Application of uncaria acidic polysaccharide URP-1 in preparation of anti-prostatic cancer drugs

Abstract

The invention discloses application of uncaria acidic polysaccharide URP-1 in preparation of anti-prostatic cancer drugs, and belongs to the technical field of antitumor of medicinal plant polysaccharides. The preparation method comprises the following steps: obtaining uncaria total polysaccharide from uncaria through boiling water extraction and ethanol precipitation methods, and then purifying the total polysaccharide through DEAE cellulose and sephadex G200 to obtain the uncaria acidic polysaccharide URP-1. The URP-1 comprises the following components in mole percent: 28.68% of arabinose, 11.08% of rhamnose, 29.55% of galactose, 19.00% of glucose, 4.47% of mannose and 7.22% of galacturonic acid. Experiments discover that the uncaria acidic polysaccharide URP-1 has remarkable anti-tumor activity on DU 145 cells, and a novel medicine raw material and a novel treatment method are provided for clinical treatment of prostatic 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-1 in the preparation of antiprostate cancer drugs. Background Technology

[0002] Uncaria rhynchophylla, also known as double hook vine or eagle claw wind, is the dried hooked stem of the Uncaria rhynchophylla plant, belonging to the Rubiaceae family. It is an important traditional Chinese medicine used to calm the liver and extinguish wind. It is slightly cold in nature, sweet in taste, and enters the liver and pericardium meridians. The most distinctive feature of Uncaria rhynchophylla is the curved hooked thorns on its branches, which is the origin of its name and the main medicinal part. Current research mainly focuses on alkaloids (rhynchophylline and isorhynchophylline), flavonoids, and triterpenoids. Research on its macromolecular active substances is relatively limited.

[0003] Traditional Chinese medicine believes that Uncaria rhynchophylla has the effects of clearing heat and calming the liver, as well as relieving wind and calming convulsions. Modern pharmacological studies have found that the core biological activities of Uncaria rhynchophylla are concentrated in two major areas: the cardiovascular system (especially antihypertensive effects) and the nervous system (sedation, anticonvulsant effects, and neuroprotective effects). Its powerful anti-inflammatory and antioxidant effects are the important foundation supporting these core activities. These activities are mainly attributed to its rich indole alkaloid components, especially rhynchophylline and isorhynchophylline. The study of the biological activities of Uncaria rhynchophylla provides modern scientific evidence for understanding its traditional effects (such as clearing heat and calming the liver, relieving wind and calming convulsions) and demonstrates its potential application value in the prevention and treatment of hypertension, stroke, epilepsy, neurodegenerative diseases, and inflammatory diseases. However, other active components in Uncaria rhynchophylla have not been fully explored, especially polysaccharide active components, which have not been reported. Polysaccharides are a class of medicinal plants with prominent active effects, mainly focusing on the mechanisms of regulating immunity and directly killing tumor cells. Currently, no research has been reported on Uncaria rhynchophylla polysaccharides, and the mechanisms and pathways of the inhibitory effects of Uncaria rhynchophylla acidic polysaccharide components on tumors have not been mentioned. Therefore, further research is needed to thoroughly analyze the pharmacological basis of Uncaria rhynchophylla, develop the pharmacological activities of other active ingredients, accurately elucidate the mechanisms of action of active ingredients, and translate them into clinical applications.

[0004] Prostate cancer is a malignant tumor originating from the glandular epithelium of the male prostate gland. It ranks second in incidence among male malignant tumors worldwide (after lung cancer), and is most common in men over 65 years of age. Pathologically, adenocarcinoma is the predominant type (accounting for over 95%), with acinar adenocarcinoma being the most common, while ductal adenocarcinoma is more aggressive. Advanced metastatic patients mainly present with bone pain (characteristic osteoblastic metastasis), anemia, and pathological fractures. Prognosis is closely related to stage: the 5-year survival rate for localized patients exceeds 99%, dropping to approximately 35% after metastasis. Therefore, inhibiting prostate cancer metastasis is an important target for basic pharmacodynamic research. Currently, there are no reports on the application of Uncaria rhynchophylla polysaccharide in inhibiting prostate cancer. Summary of the Invention

