Uncaria polysaccharide URP1-1 as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines and health foods, in particular to uncaria polysaccharide URP1-1 as well as a preparation method and application thereof. The molecular weight of the uncaria polysaccharide URP1-1 disclosed by the invention is 1000 to 100000 Da; the uncaria polysaccharide URP1-1 is a homogeneous polysaccharide; the uncaria polysaccharide URP1-1 is prepared from mannose, rhamnose, glucose, galactose and arabinose. The preparation method is simple, reaction conditions are mild, and large-scale production can be achieved. The chemical structure of the obtained high-purity uncaria polysaccharide is comprehensively identified, and a structural basis is provided for exploring the pharmacological activity mechanism of the uncaria polysaccharide. Meanwhile, the obtained uncaria polysaccharide URP1-1 lays a foundation for preparation of anti-Parkinson active drugs, health-care food and functional food from uncaria polysaccharide, quality control of the uncaria polysaccharide and deep research of the structure-function relationship and the action mechanism of the uncaria polysaccharide.

Description

Technical Field

[0001] This invention relates to the fields of medicine and health food technology, and in particular to a Uncaria rhynchophylla polysaccharide URP1-1, its preparation method and application. Background Technology

[0002] Parkinson's disease, a complex neurodegenerative disorder, is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta and the abnormal aggregation of Lewy bodies, formed from misfolded proteins, within neurons. With the increasing aging of the global population, the incidence and prevalence of Parkinson's disease have risen significantly, placing a heavy burden on patients' families and the social healthcare system. Current drug treatments, primarily levodopa, can effectively alleviate motor symptoms but cannot slow or halt the neurodegenerative process of the disease, and long-term use is often accompanied by fluctuating efficacy and adverse reactions such as dyskinesia. Therefore, exploring novel treatment strategies that can intervene in key pathological aspects of the disease at multiple targets and possess both neuroprotective and repair potential has become an urgent need and a cutting-edge direction in this research field.

[0003] Against this backdrop, bioactive molecules derived from natural products have demonstrated immense research value and application potential due to their pleiotropic effects, low toxicity, and good biocompatibility. Among them, polysaccharides, as a class of biomacromolecules widely found in animals, plants, and microorganisms, are increasingly becoming a focus of research and development for drugs treating neurodegenerative diseases. Numerous studies have shown that many natural polysaccharides not only possess well-known immunomodulatory and antioxidant activating properties but also play unique neuroprotective roles in the central nervous system.

[0004] Uncaria rhynchophylla ( Uncaria rhynchophylla (Miq.)Miq. ex Havil . Uncaria rhynchophylla is a commonly used traditional Chinese medicine in the Rubiaceae family. Its hooked stems and branches are used medicinally, possessing the effects of calming wind and relieving convulsions, clearing heat and soothing the liver. Traditionally, it is used to treat symptoms such as internal liver wind, convulsions, headaches, and dizziness. Modern pharmacological studies have confirmed that Uncaria rhynchophylla contains various active ingredients such as alkaloids, flavonoids, and polysaccharides, showing significant effects in sedation, anticonvulsant, hypotensive, and neuroprotection. However, systematic research on its main active ingredient—Uncaria rhynchophylla polysaccharide—in the prevention and treatment of Parkinson's disease is still lacking. Currently, there is a gap in research on the preparation of homogeneous components of Uncaria rhynchophylla polysaccharide, structural analysis, and its systematic pharmacological activity against key pathological links in Parkinson's disease. Therefore, it is necessary to provide a method for the extraction and purification of Uncaria rhynchophylla polysaccharide to lay the foundation for quality control and in-depth research on its Parkinson's disease activity. Summary of the Invention

[0005] The purpose of this invention is to provide a Uncaria polysaccharide URP1-1, its preparation method, and its application, in order to solve the problems in the prior art.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: One of the technical solutions of the present invention provides a Uncaria rhynchophylla polysaccharide URP1-1, wherein the molecular weight of the Uncaria rhynchophylla polysaccharide URP1- is 1000~100000 Da; The Uncaria polysaccharide URP1-1 is a homogeneous polysaccharide; the Uncaria polysaccharide URP1-1 is composed of mannose, rhamnose, glucose, galactose and arabinose.

