Application of Uncariae Ramulus Crude Polysaccharide-1 in preparation of medicine for resisting prostate cancer

Abstract

The application discloses application of Uncariae Ramulus Cruda acidic polysaccharide URP-1 in preparation of anti-prostate cancer drugs and belongs to the technical field of medicinal plant polysaccharide anti-tumor. The total Uncariae Ramulus Cruda polysaccharide is obtained from Uncariae Ramulus Cruda through a method of boiling water extraction and ethanol precipitation, then the total polysaccharide is purified through DEAE cellulose and dextran gel G200 to obtain Uncariae Ramculus Cruda acidic polysaccharide URP-1. The components of the URP-1 include arabinose, rhamnose, galactose, glucose, mannose and galacturonic acid, and the molar percentages are 28.68%, 11.08%, 29.55%, 19.00%, 4.47% and 7.22% respectively. It is found through experiments that the Uncariae Ramulus Cruda acidic polysaccharide URP-1 has significant anti-tumor activity on DU 145 cells, and provides a new drug raw material and treatment method for clinical treatment of prostate cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polysaccharide of medicinal plant anti-tumor, in particular to the application of Uncaria rhynchophylla acidic polysaccharide URP-1 in the preparation of anti-prostate cancer drugs. BACKGROUND

[0002] Uncaria rhynchophylla, also known as Shuanggouteng and Yingzhaofeng, is the dried stem branch of Uncaria rhynchophylla of Rubiaceae. It is an important traditional Chinese medicine for calming liver and wind. It is slightly cold in nature and sweet in taste, and belongs to the liver and pericardium meridians. The most distinctive feature of Uncaria rhynchophylla is the curved hooks growing on its branches, which is also the origin of its name and the main medicinal part. Current research mainly focuses on alkaloids (uncarine and isorhynchophylline), flavonoids, triterpenes and other components. The research on macromolecular active substances is less.

[0003] Traditional Chinese medicine believes that Uncaria rhynchophylla has the effects of clearing heat and calming liver, calming wind and calming. Modern pharmacological research shows that the core biological activity of Uncaria rhynchophylla is concentrated in the cardiovascular system (especially antihypertensive) and the nervous system (sedative, anticonvulsant, neuroprotective), and its strong anti-inflammatory and antioxidant effects are the important basis for supporting these core activities. These activities are mainly attributed to the rich indole alkaloids in Uncaria rhynchophylla, especially uncarine and isorhynchophylline. The study of the biological activity of Uncaria rhynchophylla provides a modern scientific basis for understanding its traditional efficacy (such as clearing heat and calming liver, calming wind and calming) and shows its potential application value in the prevention and treatment of hypertension, stroke, epilepsy, neurodegenerative diseases, inflammatory diseases and other diseases. However, other active components in Uncaria rhynchophylla have not been thoroughly explored, especially polysaccharide active components, which have not been reported. Polysaccharides are a class of active components in medicinal plants, mainly focusing on immune regulation and direct killing of tumor cells. Currently, there is no report on the study of Uncaria rhynchophylla polysaccharides, and the mode and mechanism of the inhibitory effect of Uncaria rhynchophylla acidic polysaccharides on tumors have not been mentioned. Therefore, further research is still needed on the in-depth analysis of the material basis of Uncaria rhynchophylla, the development of the pharmacological activity of other active components, the accurate elucidation of the action mechanism of active components and the transformation of clinical application.

[0004] Prostate cancer is a malignant tumor originating from the epithelium of the prostate gland of men, ranking second in the global incidence of male malignant tumors (only next to lung cancer), and is more common in the elderly over 65 years old. The pathological type is mainly adenocarcinoma (more than 95%), among which acinar adenocarcinoma is the most common, and ductal adenocarcinoma is more invasive. Patients with advanced metastasis are mainly manifested as bone pain (characteristic osteogenic metastasis), anemia, and pathological fracture. Prognosis is closely related to the stage: the 5-year survival rate of patients with localized prostate cancer is more than 99%, and after metastasis, it decreases to about 35%. Therefore, inhibiting the metastasis of prostate cancer is an important target for pharmacodynamic basic research. There is no related report on the application of Uncaria rhynchophylla polysaccharides in inhibiting prostate cancer. SUMMARY

[0005] The application aims to provide the application of Uncariae Ramulus Prodrugs-1 in the preparation of anti-prostate cancer drugs, to solve the problems in the prior art, and to provide a new treatment method for the clinical treatment of prostate cancer.

