Application of Uncariae Ramulus Crude Polysaccharide-2 in preparation of anti-esophageal cancer drugs

Abstract

The application discloses application of Uncariae Ramulus Cruda acidic polysaccharide URP-2 in preparation of anti-esophageal cancer drugs and belongs to the technical field of medicinal plant polysaccharide anti-tumor. The application adopts the method of boiling water extraction and ethanol precipitation of Uncariae Ramulus Cruda total polysaccharide, and combines DEAE DE-52 cellulose column and dextran gel column purification technology to realize separation of Uncariae Ramculus Cruda acidic polysaccharide components and obtain Uncariae Ramulus Cruda acidic polysaccharide URP-2. The components of the Uncariae Ramulus Cruda acidic polysaccharide URP-2 include arabinose, rhamnose, galactose, glucose and galacturonic acid, and the molar percentages are 14.11%, 12.64%, 6.48%, 5.00% and 61.77% respectively. The experiment finds that the Uncariae Ramulus Cruda acidic polysaccharide URP-2 has significant anti-esophageal cancer activity, which provides potential experimental basis and new strategy for application of the Uncariae Ramulus Cruda acidic polysaccharide URP-2 in clinical treatment of esophageal cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polysaccharides of medicinal plants against tumors, in particular to the application of Uncaria rhynchophylla acidic polysaccharide URP-2 in the preparation of an anti-esophageal cancer drug. BACKGROUND

[0002] Uncaria rhynchophylla is sweet in taste and cool in nature, and is attributed to the meridians of the liver and pericardium. It has the effects of clearing heat and calming the liver, and calming the wind and relieving convulsions. It is a good medicine for treating internal liver wind, convulsive seizures, and is often used for treating headache and dizziness caused by liver yang hyperactivity, delirium caused by high fever, infantile convulsions, and the like. Modern pharmacological studies have shown that alkaloids contained in Uncaria rhynchophylla, such as rhynchophylline, have significant antihypertensive, sedative and anticonvulsant effects, and have good auxiliary therapeutic effects on hypertension, cerebrovascular diseases and nervous system diseases.

[0003] The anti-tumor research of Uncaria rhynchophylla mainly focuses on the various indole alkaloids rich in Uncaria rhynchophylla, among which rhynchophylline and isorhynchophylline are the two active ingredients with the highest content and the most extensive research. In addition, corynoxeine and isocorynoxeine also have significant biological activities. However, further research is still needed on the in-depth analysis of the pharmacodynamic material basis, the development of the pharmacological activities of other active ingredients, the accurate elucidation of the action mechanism of the active ingredients, and the transformation of clinical application. In particular, there is little research on macromolecular active substances in Uncaria rhynchophylla.

[0004] Esophageal cancer is a malignant tumor originating from the esophageal mucosa, and is a common digestive tract cancer in clinical practice. Its incidence has significant regional characteristics, and the incidence in men is higher than that in women, and it mainly occurs in the elderly. Esophageal cancer is mainly divided into esophageal squamous cell carcinoma and esophageal adenocarcinoma. The early symptoms of the disease are often not obvious and are easily ignored, and the typical performance is progressive dysphagia, that is, initially difficult to swallow solid food, and gradually develops to be unable to swallow even semi-liquid and liquid, accompanied by chest pain, weight loss, hoarseness, and the like. The treatment method depends on the stage and location of the cancer, and mainly includes surgical resection, radiotherapy and chemotherapy. Early detection has a good prognosis, but most patients are diagnosed as being in the middle and late stages, and the overall five-year survival rate is still low. Therefore, early endoscopic screening for high-risk groups, changing bad living habits, and actively treating precancerous lesions are the keys to prevention.

[0005] The research of active ingredients of traditional Chinese medicine in the field of esophageal cancer treatment shows unique advantages of multiple pathways and multiple targets, and its mechanism is complex and comprehensive. A large number of studies have shown that these ingredients can effectively inhibit the proliferation of esophageal cancer cells and induce apoptosis, mainly by interfering with the cell cycle process, activating the mitochondrial or death receptor apoptosis signaling pathway. In terms of inhibiting tumor invasion and metastasis, traditional Chinese medicine ingredients can significantly reduce the migration and infiltration ability of cancer cells by down-regulating the expression of matrix metalloproteinases (MMPs) and inhibiting the epithelial-mesenchymal transition (EMT) process. At the same time, many ingredients also show anti-angiogenic activity by inhibiting related factors such as vascular endothelial growth factor (VEGF), cutting off the nutrient supply of tumors. It is worth noting that some active ingredients such as curcumin and baicalein have been confirmed to be able to reverse the multi-drug resistance of tumors, inhibit the function of drug efflux pumps such as P-glycoprotein, and increase the intracellular concentration of conventional chemotherapy drugs. In addition, traditional Chinese medicine ingredients also have a significant effect on immune regulation and "synergistic and attenuated" effects, which can not only enhance the body's anti-tumor immunity, but also reduce the adverse reactions such as myelosuppression caused by radiotherapy and chemotherapy.