[0005] The purpose of this invention is to provide the application of Uncaria rhynchophylla acidic polysaccharide URP-1 in the preparation of anti-prostate cancer drugs, in order to solve the problems existing in the prior art. Total polysaccharides from Uncaria rhynchophylla are obtained by boiling water extraction and ethanol precipitation, and then purified using a DEAE cellulose column and a molecular sieve dextran gel column to obtain Uncaria rhynchophylla acidic polysaccharide URP-1. This Uncaria rhynchophylla acidic polysaccharide URP-1 has significant anti-prostate cancer activity, providing a new treatment method for the clinical treatment of prostate cancer.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides an acidic polysaccharide URP-1 from Uncaria rhynchophylla, wherein the components of URP-1 include arabinose, rhamnose, galactose, glucose, mannose and galacturonic acid, with molar percentages of 28.68%, 11.08%, 29.55%, 19.00%, 4.47% and 7.22%, respectively.

[0008] This invention also provides a method for preparing the aforementioned Uncaria rhynchophylla acidic polysaccharide URP-1, comprising the following steps:

[0009] (1) Extract the Uncaria rhynchophylla sample with ethanol to remove impurities, centrifuge and collect the precipitate, then extract the precipitate with boiling water and precipitate with ethanol to obtain crude polysaccharide extract.

[0010] (2) After removing impurities from the crude polysaccharide extract, it was purified by ion exchange column chromatography and dextran gel column chromatography. The liquid passed through the column was collected, freeze-dried, and the Uncaria rhynchophylla acidic polysaccharide URP-1 was obtained.

[0011] Preferably, in step (1), the ratio of the Uncaria rhynchophylla sample to the ethanol is 1g:(8-10)mL.

[0012] Preferably, in step (1), the boiling water extraction and ethanol precipitation include the following steps:

[0013] The precipitate and water were extracted in a boiling water bath for 3-5 hours at a material-to-liquid ratio of 1g:(15-20)mL. After centrifugation, the supernatant extract and precipitate residue were obtained. Water was added to the precipitate residue and the extraction was repeated once. After centrifugation, the supernatants obtained from the two extractions were combined.

[0014] 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.

[0015] Preferably, in step (2), the impurity removal of the crude polysaccharide extract includes the following steps:

[0016] S1: Dissolve the crude polysaccharide extract in water, add protease for enzymatic hydrolysis, centrifuge, and collect the supernatant;

[0017] S2: Add chloroform and n-butanol to the supernatant, mix thoroughly, and collect the upper aqueous phase;

[0018] S3: Add petroleum ether to the upper aqueous phase, mix thoroughly, and collect the lower aqueous phase;

[0019] 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.

[0020] Preferably, in step S1, the amount of water used is 600mL-1000mL, and the amount of protease used is 0.4-0.6g.

[0021] 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.

[0022] And / or in step S3, 1 / 3 to 1 / 4 volume of petroleum ether is added to the upper aqueous phase;

[0023] 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.

[0024] Preferably, the purification method includes: purifying the crude polysaccharide extract after impurity removal using DEAE DE-52 cellulose, first eluting with water, then eluting with 0.1M NaCl; and then purifying with dextran gel G200, with water as the eluent.

[0025] The present invention also provides the application of the aforementioned Uncaria rhynchophylla acidic polysaccharide URP-1 in the preparation of an anti-prostate cancer drug.

[0026] The present invention discloses the following technical effects:

[0027] This invention employs hot water extraction and ethanol precipitation to separate and purify Uncaria rhynchophylla acidic polysaccharide (URP-1) using DEAE cellulose column chromatography and dextran gel electrophoresis. Experiments revealed that URP-1 exhibits significant antitumor activity against DU 145 cells. In vivo activity verification in tumor-bearing mice showed that URP-1 primarily regulates the expression of key genes and proteins involved in mitochondrial apoptosis and cell migration / invasion pathways, increasing the expression of pro-apoptotic genes and proteins like Bax and downregulating the expression of the apoptosis-inhibiting protein Bcl-2, ultimately leading to mitochondrial apoptosis and programmed cell death in tumor cells. Furthermore, by downregulating the expression of matrix metalloproteinase family members MMP-2 and MMP-9, it inhibits cell migration and invasion, thus limiting the development of prostate cancer. This invention isolates and extracts Uncaria rhynchophylla acidic polysaccharide and clarifies the mechanism of action of URP-1 in inhibiting the development and progression of prostate cancer, providing a novel pharmaceutical raw material and treatment method for the clinical treatment of prostate cancer. Attached Figure Description

[0028] 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.

