Application of uncaria acidic polysaccharide URP-2 in preparation of anti-esophageal cancer drugs

Abstract

The invention discloses application of uncaria acidic polysaccharide URP-2 in preparation of an anti-esophageal cancer drug, and belongs to the technical field of antitumor of medicinal plant polysaccharide. According to the method, a method for extracting the uncaria total polysaccharide by boiling water and precipitating the uncaria total polysaccharide by ethanol is adopted, and a DEAE DE-52 cellulose column and sephadex column purification technology is combined, so that the separation of the uncaria acidic polysaccharide component is realized, and the uncaria acidic polysaccharide URP-2 is obtained. The uncaria acidic polysaccharide URP-2 comprises the following components in mole percent: 14.11% of arabinose, 12.64% of rhamnose, 6.48% of galactose, 5.00% of glucose and 61.77% of galacturonic acid. Experiments show that the uncaria acidic polysaccharide URP-2 has remarkable anti-esophageal cancer activity, so that a potential experimental basis and a new strategy are provided for applying the uncaria acidic polysaccharide URP-2 to clinical treatment of esophageal cancer.

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 acidic polysaccharide URP-2 in the preparation of antiesophageal cancer drugs. Background Technology

[0002] Uncaria rhynchophylla is sweet in taste and cool in nature, entering the liver and pericardium meridians. It has the effects of clearing heat and calming the liver, extinguishing wind and relieving convulsions. It is an excellent medicine for treating internal liver wind, epilepsy, and convulsions, and is often used for headaches, dizziness, delirium caused by high fever, and infantile convulsions caused by liver yang hyperactivity. Modern pharmacological studies have shown that the alkaloids contained in Uncaria rhynchophylla (such as rhynchophylline) have significant antihypertensive, sedative, and anticonvulsant effects, and have good adjuvant therapeutic effects on hypertension, cerebrovascular diseases, and nervous system diseases.

[0003] Research on the antitumor effects of Uncaria rhynchophylla mainly focuses on its abundant indole alkaloids, among which rhynchophylline and isorhynchophylline are the two most abundant and widely studied active ingredients. In addition, corynoxeine and isocorynoxeine also exhibit significant biological activity. However, further research is needed to elucidate its pharmacodynamic material basis, develop the pharmacological activities of other active ingredients, precisely elucidate the mechanisms of action of active ingredients, and translate them into clinical applications. In particular, research on the macromolecular active substances in Uncaria rhynchophylla is extremely limited.

[0004] Esophageal cancer is a malignant tumor originating from the esophageal mucosal epithelium and is a common digestive tract cancer in clinical practice. Its incidence shows a significant geographical distribution, with a higher incidence in men than women, and it is more common in middle-aged and elderly people. Esophageal cancer is mainly divided into esophageal squamous cell carcinoma and esophageal adenocarcinoma. Early symptoms are often subtle and easily overlooked. A typical symptom is progressive dysphagia, initially difficulty swallowing solid food, gradually progressing to an inability to swallow even semi-liquid or liquid foods, accompanied by chest pain, weight loss, and hoarseness. Treatment depends on the stage and location of the cancer and mainly includes surgical resection, radiotherapy, and chemotherapy. Early detection leads to a good prognosis, but most patients are diagnosed at an advanced stage, and the overall five-year survival rate remains low. Therefore, early endoscopic screening for high-risk groups, changes in unhealthy lifestyle habits, and active treatment of precancerous lesions are key to prevention.

[0005] Research on the active ingredients of traditional Chinese medicine (TCM) in the treatment of esophageal cancer has demonstrated unique advantages through multiple pathways and targets, with complex and comprehensive mechanisms of action. Numerous studies have shown that these ingredients can effectively inhibit the proliferation of esophageal cancer cells and induce apoptosis, primarily by interfering with cell cycle progression and activating apoptosis signaling pathways such as mitochondria or death receptors. In inhibiting tumor invasion and metastasis, TCM ingredients significantly reduce the migration and invasion capabilities of cancer cells by downregulating the expression of matrix metalloproteinases (MMPs) and inhibiting the epithelial-mesenchymal transition (EMT) process. Simultaneously, many ingredients also exhibit anti-angiogenic activity, cutting off the tumor's nutrient supply by inhibiting vascular endothelial growth factor (VEGF) and related factors. Notably, some active ingredients, such as curcumin and baicalin, have been shown to reverse multidrug resistance in tumors by inhibiting the drug efflux pump function of P-glycoproteins, thereby increasing the intracellular concentration of conventional chemotherapy drugs. Furthermore, TCM ingredients also play a significant role in immunomodulation and "synergistic effect with reduced toxicity," enhancing the body's anti-tumor immunity while mitigating adverse reactions such as bone marrow suppression caused by radiotherapy and chemotherapy.

