Application of Uncaria rhynchophylla neutral polysaccharide URP-W in the preparation of anti-glioma drugs

Abstract

This invention discloses the application of Uncaria rhynchophylla neutral polysaccharide URP-W in the preparation of anti-glioma drugs, belonging to the field of anti-tumor technology of medicinal plant polysaccharides. The invention obtains a crude polysaccharide extract from Uncaria rhynchophylla through hot water extraction and ethanol precipitation, followed by purification using a DEAE cellulose column and G200 dextran gel to obtain Uncaria rhynchophylla neutral polysaccharide URP-W. The monosaccharide composition of URP-W includes arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, and mannuronic acid in a molar ratio of 17.99:6.32:21.71:27.17:2.56:7.57:16.20:0.49. Experiments have shown that URP-W can inhibit the proliferation of gliomas and induce apoptosis, exhibiting significant anti-tumor activity against glioma cells, providing a new drug raw material and treatment method for the clinical treatment of gliomas.

Description

Technical Field

[0001] This invention relates to the field of antitumor technology of medicinal plant polysaccharides, and in particular to the application of Uncaria rhynchophylla neutral polysaccharide URP-W in the preparation of anti-glioma drugs. Background Technology

[0002] Uncaria rhynchophylla, also known as double hook vine or eagle claw wind, is the dried hooked stem of the Uncaria rhynchophylla plant, belonging to the Rubiaceae family. It is an important traditional Chinese medicine used to calm the liver and extinguish wind. It is slightly cold in nature, sweet in taste, and enters the liver and pericardium meridians. The most distinctive feature of Uncaria rhynchophylla is the curved hooked thorns (derived from modified inflorescence stalks or lateral branches) on its branches, which is the origin of its name and the main medicinal part.

[0003] Traditional Chinese medicine believes that Uncaria rhynchophylla has the effects of clearing heat and calming the liver, relieving wind and calming convulsions. It is especially good at treating symptoms such as high fever convulsions, infantile convulsions, headaches and dizziness, and preeclampsia caused by internal liver wind.

[0004] Modern pharmacological studies have also confirmed that it contains a variety of effective components, exhibiting good sedative, anticonvulsant, and antihypertensive effects. Recent studies have shown that Uncaria rhynchophylla and its main active components (such as rhynchophylline and isorhynchophylline) demonstrate significant multi-target pharmacological activity. Its core effects are concentrated in the cardiovascular and nervous systems: First, Uncaria rhynchophylla has a clear antihypertensive effect, its mechanism involving blocking calcium ion channels, inhibiting angiotensin-converting enzyme (ACE) activity, and regulating the nitric oxide (NO) signaling pathway, thereby effectively lowering blood pressure. Second, in terms of neuroprotection, Uncaria rhynchophylla can enhance GABAergic neurotransmission and inhibit glutamate excitotoxicity, which not only endows it with anti-epileptic and anti-anxiety effects but also shows potential activity against neurodegenerative diseases such as Parkinson's disease. Furthermore, Uncaria rhynchophylla also exhibits strong anti-inflammatory and antioxidant capabilities, effectively scavenging free radicals by inhibiting the expression of key inflammatory factors such as NF-κB and COX-2, thus reducing oxidative stress and inflammatory damage to nerve tissue and the vascular system. Simultaneously, it also plays a role in improving blood circulation, achieving antithrombotic and microcirculation-improving effects by inhibiting platelet aggregation and reducing blood viscosity. Current research is delving into the molecular mechanisms of these activities, particularly their potential in treating hypertension, neurodegenerative diseases, and cerebrovascular diseases, providing important scientific evidence for the modern application of this traditional Chinese medicine and the development of innovative drugs. However, research on the antitumor activity of Uncaria rhynchophylla is limited. Studies on its antitumor activity mainly focus on its alkaloid components, particularly rhynchophylline and isorhynchophylline, but these two components have significant cardiotoxicity. Other active components in Uncaria rhynchophylla, such as polysaccharides, polypeptides, and proteins, have not been fully explored. Polysaccharides are among the most active components in medicinal plants, primarily focusing on their mechanisms of immune regulation and direct killing of tumor cells. The antitumor activity of Uncaria rhynchophylla polysaccharides has not yet been studied; therefore, current research has not fully elucidated the reasons and mechanisms by which the neutral polysaccharide components of Uncaria rhynchophylla inhibit tumor growth.

[0005] Gliomas are a type of primary intracranial tumor originating from glial cells in the central nervous system (brain and spinal cord). Glial cells are important supporting cells in the brain and spinal cord, responsible for providing nutrients to neurons, maintaining homeostasis, and forming myelin sheaths. When these cells undergo malignant transformation, gliomas are formed. Many studies have investigated glioma cells using active ingredients from plants, finding that these ingredients can inhibit the proliferation and differentiation of glioma cells and promote apoptosis of cancer cells, demonstrating significant research and application value in clinical treatment. Summary of the Invention

[0006] The purpose of this invention is to provide the application of Uncaria rhynchophylla neutral polysaccharide URP-W in the preparation of anti-glioma drugs, so as to solve the problems existing in the prior art. The Uncaria rhynchophylla neutral polysaccharide URP-W can inhibit the proliferation of glioma and induce apoptosis, and has significant anti-tumor activity against glioma cells.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a neutral polysaccharide URP-W from Uncaria rhynchophylla, wherein the monosaccharide composition of URP-W includes: arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, and mannuronic acid, with a molar ratio of 17.99:6.32:21.71:27.17:2.56:7.57:16.20:0.49.

[0009] Preferably, the URP-W includes segments of the following structure:

[0010] .

[0011] Preferably, the connection method of each segment in the URP-W is as follows:

[0012] Segment 1 is formed by interconnecting →4)-β-D-Galp-(1→); Segment 2 is formed by interconnecting →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→), with α-L-Araf-(1→5)-α-L-Araf-(1→ connected at the O-6 position of →4,6)-α-D-Glcp-(1→); Segment 3 is formed by interconnecting →4)-α-D-GalpA-6-OMe-(1→ and →6)-β-D-Galp-(1→).

[0013] This invention also provides a method for extracting and separating the neutral polysaccharide URP-W from Uncaria rhynchophylla, comprising the following steps:

[0014] (1) Extract the Uncaria rhynchophylla sample with ethanol to remove impurities, centrifuge and collect the precipitate, then mix the precipitate with water, heat to extract, centrifuge and collect the supernatant extract;

[0015] (2) The supernatant extract was precipitated with ethanol, centrifuged and the precipitate was collected and dried to obtain crude polysaccharide extract;

[0016] (3) After removing impurities from the crude polysaccharide extract, it was purified by ion exchange column chromatography and dextran gel column chromatography. The liquid passed through the column was collected, freeze-dried, and the Uncaria rhynchophylla neutral polysaccharide URP-W was obtained.