[0029] Figure 1 Here is the molecular configuration diagram of URP-1;

[0030] Figure 2 This is a chromatogram showing the absolute molecular weight analysis of URP-1.

[0031] Figure 3 The diagram shows the monosaccharide composition analysis of URP-1;

[0032] Figure 4 The image shows the FT-IR detection pattern of URP-1.

[0033] Figure 5 The effect of URP-1 on the morphology of prostate cancer DU145 cells (48h);

[0034] Figure 6 Results of the inhibition of prostate cancer DU145 cell proliferation by URP-1 at different administration times and concentrations; mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group;

[0035] Figure 7 The effect of URP-1 on the nuclear apoptosis morphology of prostate cancer DU145 cells (48h);

[0036] Figure 8 To analyze the effect of URP-1 on apoptosis in prostate cancer DU145 cells by flow cytometry (48 h);

[0037] Figure 9 The effect of URP-1 on the migration (A) and invasion (B) abilities of prostate cancer DU 145 cells (800 μg / mL, 48 h);

[0038] Figure 10 Effects of URP-1 on the expression of matrix metalloproteinase family members MMP-2 and MMP-9 in prostate cancer DU 145 cells (800 μg / mL, 48 h);

[0039] Figure 11 To track the effect of URP-1 on tumor growth in DU 145 prostate cancer-bearing mice using nuclear magnetic resonance imaging;

[0040] Figure 12 The effect of URP-1 on the survival status of DU 145 prostate 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;

[0041] Figure 13 The effect of URP-1 on the expression of proteins related to the mitochondrial apoptosis pathway in tumor tissues of tumor-bearing mice. Detailed Implementation

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] The following examples involve the main experimental materials:

[0048] Prostate cancer cell line DU 145 (CL-0075, 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.).

[0049] Example 1: Extraction, separation, and structural identification of acidic polysaccharides from Uncaria rhynchophylla.

[0050] 1. Extraction and separation method of acidic polysaccharides from Uncaria rhynchophylla

[0051] 1.1 Extraction of total polysaccharides from Uncaria rhynchophylla

[0052] (1) Crush the dried Uncaria rhynchophylla sample / raw material with a pulverizer and pass it through a 60-mesh sieve.

[0053] (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.

[0054] (3) Add pure water to the precipitate (solid-to-liquid ratio 1:20), 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.

[0055] (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.

[0056] (5) Centrifuge at 8000g for 10min, collect the precipitate solid and dry it to obtain crude polysaccharide extract.

[0057] (6) The purity of crude polysaccharide was determined by the sulfuric acid-phenol method. The specific operation was as follows: Weigh about 25 mg of crude polysaccharide extract solid, dissolve and dilute it with water, take 100 μL of polysaccharide supernatant, add 600 μL of sulfuric acid-phenol reagent (5% phenol solution: concentrated sulfuric acid = 1:5 (v / v)), mix well, let it stand for 10 min in the dark, and measure the absorbance at 490 nm.

[0058] 1.2 Removal of impurities from total polysaccharides

[0059] This process includes protein removal, fat removal, and decolorization. The purity of the crude polysaccharide is then determined using the sulfuric acid-phenol method. Detailed steps are as follows:

[0060] (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.

[0061] (2) After centrifuging the enzyme hydrolysate, 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.

[0062] (3) Add 1 / 4 volume of petroleum ether to the upper aqueous phase, mix thoroughly, and collect the lower aqueous phase.

[0063] (4) Add 1 / 2 volume of macroporous resin AB-8 to the lower aqueous phase, mix thoroughly, and allow to adsorb overnight.

[0064] (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.

[0065] 1.3 Preparation method of Uncaria rhynchophylla acidic polysaccharide (URP-1)

[0066] URP was separated and purified by molecular sieves using ion exchange column chromatography and dextran gel column chromatography. Specifically, DEAE DE-52 cellulose was used for purification, first eluted with water, then with 0.1M NaCl; then purified by dextran gel G200, with water as the eluent. The column chromatography solution was collected, lyophilized, and stored to obtain URP-1. The flow rate for the purification process was set to 1 mL / min.

