Use of a kind of chondrus ocellatus polysaccharide in preparation of medicine for regulating blood uric acid and preventing and treating hyperuricemia

Abstract

The application discloses a use of a new type of birete polysaccharide in preparation of a medicine for preventing and treating hyperuricemia. The birete polysaccharide is prepared from birete by hot water extraction, desalination and ion exchange column chromatography, and is a new type of sulfated polysaccharide mainly composed of galactose and arabinose. The hyperuricemia animal model experiment proves that the polysaccharide can significantly reduce the blood uric acid level of hyperuricemia mice, down-regulate the reabsorption protein URAT1 and GLUT9 of uric acid in the kidney, up-regulate the excretion protein OAT1 and OAT3, and can improve the liver and kidney biochemical indexes of the hyperuricemia model mice, repair the pathological damage of the liver and the kidney, and has the characteristics of mild effect, multi-target effect and high safety compared with the existing clinical medicine, and is expected to be used for long-term prevention and intervention of hyperuricemia, and has a good industrialization prospect.

Description

Technical Field

[0001] This invention belongs to the field of marine natural products, biomedicine and functional food processing technology, specifically involving the preparation process of Corylus adenophora polysaccharide (CFP) and the application of this polysaccharide in the preparation of drugs or health foods that lower uric acid and improve hyperuricemia. Background Technology

[0002] Hyperuricemia (HUA) is a chronic metabolic disease induced by purine metabolism disorder. From 2000 to 2023, the global prevalence rate increased by 51% in men and 66% in women, with the number of patients worldwide exceeding 800 million. It is the second most prevalent chronic metabolic disease after diabetes. According to the "2021 China Hyperuricemia and Gout Trend White Paper," in recent years, hyperuricemia in my country has shown a significant upward trend and a younger age of onset. The overall prevalence of hyperuricemia in my country is 13.3%, with approximately 177 million patients, nearly 60% of whom are young people aged 18-35. Hyperuricemia has become the "fourth highest" after diabetes, hypertension, and hyperlipidemia. Epidemiological statistics in my country show that the overall prevalence of hyperuricemia is approximately 13.3%, and the overall incidence rate of gout is 1.1%, with approximately 15 million gout patients in China, and the trend is towards a younger age of onset.

[0003] Hyperuricemia can induce gouty arthritis, organic kidney damage, hypertension, cardiovascular and cerebrovascular diseases, and metabolic syndrome. The majority of hyperuricemia patients are asymptomatic, and most patients currently seeking medical attention have experienced acute gout attacks. Most patients experience no obvious warning signs before a gout attack, or only fatigue, general malaise, and joint pain. The affected joints and surrounding tissues become red, swollen, hot, painful, and experience limited function. These symptoms usually subside spontaneously within a few days or two weeks. This is followed by an asymptomatic remission period lasting months, years, or even more than ten years before recurrence. Most patients experience recurrence within one year, with increasing frequency, more affected joints, and longer duration of symptoms. In the later stages of gout (which often takes about 10 years to develop), it enters the chronic tophaceous stage. Clinical manifestations include persistent joint swelling, pain, tenderness, deformity, and functional impairment. Chronic symptoms are relatively mild, but acute attacks can still occur. Gout can lead to a variety of chronic diseases, especially kidney-related diseases. The accumulation of uric acid crystals causes kidney damage, including chronic urate nephropathy, uric acid urinary tract stones, and acute uric acid nephropathy, which seriously endanger human health.

[0004] About two-thirds of uric acid in the human body is excreted through the kidneys, and one-third through the intestines. The renal tubules rely on URAT1 and GLUT9 to reabsorb uric acid, and OAT1 and OAT3 to mediate its excretion. The intestines assist in the breakdown and excretion of uric acid with the help of gut microbiota and the gut-kidney axis. When blood uric acid levels exceed 420 μmol / L, monosodium urate crystals precipitate, activating inflammatory pathways in the body and causing continuous damage to the liver, kidneys, and joint tissues.

[0005] Currently, the mainstream uric acid-lowering drugs in clinical practice are allopurinol and benzbromarone. However, these drugs have drawbacks such as single target action, and long-term use can easily lead to side effects such as liver damage, gastrointestinal discomfort, and skin rashes. Approximately 2% to 6% of patients taking allopurinol experience abnormal liver function, and severe cases can lead to a sudden increase in liver enzymes and organic lesions of the liver and kidneys. Therefore, there is an urgent need to develop new uric acid-lowering drugs.

[0006] *Pterocarya stenoptera* is a traditional marine medicinal algae found along my country's coast, known in traditional Chinese medicine as "water pine." Traditional Chinese medicine records its ability to promote diuresis, reduce swelling, and improve urinary difficulties. However, current research has not disclosed the preparation process and systematic efficacy data of *Pterocarya stenoptera* polysaccharides for the prevention and treatment of hyperuricemia. This invention discloses for the first time the use of a *Pterocarya stenoptera* sulfated polysaccharide in hyperuricemia. Summary of the Invention

[0007] Based on the problems existing in the prior art, this invention adopts a simple and effective extraction process and method for polysaccharides from *Pterocarya stenoptera* (*Pterocarya stenoptera*). Codium fragile A sulfate-containing polysaccharide (named CFP in the text) was obtained from the raw material. Animal experiments on hyperuricemia showed that CFP can effectively reduce the blood uric acid level in hyperuricemic mice and is expected to be developed into a sugar drug for treating hyperuricemia.