[0007] The second technical solution of this invention provides a method for preparing the above-mentioned Uncaria rhynchophylla polysaccharide URP1-1, comprising the following steps: 1) Mix Uncaria rhynchophylla stem and branch powder with water and extract to obtain an extract; 2) Concentrate the extract to obtain concentrate 1; 3) Mix concentrate 1 with ethanol, precipitate with alcohol, let stand, and collect the precipitate to obtain crude polysaccharide; 4) The crude polysaccharide was subjected to protein removal, dialysis, and freeze-drying to obtain Uncaria rhynchophylla polysaccharide UR1; 5) The Uncaria polysaccharide UR1 was subjected to ion exchange column chromatography with a gradient elution using 0-2M sodium chloride solution as the eluent. The elution curve was tracked using the phenol-sulfuric acid method during the elution process. The 0 M sodium chloride solution elution fraction was collected according to the elution curve, concentrated and dried to obtain Uncaria polysaccharide URP1. 6) Dissolve Uncaria polysaccharide URP1 in water, centrifuge, take the supernatant for molecular sieve gel column chromatography, elute with water, detect the elution curve using phenol-sulfuric acid method, collect the sugar fraction according to the elution curve, concentrate and freeze dry to obtain Uncaria polysaccharide URP1-1.

[0008] The third technical solution of this invention provides the application of the above-mentioned Uncaria polysaccharide URP1-1 in the preparation of anti-Parkinson's drugs or health products.

[0009] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses water extraction and alcohol precipitation to preliminarily separate Uncaria polysaccharides, with significant results. Moreover, this preparation method is simple, the reaction conditions are mild, and it can be produced on a large scale.

[0010] 2. This invention uses column chromatography to perform secondary separation and purification of crude Uncaria rhynchophylla polysaccharide, with significant results, and for the first time prepares a pure Uncaria rhynchophylla polysaccharide URP1-1.

[0011] 3. The pure Uncaria polysaccharide URP1-1 obtained by this invention has well-preserved components and controllable quality. It can restore the motor ability and motor neurons of Parkinson's zebrafish to exert anti-Parkinson's activity, providing a basis for the application of Uncaria polysaccharide in medicine, health products and other fields.

[0012] 4. This invention lays the foundation for the development of Uncaria rhynchophylla polysaccharide drugs, quality control, and in-depth research on their structure-activity relationship and mechanism of action. Attached Figure Description

[0013] Figure 1 High performance liquid chromatogram of Uncaria rhynchophylla polysaccharide URP1-1; Figure 2 Infrared spectrum of Uncaria rhynchophylla polysaccharide URP1-1; Figure 3 High-performance liquid chromatogram of the monosaccharide composition of Uncaria rhynchophylla polysaccharide URP1-1; Figure 4 Uncaria polysaccharide 1 H NMR spectrum; Figure 5 Uncaria polysaccharide 13 C NMR spectrum; Figure 6 HSQC spectrum of Uncaria rhynchophylla polysaccharide; Figure 7 HMBC map of Uncaria rhynchophylla polysaccharide; Figure 8 This is a schematic diagram of the structure of Uncaria rhynchophylla polysaccharide; Figure 9 The effects of Uncaria rhynchophylla polysaccharide URP1-1 on the behavior of Pakin zebrafish are shown in the following figures: (a) swimming trajectory diagram of zebrafish in different groups, (b) activity time ratio of zebrafish in different speed ranges, (c) total distance traveled by zebrafish, and (d) average swimming speed of zebrafish. Figure 10 The effects of Uncaria rhynchophylla polysaccharide URP1-1 on motor neurons in Parkinson's zebrafish are shown in (a) for fluorescence results of zebrafish motor neurons and (b) for quantitative statistical graph of fluorescence signal intensity. Detailed Implementation

[0014] 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.

[0015] 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. Every smaller range between any stated value or intermediate value within a stated range, and 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.

[0016] 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.

[0017] 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 obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0018] 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.

[0019] The room temperature described in this invention is 25±2℃.

[0020] All raw materials used in this invention can be obtained commercially or prepared using existing technologies.

