Uncaria polysaccharide as well as preparation method, identification method and application thereof

Abstract

The invention belongs to the technical field of medicines and health foods, and particularly relates to uncaria polysaccharide as well as a preparation method, an identification method and application thereof. Monosaccharide of the uncaria polysaccharide URP-1 consists of mannose, glucose, galactose and arabinose; the uncaria polysaccharide is separated by adopting a water extraction and alcohol precipitation method, the effect is remarkable, and the preparation method is simple, mild in reaction condition and capable of realizing large-scale production; the uncaria polysaccharide is subjected to secondary separation and purification through column chromatography, the effect is remarkable, and a pure uncaria polysaccharide product is prepared; a basis is provided for application of the uncaria polysaccharide in the fields of medicines, health care products and the like, and a foundation is laid for uncaria polysaccharide medicines, quality control and deep research on the structure-function relationship and the action mechanism of the uncaria polysaccharide medicines.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and health food technology, specifically relating to a Uncaria rhynchophylla polysaccharide and its preparation, identification and application methods. Background Technology

[0002] Immune regulation plays a central role in maintaining homeostasis and disease defense. It achieves this function by precisely regulating the functional state of immune cells (including macrophages, T cells, and natural killer cells), the dynamic balance of cytokines (such as IL-1β, TNF-α, and IL-6), and the activation levels of key signaling pathways (such as NF-κB and JAK-STAT). However, in pathological states such as aging, chemotherapy, or metabolic disorders, the body often experiences immunosuppression, which not only significantly weakens immune defense but also greatly increases the risk of infectious diseases, malignant tumors, and cardiovascular diseases. Currently used immunostimulants commonly have significant adverse reactions such as gastrointestinal irritation and flu-like symptoms. Therefore, developing novel immunomodulators with targeted regulatory properties and good safety profiles has become a key research focus.

[0003] Naturally derived polysaccharides, due to their unique low toxicity, multi-target action, and excellent biocompatibility, have become an important research subject in the field of immunomodulatory agent development. Multiple studies have confirmed that different plant polysaccharides can exert immunomodulatory effects through various mechanisms: for example, lentinan can significantly enhance the phagocytic activity of macrophages and improve the killing ability of NK cells, thus it has been approved as an adjuvant therapy for gastric cancer; astragalus polysaccharides have a bidirectional regulatory effect, promoting CD4+ and CD4+. + T lymphocyte proliferation can improve inflammatory bowel disease symptoms by regulating the Th1 / Th2 balance; while Poria cocos polysaccharides and Trametes versicolor polysaccharides mainly promote the maturation and differentiation of dendritic cells through the Toll-like receptor (TLR) signaling pathway, thereby enhancing their antigen-presenting ability. These research findings not only systematically elucidate the complex and diverse immunomodulatory mechanisms of plant polysaccharides, but also provide a solid theoretical foundation for their application in the fields of anti-tumor, antiviral, and immune-related disease treatment.

[0004] Uncaria rhynchophylla, a perennial woody vine belonging to the Rubiaceae family, is used in Traditional Chinese Medicine (TCM) for its effects of calming wind and relieving convulsions, clearing heat and soothing the liver. Clinically, it is mainly used to treat symptoms such as internal liver wind, convulsions, headaches, and dizziness. It is noteworthy that while current research on Uncaria rhynchophylla focuses primarily on the neuroprotective and anti-inflammatory effects of its small-molecule alkaloid components (such as rhynchophylline and isorhynchophylline), the potential pharmacological value of its large-molecule polysaccharide components has long been overlooked. Therefore, in-depth research on the structural analysis of Uncaria rhynchophylla polysaccharides and their immunomodulatory mechanisms can not only provide candidate molecules for the development of novel immunomodulatory drugs but also open up new avenues for the modern development and utilization of this traditional Chinese medicine. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention aims to provide a method for preparing and identifying Uncaria rhynchophylla polysaccharide, as well as its applications. It clarifies the components of Uncaria rhynchophylla polysaccharide, enables the preparation of pure Uncaria rhynchophylla polysaccharide, and provides structural evidence for exploring its pharmacological activity mechanism.