[0006] Representative traditional Chinese medicine (TCM) components currently being studied for esophageal cancer include: curcumin extracted from turmeric, which has a broad inhibitory effect on esophageal squamous cell carcinoma cells; baicalin and wogonin derived from Scutellaria baicalensis, which show outstanding performance in inducing apoptosis and inhibiting metastasis; allicin and diallyl disulfide from garlic, which exert anti-tumor effects by inducing cell cycle arrest and promoting reactive oxygen species generation; quercetin, widely found in fruits and vegetables, which can simultaneously inhibit cancer cell growth and induce autophagy and apoptosis; ginsenosides Rh2, Rg3, and other monomers, which have significant effects in anti-angiogenesis; and realgar (mainly containing arsenic tetrasulfide), a traditional mineral medicine, has also been confirmed by modern research to have a clear effect in inducing apoptosis in esophageal cancer cells. However, research on the anti-tumor effects of macromolecules in TCM on esophageal cancer has not yet been reported. Overall, most research on the anti-esophageal cancer effects of active components in TCM is still in the basic research stage, but their characteristics of "multi-target and low toxicity" give them great development potential. Future research will focus on developing these monomeric components into novel anti-tumor drugs, or as adjuvant sensitizers and toxicity reducers for chemotherapy / targeted therapy, providing esophageal cancer patients with more effective and personalized treatment options through a combination of traditional Chinese and Western medicine. Summary of the Invention

[0007] The purpose of this invention is to provide the application of Uncaria rhynchophylla acidic polysaccharide URP-2 in the preparation of anti-esophageal cancer drugs, so as to solve the problems existing in the prior art. Uncaria rhynchophylla acidic polysaccharide URP-2 has significant inhibitory effects on the proliferation of esophageal cancer cells, induces abnormal cell morphology and apoptosis, and inhibits cell migration and invasion, showing significant anti-esophageal cancer activity. This provides potential experimental evidence and new strategies for the clinical treatment of esophageal cancer using Uncaria rhynchophylla acidic polysaccharide URP-2.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] This invention provides an acidic polysaccharide URP-2 from Uncaria rhynchophylla, wherein the components of URP-2 include arabinose, rhamnose, galactose, glucose and galacturonic acid, with molar percentages of 14.11%, 12.64%, 6.48%, 5.00% and 61.77%, respectively.

[0010] Preferably, the URP-2 has a weight-average molecular weight of 2156.845 kDa.

[0011] Preferably, the glycosidic bonds in the sugar chain of the URP-2 are in the β-configuration.

[0012] The present invention also provides the application of the aforementioned Uncaria rhynchophylla acidic polysaccharide URP-2 in the preparation of anti-esophageal cancer drugs.

[0013] The present invention also provides a drug for treating esophageal cancer, the main active ingredient of which is the aforementioned Uncaria rhynchophylla acidic polysaccharide URP-2.

[0014] This invention also provides a method for preparing the aforementioned Uncaria rhynchophylla acidic polysaccharide URP-2, comprising the following steps:

[0015] (1) A crude polysaccharide extract was obtained by boiling water extraction and ethanol precipitation;

[0016] (2) The crude polysaccharide extract was purified by ion exchange chromatography. First, it was eluted with water, and then eluted with 0.1M and 0.3M NaCl solutions respectively. The 0.3M NaCl solution eluent was collected. Then, it was further purified by dextran gel chromatography. It was eluted with water, the eluent was collected, and lyophilized to obtain Uncaria rhynchophylla acidic polysaccharide URP-2.

[0017] Preferably, in step (1), the boiling water extraction and ethanol precipitation include the following steps:

[0018] The Uncaria rhynchophylla sample and ethanol were mixed and extracted at a material-to-liquid ratio of 1g:(8-10)mL, centrifuged and the precipitate was collected; the precipitate and water were mixed at a material-to-liquid ratio of 1g:(15-20)mL and extracted in a boiling water bath for 3-5 hours, centrifuged to obtain the supernatant extract and precipitate residue, water was added to the precipitate residue and the extraction was repeated once, centrifuged and the supernatants obtained from the two extractions were combined.

[0019] After concentrating the combined supernatant extract to 1 / 10 of its original volume, 3-5 times its volume of ethanol is added for precipitation. The precipitate is collected by centrifugation and dried to obtain the crude polysaccharide extract.

[0020] Preferably, in step (2), before purifying the crude polysaccharide extract using ion exchange chromatography, impurity removal is performed, which includes the following steps:

[0021] S1: Dissolve the crude polysaccharide extract in water, add protease for enzymatic hydrolysis, centrifuge, and collect the supernatant;

[0022] S2: Add chloroform and n-butanol to the supernatant, mix thoroughly, and collect the upper aqueous phase;

[0023] S3: Add petroleum ether to the upper aqueous phase, mix thoroughly, and collect the lower aqueous phase;

[0024] 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.