[0021] This invention provides a Uncaria polysaccharide URP1-1, wherein the molecular weight of Uncaria polysaccharide URP1-1 is 1000~100000 Da; The Uncaria polysaccharide URP1-1 is composed of mannose, rhamnose, glucose, galactose and arabinose; The Uncaria rhynchophylla polysaccharide URP1-1 is a homogeneous polysaccharide.

[0022] In this invention, the sugar residues of the Uncaria polysaccharide URP1-1 include α -L-Ara f (1→,→2,5)- α -L-Ara f (1→,→5)- α -L-Ara f (1→,→3,5)- α -L-Ara f (1→,→3,6)- α -D-Glc p (1→, α -L-Rha p (1→, α -D-Gal p (1→,→4)- β -D-Gal p (1→,→4,6)-β -D-Man p (1→,→2)- β -D-Gal p (1→,→6)- β -D-Gal p (1→and α -D-Glc p (4→).

[0023] This invention also provides a method for preparing the above-mentioned Uncaria rhynchophylla polysaccharide URP1-1, the obtained Uncaria rhynchophylla polysaccharide URP1-1 having a significant anti-Parkinson's disease effect, comprising the following steps: 1) Mix Uncaria rhynchophylla stem and branch powder with water and extract to obtain an extract; 2) Concentrate the extract to obtain concentrate 1; 3) Mix concentrate 1 with ethanol, precipitate with alcohol, let stand, and collect the precipitate to obtain crude polysaccharide; 4) The crude polysaccharide was subjected to protein removal, dialysis, and freeze-drying to obtain Uncaria rhynchophylla polysaccharide UR1; 5) The Uncaria polysaccharide UR1 was subjected to ion exchange column chromatography with a gradient elution using 0-2M sodium chloride solution as the eluent. The elution curve was tracked using the phenol-sulfuric acid method during the elution process. The 0 M sodium chloride solution (water) eluted fraction was collected according to the elution curve, concentrated and dried to obtain Uncaria polysaccharide URP1. 6) Dissolve Uncaria polysaccharide URP1 in water, centrifuge, take the supernatant for molecular sieve gel column chromatography, elute with water, detect the elution curve using phenol-sulfuric acid method, collect the sugar fraction according to the elution curve, concentrate and freeze dry to obtain Uncaria polysaccharide URP1-1.

[0024] This invention combines water extraction with alcohol precipitation. High-concentration ethanol can separate highly polar and water-soluble polysaccharides from less polar and poorly water-soluble polysaccharides, solving the problem of complicated and difficult separation in the later stages caused by traditional water boiling method for polysaccharide extraction.

[0025] Step 1) of this invention involves crushing the hooked stems and branches of Uncaria rhynchophylla, washing them with water, drying them to obtain Uncaria rhynchophylla stem and branch powder, adding the powder to water, heating to extract, and filtering to obtain the extract.

[0026] In this invention, the mass of water in step 1) is 6 to 10 times the mass of Uncaria rhynchophylla stem and branch powder, for example, it can be 6, 7, 8, 9 or 10 times, etc.

[0027] In this invention, the extraction temperature is 60~80℃, for example, 60℃, 70℃ or 80℃, the time is 1~3h, for example, 1h, 2h or 3h, and the number of heating extractions is 2~4 times.

[0028] Step 2 of this invention is to concentrate the extract under reduced pressure to obtain concentrate 1.

[0029] In this invention, the concentration temperature is 40~70℃, for example, it can be 40℃, 50℃, 60℃ or 70℃, the vacuum degree is -0.1MPa, and the time is 1~3 h, for example, it can be 1 h, 2 h or 3 h.

[0030] Step 3) of the present invention is to add ethanol to concentrate 1 until the ethanol volume concentration is a%, let it stand, collect the precipitate and supernatant to obtain crude polysaccharide UR1 and supernatant 1; In this invention, step 3) involves adding ethanol to concentrate 1 until the ethanol volume concentration is 50%; the standing time is 10-15 hours, for example, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours or 15 hours.

[0031] Step 4 of this invention involves removing proteins from the crude polysaccharide UR1 using the Sevag method. After protein removal, the crude polysaccharide is dialyzed using a dialysis bag with a molecular weight cutoff of 3500 Da and then freeze-dried to obtain Uncaria rhynchophylla polysaccharide UR1.