[0008] One aspect of the present invention is to provide the use of *Pteris vittata* polysaccharide or compositions comprising therein in the preparation of medicaments and health foods for treating and / or preventing hyperuricemia; The polysaccharide from *Pterocarya stenoptera* is prepared by a method comprising the following steps: (1) Polysaccharide extraction: Dried Pteris vittata was extracted with boiling water (above 100 degrees Celsius). The resulting extract was filtered, desalted, concentrated, and centrifuged to remove the precipitate. The supernatant was precipitated with alcohol and centrifuged to separate the precipitate. The precipitate was washed and dried to obtain crude polysaccharide from Pteris vittata. (2) Purification of Pterocarya spp. polysaccharide: Dissolve the crude polysaccharide of Pterocarya spp. prepared in step (1) in water, centrifuge, and perform preliminary fractionation purification of the supernatant by anion exchange column. Elute with water and 0.05-2.0M NaCl solution in sequence, collect the eluent fraction of 1.6M NaCl solution, desalt, concentrate, precipitate with ethanol, wash and dry the precipitate to obtain Pterocarya spp. polysaccharide CFP.

[0009] In a specific embodiment, the preparation method of the polysaccharide of *Pterocarya stenoptera* includes step (1): adding 15 to 30 times the weight of water to dried *Pterocarya stenoptera*, soaking, extracting at 100°C for 3 to 5 hours, extracting twice, combining the extracts, filtering, dialysis, concentrating, centrifuging to obtain supernatant, adding 3 to 5 times the volume of ethanol solution to the supernatant, centrifuging to obtain precipitate, washing the precipitate with anhydrous ethanol and acetone 3 to 6 times, and drying to obtain crude polysaccharide of *Pterocarya stenoptera*.

[0010] Step (2) includes: taking the crude polysaccharide of *Pterocarya stenoptera* prepared in step (1), dissolving it in 10 to 20 times its weight of water, and centrifuging; separating the supernatant by an anion exchange column, eluting it sequentially with water, 0.05M, 1.0M, 1.2M, 1.6M, and 2.0M NaCl solutions, detecting it with sulfuric acid and phenol, collecting and combining the eluent of 1.6M NaCl solution according to the elution curve, concentrating it, centrifuging it, dialyzing the supernatant, and freeze-drying it to obtain *Pterocarya stenoptera* polysaccharide CFP.

[0011] The polysaccharide CFP from *Pterocarya stenoptera* mainly consists of arabinose, galactose, mannose, and glucose, with a molar ratio of 50–100:30–80:1–30:1–20; preferably, the molar ratio of mannose, glucuronic acid, galactose, xylose, and fucose is 100:73:2:6. The weight-average molecular weight of the *Pterocarya stenoptera* polysaccharide CFP ranges from 2 to 1000 kDa, and the sulfate content is preferably 15-30%.

[0012] The structural unit of the *Pteris vittata* polysaccharide CFP is based on the main chain of →3)-β-L-Arap-(1→, with →4)-β-L-Arap-(1→, →3)-β-D-Galp-(1→ and →6)-β-D-Galp-(1→) segments at the C-4 position, forming a multi-branched structure. The sulfate groups mainly occur at the C-2 and / or C-4 positions of 3-Arap and the C-6 position of 3-Galp, with a small portion existing at the C-2 position of 4-Arap and the C-4 position of 6-Galp.

[0013] In a specific embodiment, the composition containing *Pterocarya stenoptera* polysaccharide contains a therapeutically effective amount of *Pterocarya stenoptera* as an active ingredient, and the composition may further include pharmaceutically acceptable excipients, such as carriers, excipients, adjuvants, and / or diluents.

[0014] The technical solution of the present invention has the following technical effects: This invention provides the use of *Platycladus orientalis* polysaccharide in the preparation of drugs and health foods for hyperuricemia. *Platycladus orientalis* polysaccharide exhibits significant uric acid-lowering activity against hyperuricemia, achieving multi-target synergistic uric acid reduction while simultaneously improving liver and kidney damage caused by hyperuricemia. Its long-term safety profile is superior to that of chemical drugs. It shows promising application prospects in the fields of foods, health products, and pharmaceuticals with hyperuricemia-regulating activity. Attached Figure Description

[0015] Figure 1 FT-IR spectra of CFP polysaccharide from *Pteris vittata* and its desulfurization product DSCF.

[0016] Figure 2 GC-MS analysis of DSCF, a desulfurization product of CFP from *Pteris vittata* polysaccharide.

[0017] Figure 3 13C NMR spectrum of DSCF, a desulfurization product of CFP.

[0018] Figure 4 CFP desulfurization product DSCF 1 H NMR spectrum.