[0025] Preferably, in step S1, the amount of water used is 600mL-1000mL, and the amount of protease used is 0.4-0.6g.

[0026] And / or, 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;

[0027] And / or, in step S3, 1 / 3 to 1 / 4 volume of petroleum ether is added to the upper aqueous phase;

[0028] And / or, in step S4, 1 / 2 to 1 / 3 volume of macroporous adsorption resin is added to the lower aqueous phase, and the dialysis conditions are dialysis with a 3000 Da dialysis bag for 24-48 hours.

[0029] Preferably, in step (2), the ion exchange chromatography uses DEAE DE-52 cellulose as the chromatography medium, and the dextran gel chromatography uses dextran gel G200 as the chromatography medium.

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

[0031] This invention employs boiling water extraction and ethanol precipitation of total polysaccharides from Uncaria rhynchophylla, combined with DEAE DE-52 cellulose column and dextran gel column purification technology, to achieve the separation of acidic polysaccharide components from Uncaria rhynchophylla, thus solving the technical challenge of polysaccharide component extraction and separation.

[0032] This invention employs monosaccharide composition analysis, molecular weight detection, and Fourier transform infrared spectroscopy to clarify the chemical composition and precise structural characteristics of the acidic polysaccharide URP-2 from Uncaria rhynchophylla. Its monosaccharide composition is: arabinose, rhamnose, galactose, glucose, and galacturonic acid, with molar percentages of 14.11%, 12.64%, 6.48%, 5.00%, and 61.77%, respectively. The anti-esophageal cancer effect of URP-2 was also evaluated using indicators such as cell proliferation, nuclear morphology, apoptosis, and tumor growth in tumor-bearing mice. Results showed that URP-2 significantly inhibited the proliferation of TE-1 cells, induced abnormal nuclear morphology and apoptosis in TE-1 cells, and inhibited the migration and invasion abilities of TE-1 cells, exhibiting significant anti-tumor activity. In vivo experiments further verified that the anti-tumor mechanism of URP-2 mainly involves regulating the mitochondrial apoptosis pathway and inhibiting cell migration and invasion. On the one hand, URP-2 can upregulate the expression of the pro-apoptotic factor Bax while downregulating the anti-apoptotic protein Bcl-2, thereby inducing apoptosis via the mitochondrial pathway. On the other hand, URP-2 can reduce the expression of matrix metalloproteinases MMP-2 and MMP-9, thereby inhibiting the migration and invasion of tumor cells and limiting the progression of esophageal cancer. This invention provides potential experimental evidence and a new strategy for the clinical application of Uncaria rhynchophylla acidic polysaccharide URP-2 in the treatment of esophageal cancer. 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 This is a chromatogram showing the absolute molecular weight analysis of URP-2;

[0035] Figure 2 The diagram shows the monosaccharide composition analysis of URP-2;

[0036] Figure 3 The image shows the FT-IR detection pattern of URP-2.

[0037] Figure 4 The effect of URP-2 on the morphology of esophageal cancer TE-1 cells (48h);

[0038] Figure 5 Results of URP-2 at different administration times and concentrations inhibiting the proliferation of esophageal cancer TE-1 cells; mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group;

[0039] Figure 6 The effect of URP-2 on the morphology of nuclear apoptosis in TE-1 esophageal cancer cells (48h);

[0040] Figure 7 To analyze the effect of URP-2 on apoptosis in esophageal cancer TE-1 cells by flow cytometry (48 h);

[0041] Figure 8 The effect of URP-2 on the migration (A) and invasion (B) abilities of esophageal cancer TE-1 cells (800 μg / mL, 48 h);

[0042] Figure 9 The effect of URP-2 on the expression of matrix metalloproteinase family members MMP-2 and MMP-9 in esophageal cancer TE-1 cells (800 μg / mL, 48 h);

[0043] Figure 10 To track the effect of URP-2 on tumor growth in TE-1 esophageal cancer-bearing mice using nuclear magnetic resonance imaging;

[0044] Figure 11 To investigate the effect of URP-2 on the blood supply around the tumor in TE-1 esophageal cancer-bearing mice using a laser speckle blood flow imaging system;

[0045] Figure 12 The effect of URP-2 on the survival status of TE-1 esophageal cancer-bearing mice; (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;

[0046] Figure 13 To investigate the effect of URP-2 on the expression of proteins related to the mitochondrial apoptosis pathway in tumor tissues of tumor-bearing mice. Detailed Implementation

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] Example 1

[0053] 1. Main experimental materials

[0054] Esophageal cancer cell line TE-1 (CL-0231, Pronosei Biotechnology Co., Ltd.); fetal bovine serum (164210, Pronosei Biotechnology Co., Ltd.); DAPI fluorescent staining solution (KGA1808-50, Jiangsu Kaiji Biotechnology Co., Ltd., China); Annexin V-FITC / PI double staining apoptosis detection kit (KGA1102-100, Jiangsu Kaiji Biotechnology Co., Ltd., China); β-actin, MMP-2, and MMP-9 antibodies (Wuhan Aibote Biotechnology Co., Ltd.).