[0006] To achieve the above-mentioned object, the application provides the following scheme:

[0007] The application provides Uncariae Ramulus Prodrugs-1, and components of the URP-1 include arabinose, rhamnose, galactose, glucose, mannose and galacturonic acid, and the molar percentages are 28.68%, 11.08%, 29.55%, 19.00%, 4.47% and 7.22% respectively.

[0008] The application further provides a preparation method of the Uncariae Ramulus Prodrugs-1, including the following steps:

[0009] (1) the Uncaria sample is extracted by ethanol to remove impurities, centrifuged and the precipitate is collected, and then the precipitate is extracted by boiling water and precipitated by ethanol to obtain a crude polysaccharide extract;

[0010] (2) after the crude polysaccharide extract is removed, ion exchange column chromatography and dextran gel column chromatography are used for purification, the column effluent is collected, and the Uncariae Ramulus Prodrugs-1 is obtained by freeze-drying.

[0011] Preferably, in step (1), the ratio of the Uncaria 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 are extracted by a boiling water bath for 3-5h according to a solid-liquid ratio of 1g:(15-20)mL, and the supernatant extract and precipitate residue are obtained by centrifugation, and the precipitate residue is repeatedly extracted once by adding water, and the supernatant extract obtained by centrifugation after the second extraction is combined;

[0014] After the combined supernatant extract is concentrated to 1 / 10 of the original volume, 3-5 times the volume of ethanol is added for precipitation, the precipitate is collected by centrifugation, and the crude polysaccharide extract is obtained by drying.

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

[0016] S1: the crude polysaccharide extract is dissolved in water, and then a protease is added for enzymolysis, centrifuged, and the supernatant is collected;

[0017] S2: trichloromethane and n-butanol are added to the supernatant, mixed thoroughly, and the upper aqueous phase is collected;

[0018] S3: petroleum ether is added to the upper aqueous phase, mixed thoroughly, and the lower aqueous phase is collected;

[0019] S4: macroporous adsorption resin is added to the lower aqueous phase, mixed thoroughly for adsorption, and then the liquid is collected, dialyzed, precipitated with ethanol, and dried.

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

[0021] and / or in step S2, 1 / 3-1 / 4 volume of trichloromethane and n-butanol is added to the supernatant, and the volume ratio of the trichloromethane and n-butanol is 4:1;

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

[0023] and / or in step S4, 1 / 2-1 / 3 volume of macroporous adsorption resin is added to the lower aqueous phase, and the dialysis condition is 3000 Da dialysis bag for 24-48 h.

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

[0025] The application also provides application of the Uncariae Ramulus Crude Acidic Polysaccharide URP-1 in preparation of a prostate cancer drug.

[0026] The application discloses the following technical effects:

[0027] The application adopts a separation method of hot water extraction and ethanol precipitation, and prepares Uncariae Ramulus Cruda acidic polysaccharide through DEAE cellulose column and dextran gel purification. It is found through experiments that Uncariae Ramulus Cruda acidic polysaccharide URP-1 has significant anti-tumor activity on DU 145 cells, and in combination with in-vivo activity verification of tumor-bearing mice, URP-1 mainly regulates the expression of key genes and proteins related to cell mitochondrial apoptosis and cell migration and invasion, increases the expression of pro-apoptotic gene and protein Bax, and down-regulates the expression of apoptosis inhibitor protein Bcl-2, so that cell mitochondrial apoptosis occurs, and tumor cells undergo programmed apoptosis. On the other hand, by down-regulating the expression of matrix metalloproteinase family protein members MMP-2 and MMP-9, the migration and invasion of cells are inhibited, and the development of prostate cancer is limited. The application separates and extracts Uncariae Ramulus Cruda acidic polysaccharide and determines the mechanism of Uncariae Ramulus Cruda acidic polysaccharide URP-1 in resisting the occurrence and development of prostate cancer, so as to provide a new drug raw material and treatment method for the clinical treatment of prostate cancer. BRIEF DESCRIPTION OF DRAWINGS

[0028] 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 effort.

