Fucoidan oligosaccharide derivative, preparation method therefor, and use thereof

Abstract

The present invention relates to a fucoidan oligosaccharide derivative, a preparation method therefor, and a use thereof. The fucoidan oligosaccharide derivative provided by the present invention is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, where n is an integer, and n=1-8, preferably n=1-3. The fucoidan oligosaccharide derivative designed and synthesized in the present invention has good therapeutic effects on inflammatory diseases such as acute kidney injury, chronic kidney disease, gout, liver injury, oral ulcers, and rhinitis, and has good stability under different acidic and basic conditions. In addition, the present invention also provides a compound represented by formula (II) or a pharmaceutically acceptable salt thereof, which is constructed by the connection of monosaccharide G and / or M by means of 1,4-glycosidic bonds, and a compound represented by formula (III) or a pharmaceutically acceptable salt thereof, which is constructed by the connection of monosaccharide Δ and G and / or M by means of 1,4-glycosidic bonds, as well as a use of said compounds or salts in the preparation of a medicament for preventing and / or treating certain inflammatory diseases.

Description

A fucoidan oligosaccharide derivative, a preparation method and use thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine. Specifically, the present application relates to a fucoidan oligosaccharide derivative, a preparation method and use thereof. BACKGROUND

[0002] Carbohydrate, nucleic acid and protein are collectively known as the three major life substances. Fucoidan mainly exists in the cell wall of kelp, sargassum and giant kelp, and is a kind of linear, unbranched and negatively charged polysaccharide compound. The high viscosity and gelation properties of fucoidan make it widely used as coagulant, thickener, stabilizer and the like in food, chemical, pharmaceutical, textile and other industrial production. In the field of medicine, fucoidan has a wide range of applications in medical biomaterials and drug controlled-release materials due to its unique physicochemical properties and good biocompatibility. Studies have also found that fucoidan has biological activities such as antioxidant, immunomodulatory and antitumor activities, but fucoidan is not easily absorbed due to its large molecular weight and strong gelation, which greatly limits its application in these fields. Oligosaccharides have attracted attention due to their clear structure, significant activity, good absorption and small side effects.

[0003] In recent years, due to the unique structure of fucoidan oligosaccharide, its activity research has become a hot spot in the research of saccharide drugs. Fucoidan is a binary linear block compound composed of β-D-(1,4)-mannuronic acid (M) and α-L-(1,4)-guluronic acid (G). There are mainly three structural fragments in its molecule: polymannuronate (PM) composed of β-D-(1,4)-mannuronic acid connected to each other; polyguluronate (PG) composed of α-L-(1,4)-guluronic acid connected to each other; and PMG fragments formed by alternating copolymerization of M and G.

[0004] The present inventors have previously developed a series of alginate lyases with high specificity, which can respectively decompose alginate into alginate disaccharides, trisaccharides or tetrasaccharides with high purity, a conjugated double bond at the non-reducing end and uniform polymerization degree. The alginate disaccharides are selected from ΔG, ΔM or a combination thereof; the alginate trisaccharides are selected from one or more of ΔGG, ΔGM, ΔMM and ΔMG; and the alginate tetrasaccharides are selected from one or more of ΔGGG, ΔGGM, ΔGMG, ΔGMM, ΔMMG, ΔMMM, ΔMGG and ΔMGM. All the oligosaccharides are connected by 1,4 glycosidic bonds; G represents α-L-guluronate; M represents β-D-mannuronate; and β-elimination occurs at the 4,5 position of guluronate and / or mannuronate to generate an unsaturated monosaccharide with a conjugated double bond at the 4,5 position of the non-reducing end, which is denoted by Δ. The structures of the corresponding monosaccharides are shown below:

[0005] Taking ΔGM as an example, the structure of the corresponding alginate trisaccharide is as follows:

[0006] Acute kidney injury (AKI), formerly known as acute renal failure, is a clinical syndrome caused by rapid decline in renal function within a short period of time due to various causes, manifested as rapid increase in serum creatinine and decrease in urine output. Although the field of nephrology has paid increasing attention to AKI, there is still no specific treatment, and the morbidity and mortality remain high. AKI has become a worldwide public health problem that threatens human health. Acute kidney injury causes great suffering to patients, so prevention and treatment of the disease is a pressing problem today.

[0007] Chronic kidney disease (CKD) is a disease that seriously endangers human health, and its incidence is high. In developed countries, more than 10% of adults have CKD of varying degrees. Although the initial evolution of CKD is related to the diversity of causes such as genetics, autoimmune-related infections, environmental factors, diet and drugs, progressive kidney disease often leads to renal fibrosis and eventually leads to renal failure.

[0008] Gout is a metabolic disease caused by disorders of purine metabolism and impaired uric acid excretion, leading to elevated blood uric acid concentration and deposition of urate crystals in joints and surrounding tissues. It is a common inflammatory arthritis disease in men, characterized by the formation of monosodium urate crystals in the synovial fluid of joints and synovium and other tissues of the body. The most common affected site is the metatarsophalangeal joint of the thumb.

[0009] Oral ulcers are a common and frequently occurring disease. Once formed, oral ulcers can not only affect a patient's normal life, work, and study, but can sometimes even trigger systemic diseases. Currently, there are many medications available for treating oral ulcers, but their effectiveness varies from person to person, and most of these products contain hormones and disinfectants, making them unsuitable for children and adolescents. Therefore, it is necessary to develop new medications for treating oral ulcers to meet the needs of different patients.

[0010] Rhinitis, or nasal inflammation, is an inflammation of the nasal mucosa caused by viruses, bacteria, allergens, various physical and chemical factors, and certain systemic diseases. The main pathological changes in rhinitis include congestion, swelling, exudation, hyperplasia, atrophy, or necrosis of the nasal mucosa.

[0011] Liver injury is a response to elevated serum transaminase levels and alterations in the physiological structure of the liver caused by one or more factors. It commonly occurs during the course of diseases such as jaundice and liver fibrosis, and in severe cases, can exacerbate liver failure. The main types of liver injury include chemical liver injury, alcoholic liver injury, cholestatic liver injury, and drug-induced liver injury.

[0012] Providing effective treatments for these diseases is also an urgent problem to be solved.

[0013] Invention Summary

[0014] To address the above problems, the present invention aims to provide a fucoidan oligosaccharide derivative, its preparation method, and its uses. Based on previous research, the inventors sequentially subjected the obtained homogeneous fucoidan oligosaccharide to a Δ-terminus removal reaction and a ring-opening oxidation reaction, synthesizing a class of fucoidan oligosaccharide derivatives. The fucoidan oligosaccharide derivatives designed and synthesized in this invention have good therapeutic effects on inflammatory diseases such as acute kidney injury, chronic kidney disease, gout, liver injury, oral ulcers, and rhinitis.

[0015] The objective of this invention is achieved through the following technical solution:

[0016] In a first aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0017] Where n is an integer, and n = 1-8, preferably n = 1-3.

[0018] According to some embodiments of the present invention, in the compound represented by formula (I), the structural unit The configuration is and / or When it originates from α-L-guluronic acid, it exhibits the following characteristics: Configuration; when it originates from β-D-mannuronic acid, it exhibits the following characteristics: Configuration.

[0019] According to some embodiments of the present invention, in the compound represented by formula (I), the end... The configuration is and / or When it originates from α-L-guluronic acid, it exhibits the following characteristics: Configuration; when it originates from β-D-mannuronic acid, it exhibits the following characteristics: Configuration.

[0020] Preferably, in the compound represented by formula (I), Structural units of configuration and The total number of ends of the configuration and Structural units of configuration and The ratio of the total number of ends of the configuration is 1-5:1, preferably 2-4:1.