[0017] Preferably, in step (1), the ratio of the Uncaria rhynchophylla sample to ethanol is 1:(8-10); and / or the ratio of the precipitate to water is 1:(15-20); and / or the heating extraction conditions are 60℃ water bath extraction for 3-5 hours.

[0018] Preferably, in step (2), after the supernatant extract is concentrated to 1 / 10 of its original volume, 4 times the volume of ethanol is added for precipitation.

[0019] Preferably, in step (3), the impurity removal of the crude polysaccharide extract includes the following steps:

[0020] S1: Dissolve the crude polysaccharide extract in water, add protease for enzymatic hydrolysis, centrifuge, and collect the supernatant;

[0021] S2: Add chloroform and n-butanol to the supernatant, mix thoroughly, and collect the upper aqueous phase;

[0022] S3: Add petroleum ether to the upper aqueous phase, mix thoroughly, and collect the lower aqueous phase;

[0023] S4: Add macroporous adsorption resin to the lower aqueous phase, mix thoroughly for adsorption, collect the liquid, dialyze it, precipitate it with ethanol, and dry the precipitate.

[0024] Preferably, in step S1, the amount of water used is 600mL-1000mL, and the amount of protease used is 0.4-0.6g.

[0025] In step S2, 1 / 3 to 1 / 4 volume of chloroform and n-butanol are added to the supernatant, wherein the volume ratio of chloroform to n-butanol is 4:1.

[0026] And / or in step S3, 1 / 3 to 1 / 4 volume of petroleum ether is added to the upper aqueous phase;

[0027] And / or in step S4, 1 / 2 to 1 / 3 volume of macroporous adsorption resin is added to the lower aqueous phase.

[0028] Preferably, the ion exchange column chromatography and dextran gel column chromatography are purified using DEAE DE-52 cellulose and dextran gel G200, respectively, with water as the eluent.

[0029] The present invention also provides the application of the aforementioned Uncaria rhynchophylla neutral polysaccharide URP-W in the preparation of an anti-glioma drug.

[0030] The present invention discloses the following technical effects:

[0031] This invention employs boiling water extraction and ethanol precipitation of Uncaria rhynchophylla polysaccharides, combined with DEAE cellulose column and molecular sieve dextran gel column purification techniques to achieve the separation of neutral Uncaria rhynchophylla polysaccharides, thus solving the technical challenge of polysaccharide component extraction and separation.

[0032] The neutral polysaccharide URP-W extracted and isolated from Uncaria rhynchophylla in this invention exhibits significant antitumor activity against U251 cells. Combined with in vivo activity verification in tumor-bearing mice, URP-W primarily regulates the expression of key genes and proteins involved in mitochondrial apoptosis-related pathways. It increases the expression of the pro-apoptotic gene and protein Bax, downregulates the expression of the apoptosis-inhibiting protein Bcl-2, and simultaneously induces the release of Cyto-c from mitochondria, triggering caspase-mediated apoptosis. Further, it cleaves PARP, significantly reducing PARP expression, ultimately leading to mitochondrial apoptosis and programmed cell death in tumor cells. Thus, URP-W inhibits the proliferation and apoptosis of gliomas. The neutral polysaccharide URP-W from Uncaria rhynchophylla of this invention provides a novel treatment method for the clinical treatment of gliomas. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 The molecular configuration diagram of URP-W;

[0035] Figure 2 The absolute molecular weight analysis chromatogram for URP-W is shown.

[0036] Figure 3 The diagram shows the monosaccharide composition analysis of URP-W.

[0037] Figure 4 The image shows the FT-IR detection pattern of URP-W.

[0038] Figure 5 This is a one-dimensional proton spectrum of URP-W;

[0039] Figure 6 This is a one-dimensional carbon spectrum of URP-W;

[0040] Figure 7 The hydrogen-hydrogen correlation (COSY) spectrum of URP-W;

[0041] Figure 8 The hydrogen-hydrogen correlation (NOESY) spectrum of URP-W;

[0042] Figure 9 The carbon-hydrogen correlation (HSQC) spectrum of URP-W;

[0043] Figure 10 The carbon-hydrogen correlation (HMBC) spectrum of URP-W;

[0044] Figure 11 Effects of URP-W on the morphology of glioma U251 cells (48 h);

[0045] Figure 12 Results of the inhibition of URP-W proliferation on glioma U251 cells by different administration times and concentrations;

[0046] Figure 13 The effect of URP-W on the morphology of nuclear apoptosis in U251 glioma cells (48 h);

[0047] Figure 14 To analyze the effect of URP-W on apoptosis in glioma U251 cells by flow cytometry (48 h);

[0048] Figure 15 To track the effect of URP-W on tumor growth in glioma U251-bearing mice using magnetic resonance imaging;

[0049] Figure 16 The effects of URP-W on the survival status of mice bearing U251 gliomas: (A) weekly water intake of tumor-bearing mice; (B) weekly food intake of tumor-bearing mice; (C) changes in body weight of tumor-bearing mice.

[0050] Figure 17 The effect of URP-W on the expression of proteins related to the mitochondrial apoptosis pathway in tumor tissues of tumor-bearing mice. Detailed Implementation

[0051] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0052] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0053] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0054] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0055] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0056] The following embodiments involve the following main materials:

[0057] Glioma cell line U251 (CL-0237, Pronosei Life Science Technology Co., Ltd.).

[0058] Fetal bovine serum (164210, Pronosei Life Sciences Co., Ltd.)

[0059] DAPI fluorescent staining solution (KGA1808-50, Jiangsu Kaiji Biotechnology Co., Ltd., China).

[0060] Annexin V-FITC / PI Double Staining Apoptosis Detection Kit (KGA1102-100, Jiangsu Kaiji Biotechnology Co., Ltd., China).

[0061] β-actin, Bax, Bcl-2, antibody (Wuhan Aibote Biotechnology Co., Ltd.)

[0062] Example 1

[0063] 1. Extraction and identification of neutral polysaccharides from Uncaria rhynchophylla

[0064] 1.1 Extraction of total polysaccharides from Uncaria rhynchophylla

[0065] (1) Crush the dried Uncaria rhynchophylla sample / raw material with a pulverizer and pass it through a 60-mesh sieve.

[0066] (2) Add anhydrous ethanol (material-to-liquid ratio 1g:10mL), stir at room temperature to extract fat-soluble pigments and some impurities, centrifuge at 6000g for 10min, and collect the precipitate.

[0067] (3) Add pure water to the precipitate (material-to-liquid ratio 1g:20mL), extract in a water bath at 60℃ for 4h, centrifuge at 6000g for 10min, and collect the supernatant extract. Repeat the extraction process on the precipitate residue.

[0068] (4) Combine the two extracts, concentrate them under vacuum to 1 / 10 of the original volume, and add four times the volume of anhydrous ethanol for overnight precipitation.