[0029] Figure 1 URP-1 molecular configuration diagram;

[0030] Figure 2 URP-1 absolute molecular weight analysis diagram;

[0031] Figure 3 URP-1 monosaccharide composition analysis diagram;

[0032] Figure 4 URP-1 FT-IR detection diagram;

[0033] Figure 5 URP-1 on prostate cancer DU 145 cell morphology (48h);

[0034] Figure 6 URP-1 on prostate cancer DU 145 cell proliferation inhibition detection results of 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 URP-1 on prostate cancer DU 145 cell nuclear apoptosis morphology (48h);

[0036] Figure 8 Effect of URP-1 on the apoptosis of prostate cancer DU 145 cells (48h) for flow cytometry analysis;

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

[0038] Figure 10 Effect 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, 48h);

[0039] Figure 11 Effect of URP-1 on the tumor growth of prostate cancer DU 145 tumor-bearing mice by nuclear magnetic resonance tracking;

[0040] Figure 12 Effect of URP-1 on the survival state of prostate cancer DU 145 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;

[0041] Figure 13 Effect of URP-1 on the expression of mitochondrial apoptosis pathway-related proteins in tumor tissues of tumor-bearing mice. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present application will now be described in detail with reference to the drawings. The detailed description is not intended to limit the present application, but rather to explain certain aspects, features, and embodiments of the present application.

[0043] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for the purposes of the present application, ranges disclosed can be understood as being inclusive of the recited endpoint, and each intermediate value. The intermediate values of any stated value or recited range, and any other stated or intermediate value in the stated range, are also expressly incorporated into the present application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0044] 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. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application.

[0045] Many modifications and variations of this application of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments 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 given are exemplary only.

[0046] 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 inclusion, but not limited to, the listed materials and methods.

[0047] The following examples relate to the main experimental materials:

[0048] Prostate cancer cell line DU 145 (CL-0075, Pons Life Science), fetal bovine serum (164210, Pons Life Science), 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 antibody (Wuhan Abot Biotech Co., Ltd.).

[0049] Example 1 Extraction and separation method and structural identification of Uncariae Ramulus Cruda acidic polysaccharide

[0050] 1. Extraction and separation method of Uncariae Ramulus Cruda acidic polysaccharide

[0051] 1.1 Uncariae Ramulus Cruda total polysaccharide extraction

[0052] (1) The dried Uncariae Ramulus Cruda sample / raw material was crushed with a crusher and passed through a 60-mesh sieve.

[0053] (2) Anhydrous ethanol was added (1 g: 10 mL), and the fat-soluble pigment and part of the impurities were extracted by stirring at room temperature. The precipitate was collected by centrifugation at 6000g for 10 min.

[0054] (3) Pure water was added to the precipitate (1:20), and the extraction was carried out in a boiling water bath for 4 h. The supernatant extract was collected by centrifugation at 6000g for 10 min. The precipitate residue was re-extracted according to the same procedure.

[0055] (4) Combine the two extracts, and concentrate to 1 / 10 of the original volume by vacuum rotary evaporation. Add four times the volume of anhydrous ethanol and let stand overnight.

[0056] (5) Collect the precipitated solids by centrifugation at 8000 g for 10 min, and dry to obtain the crude polysaccharide extract.

[0057] (6) Determine the purity of the crude polysaccharide by the sulfuric acid-phenol method. Specifically, weigh about 25 mg of the crude polysaccharide extract, dissolve and dilute in water, take 100 μL of the polysaccharide supernatant, add 600 μL of the sulfuric acid-phenol reagent (5% phenol solution: concentrated sulfuric acid = 1:5 (v / v)), mix well, avoid light, and let stand for 10 min. Measure the absorbance at 490 nm.

[0058] 1.2 Total polysaccharide impurity removal

[0059] It includes protein removal, fat removal, and decolorization, etc. Then determine the purity of the crude polysaccharide by the sulfuric acid-phenol method. The detailed steps are as follows:

[0060] (1) Add 600-1000 mL of pure water to the crude polysaccharide extract solids to fully dissolve the crude polysaccharide. Add 0.4-0.6 g of protease, respectively, and let stand overnight for enzymatic hydrolysis.