[0067] 2. Structural characteristics and identification of Uncaria rhynchophylla acidic polysaccharide (URP-1)

[0068] 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.

[0069] Secondly, infrared spectroscopy was used to detect the main functional groups present in URP-1, and the configuration of the polysaccharide was preliminarily determined, thereby understanding its structural characteristics. Further methylation was employed to detect the linkage configuration between sugar residues. Finally, the composition of Uncaria rhynchophylla acidic polysaccharide (URP-1) was confirmed by HPLC (molecular weight, homogeneity, and monosaccharide composition detection) and Fourier transform infrared spectroscopy, thus gaining a deeper understanding of its biological activity and potential application value.

[0070] 3. Functional detection of Uncaria rhynchophylla acidic polysaccharide (URP-1)

[0071] 3.1 Cell lines and cell culture

[0072] DU 145 cells were cultured in MEM medium containing 100 μg / mL streptomycin and 100 μg / mL penicillin G, with the addition of 10% fetal bovine serum, at a constant temperature and humidity of 37°C, with the carbon dioxide concentration maintained at 5%. When the cell density reached 90%, the cells were passaged at a volume ratio of 1:2–3 and cultured continuously.

[0073] 3.2 Cell proliferation inhibition detection

[0074] DU 145 cells were induced at a rate of 2 × 10⁻⁶ 3Cells were seeded at a density of 100 cells / well into 96-well plates. After 24 hours of culture, the original culture medium was replaced with a medium containing different concentrations of URP-1 (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-1 at a concentration of 5 μM. DU 145 cells were co-cultured with 5 μM DOX and different concentrations of URP-1 (0, 200, 400, and 800 μg / mL). Each treatment group had 5 replicates, and the experiment was repeated at least three times with parallel and iterative assays. Cell viability was assessed using a CCK-8 cell proliferation assay kit after 24 and 48 hours of co-incubation. The CCK-8 reagent was added to the culture medium at a 1:10 ratio, and after incubation for 30 minutes, the absorbance was read at 450 nm using a microplate reader. The calculation formula is as follows:

[0075] Proliferation inhibition rate (%) = ([A) c -A s ] / [A c -A 空白 ])×100%

[0076] 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).

[0077] 3.2 DAPI Fluorescent Staining Experiment

[0078] DU 145 cells were 1×10 4 Cells were seeded at a density of 100 cells / well in culture dishes containing DMEM medium and cultured for 24 hours. Subsequently, the original medium was replaced with drug-treated medium, and the cells were co-cultured with URP-1 for 24 and 48 hours. Afterward, the cells were washed with PBS, fixed with methanol, and washed again. 80 μL of DAPI was added to each well, stained in the dark for 5 minutes, and then washed once with PBS. Finally, images were taken using a Nikon inverted fluorescence microscope (100× and 200× magnification), with 5 images retained for each drug concentration.

[0079] 3.3 Apoptosis Assay

[0080] DU 145 cells were loaded at 2 × 10⁻⁶ 5Cells were seeded at a density of 100 cells / well in culture dishes containing DMEM medium and cultured for 24 hours. Subsequently, the original medium was replaced with the drug-treated medium, and the cells were co-cultured with URP-1 for 48 hours. 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 minutes before analysis by flow cytometry. Each sample was set up in triplicate, and the experiment was repeated at least three times.

[0081] 3.4 Cell Wound Healing Experiment

[0082] Cells in the logarithmic growth phase were seeded into 6-well plates without counting and during the cell scratch healing assay. When the cell density in the 6-well plate reached more than 80%, the cells were scratched in a cross pattern using a 1 mL pipette tip, making four horizontal lines and two vertical lines in each well. After scratching, the suspended cells were washed once with basal medium, and URP-1 drug-containing medium was added to the well plate. The cells were cultured for another 48 hours, observed, and photographed for preservation.

