Rhamnogalacturonan i, pharmaceutical compositions and methods of preparation and use thereof

Abstract

The application provides rhamnogalacturonan I, a pharmaceutical composition and a preparation method and application thereof, and belongs to the field of biotechnology and chemistry.A new rhamnogalacturonan I is separated from polygonum aviculare, and the structure and performance of the rhamnogalacturonan I are characterized.It is found that the new rhamnogalacturonan I can play an anti-kidney stone role.The new rhamnogalacturonan I has important clinical significance and application prospect in preparation of a medicine for preventing and / or treating kidney stones.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and chemistry, specifically relating to rhamnogalacturonic acid polysaccharide I, pharmaceutical compositions, their preparation methods, and applications. Background Technology

[0002] Kidney stones, clinically known as nephrolithiasis, are a prevalent urinary tract disease with a steadily rising global incidence, placing a heavy burden on healthcare systems and highlighting the urgent need for effective prevention strategies to reduce their incidence. The pathophysiology of kidney stones involves complex processes, including urinary solute supersaturation, crystal nucleation, growth, aggregation, and adhesion to renal epithelial cells. These processes are often exacerbated by oxidative stress and inflammation. Notably, crystal deposition triggers a strong inflammatory response, characterized by macrophage infiltration and the secretion of pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα). TNFα, through its receptors TnfR1 and TnfR2, upregulates adhesion molecules on renal tubular epithelial cells, such as CD44 and annexin II, promoting calcium oxalate crystal adhesion and retention, thereby driving stone formation. Current treatments primarily rely on surgical interventions, such as extracorporeal shock wave lithotripsy, ureteroscopic lithotripsy, and percutaneous nephrolithotomy. These methods effectively remove existing stones but do not address the prevention issue. Drug treatment options, such as thiazide diuretics and potassium citrate, have limited efficacy and are accompanied by side effects, including low blood pressure and gastrointestinal discomfort. This highlights the unmet need for novel therapies targeting the underlying mechanisms of stone formation, particularly inflammation and crystal adhesion.

[0003] Polysaccharides, especially those derived from medicinal plants, have become promising candidates for preventing kidney stones due to their diverse biological activities (such as anti-inflammatory, antioxidant, and gut microbiota-regulating properties) and high safety. These properties enable polysaccharides to interfere with key processes in kidney stone formation, such as crystal nucleation, growth, and adhesion. For example, polysaccharides derived from the traditional Chinese medicine Lygodium japonicum (Hydrangea macrophylla)... Lygodium japonicum The polysaccharides in this herb promote the formation of calcium oxalate dihydrate with lower adhesion, thereby reducing crystal adhesion and endocytosis. This herb is derived from the Dai ethnic medicinal herb *Shield Wing Vine* (*Shield Wing Vine*). Aspidopterys obcordata Inulin-type fructans can inhibit pro-inflammatory cytokines such as TnFA and control crystal growth, thus reducing kidney stone formation. In addition, some natural polysaccharides can act as prebiotics, regulating the gut microbiota to influence various diseases. Patients with calcium oxalate kidney stones often exhibit gut microbiota dysbiosis, characterized by oxalate-degrading bacteria (such as *Oxalocarbamate*). Oxalobacter formigenes ) and Lactobacillus spp. Lactobacillus species Decreased abundance of polysaccharides increases urinary oxalate levels and the risk of kidney stones. The significant correlation between kidney stones, polysaccharides, and the gut microbiota prompts us to consider whether polysaccharides can influence kidney stone formation by modulating the composition and function of the gut microbiota.

[0004] Polygonum aviculare ( Polygonum aviculare L. Polygonum aviculare (Kelp) is an annual herbaceous plant belonging to the Polygonaceae family, widely distributed throughout the world, especially in temperate regions. As a well-known medicinal plant, it is commonly used in traditional medicine in Asia, South America, and Africa. In China, it has long been used in traditional Chinese medicine to promote blood circulation and as a diuretic to relieve urinary tract obstruction and stones. It is also a key ingredient in modern anti-urinary tract stone preparations (such as "Shenshitong Granules" and "Qinglin Granules"). Chemical studies have shown that Polygonum aviculare is rich in various phytochemicals, including flavonoids, saponins, coumarins, alkaloids, phenols, naphthoquinones, anthraquinones, and tannins. However, the specific bioactive components responsible for its anti-kidney stone effect have not yet been fully explored. Therefore, developing new drugs that can be used to prevent and / or treat kidney stones is of great significance. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide rhamnogalacturonic acid polysaccharide I, pharmaceutical compositions, preparation methods and applications thereof.

[0006] This invention provides a rhamnogalacturonic acid polysaccharide I, which is an acidic heteropolysaccharide mainly comprising rhamnose, arabinose, galactose and galacturonic acid; wherein the molar ratio of rhamnose, arabinose, galactose and galacturonic acid is 2~8:10~15:15~20:2~8.

