She medicine snoring polysaccharide for treating knee osteoarthritis and extraction method thereof

Abstract

The invention relates to the technical field of natural pharmaceutical chemistry and biological medicine, in particular to traditional She medicine snoring polysaccharide for treating knee osteoarthritis and an extraction method thereof, the main chain of the snoring polysaccharide is composed of-1)-beta-D-Fruf-(2-and-1, 6)-beta-D-Fruf-(2-glucosidic bonds, the branch chain is connected to the O-6 site, the monosaccharide is mainly fructose (the molar ratio is larger than or equal to 70%), and the content of the polysaccharide is larger than or equal to 10%. The invention relates to a high-efficiency extraction process, which contains a small amount of glucose and galactose, promotes SUMOylation modification of HIF-1alpha at a K391, K477 or K674 site by inhibiting expression of a SUMOylation enzyme SENP1, further induces ubiquitination degradation of the HIF-1alpha and plays a role in cartilage protection, and the high-efficiency extraction process provided by the technical scheme comprises the following steps of: crushing raw materials, performing ultrasonic-assisted hot water extraction and ethanol precipitation to obtain crude polysaccharide; and performing DEAE-52 anion exchange chromatography and SephacrylS-300 gel filtration chromatography purification, and performing freeze drying to obtain the high-purity active polysaccharide which has good stability, targeting property and treatment effect.

Description

Technical Field

[0001] This invention relates to the fields of natural product chemistry and biomedicine, specifically to a polysaccharide derived from the She ethnic medicine Ga Gou Lu for treating knee osteoarthritis and its extraction method. Background Technology

[0002] Knee osteoarthritis (KOA) is a degenerative disease characterized by degeneration of articular cartilage, inflammation of the synovium, and osteophyte formation. Its global incidence exceeds 15%, and is increasing annually with population aging. KOA patients often experience joint pain and limited mobility, and in advanced stages can lead to disability, severely impacting their quality of life.

[0003] Current clinical treatment methods mainly rely on symptomatic treatment:

[0004] Nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen and celecoxib, reduce prostaglandin synthesis by inhibiting cyclooxygenase (COX), thereby relieving pain and inflammation. However, long-term use can easily lead to gastrointestinal ulcers, cardiovascular risks, and kidney damage (occurrence rate of about 10-20%).

[0005] Opioid analgesics, such as oxycodone, are only used for severe pain, but have serious side effects such as addiction and respiratory depression.

[0006] Disease-Modifying Osteoarthritis Drugs (DMOADs): such as glucosamine, have controversial efficacy and cannot reverse the degenerative process of cartilage.

[0007] Existing drugs are unable to simultaneously achieve the therapeutic goals of "anti-inflammatory and analgesic, cartilage protection, and slowing disease progression," and their long-term safety is insufficient. There is an urgent need to develop new therapeutic drugs with novel mechanisms, definite efficacy, and low toxicity.

[0008] Research foundation and shortcomings of She medicine Ga Gou Lu

[0009] Melastoma dodecandrum Lour. is a plant of the Melastomataceae family and a traditional medicinal plant of the She ethnic group in my country. Its dried whole herb has the effects of "clearing heat and dampness, reducing swelling and relieving pain, and promoting blood circulation and unblocking collaterals". It is often used in folk medicine to treat diseases such as rheumatoid arthritis and traumatic injuries, but its pharmacodynamic material basis and mechanism of action have not yet been clarified.

[0010] Existing research has the following limitations:

[0011] The active ingredients are unknown: studies on the chemical composition of Gagolu have mostly focused on small molecules such as flavonoids and tannins, while systematic research on polysaccharide components is lacking, and their chemical structures (such as glycosidic bond type, branch position, and monosaccharide composition) have not yet been clarified.

[0012] The mechanism of action is unclear: Although its anti-inflammatory activity is known, its association with key KOA targets (such as HIF-1α and SENP1) has not been revealed, especially the mechanism of "SUMOylation modification regulation" is a blank.

[0013] The extraction process is crude: existing plant polysaccharide extraction methods (such as hot water extraction and ethanol precipitation) lack targeted optimization and have problems such as low yield (<2%), insufficient purity (<60%), and destruction of active ingredients, which cannot meet the needs of drug development.

[0014] Delayed formulation development: No specific formulations suitable for the treatment of KOA (such as intra-articular targeted delivery systems) have been developed, resulting in low bioavailability and difficulty in accumulating and exerting their effects at the lesion site.

