certain downregulation effect on the release of NO and cytokines, their secretion levels were still significantly higher than those in the control group, indicating that TLR2 is also involved in the STP-3-2-induced inflammatory response to some extent, but its role is relatively weak. The overall results show that STP-3-2 mainly activates macrophages through the TLR4 signaling pathway.

[0125] 2.4.7 Effects of STP-3-2 on the expression of key proteins in the NF-κB pathway in RAW264.7 cells

[0126] like Figure 15As shown, STP-3-2 significantly increased the ratios of phosphorylated NF-κB p65 / p65 and phosphorylated IκBα / IκBα in RAW264.7 cells (p < 0.05), with this increase occurring in a dose-dependent manner with increasing polysaccharide concentration. At a concentration of 400 μg / mL, the IκBα phosphorylation ratio exceeded that of the LPS-positive control group. This suggests that STP-3-2 can activate the NF-κB signaling pathway, thereby promoting the expression of inflammation-related genes.

[0127] 3. Conclusion

[0128] The present invention verified the effects of STP-3-2 on the various biological activities of RAW264.7 cells through a series of experiments. STP-3-2 significantly promoted cell proliferation, enhanced phagocytosis, induced NO and cytokine secretion, and exerted its immunomodulatory effects by activating TLR4 and NF-κB signaling pathways. These results suggest that STP-3-2 has potential immunomodulatory and anti-inflammatory activities and may be a candidate for the development of novel immunomodulators.

[0129] Without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without expending creative work shall fall within the scope of protection of the present invention.

Claims

1. A low molecular weight Stemona polysaccharide, characterized by: The low molecular weight Stemona polysaccharide is a mixture of homologous polysaccharides having a structure of formula (I), which is as follows: In formula (I): R1 is H, R2 is R3 is H or R4 is H or -CH2CH3; R5 is H or -CH3. According to the peak area ratio of anomeric hydrogen in the hydrogen spectrum, the degree of methylation is (21.5±5.0)%. l is an integer with a mean value of 29; m is an integer with a mean value of 8±2; and n is an integer with a mean value of 3±2.

2. The low molecular weight Stemona polysaccharide according to claim 1, characterized in that: The weight average molecular weight of the low molecular weight Stemona polysaccharide ranges from 3 kDa to 20 kDa, and the polydispersity index ranges from 1.0 to 1.

8.

3. The low molecular weight Stemona polysaccharide according to claim 2, characterized in that: The weight average molecular weight of the low molecular weight Stemona polysaccharide ranges from 5 kDa to 15 kDa.

4. The low molecular weight Stemona polysaccharide according to claim 1, characterized in that: The monosaccharide composition contained in the low molecular weight Stemona polysaccharide is a mixture of galacturonic acid, rhamnose, galactose, arabinose, xylose, glucose and glucuronic acid, and the corresponding molar percentages are (41.7±5.0)%, (22.4±3.0)%, (15.1±3.0)%, (11.2±2.0)%, (4.9±1.0)%, (3.1±1.0)% and (1.5±0.5)%.

5. The method for preparing the low molecular weight Stemona polysaccharide according to any one of claims 1 to 4, characterized in that: The following steps are involved: The dried tuberous roots of Stemona radix are taken to extract and obtain the non-starch Stemona radix total polysaccharides, and the non-starch Stemona radix total polysaccharides are separated and purified to obtain the low molecular weight Stemona radix polysaccharides.

6. The method for preparing low molecular weight Stemona polysaccharide according to claim 5, characterized in that: The method of extracting non-starch total polysaccharides from Stemona radix L. specifically includes: taking the dried tuberous roots of Stemona radix L., adding solvent to extract 1 to 3 times, separating and combining the extracts, adding amylase to hydrolyze, inactivating the enzyme, ethanol precipitation, deproteinizing, ethanol precipitation, and obtaining the non-starch total polysaccharides from Stemona radix L. after precipitation and drying.

7. The method for preparing low molecular weight Stemona polysaccharide according to claim 6, characterized in that: The solvent is water.

8. The method for preparing low molecular weight Stemona polysaccharide according to claim 5, characterized in that: The separation and purification of the non-starch Stemona total polysaccharide specifically includes: using the non-starch Stemona total polysaccharide as raw material, separating by anion exchange chromatography column, collecting 0.3M sodium chloride elution components, desalting and concentrating by dialysis, and further separating and purifying by gel chromatography column, collecting each fraction of the main chromatographic peak, desalting by dialysis or ultrafiltration membrane filtration, and drying to obtain the low molecular weight Stemona polysaccharide.

9. Use of the low molecular weight Stemona polysaccharide according to any one of claims 1 to 4 in the preparation of medicines or functional foods for treating and / or preventing diseases such as immunodeficiency.

10. Use of the low molecular weight Stemona polysaccharide according to any one of claims 1 to 4 in the preparation of medicines or functional foods, wherein the medicines or functional foods can significantly activate macrophages and enhance their phagocytic function, promote the secretion of nitric oxide (NO), tumor necrosis factor-α and interleukin-6, and upregulate the expression of inducible nitric oxide synthase, IL-6 and TNF-α mRNA.

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