[0007] The total carbohydrate content of the rhamnogalacturonic acid polysaccharide I is greater than 97%, of which the content of neutral sugar is 80-85% and the content of uronic acid is 15-20%.

[0008] Furthermore, the molar ratio of rhamnose, arabinose, galactose, and galacturonic acid is 5:12:17:5.

[0009] Furthermore, the backbone of the rhamnogalacturonic acid polysaccharide I is composed of alternating →2,4)-α-Rhap-(1→ and →4)-α-GalpA-(1→) residues, wherein the C4 site of →2,4)-α-Rhap-(1→ is linked to arabinogalactan and galactan.

[0010] Furthermore, the sugar residue types in the rhamnogalacturonic acid polysaccharide I include 1,2,4-Rhap, 1,4-GalpA, T-Galp, 1,4-Galp, 1,6-Galp, 1,3,4-Galp, and 1,4,6-Galp.

[0011] Furthermore, the sugar residue types in the rhamnogalacturonic acid polysaccharide I include T-Araf, 1,5-Araf, 1,3,5-Araf, 1,2,4-Rhap, 1,4-GalpA, T-Galp, 1,4-Galp, 1,6-Galp, 1,3,4-Galp, and 1,4,6-Galp in a molar ratio of 4:7:1:5:5:4:8:3:1:1.

[0012] Furthermore, the total carbohydrate content of the rhamnogalacturonic acid polysaccharide I is greater than 99%, of which the content of neutral sugars is 83.26% and the content of uronic acid is 16.71%; the weight-average molecular weight (M) of the rhamnogalacturonic acid polysaccharide I is... w The molecular weight is 67.1 kDa, and the number-average molecular weight (M) is 67.1 kDa n The value is 62.3 kDa, and the multidispersion index (d) is 1.08.

[0013] In this invention, "total carbohydrates" refers to the sum of all carbohydrates in rhamnogalacturonic acid polysaccharide I. "Neutral sugars" refers to sugars that are neutral in aqueous solution (pH value close to 7). "Glucuronic acid" refers to sugars containing aldehyde and carboxylic acid groups.

[0014] The present invention also provides a method for preparing the above-mentioned rhamnogalacturonic acid polysaccharide I, the method comprising the following steps:

[0015] (1) Extraction of crude polysaccharide: defatted Polygonum aviculare, extracted with alkali and impurities removed; mixed with alcohol solvent, centrifuged to collect solid; added water, centrifuged to collect supernatant to obtain crude polysaccharide;

[0016] (2) Purification: The crude polysaccharide was purified by sequentially passing it through QCS / MFC, DEAE Sepharose™ Fast Flow, and Sephacryl S-200 HR gel column chromatography, and eluted to obtain rhamnogalacturonic acid polysaccharide I.

[0017] Further, in step (1), the degreasing solvent is selected from one or more of ethyl acetate, acetone, and water; the alkali is an inorganic alkali; the extraction temperature is 70~110℃; the impurity removal includes decolorization, protein removal, and filtration.

[0018] Furthermore, the defatting refers to defatting with ethyl acetate and acetone, followed by water extraction with ultrasound at room temperature, repeated three times for 1 hour each time;

[0019] The alkali is a weakly alkaline solution, preferably potassium hydroxide with pH = 9;

[0020] The extraction temperature was 90℃, and the process was repeated three times, each time for 1 hour.

[0021] The decolorization was performed using 30% hydrogen peroxide; the protein removal was performed using the Sevag method.

[0022] Further, in step (2), the elution solvent is a sodium chloride solution; in the QCS / MFC, the elution is gradient elution, and the concentration of the sodium chloride solution is 0.1, 0.3 and 0.5 mol / L;

[0023] In the DEAE Sepharose™ Fast Flow, elution is gradient elution, and the concentrations of sodium chloride solution are 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L.

[0024] In the Sephacryl S-200 HR, isocratic elution was performed, and the concentration of the sodium chloride solution was 0.2 mol / L.

[0025] The present invention also provides the use of the above-mentioned rhamnogalacturonic acid polysaccharide I in the preparation of medicaments for the prevention and / or treatment of kidney stones.

[0026] Furthermore, the drug is an anti-inflammatory drug.

[0027] Furthermore, the drug is a drug that regulates the gut microbiota, reduces inflammation, and alleviates the retention of calcium oxalate crystals in the kidneys.

[0028] Furthermore, the drug is for increasing the levels of lactobacilli ( Lactobacillus Drugs that increase taurine production and inhibit Jun-TNFα transcription axis activation in macrophages.

[0029] The present invention also provides a pharmaceutical composition, which is a formulation prepared by adding pharmaceutically acceptable excipients to the above-mentioned rhamnogalacturonic acid polysaccharide I as the active ingredient.

[0030] Furthermore, the preparation is an oral preparation.