[0021] Preferably, in the compound represented by formula (I), Structural units of configuration and The ratio of the number of structural units in the configuration is 1.0-2.5:1.

[0022] Preferably, in the compound represented by formula (I), The ends of the configuration and The ratio of the number of ends in the configuration is 3-5:1.

[0023] Preferably, the pharmaceutically acceptable salt is selected from one or more of sodium salts, potassium salts, calcium salts, magnesium salts, iron salts, zinc salts, and ammonium salts.

[0024] In a second aspect, the present invention provides a method for preparing a compound of formula (I) according to the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0025] In the presence of an oxidizing agent and a solvent, the compound represented by formula (II) or its pharmaceutically acceptable salt undergoes a ring-opening oxidation reaction to obtain the compound represented by formula (I) or its pharmaceutically acceptable salt.

[0026] According to some embodiments of the present invention, the preparation method includes the following steps:

[0027] 1) Dissolve the compound of formula (II) or its pharmaceutically acceptable salt in a solvent, then add an oxidizing agent, and react at 0-40°C, preferably 25°C, for 1-10 hours, preferably 1-3 hours, to obtain the reaction product;

[0028] 2) The reaction product is filtered, and then the filtrate is separated and purified to obtain the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0029] Preferably, in step 1), the solvent is a buffer solution; more preferably, the pH of the buffer solution is 6-10, more preferably 8-9; more preferably, the buffer solution is Na2HPO4-HCl buffer solution and / or K2HPO4-HCl buffer solution, more preferably Na2HPO4-HCl buffer solution; more preferably, the Na2HPO4 concentration in the Na2HPO4-HCl buffer solution is 0.1-1M, more preferably 0.5M.

[0030] Preferably, in step 1), the concentration of the compound represented by formula (II) or its pharmaceutically acceptable salt in the solvent is 0.1-0.5 g / mL, more preferably 0.2-0.3 g / mL.

[0031] Preferably, in step 1), the oxidant is selected from one or more of copper hydroxide, NaClO, bromine water, and H2O2.

[0032] Preferably, in step 1), the molar ratio of the compound represented by formula (II) or its pharmaceutically acceptable salt to the oxidant is 1:1-5, more preferably 1:2-4.

[0033] Preferably, in step 2), the separation and purification are performed using a gel column.

[0034] According to some embodiments of the present invention, the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is prepared by a method comprising the following steps:

[0035] In the presence of an oxidant and a solvent, the compound of formula (III) or its pharmaceutically acceptable salt undergoes a Δ-terminal removal reaction to obtain the compound of formula (II) or its pharmaceutically acceptable salt.

[0036] According to some embodiments of the present invention, the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is prepared by a method comprising the following steps:

[0037] a) Dissolve the compound of formula (III) or its pharmaceutically acceptable salt in a solvent, then add an oxidizing agent, and react at 0-40°C, preferably 25°C, for 1-10 hours, preferably 1-3 hours, to obtain the reaction product;

[0038] b) Filter the reaction product and then separate and purify the filtrate to obtain the compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0039] Preferably, in step a), the solvent is a buffer solution; more preferably, the pH of the buffer solution is 6-10, more preferably 8-9; more preferably, the buffer solution is Na2HPO4-HCl buffer solution and / or K2HPO4-HCl buffer solution, more preferably Na2HPO4-HCl buffer solution; more preferably, the Na2HPO4 concentration in the Na2HPO4-HCl buffer solution is 0.1-1M, more preferably 0.5M.

[0040] Preferably, in step a), the concentration of the compound represented by formula (III) or its pharmaceutically acceptable salt in the solvent is 0.1-1 g / mL, more preferably 0.5-0.7 g / mL.

[0041] Preferably, in step a), the oxidant is selected from one or more of copper hydroxide, NaClO, bromine water, and H2O2.

[0042] Preferably, in step a), the molar ratio of the compound represented by formula (III) or its pharmaceutically acceptable salt to the oxidant is 1:0.5-5, more preferably 1:1-1.5.

[0043] Preferably, in step b), the separation and purification are performed using a gel column.

[0044] Thirdly, the present invention provides a pharmaceutical composition comprising a preventive and / or therapeutically effective amount of a compound of formula (I) according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof.

[0045] Preferably, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is the sole active ingredient in the pharmaceutical composition.

[0046] Preferably, the pharmaceutical composition further comprises an active ingredient other than the compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0047] Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0048] Preferably, the pharmaceutical composition is an injection, tablet, capsule, granule, pill, oral solution, oral suspension, oral emulsion, effervescent tablet, ointment, cream, gel, patch, spray, external solution, external foam, suppository, or enema.

[0049] Fourthly, the present invention provides the use of a compound of formula (I) according to the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the prevention and / or treatment of inflammatory diseases; wherein the inflammatory disease is selected from one or more of acute kidney injury, chronic kidney disease, gout, oral ulcers, rhinitis, and liver injury.

[0050] Preferably, the symptoms of the chronic kidney disease are characterized by elevated levels of protein in the urine.

[0051] Preferably, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is the sole active ingredient in the drug.

[0052] Fifthly, the present invention provides the use of the compound of formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of inflammatory diseases; wherein the inflammatory disease is selected from one or more of acute kidney injury, chronic kidney disease, gout, liver injury, oral ulcers and rhinitis;

[0053] Where n is an integer, and n = 1-8, preferably n = 1-3.

[0054] In this invention, the compound represented by formula (II) is composed of monosaccharides G and / or M linked by glycosidic bonds at positions 1 and 4, where G represents α-L-guluronic acid and M represents β-D-mannuronic acid.

[0055] Preferably, the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is the sole active ingredient in the drug.

[0056] Preferably, the pharmaceutically acceptable salt is selected from one or more of sodium salts, potassium salts, calcium salts, magnesium salts, iron salts, zinc salts, and ammonium salts.

[0057] Preferably, the symptoms of the chronic kidney disease are characterized by elevated levels of protein in the urine.

[0058] In a sixth aspect, the present invention provides the use of the compound of formula (III) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of inflammatory diseases; wherein the inflammatory diseases are selected from one or more of chronic kidney disease, oral ulcers, and rhinitis;

[0059] Where n is an integer, and n = 0-8, preferably n = 0-3.

[0060] In this invention, the compound represented by formula (III) is composed of a monosaccharide Δ connected to G and / or M via glycosidic bonds at positions 1 and 4, where G represents α-L-guluronic acid, M represents β-D-mannuronic acid, and Δ represents an unsaturated monosaccharide with conjugated double bonds at positions 4 and 5, generated by β-elimination at positions 4 and 5 of α-L-guluronic acid or β-D-mannuronic acid.

[0061] Preferably, the compound represented by formula (III) or a pharmaceutically acceptable salt thereof is the sole active ingredient in the drug.

[0062] Preferably, the pharmaceutically acceptable salt is selected from one or more of sodium salts, potassium salts, calcium salts, magnesium salts, iron salts, zinc salts, and ammonium salts.

[0063] Preferably, the symptoms of the chronic kidney disease are characterized by elevated levels of protein in the urine.

[0064] According to some embodiments of the present invention, in the compounds represented by formula (II) or formula (III), the structural unit The configuration is and / or When it originates from α-L-guluronic acid, it exhibits the following characteristics: Configuration; when it originates from β-D-mannuronic acid, it exhibits the following characteristics: Configuration.

[0065] According to some embodiments of the present invention, in the compounds represented by formula (II) or (III), the end... The configuration is selected from One or more of these; when derived from α-L-guluronic acid, it exhibits the following characteristics: configuration or Configuration; when it originates from β-D-mannuronic acid, it exhibits the following characteristics: configuration or Configuration.