[0006] To solve the above problems, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a Uncaria polysaccharide URP-1, wherein the monosaccharide of Uncaria polysaccharide URP-1 is composed of mannose, glucose, galactose and arabinose; and the molecular weight of Uncaria polysaccharide URP-1 is in the range of 1000-100000 Da.

[0008] Furthermore, the mannose and glucose are in the D configuration, while the galactose and arabinose are in the L configuration.

[0009] Furthermore, the Uncaria polysaccharide URP-1 is composed of 7 sugar residues, namely α-D-Glcp-(4→,→1)-β-D-Manp-(4→,→1)-α-L-Araf-(3,5→,→1)-α-D-Glcp-(4→,t-α-D-Glcp,t-β-D-Manp and t-α-D-Galp.

[0010] Secondly, the present invention provides a method for preparing the aforementioned Uncaria polysaccharide URP-1, comprising:

[0011] Cut the Uncaria rhynchophylla to obtain Uncaria rhynchophylla segments;

[0012] The Uncaria rhynchophylla segments were subjected to water extraction to obtain an extract;

[0013] The extract was subjected to alcohol precipitation to obtain crude polysaccharide UR-A;

[0014] The crude polysaccharide UR-A was subjected to protein removal, dialysis, and freeze-drying to obtain Uncaria polysaccharide UR-1. Uncaria polysaccharide UR-1 was subjected to ion exchange column chromatography and molecular sieve gel column chromatography to collect the sugar fraction and obtain Uncaria polysaccharide URP-1.

[0015] Furthermore, the alcohol precipitation includes:

[0016] The extract was concentrated under reduced pressure to obtain concentrate 1. Ethanol was added to concentrate 1 until the ethanol volume concentration was 10-70%. The mixture was allowed to stand, and the precipitate and supernatant were collected to obtain crude polysaccharide UR-A and supernatant 1.

[0017] Furthermore, the sugar collection portion is achieved by tracking the elution curve using the phenol-sulfuric acid method, and collecting the sugar portion based on the elution curve.

[0018] Furthermore, in the water extraction, the amount of water added is 5-15 times the mass of the Uncaria rhynchophylla segment, the water extraction temperature is 60-100℃, and the extraction time is 1-10 hours.

[0019] Furthermore, the temperature for the vacuum concentration is 40-70℃, and the settling time is 10-28h.

[0020] Thirdly, the present invention provides a method for detecting the aforementioned Uncaria rhynchophylla polysaccharide URP-1, comprising: acid hydrolysis of the Uncaria rhynchophylla polysaccharide sample to be detected followed by liquid chromatography detection; derivatization after acid hydrolysis followed by D / L configuration analysis; dissolving the Uncaria rhynchophylla polysaccharide sample to be detected in water followed by ultraviolet spectroscopy detection; pressing the Uncaria rhynchophylla polysaccharide sample to be detected into a pellet followed by infrared spectroscopy detection; and methylating, hydrolyzing, reducing, and acetylating the Uncaria rhynchophylla polysaccharide sample to be detected followed by GC-MS detection.

[0021] Fourthly, the present invention provides the application of the aforementioned Uncaria polysaccharide URP-1 in the preparation of immunomodulatory drugs or health products.

[0022] Furthermore, the Uncaria polysaccharide URP-1 enhances the phagocytic capacity of phagocytes and promotes the release of nitric oxide, interleukin IL-6, IL-1β and tumor necrosis factor TNF-α.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention employs a water extraction and alcohol precipitation method for the initial separation of Uncaria rhynchophylla polysaccharides, achieving significant results. This preparation method is simple, with mild reaction conditions, and suitable for large-scale production. Furthermore, this invention utilizes column chromatography for secondary separation and purification of the crude Uncaria rhynchophylla polysaccharides, yielding a pure product. The structure of the purified polysaccharide was identified, clarifying the physicochemical properties and structure of each polysaccharide component, providing structural evidence for exploring its pharmacological activity mechanism. The pure Uncaria rhynchophylla polysaccharide obtained by this invention has well-preserved components, a clear structure, and controllable quality. It can enhance the phagocytic capacity of RAW264.7 macrophages and promote the release of molecules such as nitric oxide (NO), interleukin IL-6, IL-1β, and tumor necrosis factor TNF-α, as well as inflammatory factors. This provides a basis for the application of Uncaria rhynchophylla polysaccharides in medicine, health products, and other fields, and lays the foundation for the drug development, quality control, and in-depth research on its structure-activity relationship and mechanism of action. Attached Figure Description