[0015] (II) Technical Solution

[0016] To achieve the above objectives, the present invention provides the following technical solution: A She ethnic medicine polysaccharide for treating knee osteoarthritis, comprising gagolu polysaccharide, wherein the main chain of the gagolu polysaccharide is composed of →1)-β-D-Fruf-(2→ and →1,6)-β-D-Fruf-(2→ glycosidic bonds, with side chains linked at positions O-6; the gagolu polysaccharide can regulate the SUMOylation modification of HIF-1α at K391, K477, or K674 sites by inhibiting the expression of the deSUMOylase SENP1, thereby promoting the ubiquitination and degradation of HIF-1α.

[0017] Preferably, the monosaccharide composition of the Gaguru polysaccharide is mainly fructose, and also includes glucose and galactose, wherein the molar percentage of fructose is ≥70%.

[0018] More preferably, the weight-average molecular weight of the Gaguru polysaccharide is 50-150 kDa, and the molecular weight distribution index (Mw / Mn) is 1.2-1.8.

[0019] Further preferably, the product also includes a therapeutically effective amount of gaccharin polysaccharide and pharmaceutically acceptable excipients selected from one or more diluents, disintegrants, binders, lubricants, or solvents.

[0020] Preferably, the dosage form of the Gagolu polysaccharide is an oral preparation, an intra-articular injection, or a nanocarrier delivery system; the oral preparation includes tablets, capsules, or oral liquids, and the nanocarrier delivery system includes liposomes, chitosan nanoparticles, or hyaluronic acid nanogels.

[0021] A further preferred method for extracting polysaccharides from the She ethnic medicine Ga Gou Lu for treating knee osteoarthritis includes the following steps:

[0022] S1. Raw material pretreatment: Take the dried whole herb of the She medicine Ga Gou Lu, crush it and pass it through a 40-60 mesh sieve to obtain medicinal powder;

[0023] S2. Hot water extraction: Mix the medicinal powder with distilled water at a mass-volume ratio of 1:15-1:25, extract at 80-90℃ for 2-3 hours, and filter to collect the first filtrate;

[0024] S3. Secondary extraction: The residue is extracted once more under the conditions of step S2, and the secondary filtrate is collected.

[0025] S4. Concentration and precipitation: Combine the two filtrates, concentrate under reduced pressure to 1 / 5-1 / 4 of the original volume, add 3-5 times the volume of 95% ethanol, let stand at 4℃ for 12-24 hours, centrifuge to collect the precipitate, and obtain crude polysaccharide.

[0026] S5. Purification: The crude polysaccharide was dissolved in distilled water and purified by DEAE-52 anion exchange chromatography and Sephacryl S-300 gel filtration chromatography. The main peak fraction was collected and freeze-dried to obtain the purified polysaccharide.

[0027] Preferably, in step S2, ultrasonic assistance is used during the extraction process, with stirring every 30 minutes, and the power of the ultrasonic assistance is 300-500W and the frequency is 25-40kH.

[0028] Preferably, in step S5, the elution conditions for DEAE-52 anion exchange chromatography are: gradient elution with 0.1-0.5 mol / L NaCl solution at a flow rate of 1-2 mL / min; and the eluent for Sephacryl S-300 gel filtration chromatography is 0.1 mol / L NaCl solution at a flow rate of 0.5-1 mL / min.

[0029] More preferably, in step S4, the centrifugation temperature is 4°C, the centrifugation speed is 8000-10000 rpm, and the centrifugation time is 10-15 minutes; in step S5, the freeze-drying temperature is 50 to -40°C, and the vacuum degree is ≤10Pa.

[0030] (III) Beneficial Effects

[0031] Compared with the prior art, the present invention provides a polysaccharide derived from the She ethnic medicine Ga Gou Lu for treating knee osteoarthritis and its extraction method, which has the following beneficial effects:

[0032] This technical solution possesses significant pharmacological activity and promising clinical application prospects. The main chain of this Gaguru polysaccharide is composed of →1)-β-D-Fruf-(2→ and →1,6)-β-D-Fruf-(2→ glycosidic bonds, with branches attached at the O-6 positions, exhibiting a well-defined structural characteristic. Its monosaccharide composition is mainly fructose (molar percentage ≥70%), with small amounts of glucose and galactose. The weight-average molecular weight is 50–150 kDa, with a uniform molecular weight distribution (Mw / Mn = 1.2–1.8), demonstrating good stability and biocompatibility.

[0033] This Gaguru polysaccharide promotes SUMOylation of HIF-1α at K391, K477, or K674 sites by specifically inhibiting the expression of the deSUMOylase SENP1, thereby enhancing its ubiquitination and degradation. It effectively regulates the hypoxia signaling pathway in chondrocytes, inhibits the release of inflammatory factors and the expression of matrix degrading enzymes, and alleviates cartilage degeneration, thus playing a role in the treatment of knee osteoarthritis.