[0031] The present invention has achieved the following beneficial effects:

[0032] This invention isolates a novel rhamnogalacturonan-I (RG-I) from Polygonum aviculare, named PAPN1, and evaluates its anti-kidney stone effect using integrated multi-omics methods. The structure reveals that PAPN1 is an RG-I type pectin, its backbone composed of alternating →2,4)-α-Rhap-(1→) and →4)-α-GalpA-(1→) residues, wherein the C4 site of →2,4)-α-Rhap-(1→ is linked to arabinogalactan (composed of T / 1,5 / 1,3,5-α-Araf residues) and galactan (composed of T / 1,4 / 1,6 / 1,4,6 / 1,3,4-β-Galp residues). In in vivo experiments, oral administration of PAPN1 (20 mg / kg / day) significantly reduced calcium oxalate crystal deposition in the kidneys and improved renal function parameters. This invention reveals that the application of PAPN1 in drug development for the prevention and / or treatment of kidney stones has extremely important clinical significance and broad application prospects.

[0033] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0034] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0035] Figure 1 The following are the physicochemical and morphological properties of PAPN1. (A) Chromatographic elution profile using DEAE Sepharose™ Fast Flow. (B) Chromatographic elution profile using Sephacryl S-200 HR. (C) HPGPC chromatogram of PAPN1. (D) Chromatographic profile of the molecular weight distribution of PAPN1. (E) Monosaccharide composition analysis of PAPN1. (F) Scanning electron microscope (SEM) image of PAPN1.

[0036] Figure 2 Nuclear magnetic resonance (NMR) spectral analysis of PAPN1. (A) PAPN1 1 (B) 1H NMR spectrum of PAPN1 13 (C) CNMR spectrum of PAPN1. (D) HSQC spectrum of PAPN1. 1 H- 1 H COSY spectrum. (E) HMBC spectrum of PAPN1. (F) NOESY spectrum of PAPN1.

[0037] Figure 3 Nuclear magnetic resonance (NMR) spectral analysis of PAPN1-Ia and PAPN1-Ib. (A) Nuclear magnetic resonance (NMR) spectral analysis of PAPN1-Ia and PAPN1-Ib. 1 Comparison of H NMR spectra. (B) PAPN1-Ia and PAPN1-Ib 13 Comparison of C NMR spectra. (C) HSQC spectrum of PAPN1-Ia. (D) HSQC spectrum of PAPN1-Ib.

[0038] Figure 4 Determining the side chains of PAPN1-1a and PAPN1-1b. (A) HMBC spectrum of PAPN1-1a. (B) Connection mode of side chains in PAPN1-1a. (C) HMBC spectrum of PAPN1-1b. (D) Connection mode of side chains in PAPN1-1b.

[0039] Figure 5 Possible structures of polysaccharides. (A) Possible repeating unit of PAPN1. (B) Possible repeating unit of PAPN1-1a. (C) Possible repeating unit of PAPN1-1b.

[0040] Figure 6 PAPN1 is used to treat kidney inflammation and inhibit kidney stone formation. (A) VK staining and statistical analysis of calcium oxalate deposition in the kidneys. (B) The protective effect of PAPN1 on renal function. Detailed Implementation

[0041] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0042] The experimental materials used in the embodiments of this invention are as follows:

[0043] 1. Instruments and reagents

[0044] Scanning electron microscopy (SEM) analysis was performed using a Merlin Compact scanning electron microscope (Carl Zeiss GmbH, Germany). Nuclear magnetic resonance (NMR) spectroscopy experiments were performed on a Bruker Ascend 600 spectrometer (Bruker GmbH, Germany). Weight average molecular weight (M w ()) and number-average molecular weight (M nMeasurements were performed using an LC-20 size exclusion chromatography (SEC, Shimadzu Corporation, Japan) equipped with a DAWN HELEOS II multi-angle laser light scattering (MALLS, Wyatt Technologies, USA) and a RID-20A differential refractive index (RI, Shimadzu Corporation, Japan) detector. Ultraviolet (UV) and Fourier transform infrared (FT-IR) spectra were obtained using a UV-vis spectrophotometer (Shimadzu Corporation, Japan) and a PerkinElmer SpectrumOne FT-IR spectrophotometer (PerkinElmer Corporation, USA). Ion chromatography (IC) analysis was performed using a Thermo ICS5000 spectrometer (Thermo Fisher Scientific, USA), while gas chromatography-mass spectrometry (GC-MS) spectra were obtained using a GC-MS-QP2010 system (Shimadzu Corporation, Japan). All chemicals and reagents used in this invention were of analytical grade (Table 1).

[0045] Table 1 Chemical Reagent Information

[0046]

[0047]

[0048] 2. Experimental medicinal materials

[0049] The sphagnum moss used in the embodiments of the present invention ( Polygonum aviculare L. The whole herb was hand-harvested in July 2022 in a wild area at an altitude of 1600 to 2400 meters in Kangding City, Sichuan Province, China. The raw material was washed with water and dried indoors for storage in preparation for subsequent experiments. After the medicinal material was identified, the plant specimen was deposited at West China Hospital of Sichuan University, with the number SCIWCH-20220713.