[0019] Figure 5 Two-dimensional NMR spectrum of DSCF. Note: a AF: →3)-β-D-Gal p -(1→; →6)-β-D-Gal p -(1→; →3)-β-D-Gal p (4,6-Pyr)-(1→; T-Ara p →3)-β-L-Ara p -(1→; →3,4)-β-L-Ara p -(1→).

[0020] Figure 6 CFP 13 C10 NMR spectrum.

[0021] Figure 7 CFP 1 H NMR spectrum.

[0022] Figure 8Two-dimensional NMR spectrum of CFP. Note: a LS: →3)-β-D-Galp-(1→; →6)-β-D-Galp-(1→; →3)-β-D-Galp(6S)-(1→; →3)-β-D-Galp(4,6-Pyr)-(1→; T-Arap; →3)-β-L-Arap-(1→; →3)-β-L-Arap(2S)-(1→; →3)-β-L-Arap(4S)-(1→.

[0023] Figure 9 Effects of body weight, serum uric acid level, and serum IL-6 and IL-10 levels on hyperuricemic mice: Body weight (B), serum uric acid level (A), and serum IL-6 (C) and IL-10 (D) levels in hyperuricemic mice.

[0024] Figure 10 Serum liver function and lipid biochemical analysis results of mice with hyperuricemia.

[0025] Figure 11 Images of mouse serum creatinine, blood urea nitrogen, and HE tissue sections of the kidney.

[0026] Figure 12 RT-qPCR and immunohistochemistry were used to detect the expression levels of URAT1, GLUT9, OAT1, and OAT3 in the kidneys of hyperuricemic mice.

[0027] Figure 13 Uric acid transporter protein expression. (A) Immunohistochemical scan of mouse kidney; (B) URAT1 expression; (C) GLUT9 expression; (D) OAT family protein (OAT1 and OAT3) expression. Detailed Implementation

[0028] The present invention will be further explained and described below with reference to embodiments and accompanying drawings. It should be noted that the following embodiments are provided for illustrative purposes only and do not constitute a limitation on the scope of protection claimed by the present invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various modifications to the invention, and these equivalent forms also fall within the scope defined by the present invention.

[0029] Unless otherwise specified, the experimental methods used in the examples are conventional methods. Unless otherwise specified, the materials and reagents used in the examples are commercially available.

[0030] Example 1: Preparation of Polysaccharide from Pine Tree Algae a. Extraction of polysaccharides from *Pteris vittata*: The *Pterocarya stenoptera* was collected from Beidaihe, Qinhuangdao, Hebei Province. 2 kg of naturally dried *Pterocarya stenoptera* was chopped, ground using a high-speed mixer, and passed through a 60-mesh sieve. The algae powder was accurately weighed, and pure water was added at a ratio of 1:30. The mixture was soaked at room temperature for 2 hours, then heated to 100℃ for 3 hours. This extraction was repeated once. The extracts were combined, filtered, and the filtrate was concentrated under reduced pressure. The solution was dialyzed against running water for one day using a dialysis bag with a molecular weight cutoff of 12000 Da. The solution was concentrated again, and ethanol was added to a final concentration of 80%. The mixture was stirred to precipitate the polysaccharide, centrifuged, and the precipitate was washed with ethanol and acetone, and then vacuum dried to obtain 144 g of crude *Pterocarya stenoptera* polysaccharide, with a yield of 7.2%.

[0031] b. Purification of *Pteris vittata* polysaccharide: 10g of the prepared *Pterocarya stenoptera* polysaccharide was dissolved in 100mL of deionized water and centrifuged at 4000 r / min for 10min to remove insoluble matter. The supernatant was separated by a DEAE Sepharose Fast Flow anion exchange column, eluted sequentially with deionized water, 0.5M, 1.0M, 1.2M, 1.6M, and 2.0M NaCl solutions, and detected by the sulfuric acid-phenol method. Elution curves were plotted, and the 1.6M NaCl eluent was collected and combined according to the elution curves. The eluent was dialyzed, concentrated, centrifuged, and the supernatant was freeze-dried to obtain 3.5g of purified *Pterocarya stenoptera* polysaccharide CFP.

[0032] Example 2: Structural analysis of CFP, a polysaccharide from *Pteris vittata*. (1) The relative molecular mass of CFP polysaccharide from *Platycladus orientalis* was determined to be 377.07 kDa by high performance gel permeation chromatography (HPGPC), and its purity reached 96%.

[0033] (2) The monosaccharide composition of the completely hydrolyzed sample of *C. truncatula* polysaccharide CFP was determined by PMP derivatization. The results showed that CFP mainly contained arabinose, galactose, glucose, and mannose, with a molar ratio of 100:73:6:2. Using galactose as the standard, the total sugar content of CFP was determined to be 62.5%, and the sulfate content was 22.8%.