[0032] This invention uses dried Uncaria rhynchophylla powder as raw material, and separates crude polysaccharides using water extraction and alcohol precipitation. The extracted crude polysaccharides are then deproteinized, and purified using ion exchange chromatography and gel molecular sieve column chromatography. For the first time, a pure Uncaria rhynchophylla polysaccharide was prepared. The physicochemical properties, molecular weight, and infrared spectrum of this pure polysaccharide were systematically analyzed and identified, and the structural information of the Uncaria rhynchophylla polysaccharide was successfully obtained. The Uncaria rhynchophylla polysaccharide URP1-1 is a homogeneous polysaccharide composed of mannose, rhamnose, glucose, galactose, and arabinose. α -L-Ara f (1→,→2,5)- α -L-Ara f (1→,→5)- α -L-Ara f (1→,→3,5)- α -L-Ara f (1→,→3,6)- α -D-Glc p (1→, α -L-Rha p (1→, α -D-Gal p (1→,→4)- β -D-Gal p (1→,→4,6)- β -D-Man p (1→,→2)- β -D-Galp (1→,→6)- β -D-Gal p (1→and α -D-Glc p (4→ sugar residues).

[0033] This invention also provides the application of the above-mentioned Uncaria polysaccharide URP1-1 in the preparation of anti-Parkinson's drugs or health products.

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1 A method for preparing Uncaria rhynchophylla polysaccharide URP1-1 includes the following steps: S1, Crushing 1 kg of Uncaria rhynchophylla stems and branches were crushed, quickly washed with cold water, and dried to obtain Uncaria rhynchophylla stem and branch powder. S2, water extraction Add 8 times its weight of water to the Uncaria rhynchophylla stem and branch powder obtained in step S1, heat to 70°C and extract, extract for 1 hour each time, for a total of 3 extractions, collect the extract, and dry the residue to obtain the extract and residue. S3, graded alcohol precipitation The extract obtained in step S2 was concentrated at 60℃ under reduced pressure (vacuum degree -0.1MPa) for 1 h to obtain concentrate 1; ethanol was added to concentrate 1 until the ethanol volume concentration was 50%, and the mixture was allowed to stand for 15 h. The precipitate and supernatant were collected to obtain crude polysaccharide UR1 and supernatant 1. S4, Purification S4-01, First Purification The crude polysaccharide UR1 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 3500 Da) to obtain Uncaria rhynchophylla polysaccharide UR1. S4-02, Secondary Purification 800 mg of Uncaria rhynchophylla polysaccharide UR1 was dissolved in 10 mL of deionized water and loaded onto a DEAE-FF column. Gradient elution was performed using 0–2 M NaCl solution, resulting in one elution peak. The elution peak was the 0 M NaCl elution fraction (the elution curve was tracked using the phenol-sulfuric acid method during elution, and the sugar fractions were collected according to the elution curves). The obtained eluents were concentrated and freeze-dried to obtain Uncaria rhynchophylla polysaccharide URP1. The freeze-dried Uncaria rhynchophylla polysaccharide URP1 was dissolved in water, centrifuged, and the supernatant was loaded onto a Sephadex G-100 column and eluted with 0 M NaCl. The elution curve was tracked using the phenol-sulfuric acid method, and a single symmetrical peak appeared. The main peak was collected, concentrated, and freeze-dried to obtain Uncaria rhynchophylla polysaccharide URP1-1.

[0036] Example 2 Structural analysis of Uncaria rhynchophylla polysaccharide URP1-1 (a) Experimental material: Uncaria polysaccharide URP1-1 from Example 1.

[0037] (II) Test Methods: 1. Homogeneity and molecular weight analysis Sample processing: Accurately weigh 5 mg of Uncaria rhynchophylla polysaccharide URP1-1 and dissolve it in 1 mL of deionized water. Filter the solution through a 0.22 μm filter membrane before HPLC analysis.

[0038] Chromatographic column: TSKgel G3000PWXL, 7.8 × 300 mm, 7 μm; mobile phase: 0.02 M phosphate buffer; detector: RID; flow rate: 0.5 mL / min; injection volume: 10 μL.