[0069] (5) Centrifuge at 8000g for 10min, collect the precipitate solid and dry it to obtain crude polysaccharide extract.

[0070] (6) The purity of crude polysaccharide was determined by the sulfuric acid-phenol method. The specific operation was as follows: Weigh about 25 mg of crude polysaccharide extract solid, dissolve and dilute it with water, take 100 μL of polysaccharide supernatant, add 600 μL of sulfuric acid-phenol reagent (5% phenol solution: concentrated sulfuric acid = 1:5 (v / v)), mix well, react in the dark and let stand for 10 min, and measure the absorbance at 490 nm.

[0071] 1.2 Removal of impurities from total polysaccharides

[0072] The detailed steps, including protein removal, fat removal, and decolorization, followed by determination of crude polysaccharide purity using the sulfuric acid-phenol method, are as follows:

[0073] (1) Add 600mL-1L of pure water to the crude polysaccharide extract solid to fully dissolve the crude polysaccharide, and add 0.4-0.6g of protease to hydrolyze overnight.

[0074] (2) After centrifuging the enzyme hydrolysate, collect the aqueous phase, add 1 / 4 volume of chloroform and n-butanol (4:1, v / v) to the aqueous phase, mix thoroughly, and collect the upper aqueous phase.

[0075] (3) Add 1 / 4 volume of petroleum ether to the liquid, mix thoroughly, and collect the lower aqueous phase.

[0076] (4) Add 1 / 2 volume of macroporous adsorption resin AB-8 to the aqueous phase, mix thoroughly, and adsorb overnight.

[0077] (5) Collect the liquid and dialyze for 24-48 hours using a 3000Da dialysis bag to remove small molecule components. The polysaccharide dialysate is first precipitated with ethanol and then dried.

[0078] 1.3 Preparation method of vine neutral polysaccharide (URP-W)

[0079] URP was separated and purified by molecular sieve using ion exchange column chromatography and dextran gel column chromatography. Specifically, the total polysaccharides after impurity removal were first purified using DEAE DE-52 cellulose, with water as the eluent; then the aqueous fraction passing through the column was purified using dextran gel G200, with water as the eluent. The column pass solution was collected, lyophilized, and stored, named URP-W; the flow rate was set at 1 mL / min during the purification process.

[0080] 1.4 Structural characteristics and identification of Uncaria rhynchophylla neutral polysaccharide (URP-W)

[0081] First, the sample was dissolved in a 0.1M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL. After filtration through a 0.45 μm filter, the sample was analyzed by gel chromatography to determine molecular weight, homogeneity, and monosaccharide composition. The chromatographic system used was a gel chromatography-differential-multi-angle laser light scattering system. The liquid chromatography system was a U3000 (Thermo, USA), the differential detector was an Optilab T-rEX (Wyatt technology, CA, USA), and the laser light scattering detector was a DAWN HELEOS II (Wyatt technology, CA, USA). Specific column and elution conditions were as follows: Ohpak SB-805 HQ (300×8 mm) and Ohpak SB-803 HQ (300×8 mm) gel size exclusion columns were used in series. The column temperature was 45℃, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1M NaNO3), the flow rate was 0.6 mL / min, and the elution gradient was isocratic for 75 min.

[0082] Secondly, infrared spectroscopy was used to detect the major functional groups present in URP-W, and the configuration of the polysaccharide was preliminarily determined, thus understanding its structural characteristics. Further methylation was employed to detect the linkage configuration between sugar residues. Finally, nuclear magnetic resonance (NMR) technology was used to identify the main glycan structure of URP-W to determine its specific glycan conformation and linkage mode. These analytical methods can help confirm the chemical structure and composition of Uncaria rhynchophylla neutral polysaccharide (URP-W), thereby gaining a deeper understanding of its biological activity and potential application value.

[0083] 2. Functional verification of neutral polysaccharides from Uncaria rhynchophylla

[0084] 2.1 Cell lines and cell culture

[0085] U251 cells were cultured in DMEM medium containing 100 μg / mL streptomycin and 100 μg / mL penicillin G, with 10% fetal bovine serum added, at a constant temperature and humidity of 37°C, with the carbon dioxide concentration maintained at 5%. When the cell density reached 90%, the cells were passaged 1:5 and cultured continuously.

[0086] 2.2 Cell proliferation inhibition detection

[0087] U251 cells were used at a rate of 2 × 10 3 Cells were seeded at a density of 10 cells / well into 96-well plates. After 24 hours of cell culture, the original culture medium was replaced with a medium containing different concentrations of URP-W (0, 250, 500, and 1000 μg / mL). The experiment included negative and positive controls: the negative control group had cells cultured normally without drug addition; the positive control group received doxorubicin hydrochloride (DOX) instead of URP-W at a concentration of 5 μM. U251 cells were co-cultured with 5 μM DOX and different concentrations of URP-W (0, 250, 500, and 1000 μg / mL) after seeding. Each treatment group had 5 replicates, and the experiment was repeated at least 3 times with parallel and iterative assays. Cell viability was assessed using a CCK-8 cell proliferation assay kit after 24 and 48 hours of co-incubation. The CCK-8 reagent was added to the culture medium at a ratio of 1:10, and after incubation for 30 minutes, the absorbance was read at 450 nm using a microplate reader. The calculation formula is as follows:

[0088] Proliferation inhibition rate (%) = ([A) c -A s ] / [A c -A 空白 ])×100%

[0089] As: Absorbance of experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution); Ac: Absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but without drug); A 空白 : Absorbance of blank wells (containing culture medium and CCK-8 solution, but excluding cells and drugs).

[0090] 2.3 DAPI fluorescent staining experiment

[0091] U251 cells were used at a rate of 1×10 4Cells were seeded at a density of 100 cells / well in culture dishes containing DMEM medium and cultured for 24 hours. Subsequently, the original medium was replaced with drug-treated medium, and the cells were co-cultured with URP-W for 24 and 48 hours. Afterward, the cells were washed with PBS, fixed with methanol, and washed again. 80 μL of DAPI was added to each well, stained in the dark for 5 minutes, and then washed once with PBS. Finally, images were taken using a Nikon inverted fluorescence microscope (100× and 200× magnification), with 5 images retained for each drug concentration.

[0092] 2.4 Apoptosis Assay

[0093] U251 cells were fed at a rate of 2 × 10 5 Cells were seeded at a density of 100 cells / well in culture dishes containing DMEM medium and cultured for 24 hours. Subsequently, the original medium was replaced with the drug-treated medium, and the cells were co-cultured with URP-W for 48 hours. Afterward, the cells were washed twice with PBS, reagents were added according to the instructions and mixed thoroughly, and the mixture was incubated at room temperature in the dark for 5–15 minutes before analysis by flow cytometry. Each sample was set up in triplicate, and the experiment was repeated at least three times.