[0050] Example 1: Preparation of PAPN1

[0051] 1. Extraction of PAPN: The dried Polygonum aviculare was pulverized and defatted with ethyl acetate and acetone. The defatted powder was then extracted with water using an ultrasonic method at room temperature, repeated three times for 1 hour each time. The three extracts were mixed and concentrated, and the residue was collected for further extraction. The residue was extracted with a weakly alkaline solution (KOH, pH = 9) at heating (90°C), repeated three times for 1 hour each time. The alkaline extract was decolorized with 30% hydrogen peroxide. Proteins were removed using the Sevag method, filtered, and the supernatant was collected and concentrated. The concentrated extract was mixed with ethanol at a volume ratio of 1:9, precipitated overnight, and the solid was collected by centrifugation. The solid was redissolved in deionized water and centrifuged again. The supernatant was collected, dialyzed for 5 days, and then the solutions were combined and lyophilized to obtain the crude polysaccharide extract PAPN.

[0052] 2. Purification of PAPN1: The polysaccharide extract PAPN was purified sequentially using QCS / MFC cryogel chromatography, DEAE Sepharose™ Fast Flow, and Sephacryl S-200 HR column chromatography. First, the crude polysaccharide extract was loaded onto a QCS / MFC cryogel column and eluted with NaCl solutions of concentrations of 0.1, 0.3, and 0.5 mol / L (each gradient elution volume was 120 mL). The polysaccharide content of the extracted fraction was determined using the phenol-sulfuric acid method, and bioactivity screening was performed simultaneously. The active components were concentrated on a DEAE Sepharose™ Fast Flow column (32 × 240 mm) and eluted with gradient NaCl solutions (0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mol / L) (each gradient elution volume was 100, 100, 80, 80, 60, 60 mL), separating the active polysaccharides. Final purification was then performed on a Sephacryl S-200 HR column, eluted isocratically with 0.2 mol / L NaCl. The purified fraction was dialyzed, concentrated, and lyophilized to obtain homogeneous polysaccharide PAPN1.

[0053] Example 2: Enzymatic hydrolysis of PAPN1

[0054] The glycosidases used in this invention include β-galactosidase (EC 3.2.1.23), endo-1,4-β-galactanase (EC 3.2.1.89), α-arabinofuranase (EC 3.2.1.55), and endo-1,5-α-arabinanase (EC 3.2.1.99), all of which were purchased from Sigma-Aldrich.

[0055] 100 mg of PAPN1 was dissolved in 50 mL of acetate buffer and digested with a combination of β-galactosidase and endo-1,4-β-galactanase. The solution was stirred at 37 °C for 12 h, and then the reaction was terminated by heating at 100 °C for 10 min. After purification by dialysis and gel column chromatography, the enzymatic hydrolysate PAPN1-Ia was obtained by lyophilization.

[0056] The preparation method of PAPN1-Ia is the same as that of PAPN1-Ia, except that β-galactosidase and endo-1,4-β-galactanase are replaced with α-arabinofuranase and endo-1,5-α-arabinanase for joint enzymatic digestion to obtain the enzymatic hydrolysis product PAPN1-Ib.

[0057] The following experimental examples demonstrate the beneficial effects of the present invention.

[0058] Experimental Example 1: Structure and Performance Characterization of PAPN1

[0059] I. Experimental Methods

[0060] 1. Structural Analysis

[0061] 1.1 General Chemical Analysis

[0062] This invention uses the phenol-sulfuric acid method to determine the total neutral sugar content of PAPN1, the m-hydroxybiphenyl colorimetric method to determine the uronic acid content, and the biuret acid (BCA) method to determine the protein content.

[0063] 1.2 Ultraviolet (UV) and Fourier Transform Infrared (FT-IR) Spectroscopy Analysis

[0064] The UV-Vis absorption spectrum of PAPN1 was measured in the wavelength range of 200-600 nm using a Shimadzu UV-vis spectrophotometer (Japan), while its infrared spectral characteristics were obtained in the wavenumber range of 4000-500 cm⁻¹ using a PerkinElmer Spectrum One FT-IR spectrophotometer (USA).

[0065] 1.3 Molecular weight analysis

[0066] This invention measures the average molecular weight of PAPN1, including weight-average molecular weight (M), by using a size exclusion chromatography system combined with multi-angle light scattering and refractive index detection. w ) and number-average molecular weight (M n ), and the multi-dispersion index (M w / M n ).

[0067] 1.4 Monosaccharide Composition Analysis

[0068] Five mg of polysaccharide PAPN1 sample was treated with 2 mL of 3 M trifluoroacetic acid at 120 °C for 3 hours. After hydrolysis, the solution was evaporated under a nitrogen stream. The resulting residue was dissolved in methanol and then dried again under a nitrogen stream. The dried product was reconstituted in deionized water, centrifuged, and the supernatant was analyzed by ion chromatography on a Dionex Carbopac™ PA10 column (4 × 250 mm) to identify the monosaccharide components.