[0055] 2. Experimental Methods

[0056] 2.1 Extraction of total polysaccharides from Uncaria rhynchophylla

[0057] (1) Crush the dried Uncaria rhynchophylla sample / raw material with a pulverizer and pass it through a 60-mesh sieve.

[0058] (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.

[0059] (3) Add pure water to the precipitate (material-to-liquid ratio 1g:20mL), extract in a boiling water bath for 4 hours, centrifuge at 6000g for 10 minutes, and collect the supernatant extract. Repeat the extraction process on the precipitate residue.

[0060] (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.

[0061] (5) Centrifuge at 8000g for 10min, collect the precipitate solid and dry it to obtain crude polysaccharide extract.

[0062] (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, 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, let it stand for 10 min in the dark, and measure the absorbance at 490 nm.

[0063] 2.2 Removal of impurities from total polysaccharides

[0064] 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:

[0065] (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.

[0066] (2) After centrifuging the enzymatic hydrolysate from step (1), collect the upper 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.

[0067] (3) Add 1 / 4 volume of petroleum ether to the upper aqueous phase of step (2), mix thoroughly, and collect the lower aqueous phase.

[0068] (4) Add 1 / 2 volume of macroporous resin AB-8 to the lower aqueous phase in step (3), mix thoroughly, and allow to adsorb overnight.

[0069] (5) Collect the liquid, dialyze with a 3000Da dialysis bag for 24-48 hours to remove small molecule components, precipitate the polysaccharide dialysate with ethanol, collect the precipitate and dry it.

[0070] 2.3 Preparation method of Uncaria rhynchophylla acidic polysaccharide (URP-2)

[0071] URP was separated and purified by molecular sieves using ion exchange column chromatography and dextran gel column chromatography: DEAEDE-52 cellulose was used for purification. The sample was first eluted with water, then successively with 0.1M NaCl and 0.3M NaCl. The 0.3M NaCl fraction was collected and the resulting sample was retained after passing through the column. The sample was then purified by passing through a dextran gel G200 with water as the eluent. The column buffer was collected, lyophilized, and stored to obtain URP-2. The flow rate for the purification process was set to 1 mL / min, with 10 mL per tube.

[0072] 2.4 Structural characteristics and identification of URP-2

[0073] 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-805HQ (300×8 mm) and Ohpak SB-803HQ (300×8 mm) gel size exclusion columns were used in series. Column temperature 45℃, injection volume 100μL, mobile phase A (0.02% NaN3, 0.1M NaNO3), flow rate 0.6mL / min, elution gradient: isocratic for 75min.

[0074] Secondly, infrared spectroscopy was used to detect the main functional groups present in URP-2, and the configuration of the polysaccharide was preliminarily determined, thereby understanding its structural characteristics. Finally, the composition of Uncaria rhynchophylla acidic polysaccharide (URP-2) was confirmed by HPLC (molecular weight, homogeneity, and monosaccharide composition detection) and Fourier transform infrared spectroscopy, thereby gaining a deeper understanding of its biological activity and potential application value.

[0075] Functional identification of 2.5URP-2

[0076] (1) Cell line and cell culture: Human esophageal cancer cells (TE-1 cells) were cultured in 1640 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 carbon dioxide concentration maintained at 5%. When the cell density reached 90%, the cells were passaged at a ratio of 1:2 to 3 and cultured continuously.

[0077] (2) Cell proliferation inhibition detection: TE-1 cells were inoculated at 2×10⁻⁶ cells per cell line. 3Cells 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-2 (0, 200, 400, and 800 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-2 at a concentration of 5 μM. TE-1 cells were co-cultured with 5 μM DOX and different concentrations of URP-2 (0, 200, 400, and 800 μg / mL) after seeding. Each treatment group had 5 replicates, and the experiment was repeated at least three times with parallel and iterative assays. After 24 and 48 hours of co-incubation, cell viability was assessed using a CCK-8 cell proliferation assay kit. 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:

[0078] Proliferation inhibition rate (%) = ([A) C -A S ] / [A C -A 空白 ])×100%

[0079] 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).

[0080] (3) DAPI fluorescence staining experiment: TE-1 cells were stained with 1×10 4 Cells were seeded at a density of 100 cells / well in culture dishes containing 1640 medium and cultured for 24 h. Subsequently, the original medium was replaced with drug-treated medium, and the cells were co-cultured with URP-2 for 24 h and 48 h. 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 min, 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.