[0034] (3) Infrared spectral analysis was performed on the CFP, and the spectrum is shown in the figure. Figure 1 CFP is 3380–3390 cm. -1 Broad and strong absorption peaks appear throughout the range, corresponding to the O–H stretching vibration of the hydroxyl group, 2924–2956 cm⁻¹. -1 The C–H stretching vibration appears at 1618–1635 cm⁻¹, which is a characteristic peak for methyl / methylene groups. -1 The peak at 1214–1220 cm⁻¹ represents the stretching vibration of the C–O bond. -1S=O stretching vibration and 817–821 cm -1 The C–O–S stretching vibration indicates the presence of a sulfate group. Simultaneously, at 820 cm⁻¹... -1 The nearby signal is a stretching vibration of sulfate substitution at position 2 (primary sulfate). At 880 cm⁻¹ -1 The characteristic peaks indicate that the polysaccharide unit is in the β-configuration, which is attributed to the variable-angle vibration of CH in β-galactopyranose.

[0035] (4) Glycosidic residue linkage analysis: To further analyze the glycosidic bond linkage type and branching characteristics of CFP, CFP was desulfurized and then methylated. After multiple methylation, the desulfurized polysaccharide DSCF was examined by infrared spectroscopy at 3200 cm⁻¹. -1 The disappearance of the nearby -OH signal and 2920 cm -1 A triplet signal was generated near the -CH3 group. Partially methylated alcohol-sugar acetate was obtained through acid hydrolysis, reduction, and acetylation. The product was analyzed by GC-MS. The total ion chromatogram and corresponding mass spectra of the desulfurized and methylated CFP are shown below. Figure 2 As shown in Table 1. The mass spectrometric fragment characteristics of the main peaks were compared with the CCRC standard spectrum to determine the type of each monosaccharide derivative. Among them, the arabinose residues exist in the form of pyranose, mainly →3)-Arap-(1→, followed by →4)-Arap-(1→, with branching at the O-4 positions of 3-Arap, and the branching degree of the arabinose is about 20%. The galactose is mainly →6-Galp-(1→, followed by →3)-Galp-(1→, and abundant →3)-Galp-(1→). The presence of the β-(1→6)-Galp-(1→) branching site indicates a high degree of branching in the galactose fragment. The pyranose form of arabinose was confirmed by the assignment of anomeric hydrocarbons and C-5 in its NMR and by the DEPT135 signal. A high proportion of T-Arap was observed, which is related to the fact that arap is often attached to the β-(1→6)-D-Galp side chain as a side chain terminus or a short branch. At the same time, the high proportion of →3,6-Galp-(1→) may be related to the 3-Galp side chain being attached to the O-3 position of β-(1→6)-D-Galp.

[0036] Table 1. GC-MS analysis of DSCF, a desulfurization product of CFP from *Pteris vittata* polysaccharide.

[0037] (5) Nuclear magnetic resonance spectroscopy was used to analyze the polysaccharide CFP of *Pteris vittata* and its desulfurization product DSCF.

[0038] Structural analysis of CFP desulfurization product DSCF The NMR spectrum of DSCF is as follows Figures 3-5 As shown.13 Three anomeric carbon signal groups were observed in C, with the continuous anomeric carbon signal at 94.78–99.41 ppm attributed to β-L-Ara. p The concentrations of 103.57-104.50 ppm were attributed to β-D-Gal. p The anomeric carbon signal, at 108.17 ppm, was attributed to the O- of pyruvate-galactose. C -O signal ( Figure 3 ). 1 An anomeric hydrogen signal of 4.97–5.13 ppm was observed in H, which was attributed to β-L-Ara p , while β-D-Gal p The anomeric hydrogen signal was between 4.35 and 4.65 ppm. Figure 4 The signal range for C2-C6 in DSCF is 61.04-82.64 ppm, with the methyl carbon of acetone-acidified galactose visible at 23.81 ppm. C H3) signal, 177.81 visible pyruvate carboxyl carbon ( C =O) signal, the proton signal of H2-H6 is concentrated in 3.42-4.09 ppm, and the methyl hydrogen signal of pyruvate is visible at 1.47 ppm.

[0039] Based on HSQC, COSY and HSQC-TOCSY ( Figure 5 The sugar residues were systematically assigned, and the results are shown in Table 2. Based on the demasking effect of the glycosidic bond linkage sites and the correlation signal of the cross-peaks in the secondary spectra, the glycosidic bond linkage mode and substitution sites were determined. The anodic cross-peaks were identified as A (4.56 / 103.57), B (4.41 / 104.27), C (4.56 / 104.32), D (5.05 / 96.49), E (5.05 / 97.16), F (4.97 / 97.76), and G (5.13 / 99.41). Among them, A–D represents β-D-Gal. p Unit, E–H is β-L-Ara p Unit. Combining one-dimensional and two-dimensional information, the C3 demasking of A (4.07 / 78.46) indicates that it is →3)-β-D-Gal. p -(1→;B’s C6 demasking (3.98 / 63.38) indicates it is →6)-β-D-Gal p -(1→. The pyruvate substituent can be observed in HSQC as a methyl (- C H3) hydrocarbon signal (1.47 / 23.81 ppm), HMBC shows methyl hydrogen and O- on the pyruvate ring. C -O (1.47 / 108.17 ppm), carboxylic acid C=O (1.47 / 177.81 ppm) long-range correlation. The substitution site of pyruvate on galactose can be detected via O- on HMBC. C -O crossover signals with H4 (4.09 / 108.17) and H6 (3.59 / 108.17) confirm that residue D is →3)-β-D-Gal p (4,6-Pyr)-(1→). This is related to 1 H / 13 The triplet characteristics of the pyruvate structure at 1.47 / 23.81 (–CH3), 177.81 (C=O), and 108.17 (O–C–O) in C corroborate each other. The HSQC negative phase clusters are mainly located at 60.70, 60.96, 63.14, and 63.70 ppm, belonging to the primary carbon (–CH2), excluding typical Ara... f The presence of the -C5 signal (around 66 ppm) is consistent with the anolyte signal indicating that arabinose is predominantly pyranose. The anolyte signal of T-Ara maintains a consistent chemical shift (4.41 / 104.27) in both DSCF and CFP, and →3)-β-L-Ara p -(1→and→4)-β-L-Ara p The O-glycosidic bond linkage sites of -(1→ underwent low-field shifts, with C3 / H3 shifting to (4.09 / 78.46) and C4 shifting to (3.71 / 82.02). Further... 1 H– 1 H COSY and HSQC-TOCSY establish complete spin systems within each residue, and assign relevant signals of short-range and long-range H1-H6 / H5 and C1-C6 / C5 within sugar residues.