[0066] Preferably, in the compound represented by formula (II) or formula (III), Structural units of configuration and The total number of ends of the configuration and Structural units of configuration and The ratio of the total number of ends of the configuration is 1-5:1, preferably 2-4:1.

[0067] Preferably, in the compound represented by formula (II) or formula (III), Structural units of configuration and The ratio of the number of structural units in the configuration is 1.0-2.5:1.

[0068] Preferably, in the compound represented by formula (II) or formula (III), The total number of ends of the configuration and The ratio of the total number of ends of the configuration is 3-5:1.

[0069] The present invention has at least the following beneficial effects:

[0070] This invention synthesizes a novel alginate oligosaccharide derivative as shown in formula (I). Compared with unsaturated alginate oligosaccharides, the alginate oligosaccharide derivative designed and synthesized in this invention has better stability under different acid and alkaline conditions, which is more beneficial for the preservation and use of drugs, and helps to prolong the retention time of drugs in vivo and improve utilization.

[0071] The alginate oligosaccharide derivatives of formula (I) designed and synthesized in this invention have good therapeutic effects on inflammatory diseases such as acute kidney injury, chronic kidney disease, gout, oral ulcers, rhinitis, and liver injury. Furthermore, compared to unsaturated alginate oligosaccharides, the alginate oligosaccharide derivatives of formula (I) have a better therapeutic effect on liver injury.

[0072] The inventors have discovered that the alginate oligosaccharide derivative shown in formula (II) has good therapeutic effects on inflammatory diseases such as acute kidney injury, chronic kidney disease, gout, oral ulcers, rhinitis, and liver injury. Furthermore, compared to unsaturated alginate oligosaccharides, the alginate oligosaccharide derivative shown in formula (II) has a better therapeutic effect on liver injury.

[0073] The inventors have discovered that the alginate oligosaccharide shown in formula (III) has a good therapeutic effect on chronic kidney disease, oral ulcers and rhinitis.

[0074] The inventors have also discovered that, in the monosaccharides used to form the alginate oligosaccharides and their derivatives shown in formulas (I)-(III), when the proportion of guluronic acid (G) is greater than or equal to that of mannuronic acid (M), they have a better therapeutic effect on chronic kidney disease and oral ulcers compared to mannuronic acid oligosaccharides and their derivatives with a single configuration.

[0075] The fucoidan, trisaccharide, tetrasaccharide, and pentasaccharide derivatives with uniform degree of polymerization provided by this invention represent a revolutionary advancement in the quality control, pharmacological, and toxicological analysis of carbohydrate raw materials.

[0076] Brief description of the attached figures

[0077] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0078] Figure 1 shows the mass spectrum (LRMS(ESI)) of 1d alginate pentose;

[0079] Figure 2 shows the mass spectrum (LRMS(ESI)) of the homogeneous fucoidan derivative 2a;

[0080] Figure 3 shows the mass spectrum (LRMS(ESI)) of the homogeneous alginate derivative 2b;

[0081] Figure 4 shows the mass spectrum (LRMS(ESI)) of the homogeneous alginate derivative 2c;

[0082] Figure 5 shows the 1H NMR spectrum of the homogeneous fucoidan derivative 2a. 1 HNMR (solvent: D2O);

[0083] Figure 6 shows the 1H NMR spectrum of the homogeneous fucoidan derivative 2b. 1 HNMR (solvent: D2O);

[0084] Figure 7 shows the mass spectrum (LRMS(ESI)) of homogeneous fucoidan derivative 3a;

[0085] Figure 8 shows the mass spectrum (LRMS(ESI)) of homogeneous alginate derivative 3b;

[0086] Figure 9 shows the mass spectrum (LRMS(ESI)) of homogeneous alginate derivative 3c;

[0087] Figure 10 shows the 1H NMR spectrum of the homogeneous fucoidan derivative 3a. 1 HNMR (solvent: D2O);

[0088] Figure 11 shows the 1H NMR spectrum of the homogeneous fucoidan derivative 3b. 1 HNMR (solvent: D2O);

[0089] Figure 12 shows the effects of homogeneous alginate oligosaccharide derivatives 2 and 3 on ischemia-reperfusion (I / R) induced serum creatinine levels in rats;

[0090] Figure 13 shows the establishment and evaluation of a mouse model of acute gouty arthritis;

[0091] Figure 14 shows the effect of the brown algae oligosaccharide derivative in inhibiting joint pain symptoms in AGA model mice;

[0092] Figure 15 shows the effect of pH on the stability of different types of alginate oligosaccharides and their derivatives.

[0093] The best way to implement an invention

[0094] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0095] The homogeneous fucoidan 1 (m = 0-3) was prepared according to the methods in Examples 1-3 of the applicant's authorized patent applications CN113440532B and CN115671118B. The lysin used in the above methods was obtained from Professor Liu Weizhi's research group at Ocean University of China (patent applications CN107058423A and CN110423787A). The homogeneous fucoidan 1d (n = 3) was isolated from the mixed product after enzymatic hydrolysis, and its MS (ESI) was: m / z = 879.2 [(MH)]. - Its mass spectrum is shown in Figure 1.

[0096] The ratio of guluronic acid (G) to mannuronic acid (M) in fucoidan 1 (m = 0-3) was determined according to the following standard: YY / T 1654-2019 Sodium alginate for tissue engineering medical devices, Pharmaceutical Industry Standard of the People's Republic of China. The ratio of guluronic acid (G) to mannuronic acid (M) in the monosaccharides used to form fucoidan 1 (n = 0-3) was determined to be 1-5:1. Specifically, the ratio of G to M in the monosaccharides used to form structural units (i.e., monosaccharides excluding the two ends) was 1.0-2.5:1, and the ratio of G to M in the monosaccharides used to form the reducing end (i.e., the right end in the structural formula of fucoidan 1) was 3-5:1.

[0097] This invention also synthesized appropriate amounts of unsaturated mannuronic acid oligosaccharide 4 and mannuronic acid oligosaccharide derivative 5. Unsaturated mannuronic acid oligosaccharide 4 was prepared by first obtaining polymannuronic acid from alginate according to the method described in the paper "Purification and Activity Study of Polymannuronic Acid and Polyguluronic Acid Degradation Products" (Wang Haoxian, 2012, Ocean University of China), and then preparing it according to the methods in Examples 1-3 of the applicant's authorized patent applications CN113440532B and CN115671118B. The lysin used in the above methods was obtained from Professor Liu Weizhi's research group at Ocean University of China (patent applications CN107058423A and CN110423787A).

[0098] The ratio of guluronic acid (G) to mannuronic acid (M) in unsaturated mannuronic acid oligosaccharide 4 was determined according to the following standard: YY / T 1654-2019 Sodium alginate for tissue engineering medical devices, Pharmaceutical Industry Standard of the People's Republic of China. The determination showed that the proportion of mannuronic acid (M) in the monosaccharides used to constitute unsaturated mannuronic acid oligosaccharide 4 was above 95%.

[0099] As can be seen, the homogeneous fucoidan 1 (m = 0-3), fucoidan derivative 2, fucoidan derivative 3, unsaturated mannuronic acid oligosaccharide 4, and mannuronic acid oligosaccharide derivative 5 were obtained under near-neutral conditions, and the carboxylic acids in their structural formulas are mostly present in the form of sodium salts. Furthermore, all percentages in the following examples of this invention are by weight.

[0100] Example 1: Preparation and structural identification of homogeneous brown algae oligosaccharide derivative 2

[0101] This invention utilizes an oxidation reaction to remove the Δ end of the fucoidan oligosaccharide 1 composed of Δ, G, and M, thereby synthesizing fucoidan oligosaccharide derivative 2. The synthetic route is shown in Scheme 1.