[0025] Figure 1 High-performance liquid chromatogram of URP-1 monosaccharide composition;

[0026] Figure 2 High-performance liquid chromatography chromatogram of the D / L structure of URP-1 monosaccharide;

[0027] Figure 3 The ultraviolet spectrum of URP-1;

[0028] Figure 4 Infrared spectrum of URP-1;

[0029] Figure 5 The total ion chromatogram of the methylation products of URP-1 is shown.

[0030] Figure 6 The mass spectrum of PMAA (1,4,5-Tri-O-acetyl-1-deuterio-2,3-di-O-methyl-D-arabinitol), the methylated product of URP-1;

[0031] Figure 7 The mass spectrum of PMAA (1,5-Di-O-acetyl-1-deuterio-2,3,4,6-tetra-O-methyl-D-galactitol), the methylated product of URP-1;

[0032] Figure 8 The mass spectrum of PMAA (1,5-Di-O-acetyl-1-deuterio-2,3,4,6-tetra-O-methyl-D-mannitol), the methylated product of URP-1;

[0033] Figure 9The mass spectrum of PMAA (1,5-Di-O-acetyl-1-deuterio-2,3,4,6-tetra-O-methyl-D-glucitol), the methylated product of URP-1;

[0034] Figure 10 The mass spectrum of PMAA (1,3,4,5-Tetra-O-acetyl-1-deuterio-2-O-methyl-L-arabinitol), the methylated product of URP-1;

[0035] Figure 11 The mass spectrum of PMAA (1,4,5-Tri-O-acetyl-1-deuterio-2,3,6-tri-O-methyl-D-mannitol), the methylated product of URP-1;

[0036] Figure 12 The mass spectrum of PMAA (1,4,5-Tri-O-acetyl-1-deuterio-2,3,6-tri-O-methyl-D-glucitol), the methylated product of URP-1;

[0037] Figure 13 The effects of URP-1 on the immunomodulatory effects of RAW264.7 cells are as follows: A represents the effect of URP-1 on macrophage activity; B represents the effect of URP-1 on macrophage phagocytic function; C represents the effect of URP-1 on NO release in RAW264.7 cells; D represents the effect of URP-1 on TNF-α expression; E represents the effect of URP-1 on IL-1β expression; and F represents the effect of URP-1 on IL-6 expression. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to specific embodiments.

[0039] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0040] Example 1: Preparation method of Uncaria polysaccharide

[0041] The preparation method of Uncaria polysaccharide includes the following steps:

[0042] S1 Cutting: Cut 5kg of dried Uncaria rhynchophylla roots into 1-3cm sections with scissors, quickly wash with cold water, and air dry to obtain Uncaria rhynchophylla segments;

[0043] S2 Water Extraction: Add 10 times the weight of water to the Uncaria rhynchophylla segments obtained in step S1, heat to 80°C and extract for 3 hours. Collect the extract and dry the residue to obtain the extract and residue.

[0044] S3 alcohol precipitation:

[0045] After concentrating the extract obtained in step S2 under reduced pressure at 60℃, ethanol was added until the ethanol volume concentration was 50%. After standing at room temperature for 24 hours, the extract was centrifuged and the precipitate and supernatant were collected to obtain crude polysaccharide UR-A and supernatant.

[0046] S4 Purification: The crude polysaccharide UR-A obtained in step S3 was deproteinized using the Sevag method. After deproteinization, the crude polysaccharide was dialyzed and lyophilized using a dialysis bag (with a molecular weight cutoff of 3000 Da) to obtain Uncaria rhynchophylla polysaccharide UR-1.