[0006] The representative active ingredients of traditional Chinese medicine for esophageal cancer research at present include curcumin extracted from turmeric, which has a wide inhibitory effect on esophageal squamous cell carcinoma cells; baicalein and wogonin derived from scutellaria, which show outstanding performance in inducing apoptosis and inhibiting metastasis; allicin and diallyl disulfide in garlic can exert anti-tumor effects by causing cell cycle arrest and promoting the generation of reactive oxygen species; quercetin widely exists in fruits and vegetables, which can inhibit the growth of cancer cells and induce autophagy and apoptosis; ginsenosides Rh2, Rg3 and other monomers have significant effects on anti-angiogenesis; and traditional mineral medicine realgar (mainly containing tetraarsenic tetrasulfide) has been confirmed by modern research to have a clear effect on inducing apoptosis of esophageal cancer cells. However, the research on macromolecules in traditional Chinese medicine for anti-tumor of esophageal cancer has not been reported. Overall, the research on most active ingredients of traditional Chinese medicine for anti-esophageal cancer is still in the basic research stage, but its characteristics of "multiple targets and low toxicity" give it great development prospects. The future research direction focuses on developing these monomer ingredients into new anti-tumor drugs, or as auxiliary sensitizers and attenuators of chemotherapy / targeted therapy, providing more effective and individualized treatment options for esophageal cancer patients through the mode of traditional Chinese and Western medicine combination. SUMMARY

[0007] The purpose of the present application is to provide the application of Uncariae Ramulus Crude Polysaccharide-2 (URP-2) in the preparation of anti-esophageal cancer drugs, to solve the problems existing in the prior art, and URP-2 has significant inhibitory effect on the proliferation of esophageal cancer cells, induces cell morphological abnormalities and apoptosis, and inhibits cell migration and invasion, showing significant anti-esophageal cancer activity, providing potential experimental basis and new strategies for the application of URP-2 in the clinical treatment of esophageal cancer.

[0008] To achieve the above object, the present application provides the following scheme:

[0009] The present application provides a Uncariae Ramulus Cruda acidic polysaccharide URP-2, the components of which include arabinose, rhamnose, galactose, glucose and galacturonic acid, the molar percentages of which are 14.11%, 12.64%, 6.48%, 5.00% and 61.77% respectively.

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

[0011] Preferably, the glycosidic bond existing in the sugar chain of the URP-2 is in β-configuration.

[0012] The present application also provides the use of the Uncariae Ramulus Cruda acidic polysaccharide URP-2 in the preparation of an anti-esophageal cancer drug.

[0013] The present application also provides a drug for treating esophageal cancer, the main effective active ingredient of which is the Uncariae Ramulus Cruda acidic polysaccharide URP-2.

[0014] The present application also provides a preparation method of the Uncariae Ramulus Cruda acidic polysaccharide URP-2, comprising the following steps:

[0015] (1) obtaining a crude polysaccharide extract by using boiling water extraction and ethanol precipitation;

[0016] (2) purifying the crude polysaccharide extract by ion exchange chromatography, first eluting with water, then eluting with 0.1M and 0.3M NaCl solutions respectively, and collecting the eluent of the 0.3M NaCl solution; then further purifying by using dextran gel chromatography, eluting with water, collecting the eluent, and freeze-drying to obtain the Uncariae Ramulus Cruda acidic polysaccharide URP-2.

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

[0018] mixing Uncariae Ramulus Cruda sample and ethanol according to a solid-liquid ratio of 1g:(8-10)mL, centrifuging and collecting the precipitate; mixing the precipitate and water according to a solid-liquid ratio of 1g:(15-20)mL, extracting in a boiling water bath for 3-5h, centrifuging to obtain supernatant extract and precipitate residue, adding water to the precipitate residue to repeat the extraction once, and centrifuging to combine the supernatant extract obtained in the two times;

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

[0020] Preferably, in step (2), before the crude polysaccharide extract is purified by ion exchange chromatography, the crude polysaccharide extract is subjected to impurity removal, which comprises the following steps:

[0021] S1: after the crude polysaccharide extract is dissolved in water, a protease is added for enzymolysis, centrifuged, and the supernatant is collected;

[0022] S2: chloroform and n-butanol are added to the supernatant, mixed thoroughly, and the upper aqueous phase is collected;

[0023] S3: petroleum ether is added to the upper aqueous phase, mixed thoroughly, and the lower aqueous phase is collected;

[0024] S4: macroporous adsorption resin is added to the lower aqueous phase, mixed thoroughly for adsorption, the liquid is collected, dialyzed, precipitated with ethanol, and the precipitate is dried.