[0040] Table 2 NMR attribution data for DSCF

[0041] Note: a AG: →3)-β-D-Gal p -(1→; →6)-β-D-Gal p -(1→;→3)-β-D-Gal p (4,6-Pyr)-(1→; T-Ara p →3)-β-L-Ara p -(1→; →4)-β-L-Ara p -(1→; →3,4)-β-L-Ara p -(1→).

[0042] pass 1 H– 1The H ROESY spectrum confirms the connection order and branching sites of the main chain and branches. In the ROESY, the H1(E)–H3(E) and H1(F)–H3(F) correlation signals demonstrate →3)-β-L-Ara p -(1→、→4)-β-L-Ara p -(1→ The existence of linear segments. The correlation signals of H1(E)–H3(G) and H3(E)–H1(G) confirm →3,4)-β-L-Ara p -(1→unit as→3)-β-L-Ara p The characteristics of the main branch sites. The correlation signals of H1(E)–H3(G) and H1(F)–H4(G) confirmed →3)-β-L-Ara p -(1→、→4)-β-L-Ara p -(1→and→3,4)-β-L-Ara p -(1→ The connection between units reveals the existence of →3)-β-L-Ara on the main chain. p -(1→3,4)-β-L-Ara p -(1→4)-β-L-Ara p -(1→ branched motif. Similarly, the cross peaks of H1(E)–H3(G) and H1(A)–H4(G) further support →3)-β-L-Ara p -(1→3,4)-β-L-Ara p -(1→3)-β-D-Gal p -(1→ sequence. T-Ara p Not only connect →3)-β-L-Ara p -(1→、→4)-β-L-Ara p -(1→segment, thus forming cross signals H1(E)–H3(D) and H1(F)–H4(D), and also with →3)-β-D-Gal p -(1→、→6)-β-D-Gal p -(1→ The fragment forms cross signals H1(E)–H3(B) and H1(F)–H6(C), indicating T-Ara p As a non-reducing end motif on the side chain of arabinogalactan.

[0043] In summary, through comprehensive analysis of multidimensional NMR spectra, DSCF is characterized by →3)-β-L-Ara p -(1→ as the main chain, and rich in →3,4)-β-L-Ara p -(1→branching site, C-4 position can be →4)-β-L-Ara p -(1→、→3)-β-D-Galp -(1→and→6)-β-D-Gal p -(1→ Fragment replacement, forming a multi-branched structure. T-Ara) p The main terminal residues terminate the sugar chain segments of arabinose and galactose. (Partial → 3)-β-D-Gal p -(1→ O-4 and O-6 undergo acetone acidification to form a 4,6-ketal structure.

[0044] NMR spectral analysis of CFP polysaccharide from *Pteris vittata* The NMR spectrum of CFP is as follows Figures 6-8 As shown. 13 Three groups of anomeric carbon signals were observed in C. The continuous anomeric carbon signal in the range of 94.95-100.01 ppm was attributed to β-L-Ara. p The concentrations of 103.02–104.26 ppm were attributed to β-D-Gal. p The anomeric carbon signal, at 108.17 ppm, was attributed to the O- of pyruvate-galactose. C -O signal. 1 In the H anodic region, 5.09–5.28 ppm belong to β-L-Ara. p 4.42-4.72 ppm belongs to β-D-Gal p In CFP, the signal range of C2-C6 is 61.13-82.65 ppm, the methyl carbon (-CH3) signal of pyruvate galactose is visible at 23.71 ppm, the carboxyl carbon (C=O) signal of pyruvate is visible at 178.17 ppm, the proton signal of H2-H6 is concentrated in 3.43-4.78 ppm, and the methyl hydrogen signal of pyruvate is visible at 1.47 ppm.