[0081] (4) Apoptosis assay: TE-1 cells were inoculated at 2×10⁻⁶ cells per cell line. 5 Cells were seeded at a density of 100 cells / well in culture dishes containing 1640 medium and cultured for 24 h. Subsequently, the original medium was replaced with the drug-treated medium, and the cells were co-cultured with URP-2 for 48 h. Afterward, the cells were washed twice with PBS, reagents were added according to instructions and mixed thoroughly, and the mixture was incubated at room temperature in the dark for 5–15 min before analysis by flow cytometry. Each sample group was set up in triplicate, and the experiment was repeated at least three times.

[0082] (5) Cell scratch healing experiment: In the cell scratch healing experiment, cells in the logarithmic growth phase were seeded into 6-well plates without counting. When the cell density in the 6-well plate reached more than 80%, the cells were scratched in a cross shape with a 1 mL pipette tip, making four horizontal lines and two vertical lines in each well. After the scratching was completed, the suspended cells were washed once with basal culture medium, and URP-2 drug-containing culture medium was added to the well plate. The cells were cultured for another 48 hours, observed and photographed, and the photos were stored.

[0083] (6) Transwell chemotaxis assay: The effect of URP-2 on the invasive ability of TE-1 cells was detected using the Transwell chemotaxis assay. Before the experiment, the Transwell chambers were prepared and sterilized with alcohol and UV. The pipette tips and culture medium were pre-cooled. After coating the upper chamber membrane with Matrigel, the experiment began. TE-1 cells in the logarithmic growth phase were taken and the cell concentration was adjusted. 600 μL of culture medium containing 10% newborn calf serum (NBS) and URP-2 were added to the 12-well plate as the control group and the treatment group, respectively. The plates were tilted and placed into the chambers to avoid the formation of air bubbles in the membrane. Then, cell suspensions containing basal culture medium and URP-2 were added to the upper chamber of the control group and the treatment group, respectively. The cells were cultured for another 48 h. Remove the chamber with tweezers, discard the cell solution in the upper chamber, place the chamber on a horizontal plane, wash twice with 1 mL PBS per well, discard the PBS, add 1 mL methanol to each well for 10 min to fix, remove the methanol, add crystal violet for 10 min to 30 min to stain, wash three more times with PBS, gently wipe the cells off the upper surface of the chamber with absorbent cotton, observe and photograph five different areas under a 40x objective lens under a microscope, save the photos, and calculate the results.

[0084] (7) Immunofluorescence staining: Place sterile coverslips into the culture plate and seed cells to a density of 60-70%. Prepare 4% paraformaldehyde (PFA), 0.3% Triton X-100 permeabilization buffer, 5% BSA blocking buffer, primary antibody against the target protein, fluorescently labeled secondary antibody (e.g., Alexa Fluor 488), DAPI nuclear staining solution, and anti-quenching mounting medium. Discard the culture medium and gently wash three times with PBS (5 minutes each time) to remove serum residue; add 4% PFA and fix at room temperature for 15-20 minutes (avoid over-fixation), then wash three more times with PBS. Add 0.3% Triton X-100 for 10 minutes (this step can be omitted for membrane proteins); after washing with PBS, cover with 5% BSA blocking buffer and incubate at room temperature for 1 hour to block non-specific binding. Add primary antibody working solution (diluted with blocking buffer, commonly 1:100-1:500), and incubate in a humidified chamber at 4°C overnight (or at room temperature for 2 hours). Recover the primary antibody and rinse thoroughly with PBS three times (10 minutes each time). Add fluorescent secondary antibody (1:200-1:1000) under light-protected conditions and incubate at room temperature in a humidified chamber for 1 hour. After washing with PBS, add DAPI (1 μg / mL) to stain the nuclei for 5 minutes, followed by a final PBS wash three times. Use tweezers to blot dry the edges of the slide, add anti-quenching mounting medium to the slide, and gently press the coverslip to avoid air bubbles. Cure in the dark for 24 hours (or accelerate at 37°C for 2 hours), and observe and save the images as soon as possible using a confocal microscope.

[0085] (8) Construction and treatment of tumor-bearing mouse model: TE-1 cell suspension was inoculated into the right axilla of C57BL / 6 mice, 0.2 mL per mouse, to construct an esophageal cancer tumor-bearing mouse model. The experiment was divided into two groups, with 15 mice in each group. The control group was administered 0.2 mL of physiological saline by gavage for 28 consecutive days; the URP-2 group was administered 400 mg / kg by gavage; and the cisplatin group was administered 3 mg / kg orally by gavage every other day for 28 consecutive days. The body weight of the mice was measured 72 hours after the last administration, and then they were sacrificed. After confirming the death of the mice, they were dissected, and the tumor tissue and other organ tissues were completely removed for evaluation.

[0086] (9) Q-PCR: Total RNA was extracted from each sample and used to prepare cDNA samples. Gene expression was measured by qPCR and primers. Primer information is shown in Table 1 below.