[0034] This technical solution employs a scientifically sound and efficient extraction method, utilizing hot water extraction combined with ultrasound-assisted extraction (300–500W, 25–40kHz) to significantly improve polysaccharide extraction rate and activity retention. High-purity separation is achieved through a combination of DEAE-52 anion exchange and Sephacryl S-300 gel filtration chromatography. The obtained polysaccharides can be formulated with pharmaceutically acceptable excipients to create tablets, capsules, oral liquids, or intra-articular injections. They can also be used to construct targeted delivery systems such as liposomes, chitosan nanoparticles, or hyaluronic acid nanogels, enhancing local drug concentration and efficacy. The process is stable, safe, and allows for diverse dosage forms, possessing broad development value and industrialization potential. Attached Figure Description

[0035] Figure 1 This is a schematic diagram illustrating the extraction and structural analysis of the polysaccharide from Gaguru in this invention;

[0036] Figure 2 This is a schematic diagram of the nuclear magnetic resonance (NMR) structure of the polysaccharide gagosin of the present invention;

[0037] Figure 3 This is a schematic diagram illustrating the regulation of HIF-1α post-translational modification balance by gagosin according to the present invention;

[0038] Figure 4 This is a schematic diagram illustrating the inhibition of macrophage glycolytic metabolism by the polysaccharide of Gaguru in this invention;

[0039] Figure 5 This is a schematic diagram illustrating how the polysaccharide gaguru of the present invention improves bone and joint damage in KOA rats. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Please see Figure 1-5 The present invention discloses a herbal polysaccharide called Gagoulu for treating knee osteoarthritis, comprising Gagoulu polysaccharide, wherein the main chain of Gagoulu polysaccharide is composed of →1)-β-D-Fruf-(2→ and →1,6)-β-D-Fruf-(2→ glycosidic bonds, with side chains attached at positions O-6; the Gagoulu polysaccharide can regulate the SUMOylation modification of HIF-1α at K391, K477 or K674 sites by inhibiting the expression of deSUMOylase SENP1, thereby promoting the ubiquitination and degradation of HIF-1α.

[0042] Structure and therapeutic mechanism of polysaccharide from She medicine Ga Gou Lu

[0043] Chemical Structure Basics

[0044] The structure of gagosin (MLP) is the material basis for its therapeutic effects:

[0045] Main chain structure: The linear main chain is formed by alternating →1)-β-D-Fruf-(2→ and →1,6)-β-D-Fruf-(2→ glycosidic bonds (β-D-Fruf is β-D-fructofuranose, a five-membered ring structure). This linkage gives the polysaccharide good water solubility and molecular flexibility, enabling it to specifically bind to biological targets (such as SENP1 protein).

[0046] Monosaccharide type and configuration: "β-D-Fruf" refers to β-D-fructofuranose, which is the core monosaccharide unit that makes up this polysaccharide (furanose has a five-membered ring structure and is a common form of fructose).

[0047] Main chain glycosidic bond:

[0048] "→1)-β-D-Fruf-(2→" indicates that two adjacent β-D-fructofuranoses are connected by "carbon 1-carbon 2" (that is, the C1 of the previous fructose and the C2 of the next fructose are connected by a glycosidic bond);

[0049] “→1,6)-β-D-Fruf-(2→” indicates that some adjacent fructoses are connected by “carbon at position 1 and carbon at position 6-carbon at position 2” (the C1 and C6 of the previous fructose are simultaneously connected to the C2 of the next fructose), forming a branch point in the main chain;

[0050] Branching positions: Branches are attached to the O-6 positions of the fructose units in the main chain, forming a multi-branched three-dimensional structure, which increases the molecular surface area and improves the efficiency of interaction with target proteins.

[0051] Monosaccharide composition: Fructose is the main monosaccharide (molar percentage ≥70%), supplemented by glucose and galactose. This composition pattern has a higher affinity for sugar recognition receptors (such as mannose receptors) on the surface of macrophages, which is conducive to cellular uptake.

[0052] Molecular mechanisms of treating knee osteoarthritis

[0053] MLP intervenes in the inflammatory progression and cartilage degeneration of knee osteoarthritis (KOA) by regulating protein post-translational modification networks. The core mechanism is as follows:

[0054] Inhibition of SENP1 expression: SENP1 is a deSUMOylating enzyme that removes SUMO modification of HIF-1α and maintains its stability. MLP reduces the deSUMOylation effect on HIF-1α by inhibiting SENP1 expression;

[0055] Regulation of HIF-1α SUMOylation: HIF-1α is a key transcription factor that regulates macrophage metabolic reprogramming. SUMOylation at its K391, K477, and K674 sites can enhance its ubiquitination degradation signal (SUMOylation and ubiquitination compete for binding at lysine sites).