[0094] 2.5 Construction and Treatment of Tumor-Bearing Mouse Models

[0095] U251 cell suspension was inoculated into the right axilla of C57BL / 6 mice, 0.2 mL per mouse, to establish an osteosarcoma-bearing mouse model. The experiment was divided into two groups of five mice each. The control group received 0.2 mL of physiological saline per mouse via gavage for 28 consecutive days; the URP-W group received 400 mg / kg of the drug via gavage for 28 consecutive days. The mice were weighed 72 hours after the last administration and then sacrificed. After confirming death, the mice were dissected, and the tumor tissue was completely removed.

[0096] 2.6 Q-PCR detection

[0097] Total RNA was extracted from each sample and used to prepare cDNA samples. Gene expression was measured by qPCR and primers. The reaction conditions were as follows: heating at 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 55°C for 45 s, and 72°C for 30 s, and finally extension at 72°C for 10 min.

[0098] The primers are shown below:

[0099] Bcl-2 F: 5'-CGGTTCAGGTACTCAGTCATC-3' (SEQ ID NO. 1);

[0100] Bcl-2 R: 5'-CGGTGGGGTCATGTGTGTG-3' (SEQ ID NO. 2);

[0101] Bax F: 5'-CCTTTTCTACTTTGCCAGAAAC-3' (SEQ ID NO. 3);

[0102] Bax R: 5'-GAGGCCGTCCCAACCAC-3' (SEQ ID NO. 4);

[0103] β-actin F: 5'-TGACGTGGACATCCGCAAAG-3' (SEQ ID NO. 5);

[0104] β-actin R: 5'-CTGGAAGGTGGACAGCGAGG-3' (SEQ ID NO. 6);

[0105] Cyto-c F: 5'-AAAGGGAGGCAAGCACAAGA-3' (SEQ ID NO.7);

[0106] Cyto-c R: 5'-GATCATTTTTGTTTCCAGGGATGTA-3' (SEQ ID NO. 8);

[0107] PARP F: 5'-AACTTTGCTGGCATCCTGTC-3' (SEQ ID NO.9);

[0108] PARP R: 5'-TGCACTTTTGGACACCATGT-3' (SEQ ID NO. 10).

[0109] 2.7 Western blot for protein immunoblotting

[0110] Cell samples were collected from each culture group, and protein samples were extracted and analyzed using Western blotting. After electrophoresis, protein samples were transferred to polyvinylidene fluoride (PVDF) membranes for 1.5 hours. The membranes were then blocked with skim milk for 2 hours, incubated overnight with primary antibody, washed four times with PBST, and co-incubated with secondary antibody for 2 hours. Finally, ECL was used for color development, and protein expression was visualized and quantitatively analyzed using a Tanon-5200 imaging system (Tanon, China).

[0111] 2.8 Statistical Analysis

[0112] Each experimental design had at least three replicates, and all data were expressed as mean ± standard deviation. All data were analyzed using Microsoft Excel and SPSS software. One-way ANOVA and t-tests were used to assess the significance of the results, mean ± standard deviation, n≥3; *P < 0.05; **P < 0.01; ***P < 0.001 compared to the control group.

[0113] 3. Results and Analysis

[0114] 3.1 Molecular weight and uniformity detection of URP-W

[0115] Plotting log(Molar Mass) on the x-axis and log(RMS Radius) on the y-axis, the slope can be used as a reference for molecular configuration. A slope of 0.5-0.6 indicates a random coil. The detection value of URP-W was 0.63±0.09, indicating that the molecular configuration of URP-W is a random coil. Figure 1 The purity of URP-W was found to be 79.9% after testing.

[0116] Further HPLC analysis revealed that URP-W exhibited good homogeneity, displaying symmetrical peaks, with a weight-average molecular weight of 86.660 kDa and a number-average molecular weight of 83.241 kDa. Figure 2 .

[0117] 3.2 Monosaccharide composition analysis of URP-W

[0118] After HPLC monosaccharide composition analysis, the molar ratio of monosaccharides in URP-W is as follows: the molar ratio of arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, and mannuronic acid is 17.99:6.32:21.71:27.17:2.56:7.57:16.20:0.49.

[0119] 3.3 FT-IR Functional Group Analysis of URP-W

[0120] Functional groups of URP-W were detected by FT-IR, see [link to relevant documentation]. Figure 4 3400-3700cm -1 It contains a distinct strong absorption peak, which is the stretching vibration peak of the associated hydroxyl group, making it 3405.19 cm⁻¹. -1 There is a broad peak there, 2941.88 cm. -1 and 2830.99 cm -1 The sugar chain representing URP-W exhibits a saturated CH stretching vibration peak at 1616.53 cm⁻¹. -1 The image shows the stretching vibration of C=O. 1370.66 cm⁻¹-1 This is the stretching vibration of CO. 1049.09 cm -1 The absorption peak is due to the bending vibration of the hydroxyl group. Furthermore, at 774.28 cm⁻¹... -1 The absorption peak at that point indicates that the glycosidic bonds in the URP-W sugar chain are in the β-configuration.

[0121] 3.4 Methylation and NMR structural analysis of URP-W

[0122] (1) One-dimensional proton spectrum ( 1 H NMR)

[0123] One-dimensional proton NMR spectroscopy primarily addresses the configuration of glycosidic bonds in polysaccharide structures. Polysaccharides in... 1 The signal in 1H NMR is concentrated in the 3–6 ppm range. Typically, the anodic hydrogen signal for the β-glycosidic configuration is mainly distributed in the δ 4.3–4.8 ppm range, while that for the α-glycosidic configuration is mainly distributed in the δ 4.8–5.8 ppm range. Fructose, being a ketose, does not have an anodic hydrogen signal; therefore, the anodic carbon shift must be considered to determine the glycosidic configuration. (See [reference needed]). Figure 5 .

[0124] (2) One-dimensional carbon spectrum ( 13 (C NMR)

[0125] and 1 Compared to H NMR, polysaccharides are 13 The chemical shift signal distribution in C18NMR is relatively broad. There is little spectral overlap, which can provide information about sugar residues (polysaccharides in...). 13 The anomeric carbon signals in the 10⁻¹⁰ C NMR are concentrated in the 95–110 ppm range, which determines the linkage position of the sugar chain and certain specific groups (such as the carboxyl or acetamino group signals of hexuronic acid in the δ 170–176 range). See [link to relevant documentation]. Figure 6 .