[0045] HSQC revealed information about the direct C–H linkage. Combined with 1D NMR spectra, GC-MS results, and comparison of chemical shifts before and after desulfurization, the anodic signals can be attributed to: L (4.59 / 103.02), M (4.42 / 103.49), N (4.72 / 103.89), O (4.53 / 104.26), P (5.09 / 94.95), Q (5.09 / 95.03), R (5.28 / 95.97), and S (5.28 / 100.01). The negative signal groups in HSQC are mainly concentrated at 60.81, 61.13, 61.38 ppm, 63.59 ppm, and 67.71 ppm. The (4.22 / 67.71) signal group disappears in the DSCF spectrum, indicating that it points to the 6-sulfated galactose residue, i.e., →3)-β-D-Gal. p(6S)-(1→(residue N). Meanwhile, in the HSQC spectrum of CFP, the hydrogen signal group is at 4.45-4.83 ppm, the carbon signal group is at 71.71-78.97 ppm, and the disappearance of the 3.94 / 77.95 signal group indicates that it is assigned to the CH signal of a sulfated sugar residue. The (4.52 / 73.33) and (4.12 / 78.85) signal groups are assigned to →3)-β-L-Ara due to the low-field characteristics of the hydrogen shift. p (2S)-(1→(residue R) of (H2 / C2) and (H3 / C3). (4.64 / 73.33) and (4.71 / 77.87) are assigned to →3)-β-L-Ara p (4S)-(1→(residue S) of (H3 / C3) and (H4 / C4). This is related to the α-sulfation caused by O-sulfation. C / H The principle of deshielding effect is consistent. The methyl hydrocarbon signal (1.47 / 23.71 ppm) of the pyruvate substituent group can be observed in HSQC, and the methyl hydrogen and O- on the pyruvate ring can be seen in HMBC. C -O (1.47 / 108.17 ppm) and related signals for carboxylic acid C=O (1.47 / 177.91 ppm). HMBC shows O- C -O and H4 (4.09 / 108.17 ppm) crossover signals. Further analysis via... 1 H– 1 H COSY and HSQC-TOCSY establish complete spin systems within each residue, and assign relevant signals of short-range and long-range H1-H6 / H5 and C1-C6 / C5 within sugar residues.

[0046] Based on DSCF 1 H– 1 The repetitive sequences and branching sites confirmed by the H ROESY spectrum were further validated in the CFP ROESY. The H1(L)–H3(L) and H1(Q)–H3(Q) correlation signals proved →3)-β-D-Gal p -(1→、→3)-β-L-Ara p -(1→ The presence of linear segments. The cross signals of H1(Q)–H3(R) and H1(Q)–H3(S) reveal the substitution of C-2 and C-4 sulfation on the main chain, and the presence of →3)-β-L-Ara p -(1→3)-β-L-Ara p (2S)-(1→3)-β-L-Ara p -(1→and→3)-β-L-Ara p -(1→3)-β-L-Ara p (4S)-(1→3)-β-L-Ara p-(1→ sulfation fragment. Similarly, the cross peaks of H1(N)–H3(L) and H3(N)–H1(L) support →3)-β-D-Gal p -(1→3)-β-D-Gal p (6S)-(1→3)-β-D-Gal p The sulfation sequence of -(1→. The cross signals of H1(O)–H3(L) and H3(O)–H1(L) reveal the presence of →3)-β-D-Gal. p -(1→3)-β-D-Gal p (4,6-Pyr)-(1→3)-β-D-Gal p -(1→acetone-acidified galactose structural fragment.)

[0047] Table 3 NMR attribution data for CFP

[0048] Note: a LS: →3)-β-D-Gal p -(1→; →6)-β-D-Gal p -(1→; →3)-β-D-Gal p (6S)-(1→; →3)-β-D-Gal p (4,6-Pyr)-(1→; T-Ara p →3)-β-L-Ara p -(1→; →3)-β-L-Ara p (2S)-(1→; →3)-β-L-Ara p (4S)-(1→).

[0049] In summary, through comprehensive analysis of the multidimensional NMR spectra of samples before and after desulfurization, it was revealed that CFP is expressed as →3)-β-L-Ara p -(1→ is the main chain, and →4 is applied at position C-4)-β-L-Ara p -(1→、→3)-β-D-Gal p -(1→and→6)-β-D-Gal p -(1→ Fragment substitution, forming a multi-branched structure, with sulfate groups mainly occurring in 3-Ara p C-2 and / or C-4 bits and 3-Gal p The C-6 bit, a small portion of which exists in 4-Ara p C-2 position and 6-Gal p C-4 position. →3)-β-D-Gal p-(1→ O-4 and O-6 undergo acetone acidification to form a 4,6-ketal structure.