[0039] 2. Infrared spectroscopy detection 2.0 mg of dried Uncaria rhynchophylla polysaccharide URP1-1 test material was ground with KBr, compressed into tablets, and tested with IR Affinity-1 at 4000-400 cm⁻¹. -1 Scan within the range.

[0040] 3. Monosaccharide composition detection Sample processing: Accurately weigh 4.0 mg of each Uncaria rhynchophylla polysaccharide sample into a stoppered test tube, add 2.0 mL of 2 M trifluoroacetic acid (TFA), and place in an oil bath at 120 °C 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 0.3 M NaOH solution, then add 100 μL of 0.5 M PMP methanol solution, mix well, react in a 70 °C water bath for 30 min, cool, add 100 μL of 0.3 M 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.

[0041] Chromatographic conditions: Column: Kromasil 100-5-C18, 4.6 × 250 mm, 5 μm; Mobile phase: 0.1 M phosphate (pH = 6.9) buffer-acetonitrile (v / v = 84:16); Detection wavelength: 250 nm; Flow rate: 0.8 mL / min; Injection volume: 20 μL.

[0042] Methylation / GC-MS analysis 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.

[0043] 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 evaporated to dryness under reduced pressure, and 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, and the sample was evaporated to dryness under low pressure. Then, 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 evaporated to dryness in a fume hood for GC-MS analysis.

[0044] Nuclear magnetic resonance analysis After repeatedly lyophilizing the Uncaria rhynchophylla polysaccharide sample URP1-1, 60 mg was dissolved in 0.6 mL of D2O, placed in an NMR tube, and recorded using a 400 MHz Bruker AV-400 NMR spectrometer. 1 H NMR, 13 C NMR, HSQC, HMBC and other spectra.

[0045] (III) Test Results: 1. Structural characterization of Uncaria rhynchophylla polysaccharide URP1-1 (1) Homogeneity analysis like Figure 1 As shown in the HPLC chromatogram, the Uncaria rhynchophylla polysaccharide URP1-1 exhibits a single symmetrical peak, indicating that URP1-1 is a homogeneous polysaccharide with a molecular weight range of 1000~100000 Da.

[0046] (2) Infrared spectroscopy analysis like Figure 2As shown in the infrared spectrum of Uncaria rhynchophylla polysaccharide URP1-1, it can be seen that Uncaria rhynchophylla URP1-1 contains the characteristic infrared absorption peaks of polysaccharides.

[0047] (3) Monosaccharide composition analysis like Figure 3 As shown in the HPLC chromatogram, URP1-1 contains mannose, rhamnose, glucose, galactose, and arabinose. (Chromatographic peak order: 1: mannose, 2: rhamnose, 3: glucuronic acid, 4: galacturonic acid, 5: glucose, 6: galactose, 7: xylose, 8: arabinose, 9: fucose) (4) Methylation / GC-MS analysis Methylation analysis of URP1-1 was performed after hydrolysis and reductive acetylation, followed by GC-MS detection. The GC-MS spectrum showed that URP1-1 contained... α -L-Ara f (1→,→2,5)- α -L-Ara f (1→,→5)- α -L-Ara f (1→,→3,5)- α -L-Ara f (1→,→3,6)- α -D-Glc p (1→, α -L-Rha p (1→, α -D-Gal p (1→,→4)- β -D-Gal p (1→,→4,6)- β -D-Man p (1→,→2)- β -D-Gal p (1→,→6)- β -D-Gal p (1→and α -D-Glc p (4→ sugar residues).

[0048] (5) Nuclear magnetic resonance analysis This experiment passed 1 H NMR, 13 C NMR and HSQC were used to assign the chemical shifts of carbon and hydrogen atoms of the sugar residues in URP1-1, and then HMBC was used to confirm its linkage sequence. Figures 4-7 for 1 H NMR, 13 C NMR, HSQC, and HMBC spectra.

[0049] according to Figure 4-7 The NMR spectra of URP1-1 and their C-H assignments are shown in Table 1 below.