[0069] 1.5 Scanning Electron Microscope (SEM)

[0070] PAPN1 was dissolved in deionized water to form a homogeneous solution, rapidly frozen in liquid nitrogen, and then freeze-dried to obtain a solid sample while maintaining the structural integrity of the polysaccharide. The samples were carefully attached to double-sided conductive tape fixed to a SEM sample holder. Due to the non-conductive nature of the polysaccharide, the samples were first coated with platinum at 20 mA for 30 seconds. Subsequently, their morphology was examined using a Merlin Compact SEM (Carl Zeiss AG, Germany) at an accelerating voltage of 10 kV.

[0071] 1.6 Congo Red Experiment

[0072] Dissolve 20 mg of PAPN1 in 5 mL of deionized water to obtain a 4 mg / mL PAPN1 solution. Mix 0.7 mL of the PAPN1 solution with 0.7 mL of 0.32 mM Congo red solution. Add 1.4 mL of deionized water or NaOH solution (0.2 M, 0.4 M, 0.6 M, 0.8 M, 1.0 M) to each mixture and mix thoroughly. The final concentration of PAPN1 is 1 mg / mL, the final concentration of Congo red is 80 μM, and the final concentrations of NaOH are 0, 0.1, 0.2, 0.3, 0.4, or 0.5 M, respectively. Scan the absorption spectrum from 400 to 600 nm using a UV-Vis spectrophotometer.

[0073] 1.7 Methylation and Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

[0074] The polysaccharide PAPN1 sample was first treated with sodium borodeuteride (NaBD4) to convert the uronic acid into the corresponding neutral sugar. The carboxyl-reduced polysaccharide was then methylated in dimethyl sulfoxide using sodium hydroxide and iodomethane. Subsequently, the sample was hydrolyzed with trifluoroacetic acid, reduced with NaBD4, and acetylated with 1-methylimidazole and acetic anhydride. Partially methylated derivatives were analyzed using a Shimadzu GCMS-QP2010 system equipped with a Restek Rxi-5MS silica column (30 m × 0.25 mm, 0.25 μm) using a constant pressure split injection. The initial flow rate was 1 mL / min, and the inlet and interface temperatures were maintained at 280 °C. During injection, the column temperature was held at 80 °C for 5 min, then increased to 140 °C at 10 °C / min, then to 210 °C at 4 °C / min, and finally to 310 °C at 20 °C / min, held for 4 min. Helium was used as the carrier gas, and the ion source temperature was maintained at 200 °C. Data analysis was performed using Shimadzu GC-MS Solution software (version 2.10).

[0075] 1.8 Nuclear Magnetic Resonance (NMR) Spectroscopy Studies

[0076] The polysaccharide PAPN1 sample was dissolved in deuterated water (D₂O) and freeze-dried, a process repeated three times to achieve the substitution of volatile protons with deuterium. The sample was then redissolved in D₂O. One-dimensional (¹H and ¹³C) and two-dimensional NMR experiments, including ¹H-¹H COSY, HSQC, HMBC, and NOESY, were performed at 298 K using a Bruker Ascend™ 600 NMR spectrometer. Tetramethylsilane (TMS) was used as an internal standard for chemical shift calibration.

[0077] 2. Bioactivity assay

[0078] 2.1 Oxalate-induced kidney stone mouse model and sample collection

[0079] This invention utilizes a well-established and methodologically rigorous mouse model of kidney stones. Calcium oxalate crystal deposition was induced by daily intraperitoneal injection of dihydroxyacetate (100 mg / kg body weight) for 7 consecutive days. Each experimental group was administered PAPN1 orally via gavage at a dose of 20 mg / kg / day. After model establishment and treatment, mice were anesthetized using isoflurane inhalation. Fecal samples were collected, followed by blood collection from the retroorbital vein and urine collection. Animals were then euthanized, and kidney tissue was dissected for subsequent analysis. Blood samples were centrifuged to collect plasma, which was then subjected to non-targeted metabolomics analysis. Fecal samples were used for 16S rRNA gene sequencing. Kidney tissue was used for RNA sequencing, Von Kossa staining (calcium crystal staining), Western blotting, and other assays. The animal experiments of this invention have received ethical approval from the Animal Experiment Center of West China Hospital, Sichuan University (Approval No.: 20240402005). All procedures involving animals were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines to ensure ethical compliance.

[0080] 2.3. Von Kossa dyeing

[0081] Kidney tissue was fixed with 4% paraformaldehyde for 48 hours to maintain cell structure and prevent degradation. After fixation, the tissue was dehydrated by a gradient of ethanol solutions (70%, 95%, 100%), the alcohol was removed with xylene to enhance tissue transparency, and finally embedded in paraffin for sectioning. The tissue was sectioned into 5 μm sections using a microtome and attached to glass slides. The slides were heat-dried to ensure adhesion and stored at room temperature until staining. VK staining was used to detect calcium deposition in the kidney tissue sections. The specific steps are as follows:

[0082] (1) Dewaxing and rehydration: Paraffin-embedded sections were dewaxed three times with xylene (3 minutes each time), and then rehydrated by passing through a gradient of ethanol (100% ethanol, 95% ethanol, 70% ethanol) to distilled water.