[0083] 3.5 Transwell Chemotaxis Chamber Detection Experiment

[0084] The effect of URP-1 on the invasive ability of DU 145 cells was investigated using a Transwell chemotaxis assay. Before the experiment, Transwell chambers were prepared and sterilized with alcohol and UV light. Pipette tips and culture media were pre-cooled. After coating the upper chamber membrane with a matrigial gel, the experiment began. DU 145 cells in logarithmic growth phase were collected, and the cell concentration was adjusted. 600 μL of culture medium containing 10% newborn calf serum (NBS) and URP-1 were added to each well of a 12-well plate as the control and treatment groups, respectively. The plates were tilted to avoid membrane air bubbles. Cell suspensions containing basal culture medium and ginsenoside CK were then added to the upper chamber for the control and treatment groups, 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.

[0085] 3.6 Cell Immunofluorescence Staining

[0086] Place sterile coverslips into the culture plate and seed cells to 60-70% confluence. 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 for fixation at room temperature for 15-20 minutes (avoid over-fixation), and 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 3-wash with PBS. 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 using a confocal microscope as soon as possible.

[0087] 3.7 Construction and Treatment of Tumor-Bearing Mouse Models

[0088] DU 145 cell suspension was inoculated into the right axilla of C57BL / 6 mice, 0.2 mL per mouse, to establish a prostate cancer-bearing mouse model. The experiment was divided into two groups of 15 mice each. The control group received 0.2 mL of physiological saline per mouse via gavage; the positive control cisplatin group received cisplatin 3 mg / kg via gavage for 28 consecutive days; and the URP-1 group received cisplatin 400 mg / kg via gavage for 28 consecutive days. The mice were weighed 72 hours after the last administration and then sacrificed. After confirming death, the mice were dissected, and the tumor tissue was completely removed.

[0089] 3.8 Q-PCR detection

[0090] Total RNA was extracted from each sample and used to prepare cDNA samples. Gene expression was measured by qPCR and primers. The reaction conditions were as follows: heating at 95°C for 5 minutes; then 40 cycles were performed, with 95°C for 30 seconds, 55°C for 45 seconds, and 72°C for 30 seconds; finally, the reaction was extended to 72°C for 10 minutes.

[0091] The primers are shown in Table 1 below:

[0092] Table 1 Primer sequences

[0093]

[0094] 3.9 Cell Immunofluorescence Staining

[0095] Immunofluorescence staining was performed on prostate cancer DU 145 cells under different treatments, and the fluorescence intensity of the target protein was observed by fluorescence microscopy to evaluate the effect of URP-1 on the expression levels of MMP-2 and MMP-9 proteins in the cells.

[0096] 3.10 Western blot of proteins

[0097] Cell samples were collected from each culture group, and protein samples were extracted and analyzed using Western blotting. After electrophoresis, protein samples were transferred to polyvinylidene fluoride (PVDF) membranes for 1.5 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).

[0098] 3.11 Statistical Analysis: Each experimental design included at least three parallel experiments. All data are expressed as mean ± standard deviation. All data were analyzed using Microsoft Excel and SPSS software. One-way ANOVA and t-tests were used to assess the significance of the results, mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared to the control group.

[0099] 4. Results and Analysis

[0100] 4.1 Molecular weight determination of URP-1

[0101] Plotting log(Molar Mass) on the x-axis and log(RMS Radius) on the y-axis, the slope can be used as a reference for molecular configuration. A slope of 0.5-0.6 indicates a random coil. The detection value of URP-1 was 0.02±0.01, indicating that the molecular configuration of URP-1 is approximately spherical. Figure 1 .

[0102] Further HPLC analysis revealed that the weight-average molecular weight of URP-1 was 3962.836 kDa. Figure 2 .

[0103] 4.2 Monosaccharide composition analysis of URP-1

[0104] After HPLC monosaccharide composition analysis, the molar percentages of monosaccharides in URP-1 are as follows: arabinose, rhamnose, galactose, glucose, mannose, and galacturonic acid are 28.68%, 11.08%, 29.55%, 19.00%, 4.47%, and 7.22%, respectively. (See attached image) Figure 3 .