[0087] Table 1 Primer Information

[0088]

[0089] 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, with a final extension at 72°C for 10 min.

[0090] (10) Immunofluorescence staining: Prostate cancer DU145 cells under different treatments were stained with immunofluorescence and the fluorescence intensity of the target protein was observed by fluorescence microscopy to evaluate the effect of URP-2 on the expression levels of MMP-2 and MMP-9 proteins in the cells.

[0091] (11) Western blot analysis: Cell samples were collected from each culture group, protein samples were extracted, and analyzed using Western blot analysis. After electrophoresis, protein samples were transferred to polyvinylidene fluoride (PVDF) membranes for 1.5 h. The membranes were then blocked with skim milk for 2 h, incubated overnight with primary antibody, washed four times with PBST, and co-incubated with secondary antibody for 2 h. Finally, ECL was used for color development, and protein expression was visualized and quantitatively analyzed using a Tanon-5200 imaging system (Tanon, China).

[0092] (12) Statistical analysis: Each experimental design had at least three parallel experiments, 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 with the control group.

[0093] 3. Results and Analysis

[0094] 3.1 Molecular weight determination of URP-2

[0095] HPLC analysis of the homogeneous molecular weight revealed that URP-2 had a weight-average molecular weight of 2156.845 kDa. Figure 1 .

[0096] 3.2 Monosaccharide composition analysis of URP-2

[0097] After HPLC monosaccharide composition analysis, the molar ratios of monosaccharides in URP-2 are as follows: the molar percentages of arabinose (Ara), rhamnose (Rha), galactose (Gal), glucose (Glc), and galacturonic acid (Gla) are 14.11%, 12.64%, 6.48%, 5.00%, and 61.77%, respectively.

[0098] 3.3 FT-IR functional group analysis of URP-2

[0099] URP-2 infrared analysis showed that at 3412.90 cm⁻¹... -1 There is a distinct strong absorption peak at 2960.68 cm⁻¹, corresponding to the OH stretching vibration. -1 and 2832.44cm -1These two peaks are located in the CH stretching vibration region, indicating the presence of saturated alkyl groups in URP-2. (At 1606.41 cm⁻¹) -1 This corresponds to a relatively strong stretching vibration at C=O. 1367.76 cm -1 The absorption peak at 1068.37 cm⁻¹ corresponds to the CO bending vibration of the methyl group. -1 The absorption peak is the bending angular vibration absorption peak of -OH, and all of the above are characteristic peaks of polysaccharides. In addition, at 773.80 cm⁻¹... -1 The absorption peak at [location] indicates that the glycosidic bonds in the URP-2 sugar chain are in the β-configuration. (See [reference]) Figure 3 .

[0100] 3.4URP-2 significantly inhibited the proliferation of TE-1 cells.

[0101] Microscopic observation revealed abnormal morphology in TE-1 cells co-cultured with URP-2, exhibiting shrinkage and fragmentation. Furthermore, as the URP-2 concentration increased from 200 μg / mL to 800 μg / mL, the cell density significantly decreased; the higher the concentration, the more pronounced the morphological changes. Figure 4 The results showed that URP-2 could effectively inhibit the in vitro proliferation of esophageal cancer TE-1 cells and induce abnormal morphology in TE-1 cells.

[0102] The inhibitory effect of URP-2 on the proliferation of esophageal cancer TE-1 cells was quantitatively analyzed using the CCK-8 assay. The results showed that the inhibitory effect of URP-2 on TE-1 cell proliferation was time- and concentration-dependent. (See attached image) Figure 5 When the concentration of URP-2 was 800 μg / mL, after 48 h of treatment, the maximum inhibition rate of TE-1 cell proliferation was 63.15 ± 2.76%, which was comparable to the 63.23 ± 3.75% of the positive control drug doxorubicin hydrochloride (DOX).

[0103] 3.5URP-2 induces abnormal nuclear morphology and apoptosis in TE-1 cells.

[0104] To verify the effect of URP-2 on apoptosis in esophageal cancer TE-1 cells, TE-1 cells were treated with 200, 400, and 800 μg / mL URP-2, respectively. After 48 h, nuclear fluorescence staining with DAPI was used. The study revealed changes in the nuclear morphology of TE-1 cells after URP-2 administration, including chromatin aggregation, nuclear pyknosis, and the appearance of bright blue apoptotic bodies. The number of apoptotic bodies in the experimental groups was significantly higher than that in the control group, and this number increased with increasing drug concentration. Figure 6 .

[0105] Subsequently, flow cytometry was used to quantitatively analyze the effect of URP-2 (800 μg / mL) on apoptosis in esophageal cancer TE-1 cells. The results showed that compared with the control group, the number of viable cells decreased significantly from 85.32% to 83.00%; the apoptosis rate changed significantly (P<0.05), and the proportion of dead cells increased from 9.76% to 14.66% (P<0.05); the total apoptosis rate of URP-2 cells was higher than that of the control group, reaching 17.00%. Figure 7 The above-mentioned nuclear DAPI fluorescence staining and flow cytometry results indicate that URP-2 can significantly induce apoptosis in TE-1 cells.