[0083] (2) Silver nitrate incubation: Incubate the slices with 5% silver nitrate solution under ultraviolet light for 20-30 minutes.

[0084] (3) Rinsing: The sections are rinsed with distilled water multiple times to remove excess silver nitrate and prevent non-specific staining.

[0085] (4) Removal of unreacted silver: The sections were treated with 5% sodium thiosulfate for 5 minutes to remove unreacted silver and ensure the specificity of staining.

[0086] (5) Counterstaining: The sections were counterstained with 0.1% Nucleotide Red for 5 minutes to reveal the cell nuclei, and then rinsed with distilled water to remove excess dye and prevent turbidity during dehydration.

[0087] (6) Dehydration and clearing: The sections were dehydrated by gradient ethanol (70%, 95%, 100%, each soaked 10 times), washed in xylene three times (each soaked 10 times), and finally mounted with a permanent mounting medium.

[0088] 3. Statistical Analysis

[0089] All data are expressed as mean ± standard error (SEM). Appropriate statistical methods were used for comparisons, and a two-sided p-value <0.05 was considered statistically significant. Pearson correlation analysis was used to assess the relationship between the two groups. All statistical calculations were performed using SPSS software (version 23.0, SPSS Inc.) and GraphPad Prism (version 8.0, La Jolla, CA, USA).

[0090] II. Experimental Results

[0091] 1. Structural characterization of PAPN1

[0092] 1.1 General Analysis

[0093] Ion exchange and gel column chromatography were used for separation, and the characteristic spectral lines of the polysaccharide were analyzed by ultraviolet (UV) and Fourier transform infrared (FT-IR) spectroscopy, confirming that the active polysaccharide PAPN1 was successfully purified in this invention. Figure 1 A and B). Chemical analysis showed that PAPN1 contained over 99% total carbohydrates, of which 83.26% were neutral sugars (determined by the phenol-sulfuric acid method) and 16.71% were uronic acids (determined by the m-hydroxybiphenyl colorimetric method). High-performance gel permeation chromatography (HPGPC) was used to assess the homogeneity of PAPN1; the chromatogram showed a single symmetrical peak, indicating that the polysaccharide was homogeneous with a purity exceeding 98%. Figure 1 C).

[0094] 1.2 Molecular weight and monosaccharide composition of PAPN1

[0095] The laser scattering (LS) and dynamic light scattering (dRI) curves of PAPN1 both exhibited single-peak characteristics, further confirming that PAPN1 is a homogeneous polysaccharide. Figure 1 D). The molecular weight (Mw) of PAPN1 was determined to be 67.1 kDa, the molecular weight (Mn) to be 62.3 kDa, and the polydispersity index (d) to be 1.08. The d value being close to 1 indicates that PAPN1 has a highly consistent molecular weight distribution and a narrow molecular weight range. Monosaccharide composition analysis showed that PAPN1 is an acidic heteropolysaccharide composed of rhamnose (Rha), arabinose (Ara), galactose (Gal), and galacturonic acid (GalA), with a molar ratio of approximately 5:12:17:5. Figure 1 E).

[0096] 1.3 Morphological characteristics of PAPN1

[0097] After freeze-drying, PAPN1 appears as a white, amorphous, and fluffy solid. SEM analysis shows that PAPN1 has an irregular hollow vesicle morphology, mixed with coiled fibrous structures and twisted ribbon-like structures. Figure 1 F). The Congo red experiment showed that PAPN1 does not exhibit a triple helix structure.

[0098] 1.4. Analysis of glycosidic bond linkage through methylation experiments

[0099] Methylation and gas chromatography-mass spectrometry (GC-MS) analysis identified 10 types of methylated aldehydes (PMAAs), accounting for more than 99% of the total composition, indicating that PAPN1 is mainly composed of 10 sugar residues: T-Araf, 1,5-Araf, 1,3,5-Araf, 1,2,4-Rhap, 1,4-GalpA, T-Galp, 1,4-Galp, 1,6-Galp, 1,3,4-Galp, and 1,4,6-Galp, with molar percentages of 9.9%, 18.1%, 2.6%, 12.6%, 12.9%, 10.2%, 20.8%, 7.6%, 2.5%, and 2.4%, respectively, corresponding to an approximate molar ratio of 4:7:1:5:5:4:8:3:1:1 (Table 1). PAPN1 contains 20.1% terminal residues (T-Araf and T-Galp) and 20.1% branched residues (1,3,5-Araf, 1,2,4-Rhap, 1,3,4-Galp, and 1,4,6-Galp), indicating that it is a highly branched polysaccharide. Furthermore, in PAPN1, GalpA exists only as a 1,4-GalpA bond (12.9%), while Rha exists only as a 1,2,4-Rhap bond (12.6%), with both proportions being roughly equal. This suggests that PAPN1 may be an RG-I type pectin. In addition, PAPN1 exhibits glycosidic bonds including T / 1,5 / 1,3,5-Araf and T / 1,4 / 1,6 / 1,3,4 / 1,4,6-Galp, indicating that its side chains consist of arabinose, galactose, or two structurally different arabinogalactoses (AG-I and AG-II). Therefore, the bonding mode of PAPN1 conforms to the characteristic structural features of RG-I pectin.