[0047] Example 2: Uncaria rhynchophylla polysaccharide URP-1

[0048] Uncaria polysaccharide URP-1 was obtained by secondary purification of Uncaria polysaccharide UR-1 obtained in Example 1, and the specific method is as follows:

[0049] 1) Ion exchange column chromatography: 200 mg of Uncaria rhynchophylla polysaccharide UR-1 obtained in Example 1 was dissolved in 5 mL of deionized water and loaded onto a DEAE-Sepharose FF column. The elution fraction with NaCl at the elution peak of 0 M was collected separately (the elution curve was tracked using the phenol-sulfuric acid method during the elution process, and the sugar fraction was collected separately according to the elution curve). The obtained eluent was concentrated and freeze-dried to obtain a polysaccharide.

[0050] 2) Molecular sieve gel column chromatography: The above-mentioned lyophilized polysaccharide sample was dissolved in water, centrifuged, and the supernatant was loaded onto a Sephadex G-75 column and eluted with water. The elution curve was tracked using the phenol-sulfuric acid method. A single symmetrical peak appeared. The main peak was collected, concentrated, and lyophilized to obtain Uncaria rhynchophylla polysaccharide URP-1.

[0051] Example 3: Structural Analysis of Uncaria Polysaccharide URP-1

[0052] 1. Monosaccharide composition analysis

[0053] Sample processing:

[0054] Accurately weigh 4.0 mg of each Uncaria rhynchophylla polysaccharide sample into a stoppered test tube, add 2.0 mL of 2M trifluoroacetic acid (TFA), and place in an oil bath at 120℃ for 6 h for hydrolysis. Cool to room temperature, repeatedly add methanol to evaporate to dryness, remove TFA, dissolve in deionized water to 1 mL, centrifuge, and take 100 μL of each sample solution, add 100 μL of 0.3M NaOH solution, then add 100 μL of 0.5M PMP methanol solution, mix well, react in a 70℃ water bath for 30 min, cool, add 105 μL of 0.3M HCl solution to neutralize, add deionized water to 1 mL, then add an equal volume of chloroform solution, shake vigorously, centrifuge, remove the chloroform phase, repeat the extraction twice, and filter the aqueous phase through a 0.45 μm filter membrane for HPLC analysis.

[0055] Chromatographic conditions:

[0056] Column: Kromasil 100-5-C18, 4.6×250mm, 5μm; Mobile phase: 0.1M phosphate (pH=6.9) buffer-acetonitrile (v / v=83:17); Detection wavelength: 250nm; Flow rate: 1mL / min; Injection volume: 20μL.

[0057] 2. Monosaccharide D / L configuration analysis

[0058] URP-1 (5 mg) was added to trifluoroacetic acid (2 M, 2 mL), and the mixture was reacted in an oil bath at 120 °C for 4 h. After the reaction was completed, the mixture was extracted twice with an equal volume of chloroform. The chloroform layer was discarded, and the aqueous layer was evaporated to dryness.

[0059] After the sample was evaporated to dryness, a derivatization step was performed as follows: L-cysteine ​​methyl ester hydrochloride (2.5 mg) and anhydrous pyridine (1 mL) were added, and the mixture was reacted in a water bath at 60 °C for 1 h. Then, o-tolyl isothiocyanate (5 μL) was added, and the reaction was continued in a water bath at 60 °C for another 1 h. After the reaction was complete, the sample was filtered through a 0.45 μm microporous membrane and then analyzed by liquid chromatography.

[0060] Chromatographic conditions:

[0061] Mobile phase: 25% CH3CN-H2O (0.1% acetic acid); Flow rate: 0.8 mL / min; Injection volume: 10 μL; Detection wavelength: UV250 nm; Detection time: 60 min.

[0062] 3. Ultraviolet spectroscopy detection

[0063] Dissolve 2.0 mg of the dried sample in water, load it into a quartz cuvette, and scan the absorption spectrum in the range of 200–400 nm using a PerkinElmer Lambda 35 UV spectrophotometer. Use pure solvent as a blank control.