[0056] Promoting HIF-1α ubiquitination and degradation: MLP promotes the ubiquitination and degradation of HIF-1α by enhancing SUMOylation modification at the above sites, thereby reducing its transcriptional activity;

[0057] Improving macrophage and chondrocyte function: After HIF-1α activity was reduced, the expression of key glycolytic enzymes (HK2, GLUT1) was suppressed, lactate and ROS production was reduced, and macrophages were polarized to the anti-inflammatory M2 phenotype. At the same time, the expression of chondrocyte catabolic genes (MMP13, ADAMTS5) was downregulated, while the expression of anabolism genes (COL2A1, ACAN) was upregulated, which improved the imbalance of cartilage metabolism and delayed the progression of KOA.

[0058] The extraction method is based on the water solubility, molecular weight differences, and charge properties of polysaccharides. It achieves efficient extraction through "selective dissolution-separation purification," and its core principles include:

[0059] Hot water extraction: Taking advantage of the high solubility of polysaccharides in hot water, the cell structure of the gac cell is destroyed by hot water at 80-90℃, allowing the polysaccharides to be released from the intercellular matrix into the solution;

[0060] Ethanol precipitation: Polysaccharides are insoluble in high-concentration ethanol (≥70%). By adding 3-5 times the volume of 95% ethanol, the solubility of polysaccharides is reduced, causing them to precipitate and separate from water-soluble impurities (such as small molecule sugars and amino acids).

[0061] Chromatographic purification: DEAE-52 anion exchange chromatography utilizes the charge interaction between polysaccharides and resin (differences in charge of different polysaccharides) to achieve preliminary separation; SephacrylS-300 gel filtration chromatography further purifies based on molecular weight differences (polysaccharide molecular weight 50-150kDa), finally yielding high-purity polysaccharides.

[0062] Preferred technical solution

[0063] Monosaccharide composition (fructose molar percentage ≥ 70%)

[0064] Principle: The furan ring structure of fructose has a higher affinity for the active pocket of SENP1 protein (rich in hydrophobic amino acids), which can enhance the inhibitory effect of MLP on SENP1. Simultaneously, high fructose content increases the water solubility of polysaccharides (high fructose hydroxyl density), facilitating in vivo transport and cellular uptake. Experiments have shown that when the fructose content is <70%, the promoting effect of MLP on HIF-1α ubiquitination degradation decreases by more than 30%.

[0065] Weight-average molecular weight (50-150 kDa) and molecular weight distribution index (1.2-1.8)

[0066] Principle: Low molecular weight (<50kDa) leads to rapid metabolism and clearance of MLPs in vivo, resulting in a shortened half-life; high molecular weight (>150kDa) reduces cell membrane permeability, making it difficult for macrophages to take up. A distribution index of 1.2-1.8 indicates high molecular weight uniformity of the polysaccharide, ensuring batch-to-batch activity stability (difference <10%).

[0067] Pharmaceutical composition (containing a therapeutically effective amount of polysaccharide and excipients)

[0068] Principle: Excipients (diluents, disintegrants, etc.) can improve the physicochemical properties of polysaccharides. For example, adding microcrystalline cellulose (diluent) to tablets can increase tablet hardness, and adding croscarmellose sodium (disintegrant) can promote rapid disintegration of tablets in the gastrointestinal tract and improve oral bioavailability. Solvents (such as water for injection) can keep intra-articular injections in a sterile and clear state and avoid local irritation.

[0069] Dosage form design (oral formulations, intra-articular injections, nanocarrier delivery systems)

[0070] Oral formulations: Tablets and capsules are absorbed through the gastrointestinal tract and are suitable for long-term maintenance treatment of mild KOA. Excipients (such as enteric coating) can protect polysaccharides from gastric acid destruction.

[0071] Intra-articular injection: Directly delivers gagosin polysaccharide to the affected joint cavity, avoiding systemic metabolic depletion, resulting in high local drug concentration (5-10 times that of oral administration), rapid onset of action, and is suitable for moderate to severe KOA;

[0072] Nanocarrier delivery systems: Liposomes, chitosan nanoparticles and other carriers can enhance the targeted enrichment of polysaccharides through phagocytosis by synovial cells in the joint cavity (increasing the targeting efficiency by 40%), while simultaneously releasing drugs (extending the half-life to 24 hours) and reducing the frequency of drug administration.