[0102] Taking the preparation of homogeneous fucoidan oligosaccharide derivative 2a as an example, homogeneous fucoidan 1b (3.0 g, 5.05 mM in sodium salt form) was dissolved in 5 mL of 0.5 M Na₂HPO₄-HCl buffer (the pH was adjusted to approximately 8.5 by adding concentrated hydrochloric acid to a pre-prepared 0.5 M Na₂HPO₄ solution). Then, 3% bromine water (32.28 mL, 6.06 mM) was added dropwise, and the reaction was carried out at 25 °C for 3 hours. After the reaction, the target compound was obtained by gel column chromatography. Using the same method, 2b was obtained from 1c, and 2c was obtained from 1d.

[0103] The resulting compound 1 The H-NMR and MS data are as follows:

[0104] 2a: MS (ESI): m / z=369.1[(MH) - The mass spectrum is shown in Figure 2. 1 The H-NMR spectrum is shown in Figure 5.

[0105] 2b: MS(ESI): m / z=545.1[(MH) - The mass spectrum is shown in Figure 3; the 1H-NMR spectrum is shown in Figure 6.

[0106] 2c: MS (ESI): m / z = 721.2 [(MH)-], mass spectrum is shown in Figure 4.

[0107] Example 2: Preparation and structural identification of homogeneous brown algae oligosaccharide derivative 3

[0108] The present invention performs ring-opening oxidation on the reducing end of the above-mentioned alginate oligosaccharide derivative 2 to synthesize a series of alginate oligosaccharide acid derivatives 3, the synthetic route of which is shown in Scheme 2.

[0109] Taking the preparation of a homogeneous alginate oligosaccharide derivative 3a as an example, homogeneous alginate derivative 2a (1.18 g, 2.84 mM in sodium salt form) was dissolved in 5 mL of 0.5 M Na₂HPO₄-HCl buffer (the pH was adjusted to approximately 8.5 by adding concentrated hydrochloric acid to a pre-prepared 0.5 M Na₂HPO₄ solution). Then, 41.75 mL of 3% bromine water (7.83 mM) was added dropwise, and the reaction was carried out at 25 °C for 3 hours. After the reaction, the target compound was obtained by gel column chromatography. Using the same method, 3b was obtained from 2b, and 3c was obtained from 2c.

[0110] The resulting compound 1 The H-NMR and MS data are as follows:

[0111] 3a: MS (ESI): m / z=385.2[(MH) - The mass spectrum is shown in Figure 7. 1 The H-NMR spectrum is shown in Figure 10.

[0112] 3b: MS (ESI): m / z = 561.1 [(MH)-], mass spectrum shown in Figure 8; 1H-NMR spectrum shown in Figure 11.

[0113] 3c: MS (ESI): m / z = 737.1 [(MH)-], mass spectrum shown in Figure 9.

[0114] Example 3: Preparation of mannuronic acid oligosaccharide derivative 5

[0115] The present invention also utilizes an oxidation reaction to remove the Δ end of the unsaturated mannuronic acid oligosaccharide 4, thereby synthesizing mannuronic acid oligosaccharide derivative 5, the synthetic route of which is shown in Scheme 3.

[0116] For the specific preparation method of mannouronic acid oligosaccharide derivative 5, please refer to Example 1.

[0117] Example 4: Activity study of homogeneous fucoidan derivatives 2 and 3 in the treatment of acute kidney injury

[0118] Ischemia-reperfusion-induced kidney injury is a commonly used animal model to simulate acute kidney injury caused by clinical ischemia. The pharmacological activities of homogeneous fucoidan derivatives 2 and 3 against acute kidney injury were tested using an ischemia-reperfusion animal model.

[0119] Male Sprague Dawley rats weighing 220-250 grams were selected, with six rats in each group. Urine output was collected 24 hours prior to surgery and found to be normal. The rats were then randomly divided into three groups: a sham-operated group (Sham), a model group (I / R), and a drug administration group (I / R+2a, I / R+2b, I / R+2c, I / R+3a, I / R+3b, and I / R+3c, with a drug dosage of 0.1 g / kg / day). Ten minutes before surgery, the drug was administered via gavage. The model and sham-operated groups received the same volume of physiological saline. After intraperitoneal anesthesia with 10% chloral hydrate, the skin was routinely disinfected, and an incision was made in the abdomen to expose both kidneys. In the sham-operated group, only the kidneys were examined. The wound was then sutured layer by layer to complete the surgery. In the model and drug administration groups, the renal pedicles of both kidneys were clamped with large arterial clamps, the kidneys were repositioned, the wounds were covered with gauze, and a small amount of physiological saline was instilled for rehydration. Forty-five minutes later, the bilateral arterial clamps were released, and the wound was sutured layer by layer to complete the surgery. The rats were placed on a 37°C heating pad after surgery and returned to their metabolic cages after recovery. During this period, the rats' weight, food intake, water intake, and urine output were monitored. The rats were fed routinely post-surgery, and sacrificed 24 hours later for tissue analysis. Blood was collected from the inferior vena cava, centrifuged, and the supernatant serum was collected. Serum creatinine levels were then measured using a creatinine assay kit. The results were statistically analyzed using the p-value method, and the results are shown in Figure 12. Figure 12 shows that renal filtration function is impaired after acute ischemia-reperfusion (I / R) surgery, and the injury caused a significant increase in serum creatinine levels in rats. After administration of homogeneous alginate oligosaccharide derivatives 2 and 3, serum creatinine levels decreased to varying degrees, indicating a certain degree of recovery of glomerular function. In particular, compounds 2c, 3b, and 3c showed that serum creatinine levels could be restored to near normal, indicating that homogeneous alginate oligosaccharide derivatives 2 and 3 have significant therapeutic effects on acute kidney injury. The experimental results were statistically analyzed using the p-value method. * indicates that the data in this group were compared with the Sham group, and p < 0.05. # indicates that the data in this group were compared with the I / R group, and p < 0.05.

[0120] The results of the 24-hour urine output collected after the operation are shown in Table 1.

[0121] Table 1. Protective effects of homogeneous fucoidan derivatives 2 and 3 on I / R-induced increase in urine volume in rats.

[0122] The experimental results were statistically analyzed using the p-value method. * indicates that the data in this group were compared with the Sham group, and p < 0.05. # indicates that the data in this group were compared with the I / R group, and p < 0.05.

[0123] Table 1 shows that the urine concentration function of the kidneys was impaired after acute ischemia-reperfusion (I / R) surgery, and the urine volume increased significantly. After administration of homogeneous alginate oligosaccharide derivatives 2 and 3, the urine volume of rats was significantly reduced, suggesting that these compounds have a nephroprotective effect and that the renal tubular reabsorption function was restored to a certain extent.

[0124] Example 5: Activity study of homogeneous fucoidan and its derivatives (1, 2, 3) and mannan oligosaccharides and their derivatives (4, 5) in the treatment of chronic kidney disease.

[0125] A mouse model of chronic kidney disease induced by aristolochic acid (AA) was used as the research subject. Due to the limitation of the number of experimental animals, only one type of mannan oligosaccharide and its derivatives (4, 5) was selected for each group for study.