[0064] 4. Infrared spectroscopy detection

[0065] 2.0 mg of dried test material was ground with KBr, compressed into tablets, and heated using a Perkin EImer FT / IR-100 at 4000-4000 cm⁻¹. -1 Scan within the range.

[0066] 5. Methylation / GC-MS Analysis

[0067] Weigh 8.0 mg of dried test material into a reaction flask, add 8 mL of anhydrous DMSO, then add 800 mg of dried sodium hydroxide, sonicate for 30 min, add 3.0 mL of iodomethane in an ice bath in the dark, in three portions, sonicating in an ice bath for 30 min each time. After the reaction is complete, add 2 mL of distilled water to decompose the residual iodomethane, and add 1 mL of chloroform for extraction. Centrifuge and collect the chloroform layer.

[0068] After complete methylation, the sample was placed in a stoppered test tube and hydrolyzed in a constant temperature oil bath at 120℃ for 6 h with 2 mol / L TFA. The solution was then evaporated to dryness under reduced pressure. The process was repeated several times with the addition of methanol until the pH was neutral. The hydrolysis product was then reduced by reacting with 20 mg of NaBH4 at 40℃ for 30 min. The reaction was terminated with 100 μL of glacial acetic acid. The sample was then evaporated to dryness under low pressure, and 2 mL of acetic anhydride and pyridine were added for acetylation. The reaction was maintained at 95℃ with magnetic stirring for 2 h. Methanol was then added three times, and the solution was evaporated to dryness. The solution was dissolved in 1 mL of chloroform, washed three times with an equal volume of distilled water to remove the aqueous layer, and finally dried over anhydrous sodium sulfate. The sodium sulfate solid was removed by filtration, and the solution was concentrated under reduced pressure and evaporated to dryness for GC-MS analysis.

[0069] 6. Test Results:

[0070] 6.1. Structural analysis of Uncaria rhynchophylla polysaccharide URP-1

[0071] (1) Monosaccharide composition analysis

[0072] like Figure 1As shown in the HPLC chromatogram, URP-1 contains mannose (19.12 min), glucose (38.45 min), galactose (42.33 min), and arabinose (46.58 min). [Chromatographic peak order: 1: mannose (19–20 min), 2: rhamnose (25–26 min), 3: glucuronic acid (26–27 min), 4: galacturonic acid (31–32 min), 5: glucose (38–39 min), 6: galactose (42–43 min), 7: xylose (44–45 min), 8: arabinose (46–47 min), 9: fucose (54–55 min).]

[0073] (2) Monosaccharide D / L configuration analysis

[0074] like Figure 2 As shown in the HPLC chromatogram, mannose and glucose in URP-1 are in the D configuration, while galactose and arabinose are in the L configuration.

[0075] (3) Ultraviolet spectroscopy analysis

[0076] like Figure 3 As shown in the ultraviolet spectrum of URP-1, URP-1 does not contain nucleic acids or proteins.

[0077] (4) Infrared spectroscopy analysis

[0078] like Figure 4 As shown in the infrared spectrum of URP-1, it can be seen that all URP-1 of Uncaria rhynchophylla contains the characteristic infrared absorption peaks of polysaccharides.

[0079] (5) Methylation / GC-MS analysis

[0080] like Figure 5 and Figure 6-12 As shown, the methylation analysis of URP-1, after hydrolysis and reductive acetylation, was detected by GC-MS. The GC-MS spectrum shows that URP-1 contains α-D-Glcp-(4→,→1)-β-D-Manp-(4→,→1)-α-L-Araf-(3,5→,→1)-α-D-Glcp-(4→,t-α-D-Glcp,t-β-D-Manp and t-α-D-Galp sugar residues. Figure 5In the above, the peak at 19.568 min is 5)-L-Araf-(1→; the peak at 21.049 min is D-Galp-(1→; the peak at 21.108 min is D-Manp-(1→; the peak at 21.789 min is D-Glcp-(1→; the peak at 22.22 min is →3,5)-L-Araf-(1→; the peak at 24.369 min is →4)-D-Manp-(1→; the peak at 24.589 min is →4)-D-Glcp-(1→.