[0061] (2) Centrifuge the enzymatic hydrolysate, collect the upper aqueous phase, and add 1 / 4 volume of chloroform and n-butanol (4:1, v / v) to the aqueous phase, mix well, and collect the upper aqueous phase.

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

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

[0064] (5) Collect the liquid, dialyze in a 3000 Da dialysis bag for 24-48 h to remove small molecular components, and precipitate the polysaccharide dialysate with ethanol. Collect the precipitate and dry.

[0065] 1.3 Preparation method of Uncariae Ramulus Cruda acidic polysaccharide (URP-1)

[0066] Separate and purify URP by ion exchange column chromatography and dextran gel column chromatography. Specifically, purify by DEAE DE-52 cellulose, first elute with water, then elute with 0.1 M NaCl; then purify by dextran gel G200, the eluent is water, collect the column effluent, and freeze-dry for storage. Finally, obtain URP-1; the flow rate of the purification process is set to 1 mL / min.

[0067] 2. Structure characteristics and identification of Uncariae Ramulus Cruda acidic polysaccharide (URP-1)

[0068] First, the sample was dissolved in 0.1M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) with a final concentration of 1 mg / mL, and filtered through a filter with a pore size of 0.45 μm before being detected by gel chromatography to detect the molecular weight, uniformity and monosaccharide composition. The chromatographic system used was a gel chromatography-differential-multiple angle laser light scattering system, and the liquid phase system was U3000 (Thermo, USA), the differential detector was Optilab T-rEX (Wyatt technology, CA, USA), and the laser light scattering detector was DAWN HELEOS II (Wyatt technology, CA, USA). The specific chromatographic column and elution conditions were as follows: the gel exclusion chromatographic column Ohpak SB-805HQ (300x8mm) and Ohpak SB-803HQ (300x8mm) were connected in series. The column temperature was 45℃, the injection volume was 100 μL, the mobile phase A (0.02% NaN3, 0.1M NaNO3), the flow rate was 0.6 mL / min, and the elution gradient was isocratic for 75 min.

[0069] Secondly, the main functional groups present in URP-1 were detected by infrared spectroscopy to preliminarily judge the configuration of the polysaccharide and understand its structural characteristics. Further, methylation was used to detect the linkage configuration between sugar residues. Finally, the composition of Uncariae Ramulus Cruda Acid Polysaccharide (URP-1) was analyzed and confirmed by HPLC (molecular weight, uniformity and monosaccharide composition detection) and Fourier transform infrared spectroscopy, so as to further understand its biological activity and potential application value.

[0070] 3. Functional detection of Uncariae Ramulus Cruda Acid 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, and adding 10% fetal bovine serum, at 37℃ constant temperature and humidity, and the carbon dioxide concentration was maintained at 5%. When the cell density reached 90%, the cells were passaged at a volume ratio of 1:2-3 and continuously cultured.

[0073] 3.2 Cell proliferation inhibition detection

[0074] DU 145 cells were seeded in 96-well plates at a density of 2x10 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 1 cell / well in a culture dish containing DMEM medium and incubated for 24 hours. Then, the original medium was replaced with a drug medium and incubated with URP-1 for 48 hours. After that, the cells were washed twice with PBS, reagents were added according to the instructions and mixed well, and reacted at room temperature for 5-15 minutes in the dark. The sample was set in triplicate for each group, and the experiment was repeated more than 3 times.

[0081] 3.4 Cell wound healing experiment

[0082] The cell wound healing experiment was used to plate the cells in the logarithmic growth phase without counting, and inoculate them in a 6-well plate. When the cell density in the 6-well plate reached more than 80%, a 1 mL pipette tip was used to make a cross-shaped wound on the cells, four horizontal lines and two vertical lines were drawn in each well. After the wound was made, the suspended cells were washed once with the base medium, and URP-1 containing drug medium was added to the well plate, and the culture was continued for 48 h. Photographs were taken and saved.