[0126] Seventy-eight male C57BL / J6 mice (25-28 grams) were selected, with six mice in each group. Preoperative 24-hour urine output was normal. The mice were randomly divided into three groups: a control group (CTL), a model group (AA), and a treatment group (AA+1a, AA+1b, AA+1c, AA+2a, AA+2b, AA+2c, AA+3b, AA+3c, AA+4c, and AA+5c, with a dosage of 0.1 g / kg / 48 hours). Aristolochic acid was used to induce chronic kidney disease. During modeling, mice in each model group received intraperitoneal injections of 3 mg / kg aristolochic acid every three days for three weeks, followed by gavage administration of physiological saline every two days. After discontinuing aristolochic acid treatment, the mice were fed for another three weeks, during which time physiological saline was continuously administered via gavage. Mice in the treatment group received aristolochic acid every two days via gavage for six weeks, starting with administration of aristolochic acid. Mice in the control group received intraperitoneal injections of physiological saline for three weeks, followed by gavage administration of physiological saline for six weeks. One week before sacrifice, mice were placed in metabolic cages, and their body weight, food intake, water intake, and urine output were measured. Urine samples were collected. Under anesthesia, blood was drawn from the inferior vena cava, and the supernatant serum was collected after centrifugation. Serum and urine creatinine and urine albumin levels were measured using creatinine and urine albumin assay kits, and the urine albumin to creatinine ratio (UACR) was calculated. The experimental results were statistically analyzed using the p-value method. The results are shown in Tables 2 and 3.

[0127] Table 2. Effects of brown alginic oligosaccharide derivatives on aristolochic acid-induced serum creatinine levels in mice.

[0128] * indicates that the data in this group is p<0.05 compared with the CTL group (control group), and # indicates that the data in this group is p<0.05 compared with the AA group (model group).

[0129] Table 2 shows that aristolochic acid treatment for three weeks significantly increased serum creatinine levels in mice, while homogeneous fucoidan and its derivatives (1, 2, 3) significantly reduced serum creatinine levels, suggesting a renal protective effect. This result indicates that, compared to the control group, mice treated with intraperitoneal injection of aristolochic acid showed significantly increased serum creatinine levels. The homogeneous fucoidan and its derivatives (1, 2, 3) treatment groups showed a significant decrease in serum creatinine, although it did not completely return to normal levels, suggesting that fucoidan derivatives have a certain protective effect against the decline in renal function in mice with chronic kidney disease. The mannouronic acid and its derivatives (4, 5) treatment groups also showed a decrease in serum creatinine levels compared to the control group, but the decrease was less than that in the homogeneous fucoidan and its derivatives (1, 2, 3) treatment groups, indicating that while the single-configuration mannouronic acid oligosaccharide derivatives have a protective effect on renal function in mice with chronic kidney disease, it is weaker than that of fucoidan and its derivatives (1, 2, 3).

[0130] Table 3. Effects of fucoidan oligosaccharides and their derivatives on aristolochic acid-induced UACR in mice.

[0131] UACR: Urinary albumin to creatinine ratio; the experimental results were statistically processed using the p-value method. * indicates that the data in this group is compared with the CTL group (control group), and # indicates that the data is compared with the AA group (model group), and p is less than 0.05.

[0132] Proteinuria is an important indicator of chronic kidney injury. Table 3 shows that aristolochic acid treatment for three weeks significantly increased the urinary albumin-to-creatinine ratio (UACR) in mice. Homogeneous fucoidan and its derivatives (1, 2, 3) significantly reduced UACR, suggesting a renal protective effect. The urinary albumin levels in the mannosuric acid and its derivatives (4, 5) groups were also lower than those in the control group, but the decrease was less than that in the homogeneous fucoidan and its derivatives (1, 2, 3) groups. This indicates that while the single-configuration mannosuric acid oligosaccharide derivatives have a protective effect on renal function in mice with chronic kidney disease, it is weaker than that of fucoidan and its derivatives (1, 2, 3).

[0133] These results indicate that, compared with the control group, mice administered aristolochic acid via intraperitoneal injection showed increased urinary protein excretion. The UACR (urinary albumin reduction) was significantly decreased in the groups treated with fucoidan oligosaccharides and their derivatives (1, 2, 3), although it did not return to normal levels. This suggests that fucoidan oligosaccharide derivatives have a good protective effect against decreased renal function in mice with chronic kidney disease. However, the protective effect of mannan oligosaccharides and their derivatives (4, 5) against decreased urinary albumin was weaker than that of fucoidan oligosaccharides and their derivatives (1, 2, 3).

[0134] Example 6: Activity study of fucoidan derivatives 2 and 3 in the treatment of gout

[0135] I. Establishment of an Acute Gouty Arthritis (AGA) Mouse Model

[0136] Healthy, clean-grade male C57BL / 6 mice were randomly divided into a control group (Veh) and a model group (MSU), with six mice in each group. After disinfection with medical alcohol, the model group underwent an injection of 1 mg / 20 μl sodium urate (MSU) solution into the ankle joint cavity at a 30-40° angle along the medial side of the Achilles tendon on the posterior side of the right ankle joint. The control group received an equal volume of phosphate-buffered saline (PBS) solution in the same manner. After injection, the degree of ankle swelling and changes in the mechanical pain threshold were observed. Ankle swelling measurement: Using calipers, the diameter of the affected ankle joint was measured at four time points: before modeling, and 2 h, 6 h, 24 h, and 48 h after modeling. Three consecutive measurements were taken, and the average value was used as the final reading. Ankle swelling = current measured diameter - pre-model measured diameter. Mechanical pain behavior measurement: Mice were placed in a transparent plastic box on an elevated wire mesh and covered with transparent plexiglass for 45 minutes to acclimatize to the environment. Subsequently, following the "Up and Down" method, the pain threshold near the ankle of the right hind paw of mice was measured using von Frey wire of different specifications at four time points: before modeling, and 2h, 6h, 24h, and 48h after modeling. The paw retraction threshold was calculated using a formula.

[0137] Figure 13 illustrates the establishment and efficacy evaluation of the acute gouty arthritis mouse model. Figure 13A shows the swelling of the right ankle joint of AGA control mice after injection of phosphate buffered saline (PBS) and model mice after injection of urate (MSU) crystals. Twenty-four hours after injection of urate (MSU) crystals, the AGA model mice showed significant swelling in their right ankle joints compared to the control mice injected with phosphate buffered saline (PBS). Figure 13B compares H&E-stained sections of the ankle joints of the control and model mice. It can be observed that, compared to the control group, the AGA model mice showed extensive inflammatory cell infiltration in their ankle joint sections. Figure 13C shows the change in ankle joint diameter between mice injected with urate crystals and those injected with PBS. It can be seen that the control group mice experienced only slight swelling in their ankle joints within hours after PBS injection, which quickly subsided and remained largely unchanged after returning to normal. In contrast, the ankle joints of mice injected with urate crystals swelled rapidly, and significant swelling persisted even after 48 hours. Figure 13D shows a comparison of mechanical hyperalgesia in the right hind paw of the two groups of mice. The results show that the mechanical pain threshold of the mice injected with MSU was significantly reduced. These results are consistent with previous reports, thus indicating that the AGA mouse model has been successfully established.

[0138] II. Study on the analgesic effect of fucoidan oligosaccharide derivatives on AGA model mice

[0139] As described in "I. Establishment of an Acute Gouty Arthritis (AGA) Mouse Model" of this embodiment, an AGA mouse model was established. Mice were randomly divided into 8 groups: a control group (Veh+Veh), a model group (MSU+Veh), and a modeling drug administration group (MSU+2a, MSU+2b, MSU+2c, MSU+3a, MSU+3b, MSU+3c, with a dosage of 200 mg / kg). The drug administration group received intraperitoneal injections of the corresponding alginate oligosaccharide derivatives at 1 h before modeling, 5 h after modeling, 23 h after modeling, and 47 h after modeling, respectively. The control group and the model group received an equal volume of the control solvent PBS intraperitoneally. Mechanical pain and swelling were assessed before modeling and at 2 h, 6 h, 24 h, and 48 h after modeling. The specific detection methods were consistent with those described in "I. Establishment of an Acute Gouty Arthritis (AGA) Mouse Model" of this embodiment.