[0073] Ultrasonic-assisted extraction (300-500W, 25-40kHz)

[0074] Principle: The mechanical vibration and cavitation effect generated by ultrasound can destroy the cell wall and cell membrane of Gastrodia elata cells, increase the intercellular space, promote the penetration of hot water, and make polysaccharides easier to release. Stirring every 30 minutes can avoid excessive local temperature leading to polysaccharide degradation, while ensuring that the medicinal material is in full contact with hot water. The polysaccharide yield is 20-30% higher than that without ultrasound assistance.

[0075] Chromatographic purification conditions (DEAE-52 and Sephacryl S-300 combination)

[0076] DEAE-52 anion exchange chromatography: Gradient elution with 0.1-0.5 mol / L NaCl can sequentially elute impurity polysaccharides (such as neutral sugars and acidic sugars) with different charges by varying the ionic strength, while retaining the target MLP; a flow rate of 1-2 mL / min can ensure that the polysaccharides are fully bound and separated from the resin, avoiding peak diffusion;

[0077] Sephacryl S-300 gel filtration chromatography: 0.1 mol / L NaCl eluent maintains the solubility of polysaccharides, and a flow rate of 0.5-1 mL / min ensures that polysaccharides of different molecular weights elute in order of size. MLPs of 50-150 kDa are accurately collected with a purity of over 90%.

[0078] Centrifugation and freeze-drying parameters

[0079] Centrifugation conditions (4℃, 8000-10000rpm, 10-15 minutes): Low temperature (4℃) can reduce polysaccharide degradation, while high speed and appropriate time can ensure complete precipitation of polysaccharides (precipitation rate >95%), while avoiding the contamination of impurities (such as protein precipitation);

[0080] Freeze-drying (-50 to -40℃, vacuum degree ≤10Pa): Low-temperature freeze-drying can avoid polysaccharide denaturation due to high temperature (polysaccharides have poor thermal stability and are easily degraded at >60℃), and high vacuum degree accelerates water sublimation, resulting in loose and porous dry powder, which is beneficial for subsequent dissolution and formulation preparation.

[0081] Detailed workflow (extraction method)

[0082] Step 1: Raw material pretreatment

[0083] Procedure: Take the dried whole herb of the She medicinal herb Ga Gou Lu, crush it with a universal grinder, pass it through a 40-60 mesh sieve, and collect the medicinal powder;

[0084] Objective: Pulverization can increase the specific surface area of ​​medicinal materials and improve the dissolution efficiency of polysaccharides during subsequent extraction; sieving ensures uniform powder particle size (40-60 mesh) to avoid insufficient extraction due to excessively large particles or difficult filtration due to excessively small particles.

[0085] Step 2: Hot water extraction

[0086] Procedure: Mix the herbal powder and distilled water at a ratio of 1:15-1:25 (mass-volume ratio), place in a constant temperature water bath at 80-90℃, turn on the ultrasonic (300-500W, 25-40kHz), and extract for 2-3 hours, stirring once every 30 minutes during the extraction; after extraction, filter through three layers of gauze and collect the filtrate once.

[0087] Objective: To promote polysaccharide dissolution by hot water (80-90℃), to disrupt cell structure with ultrasound assistance, and to ensure uniform temperature and concentration of the system by stirring, thereby improving the polysaccharide dissolution rate; to remove drug residue by filtration and obtain a crude extract containing polysaccharides.

[0088] Step 3: Secondary extraction

[0089] Procedure: Extract the dregs filtered in step 2 again under the conditions of step 2 (same temperature, material-liquid ratio, and ultrasonic parameters), and filter and collect the second filtrate.

[0090] Objective: To fully utilize the polysaccharides remaining in the medicinal residue, thereby increasing the total extraction yield by 15-20% and reducing raw material waste.

[0091] Step 4: Concentration and Precipitation

[0092] Procedure: Combine the primary and secondary filtrates, and concentrate under reduced pressure (0.06-0.08 MPa) at 60-70℃ to 1 / 5-1 / 4 of the original volume; slowly add 3-5 times the volume of 95% ethanol to the concentrate while stirring, and let it stand at 4℃ for 12-24 hours; then centrifuge at 4℃ and 8000-10000 rpm for 10-15 minutes, discard the supernatant, collect the precipitate (crude polysaccharide), wash twice with anhydrous ethanol, and vacuum dry (50℃, 0.08 MPa) to remove residual ethanol;

[0093] Objective: To reduce polysaccharide degradation due to high temperature by vacuum concentration; to precipitate polysaccharides from the solution by ethanol precipitation, separating them from small molecule impurities; and to further remove residual impurities by centrifugation and washing of the precipitate.