[0105] 4.3 FT-IR functional group analysis of URP-1

[0106] Analysis of URP-1 infrared detection results showed that at 3300-3500 cm⁻¹ -1 There are obvious strong absorption peaks on both sides, and the peak shape is relatively broad. This is due to the stretching vibration of OH, which makes it reach 3405.67 cm⁻¹. -1 There is a broad peak there. 2960.68cm -1 and 2829.54cm -1 These two peaks are located in the CH stretching vibration region, at 2960.68 cm⁻¹. -1 It is an asymmetric stretching vibration, 2829.54 cm. -1 The vibration is a symmetrical stretching vibration, indicating the presence of saturated alkyl groups in GT-1. At 1602.07 cm⁻¹ -1 The interior shows strong C=O stretching vibrations. 1365.35cm -1 The absorption peak at 1067.89 cm⁻¹ corresponds to the CO stretching vibration. -1 The absorption peak is the angular vibration absorption peak of the hydroxyl group. These are all characteristic peaks of polysaccharides. In addition, there is a peak at 773.80 cm⁻¹. -1 The absorption peak at [location] indicates that the glycosidic bonds in the URP-1 sugar chain are in the β-configuration. (See [reference]) Figure 4 .

[0107] 4.4 URP-1 significantly inhibited the proliferation of DU 145 cells.

[0108] Microscopic observation revealed abnormal morphology in DU 145 cells co-cultured with URP-1, exhibiting shrunkenness and fragmentation. Furthermore, cell density significantly decreased as the drug concentration increased from 200 μg / mL to 800 μg / mL. Figure 5 The results showed that URP-1 could effectively inhibit the in vitro proliferation of prostate cancer DU 145 cells and induce abnormal morphology in DU 145 cells.

[0109] The inhibitory effect of URP-1 on the proliferation of prostate cancer DU 145 cells was quantitatively analyzed using the CCK-8 assay. The results showed that the inhibitory effect of URP-1 on the proliferation of DU 145 cells was time- and concentration-dependent. Figure 6When the concentration of URP-1 was 800 μg / mL, after 48 h of treatment, the maximum inhibition rate of DU 145 cell proliferation was 66.19 ± 2.18%.

[0110] 4.5URP-1 induces abnormal nuclear morphology and apoptosis in DU 145 cells.

[0111] To verify the effect of URP-1 on apoptosis in prostate cancer DU 145 cells, DAPI fluorescence staining was performed using URP-1 at concentrations of 200, 400, and 800 μg / mL. After drug administration, changes in cell nuclear morphology began, with chromatin aggregation, nuclear pyknosis, and the appearance of bright blue apoptotic bodies. The number of apoptotic bodies in the experimental group was significantly higher than that in the control group, and this number increased with increasing drug concentration (see [reference needed]). Figure 7 .

[0112] Subsequently, flow cytometry was used to quantitatively analyze the effect of URP-1 on apoptosis in prostate cancer DU145 cells. The results showed that the number of viable cells decreased significantly from 88.25% to 82.45%, while the apoptosis rate changed significantly (P<0.05), and the proportion of dead cells increased from 8.9% to 13.85%. The proportion of apoptotic cells increased significantly, from 2.86% to 3.7%. Figure 8 The above-mentioned nuclear DAPI fluorescence staining and flow cytometry results showed that URP-1 could significantly induce apoptosis in DU 145 cells.

[0113] 4.6URP-1 inhibits the migration and invasion of prostate cancer DU145 cells.

[0114] Furthermore, the effects of URP-1 on the migration and invasion abilities of prostate cancer DU 145 cells were investigated using a scratch healing assay and a transwell assay. The highest concentration of URP-1, 800 μg / mL, was used in the study. After 48 hours of administration, the area of ​​the scratch-healed region and the number of DU 145 cells that migrated across the membrane were observed. Figure 9 As shown in Table A and Table 1, after 48 hours, the cell migration to the scratched area in the control group decreased from 5.32 ± 1.69 mm². 2 Reduced to 3.14±1.33mm 2 In contrast, no cell migration was observed in the scratched area of ​​the URP-1 group, and the scratched area decreased from 4.12 ± 1.13 mm². 2 Increased to 7.25±0.21mm 2 (p<0.05) indicates that URP-1 significantly inhibits the migration ability of prostate cancer cells DU 145.