[0140] Figure 14 shows the effect of fucoidan oligosaccharide derivatives on inhibiting joint pain symptoms in AGA model mice. Figure 14A is a time-course graph showing the effects of the control group, model group, and various fucoidan oligosaccharide derivatives on the mechanical pain of the ankle joint in AGA model mice at a dose of 200 mg / kg. The time-course graphs show that compared with the control group, the withdrawal threshold was significantly lower in the model group, indicating significantly more severe mechanical pain; after administration of fucoidan oligosaccharide derivatives, the withdrawal threshold was significantly increased, indicating significant relief of mechanical pain. Figure 14B is a time-course graph showing the effects of each group of animals on ankle swelling in AGA model mice. Compared with the control group, the ankle swelling in the model group was very obvious; after administration of fucoidan oligosaccharide derivatives, the degree of ankle swelling in the mice was significantly reduced. The above experiments demonstrate that fucoidan oligosaccharide derivatives can effectively inhibit ankle pain and swelling in AGA model mice.

[0141] Example 7: Activity study of fucoidan and its derivatives (1, 2, 3) and mannan oligosaccharides and their derivatives (4, 5) in the treatment of oral ulcers.

[0142] This study investigated the effects of alginate oligosaccharide derivatives on oral ulcers in adults (caused by chemotherapy). Preliminary experiments revealed that direct administration of the compound powder caused irritation, leading to increased and unbearable pain at the ulcer site within a short period. Furthermore, the drug's high water solubility meant it was easily diluted by saliva, resulting in a short retention time. Therefore, this invention prepared oral ulcer patches using various candidate drugs for testing. Taking alginate oligosaccharide derivative 2b as an example, the formulation of a 6mm diameter, 60mg patch is shown in Table 4, where HPMC is hydroxypropyl methylcellulose; CP934 is carbomer 934; and MCC is microcrystalline cellulose.

[0143] Table 4 Oral Patch Prescriptions

[0144] According to the formulation ratio in Table 4, weigh out a certain amount of the drug and excipients, grind them evenly, and directly compress the powder into tablets using a 6mm diameter flat-head punch. The tablet hardness should be controlled within the range of 4-6 kgf, and the tablet thickness should be approximately 1.4mm. Oral adhesion tests showed that the resulting patches were not easily detached in the oral cavity, had minimal foreign body sensation, no unpleasant odor, and were unlikely to cause nausea or vomiting. The oral adhesion and melting time was approximately 1.5–2.0 hours, making them suitable for use as intraoral patches. The remaining alginate oligosaccharide derivatives were also prepared into 6mm diameter oral patches according to the formulation in Table 4.

[0145] Oral ulcer patch irritation and healing promotion effect test

[0146] Mannan oligosaccharides and their derivatives (4, 5) were studied using only one type per group. Adult patients with oral ulcers (caused by chemotherapy) were divided into a control group (no medication) and medication groups (groups 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b, 3c, 4c, and 5c), with 10 patients in each group. Except for the control group, which received no medication, the other groups were given oral patches made from the respective mannan oligosaccharide derivatives, 3 patches per day, applied to the ulcer until the ulcer healed. The results showed that no patients experienced irritation or pain after applying the oral patches, and the pain caused by the ulcer itself was significantly reduced after 5-10 minutes; the foreign body sensation in the mouth was not obvious, and it did not affect drinking or swallowing. The healing time of oral ulcers in each group was significantly shorter than that in the control group after medication, as shown in Table 5.

[0147] Table 5. Effect of oral patches on oral ulcer healing time.

[0148] a In the control group, only 5 patients healed within 7 days, 3 of them healed within 10 days, and 2 of them did not heal after 10 days and were treated with other methods.

[0149] b One patient in group 5c did not recover within 7 days.

[0150] As shown in Table 5, in the control group without medication, only 4 patients healed spontaneously within 7 days, and only 1 patient healed spontaneously on the 5th day. Two patients still had not healed after 10 days. In contrast, in the oral patch groups made from various fucoidan oligosaccharides and their derivatives (1, 2, 3), some patients began to heal on the 2nd day, most healed on the 3rd day, and all patients healed within 5 days. Compared with the control group, the healing rate of patients in the fucoidan oligosaccharide and its derivative (1, 2, 3) groups was significantly shorter, indicating that fucoidan oligosaccharides and their derivatives (1, 2, 3) have a significant therapeutic effect on oral ulcers. In the mannan oligosaccharide and its derivative (4, 5) groups, some patients began to heal from the 3rd day. The average healing time was significantly longer than that of the fucoidan oligosaccharide and its derivative (1, 2, 3) groups, and one patient in group 5c had not healed within 7 days. This indicates that while the structurally simple mannan oligosaccharides (4, 5) have a therapeutic effect on oral ulcers, the effect is lower than that of the fucoidan oligosaccharide and its derivative (1, 2, 3) groups.

[0151] Example 8: Activity study of fucoidan oligosaccharides and their derivatives (1, 2, 3) in the treatment of rhinitis

[0152] Rhinitis, or nasal inflammation, is an inflammation of the nasal mucosa caused by viruses, bacteria, allergens, various physical and chemical factors, and certain systemic diseases. The main pathological changes in rhinitis include congestion, swelling, exudation, hyperplasia, atrophy, or necrosis of the nasal mucosa. Allergic rhinitis (also known as hay fever) is a more common type of rhinitis caused by allergens.

[0153] A guinea pig model was established by nasal drops of 2,4-toluene diisocyanate (TDI) and olive oil solution, and the inhibitory effect of the brown algae oligosaccharide derivative on allergic rhinitis was tested.

[0154] I. Establishment of a guinea pig allergic rhinitis model induced by 10% TDI olive oil solution

[0155] Select healthy guinea pigs of similar weight, half male and half female. Add 10 μl of 10% TDI olive oil solution to both anterior nostrils of the guinea pigs (5 μl on each side) using a pipette, once a day for 5 to 7 consecutive days, then switch to every other day (maintenance period).

[0156] II. The effect of brown algae oligosaccharide derivatives in inhibiting allergic rhinitis

[0157] Guinea pigs were randomly divided into 12 groups of 8 animals each: a control group (CTL), a model group (TDI), and three model-inducing drug administration groups (TDI+1a, TDI+1b, TDI+1c, TDI+1d, TDI+2a, TDI+2b, TDI+2c, TDI+3a, TDI+3b, and TDI+3c). Drug administration began on day 7 after model initiation. Each alginate oligosaccharide derivative was diluted to 100 mg / ml with 0.9% saline and administered at a dose of 100 μl / animal, 50 μl per nostril, twice daily for 5 consecutive days. The control and model groups were fed routinely and given the same dose of saline twice daily.

[0158] Starting from the first day of administration, nasal symptoms of guinea pigs in each group were observed and scored within 30 minutes after TDI nasal drops (scoring criteria are shown in Table 6). Five days later, the guinea pigs were sacrificed, and the respiratory tract nasal mucosa was observed for congestion, redness, and swelling. Local reactions were expressed in a scoring manner according to the criteria in Table 7.

[0159] Table 6. Nasal Appearance Symptoms and Scoring Criteria in TDI Model Guinea Pigs

[0160] Table 7 Grading Criteria for Local Mucosal Irritation Response

[0161] The statistical results of allergic rhinitis symptoms in each group of guinea pigs are shown in Table 8.