[0126] (3) Hydrogen-hydrogen correlation spectrum (COSY)

[0127] Hydrogen-hydrogen correlation spectroscopy (COSY) is the most commonly used homonuclear shift correlation spectrum. It generally reflects the coupling relationship between neighboring carbons and hydrogens, thus revealing the coupling relationship between protons within the same spin system. It is a powerful tool for assigning spectral lines and deducing structures. [The text then abruptly shifts to a seemingly unrelated topic:] ...the coupling relationship between the same sugar residues... 1 The H signals will intersect diagonally, and the intersection point is called the diagonal peak. Two points symmetrically distributed on either side of the diagonal in the graph are called correlation peaks. Two / groups of mutually coupled signals... 1 The H signals will intersect at the correlation peaks; the main focus is on analyzing the correlation signals between adjacent or interphase proton hydrogens. (See...) Figure 7 .

[0128] (4) Hydrogen-hydrogen correlation spectrum (NOESY)

[0129] NOESY is a type of two-dimensional nuclear magnetic resonance (NMR) spectrum belonging to the NOE class. NOE is primarily used to determine whether two protons are close together in the three-dimensional structure of a molecule. The presence of a NOE indicates that the two protons are close; the larger the NOE value, the closer they are in space. NOE plays an important role in determining the structure, configuration, and conformation of organic compounds. (See...) Figure 8 .

[0130] (5) Carbon-hydrogen correlation spectroscopy (HSQC)

[0131] The HSQC spectrum is a CH heteronuclear single-quantum correlation spectrum. HSQC... 1 The H nucleus and its directly connected nuclei 13 The information and spectra provided are linked to the C-core. 13 C- 1 The H COSY spectrum is the same. Like HMBC, it is also measured using highly sensitive methods. 1 H-nucleus for detection 13 The sensitivity of remote coupling-related information between C-1H is higher than that of traditional remote coupling. 13 C- 1 H COSY is much higher, see Figure 9 .

[0132] (5) Carbon-hydrogen correlation spectrum (HMBC)

[0133] HMBC spectrum is a correlation spectrum of CH heteronuclear multibonds, see Figure 10 HMBC can detect long-range carbon-hydrogen coupling signals with high sensitivity, that is, the coupling of C and H between two adjacent sugar residues. By detecting the coupling of anterior hydrogen (carbon) to carbon (hydrogen) linked to another sugar residue, the linkage fragments and sequences between monosaccharide residues can be inferred.

[0134] The results showed that the 1H NMR signal of the sample was mainly concentrated between δ 3.0 and 5.5 ppm. Multiple coupled signal peaks were identified in the 4.3-5.4 ppm anodic signal region, indicating the presence of various sugar residues in the sample. The corresponding chemical shifts of the anodic hydrogens were δ 4.43, 4.59, 4.92, 5.04, 5.17, 5.2, 5.3, and 5.35. The non-anodic hydrogen signals were mainly concentrated in the 3.1-4.2 ppm region. Due to severe overlap, some signals required further analysis using COSY and HSQC spectra to assign the H2-H6 chemical shifts of each sugar residue. The strong signal peak near δ 4.71 ppm was a solvent peak, and the signal peak near δ 3.76 ppm was the signal of hydrogen from the O-CH3 group.

[0135] Multiple signal peaks were identified in the anodic carbon region of the sample, combined with13 The cross-peaks in the anodic regions of the C NMR and HSQC spectra identified the anodic signals present in the sample as follows: δ 4.59 / 104.28, 5.35 / 99.58, 5.17 / 91.86, 4.92 / 98.31, 5.2 / 109.24, 5.04 / 107.43, 5.3 / 99.75, and 4.43 / 103.32 ppm, which were denoted as sugar residues A, B, C, D, E, F, G, and H, respectively. Based on the sample's bonding structure (methylation) information, anodic signals, and comprehensive literature reports, it is inferred that sugar residue A is →4)-β-D-Galp-(1→, sugar residue B is →4)-α-D-Glcp-(1→, sugar residue C is →4)-α-D-Glcp, sugar residue D is →4)-α-D-GalpA-6-OMe-(1→, sugar residue E is α-L-Araf-(1→, sugar residue F is →5)-α-L-Araf-(1→, sugar residue G is →4,6)-α-D-Glcp-(1→, sugar residue H is →6)-β-D-Galp-(1→). Furthermore, its... 1 H and 13 The C chemical shifts were assigned, and the results are shown in Table 1. The signal peak near δ 52.82 ppm represents the carbon signal of O-CH3.

[0136] The NMR signal assignment process for major sugar residues is as follows:

[0137] Sugar residue A: The anodic signal δ 4.59 / 104.28 ppm (H1 / C1) indicates that residue A may be a β-configuration galactose residue. In the COSY spectrum, H2 (3.62 ppm) of residue A was determined based on the cross-peak δ 4.59 / 3.62 ppm, H3 (3.91 ppm) of residue A was determined based on the cross-peak δ 3.62 / 3.91 ppm, H4 (4.13 ppm) of residue A was determined based on the cross-peak δ 3.91 / 4.13 ppm, H5 (3.72 ppm) of residue A was determined based on the cross-peak δ 4.13 / 3.72 ppm, and H6 (3.71, 3.8 ppm) of residue A was determined based on the cross-peak δ 3.72 / 3.71, 3.8 ppm. Thus, the chemical shifts of hydrogen atoms on the complete sugar ring can be attributed. Then, the chemical shifts of C on the sugar ring were assigned using HSQC signals. The chemical shift of C1 of residue A was δ 104.28 ppm, the chemical shift of C2 of residue A was δ 72.5 ppm, the chemical shift of C3 of residue A was δ 73.32 ppm, the chemical shift of C4 of residue A was δ 77.66 ppm, the chemical shift of C5 of residue A was δ 72.86 ppm, and the chemical shift of C6 of residue A was δ 60.47 ppm. The chemical shifts of C1 and C4 shifted to the lower field, indicating that the residues were substituted at the O-1 and O-4 positions of the sugar ring. Based on the methylation analysis results and literature reports, it was inferred that sugar residue A might be →4)-β-D-Galp-(1→).

[0138] Sugar residue B: The anodic signal δ 5.35 / 99.58 ppm (H1 / C1) indicates that residue B may be an α-configuration glucose residue. In the COSY spectrum, H2 (3.57 ppm) of residue B was determined based on the cross-peak δ 5.35 / 3.57 ppm, H3 (3.89 ppm) of residue B was determined based on the cross-peak δ 3.57 / 3.89 ppm, H4 (3.6 ppm) of residue B was determined based on the cross-peak δ 3.89 / 3.6 ppm, H5 (3.79 ppm) of residue B was determined based on the cross-peak δ 3.6 / 3.79 ppm, and H6 (3.67, 3.77 ppm) of residue B was determined based on the cross-peak δ 3.79 / 3.67, 3.77 ppm. Thus, the chemical shifts of hydrogen atoms on the complete sugar ring can be attributed. Then, the chemical shifts of C on the sugar ring were assigned using HSQC signals. The chemical shift of C1 of residue B was δ 99.58 ppm, the chemical shift of C2 of residue B was δ 71.54 ppm, the chemical shift of C3 of residue B was δ 73.21 ppm, the chemical shift of C4 of residue B was δ 76.75 ppm, the chemical shift of C5 of residue B was δ 71.18 ppm, and the chemical shift of C6 of residue B was δ 60.68 ppm. The chemical shifts of C1 and C4 shifted to the lower field, indicating that the residues were substituted at the O-1 and O-4 positions of the sugar ring. Based on the methylation analysis results and literature reports, it is inferred that sugar residue B may be →4)-α-D-Glcp-(1→).