[0115] Transwell assays revealed that URP-1 treatment for 48 hours significantly inhibited the invasive ability of DU145 prostate cancer cells, with a significant reduction in the number of cells that crossed the membrane. The number of cells that crossed the membrane in the control group and the URP-1 group were 25.33±2.67 and 13.67±4.33, respectively (p<0.01). Figure 9 See Table B and Table 2.

[0116] Table 2. Effects of URP-1 on the migration and invasion abilities of prostate cancer DU 145 cells (48 h)

[0117]

[0118] Note: Mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0119] 4.7 URP-1 inhibits the expression of matrix metalloproteinase family proteins in prostate cancer DU145 cells.

[0120] Immunofluorescence assays were used to identify the effect of URP-1 on protein expression in prostate cancer DU145 cells. The results are as follows: Figure 10 After 48 hours, compared with the control group, the expression of MMP-2 and MMP-9 in the URP-1 treated group was significantly reduced, indicating that URP-1 inhibits the expression of matrix metalloproteinase family proteins in prostate cancer DU145 cells, thereby inhibiting cell migration and invasion.

[0121] 4.8URP-1 inhibits the proliferation of prostate cancer DU145 cells in tumor-bearing mice.

[0122] A DU 145 prostate cancer tumor-bearing mouse model was established. Mice were treated with URP-1 orally continuously, and their survival status, body weight, food intake, and tumor size were monitored. Results are as follows: Figure 11 After 4 weeks of treatment, mice in the control group were administered physiological saline by gavage, and the tumors in these mice significantly increased in size, from 34.298 mm. 2 Increased to 48.816mm 2 In contrast, the tumor volume in mice treated with URP-1 was significantly reduced, from 56.090 mm. 2 Reduced to 28.483mm 2 In contrast, the positive control group (cisplatin) showed a tumor volume in mice that was 47.150 mm². 2 Reduced to 32.690mm 2 In summary, URP-1 can significantly inhibit the proliferation of prostate cancer DU145 cells in tumor-bearing mice.

[0123] 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 experimental group mice increased. These results indicate that administration of URP-1 to the experimental group not only inhibited tumor growth in tumor-bearing mice but also effectively improved their survival status, demonstrating a significant therapeutic effect and improving their quality of life. Figure 12 .

[0124] 4.9URP-1 affects protein expression in prostate cancer-bearing mouse tumor tissues.

[0125] Through the interaction between URP-1 and DU 145 cells, this study found that URP-1 can induce apoptosis in DU 145 cells, which may be achieved by regulating multiple genes and proteins. Therefore, Q-PCR and Western blot were used to detect the expression levels of tumor-related proteins to elucidate the molecular mechanism of URP-1's anti-prostate cancer effect. First, the gene expression of apoptosis-related genes Bax, Bcl-2, MMP-2, and MMP-9 was detected, and the results are shown in Table 3.

[0126] Table 3. Effects of URP-1 on the expression of related genes in tumor tissues of DU 145 prostate cancer-bearing mice.

[0127] Gene Control URP-1 Bcl-2 1 0.22±0.13* Bax 1 4.52±0.36* MMP-2 1 0.33±0.11* MMP-9 1 0.25±0.06*

[0128] Note: Mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0129] The above results indicate that URP-1 inhibits tumor development and progression by intervening in mitochondrial apoptosis in tumors of tumor-bearing mice. Further Western blotting was used to verify the effect of URP-1 on protein expression in tumor tissues of tumor-bearing mice, see [link to relevant documentation]. Figure 13 And Table 4.

[0130] Table 4. Effects of URP-1 on the expression of apoptosis- and migration / invasion-related proteins in tumor tissues of DU 145 prostate cancer-bearing mice.

[0131] Protein Control URP-1 Bcl-2 1 0.18±0.02* Bax 1 3.17±1.12* MMP-2 1 0.31±0.16* MMP-9 1 0.26±0.11*