[0083] 3.5 Transwell chemotaxis chamber detection experiment

[0084] The Transwell chemotaxis chamber detection experiment was used to detect the effect of URP-1 on the invasion ability of DU 145 cells. Before the experiment, the Transwell chamber was prepared and sterilized with alcohol and ultraviolet light, the pipette tip and medium were pre-cooled, and matrigial was used to coat the membrane of the upper chamber. The experiment was started. The DU 145 cells in the logarithmic growth phase were taken, the cell concentration was adjusted, 600 μL of medium containing 10% newborn bovine serum (NBS) and URP-1 were added to the 12-well plate as control and drug groups, and the chamber was tilted to avoid bubbles on the membrane. Then, cell suspensions containing the base medium and ginsenoside CK were added to the upper chamber of the control and drug groups, respectively, and the culture was continued for 48 h. The chamber was removed with tweezers, the cell liquid in the upper chamber was poured out, and the chamber was placed on a horizontal plane. 1 mL of PBS / well was used to wash twice, then the PBS was poured out, 1 mL of methanol was added to each well to fix for 10 min, the methanol was aspirated, and crystal violet staining was performed for 10-30 min. Then, PBS was used to wash 3 times, the cells on the upper surface of the chamber were gently wiped off with absorbent cotton, and 5 different areas were observed under a microscope with a 40x objective lens. Photographs were taken and saved, and the results were calculated.

[0085] 3.6 Cell immunofluorescence staining

[0086] Put sterilized cover glass into the culture plate, inoculate cells to 60-70% density. Prepare 4% paraformaldehyde (PFA), 0.3% Triton X-100 permeabilization solution, 5% BSA blocking solution, target protein primary antibody, fluorescently labeled secondary antibody (such as Alexa Fluor 488), DAPI nuclear staining solution, and anti-quenching mounting agent. Aspirate the culture medium, gently rinse with PBS 3 times (5 minutes each time) to remove serum residues; add 4% PFA to fix at room temperature for 15-20 minutes (avoid over-fixation), and rinse with PBS 3 more times. Add 0.3% Triton X-100 dropwise and treat for 10 minutes (membrane proteins can be omitted); after washing with PBS, cover with 5% BSA blocking solution and incubate at room temperature for 1 hour to block non-specific binding. Add the working solution of the primary antibody (diluted with the blocking solution, usually 1:100-1:500), and place it in a wet box at 4°C overnight (or at room temperature for 2 hours). Recover the primary antibody, and wash with PBS thoroughly 3 times (10 minutes each time); add the fluorescently labeled secondary antibody (1:200-1:1000) dropwise in the dark, and incubate at room temperature in a wet box for 1 hour; after washing with PBS, add DAPI (1 μg / mL) to stain the nuclei for 5 minutes, and wash with PBS 3 more times. Use tweezers to remove the coverslip and dry the edges, add the anti-quenching mounting agent to the glass slide, and gently press the cover glass to avoid air bubbles; avoid light to cure 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.

[0087] 3.7 Construction of tumor-bearing mouse models and treatment

[0088] DU 145 cell suspension was inoculated into the right axillary of C57BL / 6 mice, 0.2 mL per mouse, to construct a prostate cancer tumor-bearing mouse model. The experiment was divided into 2 groups, 15 mice in each group. The control group was given 0.2 mL of normal saline per mouse by gavage, and the positive control cisplatin group was given cisplatin 3 mg / kg by gavage, for 28 consecutive days; the URP-1 group was given 400 mg / kg by gavage, for 28 consecutive days. The body weight of the mice was measured 72 hours after the last administration, and then the mice were sacrificed. After confirming the death of the mice, the mice were dissected and the tumor tissue was completely peeled off.

[0089] 3.8 Q-PCR detection

[0090] Total RNA was extracted from each sample and used to prepare a cDNA sample, and gene expression was measured by qPCR and primers. The reaction conditions were as follows: heating at 95°C for 5 minutes; then 40 cycles of 95°C for 30 seconds, 55°C for 45 seconds, and 72°C for 30 seconds; and finally extension at 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] Cell immunofluorescence staining was performed on different treated prostate cancer DU 145 cells to evaluate the effect of URP-1 on the expression level of MMP-2 and MMP-9 protein in cells by observing the fluorescence intensity of the target protein under fluorescence microscope.

[0096] 3.10 Western blotting

[0097] Cell samples were collected from each group of culture conditions, protein samples were extracted and analyzed by Western blotting. After electrophoretic separation, the protein samples were transferred to polyvinylidene fluoride (PVDF) membrane for 1.5 h. Then, the membrane was blocked with skim milk for 2 h, incubated with primary antibody overnight, washed with PBST 4 times, and co-incubated with secondary antibody for 2 h. Finally, color development was performed with ECL kit, and the protein expression was visualized and quantitatively analyzed using Tanon-5200 imaging system (Tanon, China).