[0050] Example 3: The therapeutic effect of *Platycladus orientalis* polysaccharide on mice with hyperuricemia. Forty healthy male Kunming mice aged 5 weeks (SPF grade) were selected and randomly divided into four groups (n=10 per group): blank, model, allopurinol, and CFP. The CFP sample was prepared as described in Example 1. Modeling was initiated after 7 days of acclimatization. A hyperuricemia model was established by gavage administration of potassium oxonate (380 mg / kg) and yeast extract (5 g / kg). After one week of modeling, except for the blank group, the average serum uric acid concentration of mice significantly increased to over 200 μmol / L, indicating successful modeling and meeting the experimental drug requirements. Following successful modeling, continuous gavage administration was administered for 5 weeks. Mice in the model and blank control groups received the same volume of saline, allopurinol at 30 mg / kg, and CFP at 150 mg / kg. Body weight and serum uric acid levels were monitored weekly. After three weeks of modeling, only potassium oxonate was used, and yeast extract was discontinued. Five weeks after administration, the mice were sacrificed, and liver function, kidney function, inflammatory factors, kidney gene expression, and histopathology were measured.

[0051] During the first week of administration, allopurinol rapidly reduced serum uric acid, while CFP slowly and gradually reduced uric acid. After three weeks of continuous administration, serum uric acid in the CFP group returned to the normal level of the control group, indicating that CFP has a significant uric acid-lowering effect, with a mild and long-lasting effect, suitable for long-term management of chronic diseases. Figure 9 ).

[0052] Persistent hyperuricemia-induced urate crystal (MSU) deposition activates inflammatory pathways, induces the release of pro-inflammatory factors, and thus exacerbates tissue damage and induces kidney injury. Therefore, inflammation suppression is an important complementary dimension in the treatment of hyperuricemia. Serum IL-6 and IL-10 inflammatory factors were detected using ELISA. Results showed that serum IL-6 levels were significantly increased and serum IL-10 levels were significantly decreased in the model group mice. After CFPS treatment, compared with the model group, the level of pro-inflammatory factor IL-6 was significantly decreased, while the level of anti-inflammatory factor IL-10 was increased. This indicates that *Platycladus orientalis* polysaccharide CFP can inhibit the inflammatory response in hyperuricemic mice. Figure 9 ).

[0053] Hyperuricemia is not only a risk factor for gout and kidney damage, but it is also closely related to the occurrence and development of liver damage. Figure 10 and Figure 11The results showed that liver and kidney function indicators in the model group mice were abnormal to some extent, indicating that persistent hyperuricemia can damage liver and kidney function. After CFP treatment, the abnormal liver and kidney function indicators in HUA mice significantly decreased, showing no significant difference from the BLANK group, indicating that CFP can effectively protect liver and kidney function in the treatment of hyperuricemia mice.

[0054] Mouse kidney HE staining ( Figure 11 The results showed that, compared with the control group, the model group mice had increased intercellular spaces in the kidneys, tissue damage, and decreased and severely damaged functional cell density. Thickened and proliferated blood vessel walls (red arrows) may have led to a decline in blood circulation and metabolic capacity. Glomeruli (green arrows) showed varying degrees of damage, reduced internal active cells, and proliferation, swelling, and infiltration of inflammatory factors, resulting in decreased filtration capacity. CFP exhibited good renal protective activity; after administration, morphology significantly recovered, with a uniform and moderate distribution of renal tubules, and an increased number and fullness of glomeruli, containing mostly active filtering cells, with less matrix embolism and proliferation, promoting normal kidney function. Simultaneously, the tightly packed and highly active blood vessel wall cells ensured normal blood metabolic exchange in the kidneys. Overall, CFP demonstrated good biocompatibility in the treatment of hyperuricemia, providing good protection for the liver and kidneys, two important metabolic and excretory organs, laying a structural and functional foundation for the recovery of uric acid levels.

[0055] Uric acid reabsorption plays a crucial role in renal uric acid metabolism. URAT1, an organic anion transporter encoded by the SLC22A12 gene, is mainly distributed on the apical membrane side of proximal convoluted tubule epithelial cells in the kidneys. It is responsible for uploading uric acid from the primary urine into the renal tubular epithelial cells, mediating the initiation of urate reabsorption. URAT1 expression was significantly elevated in the MODEL group, indicating a substantial increase in uric acid reabsorption in the mouse kidneys, activating the mechanism of elevated serum uric acid and inducing uric acid absorptive uptake (HUA). CFP administration significantly inhibited URAT1 expression, demonstrating that CFP effectively inhibits uric acid reabsorption. GLUT9 is another key transporter regulating renal reabsorption, responsible for transporting intracellular uric acid into the blood. High expression of GLUT9 protein was observed in MODEL model mice, indicating that its reabsorption of uric acid contributes to an increase in serum uric acid concentration. CFP treatment significantly decreased GLUT9 protein expression. CFP has a good inhibitory effect on GLUT9, promoting uric acid excretion and reducing the damaging effects of uric acid crystal precipitation on the kidneys. This may be an important pathway by which CFP protects the kidneys. Two members of the OAT protein family, OAT1 and OAT3, are expressed in the basement membrane of renal tubular epithelial cells, transporting urate from the renal interstitium to the renal tubular epithelial cells, promoting uric acid excretion. The MODEL group showed low expression of OAT1 and OAT3, indicating obstructed uric acid transport and exacerbated uric acid accumulation in the body. CFP significantly upregulated the expression of OAT1 and OAT3 proteins, promoting uric acid excretion and enhancing renal uric acid excretion function. Compared to allopurinol, the upregulation of OAT3 expression in the CFP group was more significant than that in the MODEL group. Figure 12 ).