[0106] 3.6URP-2 inhibits the migration and invasion of esophageal cancer TE-1 cells.

[0107] Furthermore, the effects of URP-2 on the migration and invasion abilities of esophageal cancer TE-1 cells were investigated using a scratch healing assay and a transwell assay. The highest concentration of URP-2, 800 μg / mL, was used in the study. After 48 hours of administration, the area of ​​the scratch-healed region and the number of TE-1 cells that migrated across the membrane were observed. Figure 8 As shown in Table A and Table 1, after 48 hours, the cell migration area of ​​the control group towards the scratched area decreased from 6.92 ± 1.34 mm². 2 Reduced to 3.87±1.69mm 2 In contrast, no cell migration was observed in the scratched area of ​​the URP-2 group, and the scratched area increased from 5.16 ± 1.57 mm². 2 Increased to 7.93±1.48mm 2 (p<0.05) indicates that URP-2 significantly inhibits the migration ability of TE-1 esophageal cancer cells.

[0108] Transwell assays revealed that URP-2 treatment for 48 hours significantly inhibited the invasive ability of TE-1 esophageal cancer cells, resulting in a significant reduction in the number of cells that invasive across the membrane. The number of cells that invasive across the membrane in the control group and the URP-2 group were 27.67±2.33 and 11.33±1.67, respectively (p<0.01). Figure 8 See Table B and Table 2.

[0109] Table 2. Effects of URP-2 on the migration and invasion ability of esophageal cancer TE-1 cells (48h)

[0110]

[0111] Note: Mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0112] 3.7 URP-2 inhibits the expression of matrix metalloproteinase family proteins in esophageal cancer TE-1 cells.

[0113] Immunofluorescence assays were used to identify the effect of URP-2 on protein expression in esophageal cancer TE-1 cells. The results are as follows: Figure 9 As shown in the figure. After 48 hours, compared with the control group, the expression of MMP-2 and MMP-9 in the URP-2 treated group was significantly reduced, indicating that URP-2 inhibits the expression of matrix metalloproteinase family proteins in esophageal cancer TE-1 cells, thereby inhibiting cell migration and invasion.

[0114] 3.8 In vivo validation of URP-2 function

[0115] An esophageal cancer TE-1 tumor-bearing mouse model was established. The mice were treated with URP-2 orally continuously, and their survival status, body weight, food intake, tumor size, and blood flow around the tumor were monitored. Results are as follows: Figure 10 As shown, after 4 weeks of treatment, mice in the control group were administered physiological saline by gavage, and the tumor volume of these mice increased significantly, from 68.359 mm. 2 Increased to 76.054mm 2 In contrast, the tumor volume in mice treated with URP-2 was significantly reduced, from 56.854 mm. 2 Reduced to 49.159mm 2 In contrast, the positive control group (cisplatin) showed a tumor volume in mice that was 58.871 mm². 2 Reduced to 52.371mm 2 Meanwhile, the blood supply around the tumor changed significantly. After 4 weeks of treatment, laser speckle blood flow imaging showed that the blood supply around the tumor in the control group mice increased from 201.13±11.24 to 243.36±21.46, while that in the URP-2 group decreased from 183.02±21.41 to 135.69±11.36. Figure 11 As shown in the figure. In summary, URP-2 exhibits superior tumor-suppressive effects compared to the positive control drug cisplatin. URP-2 significantly inhibits the proliferation of esophageal cancer TE-1 cells in tumor-bearing mice.

[0116] Meanwhile, observation of the mice's survival status revealed that the average body weight and food and water intake of mice in the control and cisplatin groups decreased as the tumor progressed, while all indicators of the mice in the URP-2 administration group increased. These results indicate that URP-2 not only inhibits tumor growth in tumor-bearing mice but also effectively improves their survival status, demonstrating significant therapeutic effects and improving their quality of life. Figure 12 .

[0117] 3.9 URP-2 affects protein expression in tumor tissues of esophageal cancer-bearing mice

[0118] Through the interaction between URP-2 and TE-1 cells, this study found that URP-2 can induce apoptosis in TE-1 cells, possibly through the regulation of multiple genes and proteins. Therefore, we used Q-PCR and Western blot to detect the expression levels of tumor-related genes and proteins to elucidate the molecular mechanism of URP-2's anti-esophageal cancer effect. First, we detected the gene expression of apoptosis-related genes Bax, Bcl-2, MMP-2, and MMP-9, and the results are shown in Table 3.

[0119] Table 3. Effects of URP-2 on the expression of related genes in tumor tissues of TE-1 esophageal cancer-bearing mice.