[0098] 3.11 Statistical analysis: At least three parallel experiments were designed for each experiment, and all data were expressed as mean ± standard deviation. All data were analyzed using Microsoft Excel and SPSS software. Single factor analysis of variance (ANOVA) and t-test were used to evaluate the significance of the results, mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0099] 4. Results and analysis

[0100] 4.1 Molecular weight detection of URP-1

[0101] The slope can be used as a reference for the molecular configuration, with a slope of 0.5-0.6 indicating a random coil. The detected value of URP-1 was 0.02±0.01, indicating that the molecular configuration of URP-1 was approximately spherical, as shown in Figure 1 .

[0102] Further analysis of the uniform molecular weight by HPLC found that the weight average molecular weight of URP-1 was 3962.836 kDa, as shown in Figure 2 .

[0103] 4.2 Analysis of monosaccharide composition of URP-1

[0104] After HPLC monosaccharide composition analysis, the molar ratio information of monosaccharide composition of URP-1 is as follows: the molar percentage of arabinose, rhamnose, galactose, glucose, mannose and galacturonic acid is 28.68%, 11.08%, 29.55%, 19.00%, 4.47% and 7.22%, respectively, as shown in Table 1. Figure 3 .

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

[0106] The FT-IR analysis of URP-1 shows that there is a strong absorption peak at 3300-3500 cm -1 , and the peak type is wide, which is due to the O-H stretching vibration, so there is a wide peak at 3405.67 cm -1 . The two peaks at 2960.68 cm -1 and 2829.54 cm -1 are in the C-H stretching vibration region, 2960.68 cm -1 is asymmetric stretching vibration, and 2829.54 cm -1 is symmetric stretching vibration, which indicates that GT-1 contains saturated alkyl groups. The strong C=O stretching vibration is shown at 1602.07 cm -1 . The absorption peak at 1365.35 cm -1 is C-O stretching vibration. The absorption peak at 1067.89 cm -1 is the angular vibration absorption peak of hydroxyl group. These are all characteristic peaks of polysaccharide substances. In addition, the absorption peak at 773.80 cm -1 indicates that the glycosidic bond in the sugar chain of URP-1 is in β-conformation, as shown in Table 2. Figure 4 .

[0107] 4.4 URP-1 significantly inhibits the proliferation ability of DU 145 cells

[0108] Microscopic observation shows that the morphology of DU 145 cells co-cultured with URP-1 is abnormal, showing shrinkage and fragmentation, and as the concentration of URP-1 increases from 200 to 800 μg / mL, the cell density significantly decreases, as shown in Table 3. Figure 5 The results show that URP-1 can effectively inhibit the in vitro proliferation ability of prostate cancer DU 145 cells and induce abnormal morphology of DU 145 cells.

[0109] The CCK-8 method was used to quantitatively analyze the proliferation inhibition ability of URP-1 on prostate cancer DU 145 cells, and the results showed that the proliferation inhibition effect of URP-1 on DU 145 cells showed time-dependent and concentration-dependent characteristics, as shown in Table 4. Figure 6The maximum inhibition rate of DU 145 cell proliferation was 66.19±2.18% after 48h of URP-1 administration at a concentration of 800μg / mL.

[0110] 4.5 URP-1 induces DU 145 cell nuclear morphological abnormalities and apoptosis

[0111] To verify the effect of URP-1 on the apoptosis of prostate cancer DU 145 cells, DAPI fluorescent staining was performed using 200, 400 and 800μg / mL of URP-1, respectively. After drug administration, the nuclear morphology began to change, the chromatin aggregated, the nucleus pyknosis, and the blue light apoptotic bodies appeared. The number of apoptotic bodies in the experimental group was significantly more than that in the control group, and increased with the increase of drug concentration, see Figure 7 .

[0112] Subsequently, flow cytometry was used to quantitatively analyze the effect of URP-1 on the apoptosis of prostate cancer DU 145 cells, and the results showed that the number of living cells decreased from 88.25% to 82.45%, significantly reduced, 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%, see Figure 8 . The above nuclear DAPI fluorescent staining and flow cytometry results showed that URP-1 could significantly induce DU 145 cells to undergo apoptosis.