[0139] Sugar residue C: The anodic signal δ 5.17 / 91.86 ppm (H1 / C1) indicates that residue C may be an α-configuration glucose residue. In the COSY spectrum, H2 (3.52 ppm) of residue C was determined based on the cross peak δ 5.17 / 3.52 ppm, H3 (3.93 ppm) of residue C was determined based on the cross peak δ 3.52 / 3.93 ppm, and H4 (3.71 ppm) of residue C was determined based on the cross peak δ 3.93 / 3.71 ppm. Then, the chemical shifts of C on the sugar ring were assigned using HSQC signals. The C1 chemical shift of residue C was δ 91.86 ppm, the C2 chemical shift of residue C was δ 71.18 ppm, the C3 chemical shift of residue C was δ 73.4 ppm, and the C4 chemical shift of residue C was δ 76.18 ppm. Due to the weak spectral signal of sugar residue C, the chemical shifts could not be completely assigned. Based on the methylation analysis results and literature reports, it was inferred that sugar residue C might be →4)-α-D-Glcp.

[0140] Sugar residue D: The anodic signal δ 4.92 / 98.31 ppm (H1 / C1) indicates that residue D may be an α-configuration galacturonic acid residue. In the COSY spectrum, H2 (3.67 ppm) of residue D was determined based on the cross peak δ 4.92 / 3.67 ppm, H3 (3.95 ppm) of residue D was determined based on the cross peak δ 3.67 / 3.95 ppm, and H4 (4.41 ppm) of residue D was determined based on the cross peak δ 3.95 / 4.41 ppm. Then, the chemical shifts of C on the sugar ring were assigned using HSQC signals. The C1 chemical shift of residue D was δ 98.31 ppm, the C2 chemical shift of residue D was δ 72.86 ppm, the C3 chemical shift of residue D was δ 68.65 ppm, and the C6 chemical shift of residue D was δ 170.8 ppm. Since the spectral signal of sugar residue D was weak, the chemical shifts could not be completely assigned. Based on the methylation analysis results and literature reports, it was inferred that sugar residue D might be →4)-α-D-GalpA-6-OMe-(1→).

[0141] Sugar residue E: The anodic signal δ 5.2 / 109.24 ppm (H1 / C1) indicates that residue E may be an α-configuration arabinose residue. In the COSY spectrum, H2 (4.16 ppm) of residue E was determined based on the cross peak δ 5.2 / 4.16 ppm, H3 (3.9 ppm) of residue E was determined based on the cross peak δ 4.16 / 3.9 ppm, H4 (4.03 ppm) of residue E was determined based on the cross peak δ 3.9 / 4.03 ppm, and H5 (3.86 ppm) of residue E was determined based on the cross peak δ 4.03 / 3.86 ppm, thus allowing the chemical shift of hydrogen on the complete sugar ring to be assigned. Then, the chemical shifts of C on the sugar ring were assigned using HSQC signals. The chemical shift of C1 for residue E was δ 109.24 ppm, the chemical shift of C2 for residue E was δ 82.03 ppm, the chemical shift of C3 for residue E was δ 76.7 ppm, the chemical shift of C4 for residue E was δ 83.9 ppm, and the chemical shift of C5 for residue E was δ 60.68 ppm. The chemical shift of C1 shifted to a lower field, indicating that the residue was substituted at the O-1 position of the sugar ring. Based on the methylation analysis results and literature reports, it was inferred that sugar residue E may be α-L-Araf-(1→).

[0142] Sugar residue F: The anodic signal δ 5.04 / 107.43 ppm (H1 / C1) indicates that residue F may be an α-configuration arabinose residue. In the COSY spectrum, H2 (4.08 ppm) of residue F was determined based on the cross-peak δ 5.04 / 4.08 ppm, H3 (3.96 ppm) of residue F was determined based on the cross-peak δ 4.08 / 3.96 ppm, H4 (3.99 ppm) of residue F was determined based on the cross-peak δ 3.96 / 3.99 ppm, and H5 (3.78, 3.89 ppm) of residue F was determined based on the cross-peak δ 3.99 / 3.78, 3.89 ppm. Thus, the chemical shift of hydrogen on the sugar ring can be attributed. Then, the chemical shifts of C on the sugar ring were assigned using HSQC signals. The C1 chemical shift of residue F was δ 107.43 ppm, the C2 chemical shift of residue F was δ 80.8 ppm, the C3 chemical shift of residue F was δ 76.54 ppm, the C4 chemical shift of residue F was δ 83.86 ppm, and the C5 chemical shift of residue F was δ 66.44 ppm. The chemical shifts of C1 and C5 shifted to the lower field, indicating that the residues were substituted at the O-1 and O-5 positions of the sugar ring. Based on the methylation analysis results and literature reports, it was inferred that sugar residue F may be →5)-α-L-Araf-(1→).

[0143] Sugar residue G: The anodic signal δ 5.3 / 99.75 ppm (H1 / C1) indicates that residue G may be an α-configuration glucose residue. In the COSY spectrum, H2 (3.59 ppm) of residue G was determined based on the cross-peak δ 5.3 / 3.59 ppm, H3 (3.86 ppm) of residue G was determined based on the cross-peak δ 3.59 / 3.86 ppm, H4 (3.63 ppm) of residue G was determined based on the cross-peak δ 3.86 / 3.63 ppm, H5 (3.83 ppm) of residue G was determined based on the cross-peak δ 3.63 / 3.83 ppm, and H6 (3.75, 3.84 ppm) of residue G was determined based on the cross-peak δ 3.83 / 3.75, 3.84 ppm. Thus, the chemical shifts of hydrogen atoms on the complete sugar ring can be attributed. Then, the chemical shifts of C on the sugar ring were assigned using HSQC signals. The chemical shift of residue G was δ 99.75 ppm for C1, δ 71.97 ppm for C2, δ 73.26 ppm for C3, δ 76.89 ppm for C4, δ 70.73 ppm for C5, and δ 66.82 ppm for C6. The chemical shifts of C1, C4, and C6 shifted to a lower field, indicating that the residues were substituted at positions O-1, O-4, and O-6 on the sugar ring. Based on the methylation analysis results and literature reports, it was inferred that sugar residue G might be →4,6)-α-D-Glcp-(1→).