[0100] To further elucidate the structural composition of PAPN1, enzymatic treatment targeting the cleavage of arabinose and galactose residues was employed (Example 2). Methylation experiments showed that PAPN1-Ia is mainly composed of sugar residues T-Araf, 1,5-Araf, 1,3,5-Araf, 1,4-GalpA, 1,2,4-Rhap, and 1,2-Rhap, with molar percentages of 17.6%, 31.2%, 4.8%, 23.0%, 13.1%, and 8.7%, respectively, corresponding to an approximate molar ratio of 4.7:1.5:3.2 (Table 1). The similar content of Rha residues (1,2,4-Rhap and 1,2-Rhap) and Gala residues (1,4-GalpA) in PAPN1-Ia further confirms that PAPN1 belongs to the RG-I type pectin. Furthermore, the molar ratios of T-Araf, 1,5-Araf, and 1,3,5-Araf / 1 in PAPN1-Ia are also consistent with the methylation results of PAPN1. The glycosidic bonds in PAPN1-1b were identified as T-Galp, 1,4-Galp, 1,6-Galp, 1,3,4-Galp, 1,4,6-Galp, 1,4-GalpA, 1,2,4-Rhap, and 1,2-Rhap, with molar percentages of 14.2%, 29.1%, 11.0%, 3.9%, 3.5%, 18.7%, 7.9%, and 10.6%, respectively, corresponding to an approximate molar ratio of 4:8:3:1:1:5:2:3 (Table 1). Molar ratio analysis of residues in PAPN1, PAPN1-Ia, and PAPN1-Ib indicates that the side chains of PAPN1 belong to only two types: arabinogalactan and galactan. Furthermore, based on the molar ratio of 1,2-Rhap residues in PAPN1-Ia and PAPN1-Ib, the estimated ratio of arabinogalactan to galactan side chains in PAPN1 is 3:2 (Table 1).

[0101] Table 1. Methylation analysis of PAPN1, PAPN1-Ia, and PAPN1-Ib using gas chromatography-mass spectrometry.

[0102]

[0103] a ND = Not detected.

[0104] 1.5 Nuclear Magnetic Resonance (NMR) Spectroscopic Analysis of PAPN1

[0105] The precise structure of PAPN1 was elucidated through comprehensive analysis of its one-dimensional and two-dimensional NMR spectra of PAPN1 and its enzymatic hydrolysis products (PAPN1-Ia and PAPN1-Ib). 1 H and 13The C NMR chemical shift distributions are as follows: Figure 2 , 3 As shown in Tables 2, 3, and 4.

[0106] Table 2. Nuclear magnetic resonance chemical shifts (δ, ppm) of PAPN1 at ¹H and ¹³C.

[0107]

[0108] Table 3. ¹H and ¹³C NMR chemical shifts (δ, ppm) of PAPN1-Ia

[0109]

[0110] Table 4. ¹H and ¹³C NMR chemical shifts (δ, ppm) of PAPN1-Iba

[0111]

[0112] The connection sequences and substitution sites of the PAPN1 main chain and branches were elucidated using HMBC and NOESY spectra. Figure 2 E and 2F). HMBC and NOESY spectra further confirm that the PAPN1 backbone is composed of repeating disaccharide units [→2)-α-Rhap-(1→4)-α-GalpA-(1→] ( Figure 2 F). The HMBC spectra of PAPN1-Ia and PAPN1-Ib provide further evidence for the PAPN1 backbone structure. Figure 4 Studies have confirmed that a single repeating unit of PAPN1 consists of five [→2,4)-α-Rhap-(1→4)-α-GalpA-(1→] disaccharide units, with three arabinose and two galactose side chains attached to the C4 position of →2,4)-α-Rhap-(1→). HMBC and NOESY spectra ( Figure 2 E, Figure 2F) revealed that the PAPN1 side chain contains α-Araf-(1→5)-α-Araf-(1→]n, α-Araf-(1→5)-[α-Araf-(1→3)]-α-Araf-(1→5)-α-Araf-(1→]m, β-Galp-(1→[4)-β-Galp-(1→]x and β-Galp-(1→[6)-β-Galp-(1→)6)-(β-Galp-(1→4)-β -Galp-(1→3)-β-Galp-(1→[4)-β-Galp-(1→]y. Furthermore, based on the Ara residue ratio determined by methylation experiments, it is inferred that each PAPN1 repeating unit contains two α-Araf-(1→5)-α-Araf-(1→]n side chains and one α-Araf-(1→5)-[α-Araf-(1→3)]-α-Araf-(1→5)-α-Araf-(1→]m side chain, where m... + 2n = 7; it is inferred that each PAPN1 repeating unit contains a β-Galp-(1→[4)-β-Galp-(1→]x sidechain and a β-Galp-(1→[6)-β-Galp-(1→)6)-(β-Galp-(1→4)-β-Galp-(1→3)-β-Galp-(1→[4)-β-Galp-(1→]y sidechain, where x + y equals 8. Figure 5 The proposed glycosidic bond structure of PAPN1 is shown.