[0132] Note: Mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0133] As can be seen from the above embodiments, this invention employs boiling water extraction and ethanol precipitation of total polysaccharides from Uncaria rhynchophylla, combined with DEAE cellulose column and molecular sieve dextran gel column purification techniques to achieve the separation of acidic polysaccharides from Uncaria rhynchophylla, solving the technical challenge of polysaccharide component extraction and separation. Monosaccharide composition analysis, molecular weight detection, and Fourier transform infrared spectroscopy were used to clarify the chemical composition and precise structural characteristics of the Uncaria rhynchophylla acidic polysaccharide URP-1. Through CCK-8 cell proliferation assay, the highly water-soluble orange-yellow formazan was generated by the reducing action of WST-8 in mitochondria, thereby quantitatively analyzing cell proliferation capacity and resolving the influence of URP-1 polysaccharide components on the proliferation capacity of prostate cancer cell line DU 145. Furthermore, cell staining with the fluorescent dye DAPI and observation of nuclear apoptosis morphology were combined with inverted fluorescence microscopy to assess the preliminary effect of URP-1 on DU 145 cell apoptosis. Flow cytometry was then used to quantitatively analyze the proportion of apoptotic cells, solving the problem of URP-1's ability to induce and quantify apoptosis. Finally, by measuring and quantifying the effects of URP-1 on apoptosis of the prostate cancer cell line DU 145, the expression of related genes and proteins in tumor tissues of tumor-bearing mice, and simultaneously using small animal MRI to track tumor size and survival status in mice, the technical challenges of the inhibitory mechanism of the Uncaria rhynchophylla acidic polysaccharide URP-1 component on prostate cancer were resolved. These research findings provide important references for the development of Uncaria rhynchophylla acidic polysaccharide URP-1 anti-prostate cancer drugs.

[0134] 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-1, characterized in that, The components of URP-1 include arabinose, rhamnose, galactose, glucose, mannose, and galacturonic acid, with molar percentages of 28.68%, 11.08%, 29.55%, 19.00%, 4.47%, and 7.22%, respectively.

2. A method for preparing the Uncaria rhynchophylla acidic polysaccharide URP-1 according to claim 1, characterized in that, Includes the following steps: (1) Extract the Uncaria rhynchophylla sample with ethanol to remove impurities, centrifuge and collect the precipitate, then extract the precipitate with boiling water and precipitate with ethanol to obtain crude polysaccharide extract. (2) After removing impurities from the crude polysaccharide extract, it was purified by ion exchange column chromatography and dextran gel column chromatography. The liquid passed through the column was collected, freeze-dried, and the Uncaria rhynchophylla acidic polysaccharide URP-1 was obtained.

3. The preparation method according to claim 2, characterized in that, In step (1), the ratio of the Uncaria rhynchophylla sample to the ethanol is 1g:(8-10)mL.

4. The preparation method according to claim 2, characterized in that, In step (1), the boiling water extraction and ethanol precipitation include the following steps: The precipitate and water were extracted in a boiling water bath for 3-5 hours at a material-to-liquid ratio of 1g:(15-20)mL. After centrifugation, the supernatant extract and precipitate residue were obtained. Water was added to the precipitate residue and the extraction was repeated once. After centrifugation, 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.

5. The preparation method according to claim 2, characterized in that, In step (2), the impurity removal of the crude polysaccharide extract includes the following steps: S1: Dissolve the crude polysaccharide extract in water, add protease for enzymatic hydrolysis, centrifuge, and collect the supernatant; S2: Add chloroform and n-butanol to the supernatant, mix thoroughly, and collect the upper aqueous phase; S3: Add petroleum ether to the upper aqueous phase, mix thoroughly, and collect the lower aqueous phase; S4: Add macroporous adsorption resin to the lower aqueous phase, mix thoroughly for adsorption, collect the liquid, dialyze it, precipitate it with ethanol, and dry the precipitate.

6. The preparation method according to claim 5, characterized in that, In step S1, the amount of water used is 600mL-1000mL, and the amount of protease used is 0.4-0.6g. In step S2, 1 / 3 to 1 / 4 volume of chloroform and n-butanol are added to the supernatant, wherein the volume ratio of chloroform to n-butanol is 4:

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

7. The preparation method according to claim 2, characterized in that, The purification method includes: purifying the crude polysaccharide extract after impurity removal using DEAE DE-52 cellulose, first eluting with water, then eluting with 0.1M NaCl; and then purifying with dextran gel G200, with water as the eluent.

8. The use of the Uncaria rhynchophylla acidic polysaccharide URP-1 according to claim 1 in the preparation of an anti-prostate cancer drug.

Images