[0050] Table 1. Results of URP1-1 nuclear magnetic resonance analysis

[0051] In summary, URP1-1 is composed of mannose, rhamnose, glucose, galactose, and arabinose, and methylation analysis indicates that it contains... α -L-Ara f (1→,→2,5)- α -L-Ara f (1→,→5)- α -L-Ara f (1→,→3,5)- α -L-Ara f (1→,→3,6)- α -D-Glc p (1→, α -L-Rha p (1→, α -D-Gal p (1→,→4)- β -D-Gal p (1→,→4,6)- β -D-Man p (1→,→2)- β -D-Gal p (1→,→6)- β -D-Gal p (1→and α -D-Glc p (4→Isoglycolic residues, the connection order between different sugar residues was determined by two-dimensional NMR HMBC spectrum analysis, and the structure of URP1-1 was obtained from the above analysis as follows) Figure 8 As shown, where 3≤m+n+r≤10, 1≤z≤y≤x≤6.

[0052] Example 3 Study on the anti-Parkinson's effect of pure Uncaria polysaccharide URP1-1 on zebrafish (a) Experimental material: Uncaria polysaccharide URP1-1 from Example 1.

[0053] (ii) Experimental subject: Zebrafish (purchased from Nanjing Yishu Lihua Technology Co., Ltd.).

[0054] (III) Test Methods: 1. Zebrafish culture: Adult zebrafish were reared in an automated system at 28 ± 0.5 ℃ with alternating light and dark cycles of 14 h and 10 h. Spawning was induced and eggs were collected, then transferred to E3 medium (0.33 mmol / L CaCl2, 5.00 mmol / L NaCl, 0.18 mmol / L KCl, 0.34 mmol / L MgSO4, pH 7.40) and placed in an incubator at 28.5 ℃.

[0055] 2. Administration Fertilized AB zebrafish larvae at 2 dpf and Tg(HuC:GFP) zebrafish larvae were randomly transferred to each well of a 24-well plate, with 20 larvae placed in each well. A blank control group, a model group, a positive control group, and different concentrations of polysaccharide-treated groups were established, with 3 wells in each group. The fish were cultured in a 28.5 ℃ incubator until 5 dpf (14 h light / 10 h dark).

[0056] Blank group: Add 2 mL of E3 culture medium.

[0057] Model group: Add 2 mL of 250 μg / ml 6-hydroxydopamine (6-OHDA) solution.

[0058] Polysaccharide group: 2 mL of 6-OHDA at a final concentration of 250 μg / mL and polysaccharide solutions of different concentrations (100, 200 and 400 μg / mL) were added.

[0059] 3. Ethical Evaluation of Zebrafish After the 5dpf experiment, the drugs were washed off, and the zebrafish were placed in 96-well plates, one per well. They were then placed in the Zebrabox zebrafish behavior analyzer and allowed to acclimatize for 10 minutes. The analysis then began, lasting 20 minutes. Data processing was performed using Zeblab software to calculate the total swimming distance and average speed of each fish.

[0060] 4. Development of neurons in zebrafish After the 5dpf experiment, the drugs were washed off, and the development of zebrafish neurons with labeled neurons was observed using a fluorescence microscope. Stereoscopic fluorescence microscope images were taken, and ImageJ was used for quantification.

[0061] (iv) Experimental Results like Figure 9-10 As shown, Uncaria rhynchophylla polysaccharide URP1-1 can restore the motor function and neuronal development of Parkinson's disease zebrafish, thus exerting anti-Parkinson's disease activity. Figure 9The effects of Uncaria rhynchophylla polysaccharide URP1-1 on the behavior of Parkinson's disease zebrafish were investigated. (a) shows the swimming tracks of zebrafish in different groups. The tracks in the blank group were dense and covered a large area, indicating that the zebrafish were active. The tracks in the 6-OHDA model group were sparse, short and messy, reflecting the bradykinesia and stereotyped behavior of the Parkinson's disease model. In the URP1-1 intervention group, the tracks gradually became denser and the range expanded with the increase of dose, indicating that the motor ability was improved. (b) is a graph showing the proportion of activity time in zebrafish at different speed ranges. The proportion of slow movement time in the 6-OHDA model group increased significantly, while the proportion of fast movement time decreased. After URP1-1 intervention, this trend was reversed and showed a dose-dependent effect. (c) is a graph showing the total movement distance of zebrafish. The total distance in the model group was significantly shortened, while after URP1-1 treatment, the total distance increased with increasing dose, demonstrating that polysaccharides can improve bradykinesia. (d) is a graph showing the average swimming speed of zebrafish. The speed in the model group was significantly reduced, while the speed rebounded after URP1-1 intervention, further verifying its effect on improving motor function.