[0113] 2. Bioactivity

[0114] 2.1 PAPN1 can inhibit the formation of kidney stones.

[0115] VK staining showed a large number of black-stained calcium oxalate crystals in the kidneys of mice in the model group. Compared with the model group, the crystal area in the PAPN1 treatment group was significantly reduced. Figure 6 A) indicates that PAPN1 can effectively inhibit the formation or retention of crystals in the kidneys. Renal function was assessed by measuring blood and urine biochemical parameters standardized to creatinine levels. The model group showed elevated urea / creatinine ratios, sodium / creatinine ratios, chloride / creatinine ratios, and total protein / creatinine ratios in urine, indicating impaired glomerular filtration function and renal tubular dysfunction. PAPN1 treatment significantly improved these indicators, restoring them to levels close to those of the control group ( Figure 6 B).

[0116] III. Conclusion

[0117] This invention utilizes a bioassay-guided fractionation method to isolate a novel RG-I type pectin—PAPN1—from *Polygonum aviculare*. The chemical structure of PAPN1 is as follows: the main chain consists of alternating →2,4)-α-Rhap-(1→) and →4)-α-GalpA-(1→) residues, wherein the C4 site of →2,4)-α-Rhap-(1→) is linked to arabinogalactan (composed of T / 1,5 / 1,3,5-α-Araf residues) and galactan (composed of T / 1,4 / 1,6 / 1,4,6 / 1,3,4-β-Galp residues). Mechanistic studies have shown that PAPN1 can inhibit kidney stone formation and protect kidney function.

Claims

1. A method for preparing rhamnogalacturonic acid polysaccharide I, characterized in that, The method includes the following steps: (1) Extraction of crude polysaccharides: After crushing the Polygonum aviculare, it was defatted with ethyl acetate and acetone to obtain defatted powder; The defatted powder was extracted with water using an ultrasonic method at room temperature. The extraction was repeated three times, each time for 1 hour. The water extracts were combined and concentrated to obtain the water extraction residue. The water extraction residue was extracted using a potassium hydroxide solution with pH=9 at 90°C. The extraction was repeated three times, each time for 1 hour, and the alkaline extracts were combined. The alkaline extract was subjected to the following treatments in sequence: decolorization with 30% hydrogen peroxide, removal of protein using the Sevag method, filtration, collection of supernatant and concentration. The concentrated extract was mixed with an alcohol solvent, precipitated, and the solid was collected by centrifugation. The solid was dissolved in water, centrifuged, and the supernatant was collected. The supernatant was then dialyzed and freeze-dried to obtain a crude polysaccharide extract. (2) Purification: The crude polysaccharide extract was sequentially purified by QCS / MFC, DEAE Sepharose™ Fast Flow, and Sephacryl S-200 HR gel column chromatography. (i) A QCS / MFC gel column was used for gradient elution with sodium chloride solutions of concentrations of 0.1, 0.3 and 0.5 mol / L, with each gradient elution volume being 120 mL. The polysaccharide content of the extracted fraction was determined by the phenol-sulfuric acid method, and bioactivity screening was performed to obtain the polysaccharides. (ii) The polysaccharide obtained in step (i) was subjected to gradient elution with sodium chloride solutions of concentrations of 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mol / L using a DEAE Sepharose™ Fast Flow gel column. The elution volumes for each gradient were 100, 100, 80, 80, 60 and 60 mL, respectively. The active polysaccharide obtained by elution with 0.1 mol / L sodium chloride solution was then separated and collected. (iii) The polysaccharide obtained in step (ii) was eluted isocratically with a 0.2 mol / L sodium chloride solution using a Sephacryl S-200 HR gel column; (iv) Collect the eluent from step (iii), and then dialyze, concentrate, and freeze-dry to obtain the rhamnogalacturonic acid polysaccharide I.

2. A rhamnogalacturonic acid polysaccharide I, characterized in that, The rhamnogalacturonic acid polysaccharide I is prepared by the method described in claim 1.

3. Use of the rhamnogalacturonic acid polysaccharide I according to claim 1 in the preparation of a medicament for the prevention and / or treatment of kidney stones.

4. The use according to claim 3, characterized in that, The drug in question is an anti-inflammatory drug.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition is a formulation prepared by adding pharmaceutically acceptable excipients to rhamnogalacturonic acid polysaccharide I as the active ingredient as described in claim 2.

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