[0162] Table 8. Effects of each drug administration group on TDI-induced allergic rhinitis symptoms in guinea pigs (x±s, n=8)

[0163] Table 8 shows that, compared with the control group, the model group guinea pigs experienced significantly worsened symptoms of nasal itching, sneezing, and runny nose before and within 5 days after drug administration. Compared with the model group, the symptoms in all groups treated with the alginate oligosaccharide derivatives began to significantly decrease from the second day after administration. On the fifth day, except for the TDI+1d, TDI+2c, and TDI+3a groups (which had scores above 4, and the TDI+1a group (close to 4)), the scores in the remaining groups were between 2 and 3. This indicates that the alginate oligosaccharide derivatives all have a certain therapeutic effect on allergic rhinitis.

[0164] Table 9. Effects of each drug administration group on the nasal mucosal irritation response score of TDI-treated guinea pigs (x±s, n=8)

[0165] Table 9 shows the scores of nasal mucosal irritation response in each group of guinea pigs. Compared with the control group, the score of the model group was significantly higher, indicating that the nasal mucosal irritation was very severe. Compared with the model group, the nasal mucosal irritation scores of all treatment groups of the alginate oligosaccharide derivative were significantly lower. Except for the TDI+1d, TDI+2c, and TDI+3a groups, whose scores were slightly higher (between 0.4 and 0.6), the scores of the other treatment groups were basically around 0.2 to 0.3, slightly higher than the control group. This indicates that these compounds have a good repair effect on nasal mucosal irritation in the guinea pig model of allergic rhinitis.

[0166] Example 9: Study on the therapeutic activity of fucoidan oligosaccharides and their derivatives (1, 2, 3) for liver injury.

[0167] Liver injury is a response to elevated serum transaminases and altered liver physiological structure caused by one or more factors. It commonly occurs during the course of diseases such as jaundice and liver fibrosis, and in severe cases, can exacerbate liver failure. Liver injury is mainly classified into chemically induced liver injury, alcoholic liver injury, cholestatic liver injury, and drug-induced liver injury. Chemically induced liver injury is a very important type, and CCl4 is a commonly used modeling agent for it. The inventors used a CCl4-induced mouse model of acute liver injury to study the protective effect of certain fucoidan oligosaccharide derivatives against acute liver injury, and compared them with unsaturated fucoidan oligosaccharide 1.

[0168] Sixty C57BL / 6 mice, weighing 18–22 g, with half males and half females, were randomly divided into 10 groups of 6 mice each. These included a blank control group (CTL), a model group (CCl4), and model-induced drug administration groups (CCl4+1b, CCl4+1c, CCl4+2a, CCl4+2b, CCl4+2c, CCl4+3a, CCl4+3b, and CCl4+3c). Administration was by gavage at a dose of 200 mg / kg. Unsaturated fucoidan 1 and fucoidan derivatives 2 and 3 were diluted with 0.9% physiological saline to 20 mg / ml and administered by gavage at a dose of 0.1 ml / 10 g body weight (dose 200 mg / kg). The blank control group and the model control group were given 0.1 ml / 10 g body weight of physiological saline daily. Mice in the treatment groups were administered the drug via gavage daily (0.1 ml / 10 g) for 7 days. Two hours after the last administration, except for the blank control group which received an intraperitoneal injection of a blank oil solution, all other groups received an intraperitoneal injection of 0.1 ml / 10 g of 0.1% CCl4 olive oil solution. Blood was collected 24 hours later, centrifuged for 8 minutes at 3500 rpm, and serum was separated. Simultaneously, liver tissue from the same location was harvested from the mice, homogenized in physiological saline solution to prepare a 10% homogenate, centrifuged for 15 minutes at 3500 rpm, and the supernatant was separated. The separated serum and liver homogenate were stored at -20℃ for later analysis. Various indicators in the serum and liver were detected using a fully automated biochemical analyzer, following the instructions for each kit. The results of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice are shown in Table 10.

[0169] Table 10 Effects of the drug on serum ALT and AST in mice (x±s, n=6)

[0170] In hepatocytes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are distributed in the cytoplasm and mitochondria. Damage to hepatocytes can lead to leakage, resulting in a significant increase in serum ALT and AST levels, indicating hepatocyte damage. As shown in Table 10, compared to the control group, the model group showed a significant increase in serum ALT and AST, indicating hepatocyte damage. In the fucoidan derivative 2 and 3 treatment groups (CCl4+2a, CCl4+2b, CCl4+2c, CCl4+3a, CCl4+3b, and CCl4+3c), serum ALT and AST levels were significantly lower than in the model group, indicating that these two compounds significantly improved CCl4-induced liver injury. However, in the unsaturated fucoidan 1 treatment groups (CCl4+1b and CCl4+1c), although serum ALT and AST levels decreased compared to the model group, the changes were minimal, indicating that these compounds had little protective effect against liver injury in mice.

[0171] The results of the detection of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) in mouse liver tissue are shown in Table 11.

[0172] Table 11 Effects of the drug on GSH-Px, SOD and MDA in mouse liver tissue (x±s, n=6)

[0173] GSH-Px is an important peroxidase widely present in the body, whose main function is to remove various hydrogen peroxides; SOD is an oxygen free radical scavenging enzyme. Measuring GSH-Px and SOD in mouse liver homogenate can directly reflect the degree of liver damage. Table 11 shows that compared with the control group, both GSH-Px and SOD in mouse liver tissue were significantly reduced, indicating liver damage. After drug administration, in all groups, the groups treated with fucoidan derivatives 2 and 3 (CCl4+2a, CCl4+2b, CCl4+2c, CCl4+3a, CCl4+3b, and CCl4+3c) showed significantly increased GSH-Px and SOD in mouse liver tissue compared with the model group, indicating that these two compounds can significantly improve CCl4-induced liver damage. However, compared with the model group, the levels of GSH-Px and SOD in the liver tissue of mice in the unsaturated fucoidan-1 treatment groups (CCl4+1b and CCl4+1c) were increased, but the changes were not significant, indicating that these compounds have a weak protective effect on the liver of mice.

[0174] Malondialdehyde (MDA) is a product of the reaction between oxygen free radicals and unsaturated fatty acids on the cell membrane surface, and is a cause of cell swelling and necrosis. Measuring the MDA content in liver tissue can reflect the degree of oxidative damage to tissues when cells are attacked by free radicals. Table 11 shows that compared with the control group, the model group had a significantly increased MDA content in mouse liver tissue, indicating liver damage. After drug administration, the groups treated with fucoidan derivatives 2 and 3 (CCl4+2a, CCl4+2b, CCl4+2c, CCl4+3a, CCl4+3b, and CCl4+3c) showed varying degrees of decrease in MDA in mouse liver tissue compared with the model group, indicating that these two types of compounds can significantly improve CCl4-induced liver damage. However, the MDA content in the liver tissue of mice treated with unsaturated fucoidan derivative 1 (CCl4+1b and CCl4+1c) was basically the same as that in the model group, indicating that this type of compound had almost no protective effect on the mouse liver.

[0175] Example 10: Stability Study of Different Types of Fucoidan Oligosaccharides and Their Derivatives (1, 2, 3)

[0176] In the process of preparing various types of brown algae oligosaccharide compounds and conducting activity tests, the inventors found that their stability varied significantly under different acidic and alkaline conditions, which may be one of the reasons for the differences in their activity.