[0062] Figure 10 The effects of Uncaria rhynchophylla polysaccharide URP1-1 on motor neurons in zebrafish with Parkinson's disease were investigated. (a) showed the fluorescence results of zebrafish motor neurons. As the polysaccharide dose increased, the fluorescence signal gradually increased and the continuity was restored, indicating that the neuronal damage was significantly improved. (b) showed the quantitative statistical graph of the fluorescence signal intensity, which quantitatively proved that the protective effect of URP1-1 on neurons was dose-dependent.

[0063] In summary, the pure Uncaria rhynchophylla polysaccharide URP1-1 prepared by this invention is a homogeneous polysaccharide, and it can restore the motor ability and neuronal development of Parkinson's disease zebrafish to exert anti-Parkinson's disease activity.

[0064] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of Uncaria polysaccharide URP1-1, characterized in that, The molecular weight of the Uncaria polysaccharide URP1-1 is 1000~100000 Da; The Uncaria polysaccharide URP1-1 is a homogeneous polysaccharide; the Uncaria polysaccharide URP1-1 is composed of mannose, rhamnose, glucose, galactose and arabinose.

2. The Uncaria polysaccharide URP1-1 according to claim 1, characterized in that, The sugar residues of the Uncaria rhynchophylla polysaccharide URP1-1 include α -L-Ara f (1→,→2,5)- α -L-Ara f (1→,→5)- α -L-Ara f (1→,→3,5)- α -L-Ara f (1→,→3,6)- α -D-Glc p (1→, α -L-Rha p (1→, α -D-Gal p (1→,→4)- β -D-Gal p (1→,→4,6)- β -D-Man p (1→,→2)- β -D-Gal p (1→,→6)- β -D-Gal p (1→and α -D-Glc p (4→).

3. A method for preparing Uncaria rhynchophylla polysaccharide URP1-1 according to claim 1 or 2, characterized in that, Includes the following steps: 1) Mix Uncaria rhynchophylla stem and branch powder with water and extract to obtain an extract; 2) Concentrate the extract to obtain concentrate 1; 3) Mix concentrate 1 with ethanol, precipitate with alcohol, let stand, and collect the precipitate to obtain crude polysaccharide; 4) The crude polysaccharide was subjected to protein removal, dialysis, and freeze-drying to obtain Uncaria rhynchophylla polysaccharide UR1; 5) The Uncaria polysaccharide UR1 was subjected to ion exchange column chromatography with a gradient elution using 0-2M sodium chloride solution as the eluent. The elution curve was tracked using the phenol-sulfuric acid method during the elution process. The 0 M sodium chloride solution elution fraction was collected according to the elution curve, concentrated and dried to obtain Uncaria polysaccharide URP1. 6) Dissolve Uncaria polysaccharide URP1 in water, centrifuge, take the supernatant for molecular sieve gel column chromatography, elute with water, detect the elution curve using phenol-sulfuric acid method, collect the sugar fraction according to the elution curve, concentrate and freeze dry to obtain Uncaria polysaccharide URP1-1.

4. The preparation method according to claim 3, characterized in that, In step 1), the mass of water should be 6 to 10 times the mass of the Uncaria rhynchophylla stem and branch powder.

5. The preparation method according to claim 3, characterized in that, The extraction temperature is 60~80℃, the time is 1~3h, and the number of extractions is 2~4.

6. The preparation method according to claim 3, characterized in that, The concentration is carried out at a temperature of 40~70℃, a vacuum degree of -0.1MPa, and a time of 1~3h.

7. The preparation method according to claim 3, characterized in that, Step 3) involves adding ethanol to concentrate 1 until the ethanol volume concentration reaches 50%; the standing time is 10-15 hours.

8. The use of Uncaria rhynchophylla polysaccharide URP1-1 as described in claim 1 or 2 in the preparation of anti-Parkinson's drugs or health products.

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