[0056] Further immunohistochemical techniques were used to observe the expression of three key transport proteins, URAT1, GLUT9, and OAT, in the kidney. Electron microscopy results in the allopurinol group showed larger intercellular spaces, indicating some damage. The protein expression in the MODEL group was significantly different from the control group; OAT excretion-promoting proteins stained lighter, indicating downregulation, while reabsorption proteins URAT1 and GLUT9 stained darker, indicating upregulation, consistent with the basic characteristics of hyperuricemia. After drug administration, the expression of each protein tended to normalize, largely consistent with the previous PCR results. Overall, CFPS can simultaneously regulate both uric acid reabsorption and excretion pathways, inhibiting URAT1 and GLUT9 to reduce uric acid reabsorption, while upregulating and activating OAT family proteins to promote uric acid excretion, thus restoring the dynamic balance between the two pathways to a certain extent, exerting a uric acid-lowering effect, and improving hyperuricemia. Figure 13 ).

[0057] The above embodiments are merely illustrative of the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and substance defined by the claims of the present invention; and such modifications or substitutions are still within the scope defined by the claims of the present invention.

Claims

1. The use of *Platycladus orientalis* polysaccharide in the preparation of drugs or pharmaceutical formulations for the treatment and / or prevention of hyperuricemia, characterized in that: in, The polysaccharide from *Pterocarya stenoptera* is prepared by a method comprising the following steps: (1) Polysaccharide extraction: The dried *Pterocarya stenoptera* was extracted with hot water. The resulting extract was filtered, desalted, concentrated, and centrifuged to remove the precipitate. The supernatant was precipitated with alcohol and centrifuged. The precipitate was washed and dried to obtain crude polysaccharide from *Pterocarya stenoptera*. (2) Purification of polysaccharides from *Pteris vittata*: Dissolve the crude polysaccharide of *Pterocarya stenoptera* prepared in step (1) in water, centrifuge, and preliminarily purify the supernatant by anion exchange column. Elute with water and 0.05–2.0 M NaCl solution in sequence, collect the eluent fraction of 1.6 M NaCl solution, desalt, concentrate, precipitate with ethanol, wash and dry the precipitate to obtain *Pterocarya stenoptera* polysaccharide CFP.

2. The application according to claim 1, characterized in that, Step (1) includes: adding 15 to 30 times the weight of water to dried *Pterocarya stenoptera*, soaking, extracting at 100°C for 3 to 5 hours, extracting twice, combining the extracts, filtering, dialysis, concentrating, centrifuging to obtain supernatant, adding 3 to 5 times the volume of ethanol solution to supernatant, centrifuging to obtain precipitate, washing the precipitate with anhydrous ethanol and acetone 3 to 6 times, and drying to obtain crude polysaccharide from *Pterocarya stenoptera*. Step (2) includes: taking the crude polysaccharide of *Pterocarya stenoptera* prepared in step (1), dissolving it in 10 to 20 times its weight of water, and centrifuging; separating the supernatant by an anion exchange column, eluting it sequentially with water, 0.05M, 1.0M, 1.2M, 1.6M, and 2.0M NaCl solutions, detecting it with sulfuric acid and phenol, collecting and combining the eluent of 1.6M NaCl solution according to the elution curve, concentrating it, centrifuging it, dialyzing the supernatant, and freeze-drying it to obtain *Pterocarya stenoptera* polysaccharide CFP.

3. The application according to any one of claims 1-2, characterized in that, The polysaccharide CFP from *Pterocarya stenoptera* is mainly composed of arabinose, galactose, glucose, and mannose, with a molar ratio of 50–100:30–80:1–30:1–20; preferably, the molar ratio of mannose, glucuronic acid, galactose, xylose, and fucose is 100:73:6:

2.

4. The application according to any one of claims 1-2, characterized in that, The weight-average molecular weight of the *Pterocarya stenoptera* polysaccharide CFP ranges from 2 to 1000 kDa, and the sulfate content is preferably 15-30%.

5. The application according to any one of claims 1-2, characterized in that, The structural unit of the CFP polysaccharide from *Pteris vittata* is →3)-β-L-Ara p -(1→ is the main chain, and →4 is applied at position C-4)-β-L-Ara p -(1→、→3)-β-D-Gal p -(1→and→6)-β-D-Gal p -(1→ Fragment substitution, forming a multi-branched structure, with sulfate groups mainly occurring in 3-Ara p C-2 and / or C-4 bits and 3-Gal p The C-6 bit, a small portion of which exists in 4-Ara p C-2 position and 6-Gal p The C-4 bit.

6. Use of a composition comprising the polysaccharide of *Pteris vittata* according to claim 1 in the preparation of a medicament or pharmaceutical preparation for treating and / or preventing hyperuricemia.

7. The application according to claim 6, characterized in that, The composition containing *Platycladus orientalis* polysaccharide contains a therapeutically effective amount of the *Platycladus orientalis* polysaccharide as an active ingredient; preferably, the composition containing *Platycladus orientalis* polysaccharide further includes pharmaceutically acceptable excipients.

Images