[0094] Step 5: Purification

[0095] operate:

[0096] (1) Dissolve the crude polysaccharide in distilled water (concentration 5-10 mg / mL) and remove insoluble matter by filtering through a 0.45 μm filter membrane;

[0097] (2) Load the sample into a DEAE-52 anion exchange chromatography column (column size 5×30cm), elute with distilled water until the baseline is stable, then elute with a gradient of 0.1-0.5mol / L NaCl solution (flow rate 1-2mL / min), monitor with a UV detector (280nm), and collect the main peak component;

[0098] (3) After concentrating the main peak component, load it onto a Sephacryl S-300 gel filtration chromatography column (column specification 2.6×100cm), elute with 0.1mol / L NaCl solution (flow rate 0.5-1mL / min), and collect the main peak;

[0099] (4) The purified polysaccharide solution was dialyzed (molecular weight cutoff 10kDa) for 24 hours to remove NaCl, and then freeze-dried at -50 to -40℃ and vacuum degree ≤10Pa to obtain purified polysaccharide;

[0100] Objective: To remove impurities with different charges (such as acidic polysaccharides and proteins) by DEAE-52 chromatography, remove impurities with different molecular weights (such as small oligosaccharides) by SephacrylS-300 chromatography, and obtain high-purity (≥90%) and structurally stable MLP by freeze drying.

[0101] The specific application examples for verifying the efficacy and mechanism of action of Gaguru polysaccharide in the treatment of knee osteoarthritis (KOA) are described in detail below:

[0102] Modeling of Osteoarthritis (Example 1)

[0103] KOA Animal Model Construction and Intervention: An SD rat KOA model was constructed using anterior cruciate ligament transection (ACLT) to simulate the pathological state of clinical KOA (articular cartilage degeneration, osteophyte formation, etc.). The degree of inflammation, fibrosis level, and cartilage matrix synthesis in the joint tissues after polysaccharide intervention were assessed using X-ray, micro-CT, and tissue staining (HE, Masson's stain, Alsin Blue stain), directly verifying the therapeutic effect of polysaccharides in live animals.

[0104] Macrophage Inflammation Model: An inflammation model was constructed by stimulating THP-1 macrophages with LPS to simulate the local inflammatory microenvironment of the KOA (Korean Oleoma). By detecting the expression of key glycolytic enzymes (HK2, PKM2) and the secretion of inflammatory factors (TNF-α, IL-6), the regulatory role of polysaccharides on macrophage metabolic reprogramming and inflammatory phenotype was analyzed, providing cellular-level validation of the mechanism of action of polysaccharides.

[0105] Mechanism study of SUMOylation regulating HIF-1α activity (Example 2)

[0106] This section is a mechanism verification experiment. By detecting the SUMOylation and ubiquitination modification levels of HIF-1α, constructing a SENP1 knockdown / overexpression model, and verifying the competitive relationship between SUMOylation and ubiquitination, the regulatory rules of HIF-1α post-translational modification in KOA are clarified, laying a theoretical foundation for the subsequent verification of the polysaccharide mechanism.

[0107] Efficacy study of purified polysaccharides in treating KOA (Example 3)

[0108] Animal model validation: Different doses of purified polysaccharide were directly administered to an ACLT rat model. The effects of polysaccharide on osteophyte formation and cartilage degeneration were evaluated by imaging and histological methods to clarify the in vivo therapeutic effect and dose-response relationship of polysaccharide.

[0109] Cell model validation: Chondrocytes were treated with macrophage conditioned medium, and the expression changes of chondrocyte metabolism-related genes (catabolism genes MMP13, ADAMTS5; anabolism genes COL2A1, ACAN) were detected. Combined with the detection of HIF-1α SUMOylation level, the molecular mechanism by which polysaccharides improve cartilage metabolism by regulating HIF-1α SUMOylation modification was directly verified, which is experimental evidence for the core mechanism of the technical solution.

[0110] In summary, these findings, through animal experiments, cell experiments, and molecular mechanism studies, systematically validated the effectiveness and mechanism of action of She ethnic medicine Ga Gou Lu polysaccharide in treating KOA, and represent a specific application embodiment of the technical solution.

[0111] Appendix Figure 1-5 It serves as key experimental evidence to verify the core content of this technical solution, providing visual support from three dimensions: polysaccharide structural characterization, mechanism of action, and efficacy. Its specific functions are as follows:

[0112] like Figure 1 The image shows the extraction and structural analysis of Gagolu polysaccharide.

[0113] Core objective: To clarify the extraction process and chemical structural basis of Gaguru polysaccharide, and to prove the uniqueness and reproducibility of its structure.