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

[0026] and / or, in step S2, 1 / 3-1 / 4 volume of chloroform and n-butanol is added to the supernatant, and the volume ratio of chloroform to n-butanol is 4:1;

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

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

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

[0030] The present application has the following technical effects:

[0031] The present application uses boiling water extraction and ethanol precipitation of total polysaccharides from Uncaria, and combines DEAE DE-52 cellulose column and dextran gel column purification technology to realize the separation of Uncaria acidic polysaccharide components, solving the technical problem of polysaccharide component extraction and separation.

[0032] The present application adopts monosaccharide composition analysis, molecular weight detection, Fourier transform infrared spectrum analysis and the like methods to determine the chemical composition and accurate structure characteristics of Uncariae Ramulus Proximalis Polysaccharide-2 (URP-2). The monosaccharide composition of URP-2 is arabinose, rhamnose, galactose, glucose and galacturonic acid, and the molar percentage is 14.11%, 12.64%, 6.48%, 5.00% and 61.77% respectively. The anti-esophageal cancer effect of URP-2 is evaluated by indexes such as cell proliferation, cell nucleus morphology, cell apoptosis and tumor growth of tumor-bearing mice. The results show that URP-2 significantly inhibits the proliferation of TE-1 cells, induces the abnormal morphology of TE-1 cell nucleus and cell apoptosis, and inhibits the migration and invasion ability of TE-1 cells, and shows significant anti-tumor activity. The present application also verifies the anti-tumor mechanism of URP-2 through in-vivo experiments. The anti-tumor mechanism of URP-2 mainly involves regulating the cell mitochondrial apoptosis pathway and inhibiting cell migration and invasion. On the one hand, URP-2 can up-regulate the expression of pro-apoptotic factor Bax, and down-regulate the expression of anti-apoptotic protein Bcl-2, thereby inducing the cell apoptosis of 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 ability of tumor cells and limiting the progression of esophageal cancer. The present application provides potential experimental basis and new strategies for the application of Uncariae Ramulus Proximalis Polysaccharide-2 (URP-2) in the clinical treatment of esophageal cancer. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below only show some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0034] Figure 1 the absolute molecular weight analysis diagram of URP-2;

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

[0036] Figure 3 the FT-IR detection diagram of URP-2;

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

[0038] Figure 5 the detection results of URP-2 with different administration time and concentration on the proliferation inhibition 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 Effect of URP-2 on the apoptosis morphology of esophageal cancer TE-1 cells (48h);

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

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

[0042] Figure 9 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, 48h);

[0043] Figure 10 Nuclear magnetic resonance tracking of the effect of URP-2 on the growth of esophageal cancer TE-1 tumor-bearing mice;

[0044] Figure 11 Laser speckle blood flow imaging system detection of the effect of URP-2 on the blood supply around esophageal cancer TE-1 tumor-bearing mice;

[0045] Figure 12 Effect of URP-2 on the survival state of esophageal cancer TE-1 tumor-bearing mice; (A) weekly water consumption of tumor-bearing mice; (B) weekly grain consumption of tumor-bearing mice; (C) body weight change of tumor-bearing mice;

[0046] Figure 13 Effect of URP-2 on the expression of mitochondrial apoptosis pathway-related proteins in tumor tissues of tumor-bearing mice. DETAILED DESCRIPTION

[0047] Various exemplary embodiments of the present application will now be described in detail with reference to the drawings. Such description, however, is to be considered in all aspects as illustrative and not restrictive, understood to be a description of certain aspects, features and embodiments of the present application.

[0048] It is to be understood that the terms used in the present application are merely used to describe particular embodiments and are not intended to limit the present application. In addition, for numerical ranges in the present application, it is to be understood that each intermediate value between the upper limit and the lower limit of the range is specifically disclosed. Each intermediate value within any stated value or stated range, and any other stated value or intermediate value within the stated range, is also included within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the ranges.

[0049] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains unless otherwise specifically defined herein. Although preferred methods and materials are described herein, any method and material similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described herein. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0050] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0051] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0052] Example 1

[0053] 1. Main experimental materials

[0054] Esophageal cancer cell line TE-1 (CL-0231, Pons Life Science Co., Ltd.); Fetal bovine serum (164210, Pons Life Science Co., Ltd.); DAPI fluorescent staining solution (KGA1808-50, Jiangsu KeyGen Biotech Co., Ltd., China); Annexin V-FITC / PI double staining cell apoptosis detection kit (KGA1102-100, Jiangsu KeyGen Biotech Co., Ltd., China); β-actin, MMP-2, MMP-9 antibodies (Wuhan Aibiotek Biological Technology Co., Ltd.).