[0081] Example 4: Study on the immunomodulatory effect of pure Uncaria polysaccharide on macrophages

[0082] RAW264.7 cells (provided by Shanghai Cell Bank, Chinese Academy of Sciences).

[0083] Test method:

[0084] 1. Experimental Design and Grouping:

[0085] This invention uses a macrophage RAW 264.7 model and sets up a blank group, a positive drug group, and a Uncaria rhynchophylla polysaccharide administration group. The concentrations of Uncaria rhynchophylla polysaccharide URP-1 are 50 μg / mL, 100 μg / mL, and 200 μg / mL. The positive drug is lipopolysaccharide (LPS) at a concentration of 1 μg / mL.

[0086] 2. Preparation of stock solution and cell counting

[0087] Take 20 mg of URP-1 and dissolve it in 1 ml of PBS buffer. Filter the prepared stock solution through a 0.22 μm filter and sterilize it by UV irradiation for 30 min. Then store it in a -20°C refrigerator for later use.

[0088] Place the culture flask in a clean bench, remove the old culture medium, add 5 mL of PBS buffer to wash the cells, then remove the PBS buffer and add 600 μL of trypsin to digest the RAW 264.7 cells for 1 min. Add DMEM medium and repeatedly pipette to form a homogeneous single-cell suspension. Take 20 μL of this single-cell suspension and add it to 180 μL of DMEM medium. After repeated pipetting, take 10 μL and count the cells in a cell counting chamber. Dilute the cells to 5 × 10⁶ cells / mL. 4 Cells / mL, plated.

[0089] 3. Administration

[0090] RAW264.7 cells were seeded into 96-well plates and cultured for 24 h. The test samples were then added. The URP-1 concentrations were 50 μg / mL, 100 μg / mL and 200 μg / mL, respectively. After administration, the RAW264.7 cells were placed in a CO2 incubator and cultured for 24 h.

[0091] 4. MTT Experiment

[0092] Remove the culture medium from the 96-well plate, then add 20 μL of 5 mg / mL thiazolyl blue (MTT) solution to each well and incubate in a CO2 incubator for 4 h. After 4 h, remove the MTT solution from the 96-well plate, add 150 μL of dimethyl sulfoxide (analytical grade) to each well to dissolve formazan crystals, and measure the absorbance (OD value) at 492 nm using a microplate reader. Calculate the proliferation-promoting effect of the sample on RAW264.7 cells. The experiment was repeated three times.

[0093] 5. Neutral red phagocytosis experiment

[0094] RAW264.7 cells were treated with different concentrations of URP-1, washed three times with PBS, and then incubated with 100 μL of neutral red solution (0.1%) for 1 h. The neutral red solution was discarded, and the cells were washed three times with PBS, dried, and then 150 μL of cell lysis buffer (glacial acetic acid: ethanol = 1:1) was added. The cells were incubated at room temperature for 1 h, and the OD value was measured at 540 nm using a microplate reader. The experiment was repeated three times.

[0095] 6. NO Release Test

[0096] RAW264.7 cells were seeded in 96-well plates. After 4 hours, the cells were treated with different concentrations of URP-1 (50 μg / mL, 100 μg / mL and 200 μg / mL) and 1 μg / mL LPS for 24 hours. The supernatant was collected and reacted with Griess reagent. The OD value was measured at 550 nm using an ELISA reader.

[0097] 7. Effects of URP-1 on inflammatory factors in RAW264.7 cells

[0098] RAW264.7 cells were seeded in 24-well plates and incubated for 4 h. After treatment with URP-1 (50 μg / mL, 100 μg / mL and 200 μg / mL) and LPS (1 μg / mL) for 24 h, the supernatant was collected and inflammatory factors such as TNF-α, IL-6 and IL-1β were detected using an ELISA kit.