[0114] Specific value:

[0115] The complete process of extracting and purifying polysaccharides from the She ethnic medicine Ga Gou Lu (such as the chromatographic column purification step) is demonstrated, providing a methodological basis for the preparation of polysaccharides in subsequent experiments;

[0116] Combined with subsequent nuclear magnetic resonance data ( Figure 2The preliminary structural framework of MLP was presented, providing intuitive extraction and separation evidence for the structural features that "the main chain is composed of →1)-β-D-Fruf-(2→ and →1,6)-β-D-Fruf-(2→ glycosidic bonds, and the side chains are connected at the O-6 positions".

[0117] like Figure 2 The image shows the NMR structure analysis of Gaguru polysaccharide.

[0118] Core function: The precise determination of the chemical structure of MLP using multidimensional nuclear magnetic resonance (NMR) technology is a key basis for proving its structural novelty;

[0119] Specific Value

[0120] (A) Proton NMR: No significant absorption peaks were detected in the range of δ4.4–5.3 ppm (δ4.71 ppm is the HOD solvent peak), consistent with the structural characteristics of fructose; (B) Carbon NMR: The methylene signal is distributed in the range of δ60.45–62.58 ppm, supporting the furan ring structure of fructose; (C) Heteronuclear single quantum coherence spectroscopy (HSQC): No corresponding hydrogen cross-peaks were found for the anomeric carbons (δ~103 ppm) of sugar residues A, B, and C, consistent with the characteristic that the anomeric carbon (C2) of fructose is a quaternary carbon; (D) Chemical shift correlation spectroscopy (COSY) (E) H-1 / H-2 (δ5.32 / 3.89) continuum correlation reveals the proton coupling network of the sugar ring; (E) Heteronuclear multi-bond correlation spectrum (HMBC): glycosidic bonds exist between sugar residue AC2 and sugar residue AH1, sugar residue AC2 and sugar residue BH1, sugar residue BC2 and sugar residue AH1, and sugar residue CC2 and sugar residue BH6; (F) Nuclear Overhouse effect spectrum (NOESY): verifies the sugar ring conformation; (G) AM-1 component deduced chemical structure: constructed based on multidimensional spectral data, showing the main chain of the gagosin polysaccharide;

[0121] like Figure 3 As shown, this illustrates the balance of HIF-1α post-translational modifications regulated by gagosin.

[0122] Core function: To verify the molecular mechanism by which MLP exerts its effects through the regulation of HIF-1α SUMOylation and ubiquitination, which is key experimental evidence for discovering the mechanism of action.

[0123] Specific value:

[0124] Western blot results (A) and quantitative analysis (BD) directly demonstrate that MLP can upregulate the expression of pVHL (ubiquitination-related protein) and downregulate the expression of HIF-1α and SENP1 (de-SUMOylase), providing protein-level evidence for the mechanism of "MLP inhibiting SENP1 and promoting HIF-1α ubiquitination degradation".

[0125] The mechanism diagram (E) visually illustrates the competitive regulatory relationship between SUMOylation and ubiquitination of HIF-1α, explaining how MLP inhibits HIF-1α activity by disrupting this balance, thus supporting the description of its mechanism of action in the claims.

[0126] like Figure 4 As shown, this demonstrates how gagosin inhibits macrophage glycolysis.

[0127] Key role: To verify the regulatory role of Gaguru polysaccharide in macrophage metabolic reprogramming, providing crucial intermediate evidence linking molecular mechanisms and anti-inflammatory effects.

[0128] Specific value:

[0129] Reactive oxygen species (ROS) and lactate assays (A, B) showed that MLP could reduce oxidative stress and accumulation of metabolites in macrophages under inflammatory conditions, demonstrating its anti-inflammatory effect.

[0130] Western blot and qPCR results (CF) confirmed that MLP can downregulate the protein and mRNA levels of key glycolytic enzymes (GLUT1 and PKM2), directly supporting the mechanism of "MLP inhibiting the expression of key glycolytic enzymes" and explaining how it inhibits pro-inflammatory phenotypes by improving macrophage metabolism.

[0131] like Figure 5 As shown, this demonstrates how gaguru polysaccharide improves bone and joint damage in KOA rats.

[0132] Core function: Validating the therapeutic effect of MLP on knee osteoarthritis (KOA) in animal models provides the final evidence of the invention's practicality.

[0133] Specific value:

[0134] X-ray and Micro-CT images (A, B) visually demonstrate that MLP can reduce bone and joint damage in KOA rats by comparing the joint structures (such as osteophyte formation and cartilage degeneration) of the intervention group and the control group.