[0113] 4.6 URP-1 inhibits the migration and invasion ability of prostate cancer DU 145 cells

[0114] Further, wound healing and transwell chamber experiments were used to explore the effect of URP-1 on the migration and invasion ability of prostate cancer DU 145 cells, and the highest concentration of 800μg / mL of URP-1 was used to carry out the study, and the wound healing area and the number of DU 145 cells migrated through the membrane after URP-1 action were observed after 48h of administration, respectively. From Figure 9 Table A and Table 1, it can be seen that the wound area of the control group migrated to the wound area after 48h, and the blank area decreased from 5.32±1.69mm 2 to 3.14±1.33mm 2 , while the wound area of the URP-1 group still had no cell migration, and the wound area increased from 4.12±1.13mm 2 to 7.25±0.21mm 2 (p<0.05), indicating that URP-1 significantly inhibited the migration ability of prostate cancer cells DU 145 cells.

[0115] Transwell chamber experiment found that URP-1 can significantly inhibit the invasion ability of prostate cancer DU 145 cells after 48h, and the number of cells invading the membrane was significantly reduced. The number of cells invading the membrane in the control group and the URP-1 group was 25.33±2.67 and 13.67±4.33 respectively (p<0.01), see Figure 9 Table 2.

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

[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 DU 145 cells

[0120] Cellular immunofluorescence experiment was used to identify the effect of URP-1 on protein expression in prostate cancer DU 145 cells, and the results are as follows Figure 10 . After 48h, compared with the control group, the expression of MMP-2 and MMP-9 in URP-1 treated cells was significantly reduced, indicating that URP-1 inhibits the expression of matrix metalloproteinase family proteins in prostate cancer DU 145 cells, thereby inhibiting the occurrence of cell migration and invasion.

[0121] 4.8 URP-1 inhibits the proliferation of prostate cancer DU 145 cells in tumor-bearing mice

[0122] A prostate cancer DU 145 tumor-bearing mouse model was constructed, and URP-1 was used for continuous oral treatment of tumor-bearing mice. During the treatment, the survival status, body weight, food intake and tumor size of tumor-bearing mice were tracked. The results are as follows Figure 11 After 4 weeks of treatment, the control group mice were given normal saline, and the tumor volume of the mice increased significantly from 34.298mm 2 to 48.816mm 2 , while the tumor volume of the URP-1 treated mice decreased significantly from 56.090mm 2 to 28.483mm 2 . While the positive control drug cisplatin group, the tumor volume of the mice decreased from 47.150mm 2 to 32.690mm 2 . In summary, URP-1 can significantly inhibit the proliferation of prostate cancer DU 145 cells in tumor-bearing mice.

[0123] Meanwhile, the survival status of mice was observed, and it was found that the average weight, diet and water consumption of mice in the control group and the cisplatin group decreased with the development of tumors, and the indicators of mice in the experimental group all developed towards increase, which indicated that the administration of URP-1 in the experimental group not only inhibited the growth of tumors in tumor-bearing mice, but also effectively improved the survival status of mice, had a significant therapeutic effect, improved the survival quality of mice, and the results are shown in Figure 12 .

[0124] 4.9 URP-1 affects the protein expression in the tumor tissue of prostate cancer-bearing mice

[0125] Through the above interaction between URP-1 and DU 145 cells, it was found that URP-1 could induce apoptosis of DU 145 cells, which might be achieved by regulating multiple genes and proteins. Therefore, Q-PCR and Western blot were used to detect the expression level of tumor-related proteins to clarify the molecular mechanism of URP-1 against prostate cancer. 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 Effect of URP-1 on the expression of related genes in the tumor tissue of prostate cancer DU 145-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 show that URP-1 plays a role in inhibiting the occurrence and development of tumors in tumor-bearing mice by interfering with mitochondrial apoptosis in tumors. Further, Western Blot was used to verify the effect of URP-1 on the protein expression in the tumor tissue of tumor-bearing mice, and the results are shown in Figure 13 and Table 4.