[0099] 8. Experimental Results

[0100] like Figure 13As shown, after treatment with different concentrations of Uncaria rhynchophylla polysaccharide URP-1, the activity of RAW264.7 cells was not significantly different from that of the control group (P>0.05), indicating that Uncaria rhynchophylla polysaccharide URP-1 is safe and non-cytotoxic at concentrations of 50 μg / mL-200 μg / mL. Furthermore, after treatment with different concentrations of URP-1, the phagocytic activity of RAW264.7 cells significantly increased (P<0.01), and the expression levels of inflammatory factors such as NO, TNF-α, IL-6, and IL-1β significantly increased (P<0.05, P<0.001, or P<0.0001), indicating that Uncaria rhynchophylla polysaccharide URP-1 can enhance the body's immune regulation by improving the phagocytic capacity of macrophages, increasing NO release, and promoting the release of inflammatory factors such as TNF-α, IL-6, and IL-1β.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A type of Uncaria polysaccharide URP-1, characterized in that, The monosaccharide of the Uncaria rhynchophylla polysaccharide URP-1 is composed of mannose, glucose, galactose and arabinose; the molecular weight of the Uncaria rhynchophylla polysaccharide URP-1 ranges from 1000 to 100000 Da.

2. The Uncaria polysaccharide URP-1 according to claim 1, characterized in that, The Uncaria rhynchophylla polysaccharide URP-1 is a mannose-glucose-galactose-arabinoglycosaccharide composed of seven sugar residues, namely α-D-Glcp-(4→,→1)-β-D-Manp-(4→,→1)-α-L-Araf-(3,5→,→1)-α-D-Glcp-(4→,t-α-D-Glcp,t-β-D-Manp and t-α-D-Galp.

3. The method for preparing Uncaria rhynchophylla polysaccharide URP-1 according to claim 1 or claim 2, characterized in that, include: Cut the Uncaria rhynchophylla to obtain Uncaria rhynchophylla segments; The Uncaria rhynchophylla segments were subjected to water extraction to obtain an extract; The extract was subjected to alcohol precipitation to obtain crude polysaccharide UR-A; The crude polysaccharide UR-A was subjected to protein removal, dialysis, and freeze-drying to obtain Uncaria polysaccharide UR-1. Uncaria polysaccharide UR-1 was subjected to ion exchange column chromatography and molecular sieve gel column chromatography to collect the sugar fraction and obtain Uncaria polysaccharide URP-1.

4. The preparation method according to claim 3, characterized in that, The fractional alcohol precipitation includes: The extract was concentrated under reduced pressure to obtain concentrate 1. Ethanol was added to concentrate 1 until the ethanol volume concentration was 10-70%. The mixture was allowed to stand, and the precipitate and supernatant were collected to obtain crude polysaccharide UR-A and supernatant 1.

5. The preparation method according to claim 3, characterized in that, The sugar fraction is collected by tracking the elution curve using the phenol-sulfuric acid method, and the sugar fraction is collected based on the elution curve.

6. The preparation method according to claim 3, characterized in that, In the water extraction, the amount of water added is 5-15 times the mass of the Uncaria rhynchophylla segments, the water extraction temperature is 60-100℃, and the extraction time is 1-10 hours.

7. The preparation method according to claim 4, characterized in that, The vacuum concentration is carried out at a temperature of 40-70℃ and the settling time is 10-28 hours.

8. The method for detecting Uncaria rhynchophylla polysaccharide URP-1 according to claim 1 or claim 2, characterized in that, include: The samples of Uncaria rhynchophylla polysaccharides to be tested were subjected to acid hydrolysis and then detected by liquid chromatography; after acid hydrolysis and derivatization, D / L configuration analysis was performed. The samples of Uncaria rhynchophylla polysaccharides to be tested were dissolved in water and then subjected to ultraviolet spectroscopy; the samples of Uncaria rhynchophylla polysaccharides to be tested were pressed into tablets and then subjected to infrared spectroscopy; the samples of Uncaria rhynchophylla polysaccharides to be tested were subjected to methylation, hydrolysis reduction and acetylation and then subjected to GC-MS.

9. The use of the Uncaria polysaccharide URP-1 according to claim 1 in the preparation of immunomodulatory drugs or health products.

10. The application according to claim 9, characterized in that, The Uncaria polysaccharide URP-1 enhances the phagocytic capacity of phagocytes and promotes the release of nitric oxide, interleukin IL-6, IL-1β and tumor necrosis factor TNF-α.

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