[0144] Sugar residue H: The anodic signal δ 4.43 / 103.32 ppm (H1 / C1) indicates that residue H may be a β-configuration galactose residue. In the COSY spectrum, H2 (3.5 ppm) of residue H was determined based on the cross-peak δ 4.43 / 3.5 ppm, H3 (3.64 ppm) based on the cross-peak δ 3.5 / 3.64 ppm, H4 (3.77 ppm) based on the cross-peak δ 3.64 / 3.77 ppm, H5 (3.97 ppm) based on the cross-peak δ 3.77 / 3.97 ppm, and H6 (4 ppm) based on the cross-peak δ 3.97 / 4 ppm. Thus, the chemical shift of the hydrogen on the sugar ring can be attributed. Then, the chemical shifts of C on the sugar ring were assigned using HSQC signals. The C1 chemical shift of residue H was δ 103.32 ppm, the C2 chemical shift of residue H was δ 70.73 ppm, the C3 chemical shift of residue H was δ 73.01 ppm, the C4 chemical shift of residue H was δ 70.75 ppm, the C5 chemical shift of residue H was δ 73.07 ppm, and the C6 chemical shift of residue H was δ 69.3 ppm. The chemical shifts of C1 and C6 shifted to the lower field, indicating that the residues were substituted at the O-1 and O-6 positions of the sugar ring. Based on the methylation analysis results and literature reports, it was inferred that sugar residue I might be →6)-β-D-Galp-(1→).

[0145] Following similar steps as described above, the assignment of all sugar residue shifts can be completed, and the results are shown in Table 1:

[0146] Table 1 Sugar residues 1 H and 13 Chemical shift of C

[0147]

[0148] Note: nd is an abbreviation for "not detected".

[0149] Based on each sugar residue in the sample 13 C and 1The chemical shift of H, combined with HMBC and NOESY spectra, was used to analyze the structure and linkage mode of this polysaccharide. The HMBC spectrum showed no connection signal. According to the NOESY spectrum, there were cross-peaks between H1 and H4 of sugar residue A (δ 4.59 / 4.13 ppm), H1 and H4 of sugar residue B (δ 5.35 / 3.6 ppm), H1 and H4 of sugar residue G (δ 5.35 / 3.63 ppm), H1 and H4 of sugar residue D (δ 4.92 / 4.41 ppm), H1 and H6 of sugar residue H (δ 4.92 / 4.0 ppm), H1 and H5 of sugar residue E (δ 5.2 / 3.78 ppm), H1 and H5 of sugar residue F (δ 5.2 / 3.89 ppm), and H1 and H6 of sugar residue G (δ 5.2 / 3.89 ppm). At 5.04 / 3.75 ppm, there is a cross-peak between H1 of sugar residue F and H6 of sugar residue G (δ 5.04 / 3.84 ppm), a cross-peak between H1 of sugar residue G and H4 of sugar residue B (δ 5.3 / 3.6 ppm), and a cross-peak between H1 of sugar residue H and H4 of sugar residue D (δ 4.43 / 4.41 ppm). Sugar residue C is not represented in the glycan linkage due to its weak spectral signal.

[0150] Therefore, based on the analysis of one-dimensional and two-dimensional NMR information and methylation results, it is inferred that the polysaccharide may contain the following fragments: fragment 1 is formed by the interconnection of →4)-β-D-Galp-(1→); fragment 2 is formed by the interconnection of →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→) to form the main chain, with α-L-Araf-(1→5)-α-L-Araf-(1→) attached to the O-6 position of →4,6)-α-D-Glcp-(1→); fragment 3 is formed by the interconnection of →4)-α-D-GalpA-6-OMe-(1→ and →6)-β-D-Galp-(1→).

[0151] Finally, the chemical structure of URP-W was determined as follows:

[0152]

[0153] 3.5 URP-W significantly inhibited the proliferation of U251 cells.

[0154] Microscopic observation revealed that U251 cells co-cultured with URP-W exhibited abnormal morphology, showing shrinkage and fragmentation, and a significantly reduced cell density. Figure 11 The results showed that URP-W could effectively inhibit the in vitro proliferation of glioma U251 cells and induce abnormal morphology in U251 cells.

[0155] The inhibitory effect of URP-W on the proliferation of U251 glioma cells was quantitatively analyzed using the CCK-8 assay. The results showed that the inhibitory effect of URP-W on the proliferation of U251 cells was time- and concentration-dependent. (See attached figure) Figure 12 When the concentration of URP-W was 1000 μg / mL, the maximum inhibition rate of U251 cell proliferation was 58.25±0.91% after 48 h of treatment.

[0156] 3.6 URP-W induces abnormal nuclear morphology and apoptosis in U251 cells

[0157] To verify the effect of URP-W on apoptosis in U251 glioma cells, DAPI fluorescence staining was performed using URP-W at concentrations of 250, 500, and 1000 µg / mL. After drug administration, cell nuclear morphology began to change, with chromatin aggregation, nuclear pyknosis, and the appearance of bright blue apoptotic bodies. The number of apoptotic bodies in the experimental group was significantly higher than that in the control group, and this number increased with increasing drug concentration (see [reference needed]). Figure 13 .

[0158] Subsequently, flow cytometry was used to quantitatively analyze the effect of URP-W on apoptosis in glioma U251 cells. The results showed that the number of viable cells decreased significantly from 93.24% to 83.91%, while the apoptosis rate changed significantly (P<0.05). The proportions of early and late apoptotic cells increased significantly, from 0.45% and 0.43% to 2.78% and 4.24%, respectively, and the proportion of dead cells increased from 5.89% to 9.07%. Figure 14 The above-mentioned nuclear DAPI fluorescence staining and flow cytometry results showed that URP-W could significantly induce apoptosis in U251 cells.

[0159] 3.7 URP-W Effects on Tumor Growth and Survival in U251 Glioma-Bearing Mice

[0160] A mouse model of glioma U251 was established. Mice were treated with URP-W orally continuously, and their survival status, body weight, food intake, and tumor size were monitored. Results are as follows: Figure 15 After 4 weeks of treatment, mice in the control group were administered physiological saline by gavage, and the tumors in these mice significantly increased in size, from 30.762 mm. 2 Increased to 70.813 mm 2 In contrast, the tumor volume in the URP-W treatment group mice was significantly reduced, from 39.789 mm. 2 Reduced to 30.849 mm 2 Based on the above results, the proliferation of glioma U251 cells in tumor-bearing mice can be inhibited.

[0161] Meanwhile, observation of the mice's survival status revealed that the average body weight and food and water intake of the control group mice decreased as the tumor progressed, while all indicators of the treatment group mice increased. These results indicate that URP-W not only inhibits tumor growth in tumor-bearing mice but also effectively improves their survival status, demonstrating significant therapeutic effects. Figure 16 .