[0130] Table 4 Effect of URP-1 on the expression of apoptosis and migration and invasion related proteins in the tumor tissue of prostate cancer DU 145-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, the present application adopts a method of boiling water extraction and ethanol precipitation of Uncaria total polysaccharides, and combines DEAE cellulose column and molecular sieve dextran gel column purification technology to realize the separation of Uncaria acidic polysaccharides, and solves the technical problems of polysaccharide component extraction and separation. The chemical composition and accurate structure characteristics of Uncaria acidic polysaccharide URP-1 are determined by methods such as monosaccharide composition analysis, molecular weight detection, and Fourier transform infrared spectrum analysis; the influence of URP-1 polysaccharide component on the proliferation ability of prostate cancer cell line DU 145 cells is solved by CCK-8 cell proliferation ability detection, using WST-8 in the reagent to generate highly water-soluble orange formazan through the reduction of cell mitochondrial dehydrogenase, thereby quantitatively analyzing the cell proliferation ability. In addition, the preliminary effect of URP-1 on DU 145 cell apoptosis is evaluated by staining the cells with fluorescent dye DAPI and observing the nuclear apoptosis morphology, combined with inverted fluorescence microscopic imaging technology, and the problems of URP-1 induction and quantitative analysis ability on cell apoptosis are further solved by flow cytometry quantitative analysis of the proportion of apoptotic cells. Finally, the technical problems of Uncaria acidic polysaccharide URP-1 component on the inhibition mechanism of prostate cancer are solved by determining the expression and realizing quantitative analysis of related genes and proteins in prostate cancer cell line DU 145 cell apoptosis, tumor tissue of tumor-bearing mice, and simultaneously using small animal nuclear magnetic resonance to track the size and survival state of the mouse tumor. These research results provide an important reference for the development of Uncaria acidic polysaccharide URP-1 anti-prostate cancer drugs.

[0134] The above-described embodiments only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. Uncaria acid polysaccharide URP-1, characterized in that, The components of the URP-1 include: arabinose, rhamnose, galactose, glucose, mannose and galacturonic acid, and the molar percentages are: 28.68%, 11.08%, 29.55%, 19.00%, 4.47% and 7.22% respectively.

2. The preparation method of Uncariae Ramulus Crude Polysaccharide-1 (URP-1) according to claim 1, characterized in that, The method comprises the following steps: (1) the Uncaria sample is extracted with ethanol to remove impurities, centrifuged and the precipitate is collected, and then the precipitate is extracted with boiling water and precipitated with ethanol to obtain a crude polysaccharide extract; (2) after the crude polysaccharide extract is removed of impurities, ion exchange column chromatography and dextran gel column chromatography are used for purification, the column effluent is collected, freeze-dried to obtain the Uncaria acidic polysaccharide URP-1.

3. The production method according to claim 2, wherein In step (1), the Uncaria sample and the ethanol have a ratio of 1g:(8-10)mL.

4. The production method according to claim 2, wherein In step (1), the boiling water extraction and ethanol precipitation comprise the following steps: The precipitate and water are mixed according to a ratio of 1g:(15-20)mL, extracted in a boiling water bath for 3-5h, centrifuged to obtain supernatant extract and precipitate residue, water is added to the precipitate residue for repeated extraction, and after centrifugation, the supernatant extract obtained in the two times is combined; After the combined supernatant extract is concentrated to 1 / 10 of the original volume, 3-5 times the volume of ethanol is added for precipitation, centrifuged to collect the precipitate, and dried to obtain the crude polysaccharide extract.

5. The production method according to claim 2, wherein In step (2), the removal of impurities from the crude polysaccharide extract comprises the following steps: S1: the crude polysaccharide extract is dissolved in water, and a protease is added for enzymolysis, centrifuged, and the supernatant is collected; S2: trichloromethane 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, precipitated with ethanol, and the precipitate is dried.

6. The production method according to claim 5, wherein 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 / 3-1 / 4 volume of trichloromethane and n-butanol is added to the supernatant, and the volume ratio of trichloromethane 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 conditions are 3000Da dialysis bag for 24-48h.

7. The production method according to claim 2, wherein The purification method comprises: using DEAE DE-52 cellulose to purify the crude polysaccharide extract after removing impurities, first eluting with water, and then eluting with 0.1M NaCl; and then using dextran gel G200 for purification, and the eluent is water.

8. The Uncaria acidic polysaccharide URP-1 of claim 1 in the preparation of a drug for treating prostate cancer.

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