[0177] For unsaturated fucoidan 1 and fucoidan derivatives (2 and 3), one of each type (1b, 2b, and 3b) was dissolved in purified water to prepare a solution with a concentration of 100 mg / ml. The pH of the solution was adjusted to 3, 5, 7, 9, 11, and 13 with hydrochloric acid and / or sodium hydroxide. After standing at room temperature for 1, 3, 5, 7, and 9 days, the concentration changes were measured. The results are shown in Figure 15. As can be seen from Figure 15A, unsaturated fucoidan 1b has good stability at moderate acidity and alkalinity (pH 5–9); at pH values ​​of 3 and 11, the acidity and alkalinity are relatively strong, and about 10% of the product is destroyed after 9 days; with increasing pH value, especially when the pH rises above 11, the destruction rate is significantly accelerated. Figures 15B and 15C show that saturated brown algae oligosaccharide derivatives 2b and 3b exhibit significantly enhanced stability under the same acidic or alkaline conditions. Within a pH range of 3–13, degradation did not exceed 5% after 9 days at room temperature. This enhanced stability in acidic and alkaline environments is more beneficial for drug preservation and use, extending the drug's retention time in the body and improving its utilization rate.

[0178] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or related embodiments obtained by those skilled in the art through some modifications or variations made to the above-disclosed technical content without departing from the scope of the present invention are within the scope of the present invention.

Claims

1. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof, in, n is an integer, and n = 1-8, preferably n = 1-3.

2. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein, In the compound shown in formula (I), the structural unit The configuration is and / or end The configuration is and / or Preferably, in the compound represented by formula (I), Structural units of configuration and The total number of ends of the configuration and Structural units of configuration and The ratio of the total number of ends of the configuration is 1-5:1, preferably 2-4:1; Preferably, in the compound represented by formula (I), Structural units of configuration and The ratio of the number of structural units in the configuration is 1.0-2.5:1; Preferably, in the compound represented by formula (I), The ends of the configuration and The ratio of the number of ends in the configuration is 3-5:1; Preferably, the pharmaceutically acceptable salt is selected from sodium salts, potassium salts, calcium salts, magnesium salts, and iron salts. One or more of the following: salts, zinc salts, and ammonium salts.

3. A method for preparing the compound of formula (I) according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, comprising the following steps: In the presence of an oxidizing agent and a solvent, the compound represented by formula (II) or its pharmaceutically acceptable salt undergoes a ring-opening oxidation reaction to obtain the compound represented by formula (I) or its pharmaceutically acceptable salt.

4. The preparation method according to claim 3, wherein, The preparation method includes the following steps: 1) Dissolve the compound of formula (II) or its pharmaceutically acceptable salt in a solvent, then add an oxidizing agent, and react at 0-40°C, preferably 25°C, for 1-10 hours, preferably 1-3 hours, to obtain the reaction product; 2) The reaction product is filtered, and then the filtrate is separated and purified to obtain the compound of formula (I) or a pharmaceutically acceptable salt thereof; Preferably, in step 1), the solvent is a buffer solution; more preferably, the pH of the buffer solution is 6-10, more preferably 8-9; more preferably, the buffer solution is Na2HPO4-HCl buffer and / or K2HPO4-HCl buffer, more preferably Na2HPO4-HCl buffer; more preferably, the Na2HPO4 concentration in the Na2HPO4-HCl buffer solution is 0.1-1M, more preferably 0.5M; Preferably, in step 1), the concentration of the compound represented by formula (II) or its pharmaceutically acceptable salt in the solvent is 0.1-0.5 g / mL, more preferably 0.2-0.3 g / mL; Preferably, in step 1), the oxidant is selected from one or more of copper hydroxide, NaClO, bromine water, and H2O2; Preferably, in step 1), the molar ratio of the compound represented by formula (II) or its pharmaceutically acceptable salt to the oxidant is 1:1-5, preferably 1:2-4; Preferably, in step 2), the separation and purification are performed using a gel column.

5. The preparation method according to claim 3 or 4, wherein, The compound represented by formula (II) or a pharmaceutically acceptable salt thereof is prepared by a method comprising the following steps: In the presence of an oxidizing agent and a solvent, the compound represented by formula (III) or thereof becomes pharmaceutically acceptable. The salt undergoes a Δ-terminal removal reaction to give the compound shown in formula (II) or a pharmaceutically acceptable salt thereof.

6. The preparation method according to any one of claims 3 to 5, wherein, The compound represented by formula (II) or a pharmaceutically acceptable salt thereof is prepared by a method comprising the following steps: a) Dissolve the compound of formula (III) or its pharmaceutically acceptable salt in a solvent, then add an oxidizing agent, and react at 0-40°C, preferably 25°C, for 1-10 hours, preferably 1-3 hours, to obtain the reaction product; b) Filter the reaction product and then separate and purify the filtrate to obtain the compound of formula (II) or a pharmaceutically acceptable salt thereof; Preferably, in step a), the solvent is a buffer solution; more preferably, the pH of the buffer solution is 6-10, more preferably 8-9; more preferably, the buffer solution is Na2HPO4-HCl buffer and / or K2HPO4-HCl buffer, more preferably Na2HPO4-HCl buffer; more preferably, the Na2HPO4 concentration in the Na2HPO4-HCl buffer solution is 0.1-1M, more preferably 0.5M; Preferably, in step a), the concentration of the compound represented by formula (III) or its pharmaceutically acceptable salt in the solvent is 0.1-1 g / mL, more preferably 0.5-0.7 g / mL; Preferably, in step a), the oxidant is selected from one or more of copper hydroxide, NaClO, bromine water, and H2O2; Preferably, in step a), the molar ratio of the compound represented by formula (III) or its pharmaceutically acceptable salt to the oxidant is 1:0.5-5, more preferably 1:1-1.5; Preferably, in step b), the separation and purification are performed using a gel column.

7. A pharmaceutical composition comprising a preventive and / or therapeutically effective amount of the compound of formula (I) according to claim 1 or 2 or a pharmaceutically acceptable salt thereof; Preferably, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is the sole active ingredient in the pharmaceutical composition; Preferably, the pharmaceutical composition further comprises an active ingredient other than the compound represented by formula (I) or a pharmaceutically acceptable salt thereof; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients; Preferably, the pharmaceutical composition is an injection, tablet, capsule, granule, pill, oral solution, oral suspension, oral emulsion, effervescent tablet, ointment, cream, gel, patch, spray, external solution, external foam, suppository, or enema.

8. Use of the compound of formula (I) according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the prevention and / or treatment of inflammatory diseases; wherein, The inflammatory disease is selected from one or more of the following: acute kidney injury, chronic kidney disease, gout, oral ulcers, rhinitis, and liver injury; Preferably, the symptoms of the chronic kidney disease include elevated levels of protein in the urine; Preferably, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is the sole active ingredient in the drug.

9. Use of the compound represented by formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of inflammatory diseases; wherein, The inflammatory disease is selected from one or more of the following: acute kidney injury, chronic kidney disease, gout, liver injury, oral ulcers, and rhinitis; Where n is an integer, and n = 1-8, preferably n = 1-3.

10. The use according to claim 9, wherein, The compound represented by formula (II) or a pharmaceutically acceptable salt thereof shall be the sole active ingredient in the drug; Preferably, the pharmaceutically acceptable salt is selected from one or more of sodium salts, potassium salts, calcium salts, magnesium salts, iron salts, zinc salts, and ammonium salts; Preferably, the symptoms of the chronic kidney disease are characterized by elevated levels of protein in the urine.

11. Use of the compound represented by formula (III) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of inflammatory diseases; wherein, The inflammatory disease is selected from one or more of chronic kidney disease, oral ulcers, and rhinitis; Where n is an integer, and n = 0-8, preferably n = 0-3.

12. The use according to claim 11, wherein, The compound represented by formula (III) or a pharmaceutically acceptable salt thereof shall be the sole active ingredient in the drug; Preferably, the pharmaceutically acceptable salt is selected from one or more of sodium salts, potassium salts, calcium salts, magnesium salts, iron salts, zinc salts, and ammonium salts; Preferably, the symptoms of the chronic kidney disease are characterized by elevated levels of protein in the urine.

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