[0055] 2. Experimental methods

[0056] 2.1 Extraction of Uncaria total polysaccharides

[0057] (1) The dried Uncaria sample / material was crushed with a crusher and passed through a 60-mesh sieve.

[0058] (2) Anhydrous ethanol was added (1 g: 10 mL), and the fat-soluble pigments and part of the impurities were extracted by stirring at room temperature. The precipitate was collected by centrifugation at 6000 g for 10 min.

[0059] (3) Pure water was added to the precipitate (1 g: 20 mL), and the extraction was carried out in a boiling water bath for 4 h. The supernatant extract was collected by centrifugation at 6000 g for 10 min. The precipitate residue was re-extracted according to the same procedure.

[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 blotting 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, as shown in [the following text is missing]. 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. Uncariae Ramulus Proximalis Polysaccharide-2 (URP-2) characterized in that, The components of the URP-2 include: arabinose, rhamnose, galactose, glucose and galacturonic acid, and the molar percentages are 14.11%, 12.64%, 6.48%, 5.00% and 61.77% respectively.

2. Uncaria acid polysaccharide URP-2 according to claim 1, characterized in that, The weight average molecular weight of the URP-2 is 2156.845 kDa.

3. Uncaria acid polysaccharide URP-2 according to claim 1, characterized in that, The glycosidic bond existing in the sugar chain of the URP-2 is in a beta-configuration.

4. The use of the Uncaria acidic polysaccharide URP-2 in claim 1 in the preparation of an anti-esophageal cancer drug.

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

6. A method for preparing Uncaria acid polysaccharide URP-2 according to claim 1, characterized in that, The method comprises the following steps: (1) crude polysaccharide extract is obtained by boiling water extraction and ethanol precipitation; (2) the crude polysaccharide extract is purified by ion exchange chromatography, first eluted by water, then eluted by 0.1M and 0.3M NaCl solution respectively, and the eluate of 0.3M NaCl solution is collected; then further purified by dextran gel chromatography, eluted by water, and the eluate is collected, freeze-dried to obtain the Uncaria acidic polysaccharide URP-2.

7. The production method according to claim 6, wherein In step (1), the boiling water extraction and ethanol precipitation comprise the following steps: Uncaria sample and ethanol are mixed for extraction according to the solid-liquid ratio of 1g:(8-10)mL, centrifuged and the precipitate is collected; the precipitate and water are mixed according to the solid-liquid ratio of 1g:(15-20)mL, extracted in a boiling water bath for 3-5h, centrifuged to obtain supernatant extract and precipitate residue, and the precipitate residue is added with water for repeated extraction once, and after centrifugation, the supernatant extract obtained in the two times is combined; The combined supernatant extract is concentrated to 1 / 10 of the original volume, then 3-5 times volume of ethanol is added for precipitation, centrifuged to collect the precipitate, and dried to obtain the crude polysaccharide extract.

8. The production method according to claim 6, wherein In step (2), before the crude polysaccharide extract is purified by ion exchange chromatography, impurities are removed, and the removing of impurities comprises the following steps: S1: the crude polysaccharide extract is dissolved in water, then protease is added for enzymolysis, centrifuged, and the supernatant is collected; S2: toluene and n-butanol are added to the supernatant, mixed thoroughly, and the upper aqueous phase is collected; S3: petroleum ether is added to the upper aqueous phase, mixed thoroughly, and the lower aqueous phase is collected; S4: macroporous adsorption resin is added to the lower aqueous phase, mixed thoroughly for adsorption, the liquid is collected, dialyzed, then precipitated with ethanol, and the precipitate is dried.

9. The production method according to claim 8, wherein In step S1, the amount of water is 600mL-1000mL, and the amount of protease is 0.4-0.6g; In step S2, 1 / 3-1 / 4 volume of toluene and n-butanol is added to the supernatant, and the volume ratio of toluene to n-butanol is 4:1; In step S3, 1 / 3-1 / 4 volume of petroleum ether is added to the upper aqueous phase; In step S4, 1 / 2-1 / 3 volume of macroporous adsorption resin is added to the lower aqueous phase, and the dialysis condition is 3000Da dialysis bag for 24-48h.

10. The production method according to claim 6, wherein In step (2), the chromatography medium used in the ion exchange chromatography is DEAE DE-52 cellulose, and the chromatography medium used in the dextran gel chromatography is dextran gel G200.

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