[0120] Gene control group URP-2 group Bcl-2 1 0.43±0.12* Bax 1 3.31±0.26* MMP-2 1 0.46±0.28* MMP-9 1 0.36±0.14*

[0121] Note: Mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0122] The above results indicate that URP-2 inhibits tumor development and progression by intervening in mitochondrial apoptosis in tumors of tumor-bearing mice. Further Western blot analysis was used to verify the effects of URP-2 on the expression of apoptosis, cell migration, and invasion-related proteins in tumor tissues of tumor-bearing mice. Figure 13 And Table 4.

[0123] Table 4. Effects of URP-2 on the expression of apoptosis- and migration / invasion-related proteins in tumor tissues of TE-1 esophageal cancer-bearing mice.

[0124] protein control group URP-2 group Bcl-2 1 0.10±0.03* Bax 1 1.78±0.35* MMP-2 1 0.54±0.11* MMP-9 1 0.48±0.16*

[0125] Note: Mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0126] The above results demonstrate that the acidic polysaccharide URP-2 from Uncaria rhynchophylla exhibits significant antitumor activity against TE-1 cells, which has been validated in vivo in a tumor-bearing mouse model. Its mechanism of action primarily involves regulating the mitochondrial apoptosis pathway and inhibiting cell migration and invasion. On one hand, URP-2 upregulates the expression of the pro-apoptotic factor Bax while downregulating the anti-apoptotic protein Bcl-2, thereby inducing mitochondrial apoptosis. On the other hand, URP-2 reduces the expression of matrix metalloproteinases MMP-2 and MMP-9, thereby inhibiting the migration and invasion of tumor cells and limiting the progression of esophageal cancer. This invention provides potential experimental evidence and a novel strategy for the clinical application of Uncaria rhynchophylla acidic polysaccharide URP-2 in the treatment of esophageal cancer.

[0127] 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 type of Uncaria rhynchophylla acidic polysaccharide URP-2, characterized in that, The components of URP-2 include arabinose, rhamnose, galactose, glucose, and galacturonic acid, with molar percentages of 14.11%, 12.64%, 6.48%, 5.00%, and 61.77%, respectively.

2. The Uncaria rhynchophylla acidic polysaccharide URP-2 as described in claim 1, characterized in that, The weight-average molecular weight of the URP-2 is 2156.845 kDa.

3. The Uncaria rhynchophylla acidic polysaccharide URP-2 as described in claim 1, characterized in that, The glycosidic bonds in the sugar chain of URP-2 are in the β-configuration.

4. The use of Uncaria rhynchophylla acidic polysaccharide URP-2 as described in claim 1 in the preparation of anti-esophageal cancer drugs.

5. A drug for treating esophageal cancer, characterized in that, Its main active ingredient is the Uncaria rhynchophylla acidic polysaccharide URP-2 as described in claim 1.

6. A method for preparing the Uncaria rhynchophylla acidic polysaccharide URP-2 according to claim 1, characterized in that, Includes the following steps: (1) A crude polysaccharide extract was obtained by boiling water extraction and ethanol precipitation; (2) The crude polysaccharide extract was purified by ion exchange chromatography. First, it was eluted with water, and then eluted with 0.1M and 0.3M NaCl solutions respectively. The 0.3M NaCl solution eluent was collected. Then, it was further purified by dextran gel chromatography. It was eluted with water, the eluent was collected, and lyophilized to obtain Uncaria rhynchophylla acidic polysaccharide URP-2.

7. The preparation method according to claim 6, characterized in that, In step (1), the boiling water extraction and ethanol precipitation include the following steps: The Uncaria rhynchophylla sample and ethanol were mixed and extracted at a material-to-liquid ratio of 1g:(8-10)mL, centrifuged and the precipitate was collected; the precipitate and water were mixed at a material-to-liquid ratio of 1g:(15-20)mL and extracted in a boiling water bath for 3-5 hours, centrifuged to obtain the supernatant extract and precipitate residue, water was added to the precipitate residue and the extraction was repeated once, centrifuged and the supernatants obtained from the two extractions were combined. After concentrating the combined supernatant extract to 1 / 10 of its original volume, 3-5 times its volume of ethanol is added for precipitation. The precipitate is collected by centrifugation and dried to obtain the crude polysaccharide extract.

8. The preparation method according to claim 6, characterized in that, In step (2), before the crude polysaccharide extract is purified by ion exchange chromatography, impurity removal is performed, which 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.

9. The preparation method according to claim 8, 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. And / or, 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, and the dialysis conditions are dialysis with a 3000 Da dialysis bag for 24-48 hours.

10. The preparation method according to claim 6, characterized in that, In step (2), the ion exchange chromatography uses DEAE DE-52 cellulose as the chromatography medium, and the dextran gel chromatography uses dextran gel G200 as the chromatography medium.

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