[0135] Quantitative analysis (such as bone pathological changes in the ROI region) demonstrates that the efficacy of MLP is dose-dependent, providing in vivo experimental evidence for its clinical application potential and echoing the application scenario of "treatment of knee osteoarthritis" in the claims.

[0136] In summary, attached Figure 1-5 The complete chain of evidence, from structure and mechanism to efficacy, not only verifies the scientific validity and innovation of the invention, but also provides direct experimental support for the technical features in the claims (such as structure, mechanism, and application).

[0137] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A polysaccharide derived from the She ethnic medicine Ga Gou Lu for treating knee osteoarthritis, characterized in that, The product includes Gagosin, whose main chain is composed of →1)-β-D-Fruf-(2→ and →1,6)-β-D-Fruf-(2→ glycosidic bonds, with side chains attached at positions O-6; Gagosin can regulate the SUMOylation modification of HIF-1α at K391, K477 or K674 sites by inhibiting the expression of the deSUMOylase SENP1, thereby promoting the ubiquitination and degradation of HIF-1α.

2. The herbal polysaccharide Gagolu for treating knee osteoarthritis according to claim 1, characterized in that, The monosaccharide composition of the Gagolu polysaccharide is mainly fructose, and also includes glucose and galactose, with fructose accounting for ≥70% of the molar content.

3. The herbal polysaccharide Gagolu for treating knee osteoarthritis according to claim 2, characterized in that, The weight-average molecular weight of the Gagolu polysaccharide is 50-150 kDa, and the molecular weight distribution index (Mw / Mn) is 1.2-1.

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4. The herbal medicine Ga Gou Lu polysaccharide for treating knee osteoarthritis according to claim 3, characterized in that, It also includes a therapeutically effective amount of gaccharin polysaccharide and pharmaceutically acceptable excipients selected from one or more of diluents, disintegrants, binders, lubricants, or solvents.

5. The herbal medicine Ga Gou Lu polysaccharide for treating knee osteoarthritis according to claim 4, characterized in that, The dosage form of the Gagolu polysaccharide is an oral preparation, an intra-articular injection, or a nanocarrier delivery system; the oral preparation includes tablets, capsules, or oral liquids, and the nanocarrier delivery system includes liposomes, chitosan nanoparticles, or hyaluronic acid nanogels.

6. A method for extracting polysaccharides from the She ethnic medicine *Gagolu* for treating knee osteoarthritis, characterized in that, Includes the following steps: S1. Raw material pretreatment: Take the dried whole herb of the She medicine Ga Gou Lu, crush it and pass it through a 40-60 mesh sieve to obtain medicinal powder; S2. Hot water extraction: Mix the medicinal powder with distilled water at a mass-volume ratio of 1:15-1:25, extract at 80-90℃ for 2-3 hours, and filter to collect the first filtrate; S3. Secondary extraction: The residue is extracted once more under the conditions of step S2, and the secondary filtrate is collected. S4. Concentration and precipitation: Combine the two filtrates, concentrate under reduced pressure to 1 / 5-1 / 4 of the original volume, add 3-5 times the volume of 95% ethanol, let stand at 4℃ for 12-24 hours, centrifuge to collect the precipitate, and obtain crude polysaccharide. S5. Purification: The crude polysaccharide was dissolved in distilled water and purified by DEAE-52 anion exchange chromatography and Sephacryl S-300 gel filtration chromatography. The main peak fraction was collected and freeze-dried to obtain the purified polysaccharide.

7. The method for extracting polysaccharides from the She ethnic medicine *Gagolu* for treating knee osteoarthritis according to claim 6, characterized in that... In step S2, ultrasonic assistance is used during the extraction process, with stirring every 30 minutes. The power of the ultrasonic assistance is 300-500W and the frequency is 25-40kH.

8. The method for extracting polysaccharides from the She ethnic medicine *Gagolu* for treating knee osteoarthritis according to claim 6, characterized in that... In step S5, the elution conditions for DEAE-52 anion exchange chromatography are: gradient elution with 0.1-0.5 mol / L NaCl solution at a flow rate of 1-2 mL / min; the eluent for Sephacryl S-300 gel filtration chromatography is 0.1 mol / L NaCl solution at a flow rate of 0.5-1 mL / min.

9. The method for extracting polysaccharides from the She ethnic medicine Ga Gou Lu for treating knee osteoarthritis according to claim 6, characterized in that, In step S4, the centrifugation temperature is 4℃, the centrifugation speed is 8000-10000rpm, and the centrifugation time is 10-15 minutes; in step S5, the freeze-drying temperature is 50 to -40℃, and the vacuum degree is ≤10Pa.

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