[0162] 3.8 URP-W affects protein expression in tumor tissues of glioma-bearing mice

[0163] Through the interaction between URP-W and U251 cells, this study found that URP-W can induce apoptosis in U251 cells, possibly through the regulation of multiple genes and proteins. Therefore, Q-PCR and Western blot were used to detect the expression levels of tumor-related proteins to elucidate the molecular mechanism of URP-W's anti-glioma effect. First, the gene expression of apoptosis-related genes Bax, Bcl-2, Cyto-c, and PARP was detected, and the results are shown in Table 2.

[0164] Table 2. Effects of URP-W on gene expression in tumor tissues of U251 glioma-bearing mice.

[0165]

[0166] Note: Mean ± standard deviation, n≥3; *P < 0.05; **P < 0.01; ***P < 0.001 compared with the control group.

[0167] The above results indicate that URP-W exerts its inhibitory effect on tumor development and progression by intervening in mitochondrial apoptosis in tumor-bearing mice. Further Western blotting was used to verify the effect of URP-W on protein expression in tumor tissues of tumor-bearing mice, see [link to relevant documentation]. Figure 17 And Table 3.

[0168] Table 3. Effects of URP-W on the expression of mitochondrial apoptosis pathway-related proteins in tumor tissues of U251 glioma-bearing mice.

[0169]

[0170] Note: Mean ± standard deviation, n≥3; *P < 0.05; **P < 0.01; ***P < 0.001 compared with the control group.

[0171] As can be seen from the above embodiments, this invention employs boiling water extraction and ethanol precipitation of Uncaria rhynchophylla polysaccharides, combined with DEAE cellulose column and molecular sieve dextran gel column purification techniques to achieve the separation of neutral Uncaria rhynchophylla polysaccharides, solving the technical challenge of polysaccharide component extraction and separation. Monosaccharide composition analysis, molecular weight detection, Fourier transform infrared spectroscopy, methylation analysis, and nuclear magnetic resonance detection were used to clarify the chemical composition and precise structural characteristics of the neutral Uncaria rhynchophylla polysaccharide URP-W. Through CCK-8 cell proliferation assay, the highly water-soluble orange-yellow formazan was generated by the reduction of WST-8 in mitochondria by dehydrogenases, thereby quantitatively analyzing cell proliferation capacity and resolving the influence of URP-W polysaccharide components on the proliferation capacity of the glioma cell line U251. Furthermore, cell staining with fluorescent dyes and observation of nuclear apoptosis morphology were combined with inverted fluorescence microscopy to assess the preliminary effect of URP-W on U251 cell apoptosis. Flow cytometry was then used to quantitatively analyze the proportion of apoptotic cells, solving the problem of URP-W's ability to induce and quantify apoptosis. Finally, protein quantification analysis was employed. Based on the principle of antigen-antibody immunoreaction, the effects of URP-W on apoptosis of U251 glioma cell line and the expression of related proteins in tumor tissues of tumor-bearing mice were measured and quantified. Simultaneously, survival status monitoring was conducted, resolving the technical challenges in understanding the inhibitory mechanism of the Uncaria rhynchophylla neutral polysaccharide URP-W component on glioma. These research findings provide important reference for the development of Uncaria rhynchophylla neutral polysaccharide URP-W anti-glioma drugs.

[0172] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A neutral polysaccharide URP-W from Uncaria rhynchophylla, characterized in that, The monosaccharide composition of the URP-W includes arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, and mannuronic acid, with a molar ratio of 17.99:6.32:21.71:27.17:2.56:7.57:16.20:0.

49.

2. The Uncaria rhynchophylla neutral polysaccharide URP-W as described in claim 1, characterized in that, The URP-W includes segments of the following structure: 。 3. The Uncaria rhynchophylla neutral polysaccharide URP-W as described in claim 2, characterized in that, The connection method for each segment in the URP-W is as follows: Segment 1 is formed by interconnecting →4)-β-D-Galp-(1→); Segment 2 is formed by interconnecting →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→), with α-L-Araf-(1→5)-α-L-Araf-(1→ connected at the O-6 position of →4,6)-α-D-Glcp-(1→); Segment 3 is formed by interconnecting →4)-α-D-GalpA-6-OMe-(1→ and →6)-β-D-Galp-(1→).

4. A method for extracting and separating the neutral polysaccharide URP-W from Uncaria rhynchophylla according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Extract the Uncaria rhynchophylla sample with ethanol to remove impurities, centrifuge and collect the precipitate, then mix the precipitate with water, heat to extract, centrifuge and collect the supernatant extract; (2) The supernatant extract was precipitated with ethanol, centrifuged and the precipitate was collected and dried to obtain crude polysaccharide extract; (3) After removing impurities from the crude polysaccharide extract, it was purified by ion exchange column chromatography and dextran gel column chromatography. The liquid passed through the column was collected, freeze-dried, and the Uncaria rhynchophylla neutral polysaccharide URP-W was obtained.

5. The method as described in claim 4, characterized in that, In step (1), the ratio of the Uncaria rhynchophylla sample to ethanol is 1:(8-10); and / or the ratio of the precipitate to water is 1:(15-20); and / or the heating extraction conditions are 60℃ water bath extraction for 3-5 hours.

6. The method as described in claim 4, characterized in that, In step (2), after the supernatant extract is concentrated to 1 / 10 of its original volume, 4 times the volume of ethanol is added for precipitation.

7. The method as described in claim 4, characterized in that, In step (3), the impurity removal of the crude polysaccharide extract includes the following steps: S1: Dissolve the crude polysaccharide extract in water, add protease for enzymatic hydrolysis, centrifuge, and collect the supernatant; S2: Add chloroform and n-butanol to the supernatant, mix thoroughly, and collect the upper aqueous phase; S3: Add petroleum ether to the upper aqueous phase, mix thoroughly, and collect the lower aqueous phase; S4: Add macroporous adsorption resin to the lower aqueous phase, mix thoroughly for adsorption, collect the liquid, dialyze it, precipitate it with ethanol, and dry the precipitate.

8. The method as described in claim 7, characterized in that, In step S1, the amount of water used is 600mL-1000mL, and the amount of protease used is 0.4-0.6g. In step S2, 1 / 3 to 1 / 4 volume of chloroform and n-butanol are added to the supernatant, wherein the volume ratio of chloroform to n-butanol is 4:

1. And / or in step S3, 1 / 3 to 1 / 4 volume of petroleum ether is added to the upper aqueous phase; And / or in step S4, 1 / 2 to 1 / 3 volume of macroporous adsorption resin is added to the lower aqueous phase.

9. The method as described in claim 4, characterized in that, The ion exchange column chromatography and dextran gel column chromatography were purified using DEAE DE-52 cellulose and dextran gel G200, respectively, with water as the eluent.

10. The use of the Uncaria rhynchophylla neutral polysaccharide URP-W according to any one of claims 1-3 in the preparation of an anti-glioma drug.

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