Dehydroxylated brown algae oligosaccharide derivative, and preparation method therefor and use thereof

Abstract

The present invention relates to a dehydroxylated brown algae oligosaccharide derivative, and a preparation method therefor and a use thereof. The dehydroxylated brown algae oligosaccharide derivative provided by the present invention is formed by linking G or M with dihydroxylation at the 4-position of a non-reducing end to monosaccharide G and / or M by means of a 1,4-glycosidic bond. A dehydroxylated brown algae oligosaccharide acid derivative provided by the present invention is formed by linking G or M with dihydroxylation at the 4-position of a non-reducing end, G or M with oxidization into carboxyl at the 1-position of a reducing end, and monosaccharide G and / or M by means of a 1,4-glycosidic bond. The dehydroxylated brown algae oligosaccharide derivative and the dehydroxylated brown algae oligosaccharide acid derivative designed and synthesized in the present invention both have relatively good treatment effects on inflammatory diseases such as acute kidney injury, chronic kidney diseases, gout, ulcerative colitis, Crohn's disease, oral ulcer, rhinitis, and liver injury.

Description

A dehydroxylated brown algal oligosaccharide derivative, its preparation method and uses Technical Field

[0001] This invention belongs to the field of pharmaceutical technology. Specifically, this invention relates to a dehydroxylated brown algae oligosaccharide derivative, its preparation method, and its uses. Background Technology

[0002] Carbohydrates, along with nucleic acids and proteins, are considered the three major substances of life. Alginate, mainly found in the cell walls of kelp, Sargassum, and giant kelp, is a type of linear, unbranched polysaccharide compound with a negative charge. Its high viscosity and gelling properties make it widely used as a coagulant, thickener, and stabilizer in food, chemical, pharmaceutical, and textile industries. In the pharmaceutical field, alginate has broad applications in medical biomaterials and drug sustained-release materials due to its unique physicochemical properties and good biocompatibility. Studies have also found that alginate possesses antioxidant, immunomodulatory, and antitumor biological activities; however, its large molecular weight, strong gelling properties, and poor absorption significantly limit its applications in these areas. Oligosaccharides, on the other hand, have attracted attention due to their well-defined structure, significant activity, good absorption, and minimal side effects.

[0003] In recent years, due to the unique structure of alginate oligosaccharides, their activity research has become a hot topic in carbohydrate drug research. Alginate is a binary linear block compound composed of β-D-(1,4)-mannuronic acid (M) and α-L-(1,4)-guluronic acid (G). Its molecule mainly contains three structural segments: polymannuronate (PM) formed by interconnected β-D-(1,4)-mannuronic acids; polyguluronate (PG) formed by interconnected α-L-(1,4)-guluronic acids; and PMG segments formed by alternating copolymerization of M and G.

[0004] In its previous research, the inventors developed a series of highly specific alginate lyases to address the current challenges in carbohydrate research. These lyases can decompose alginate into highly pure, non-reducing, and uniformly polymerized fucoidan, trisaccharide, or tetrasaccharide. Specifically, the fucoidan is selected from ΔG, ΔM, or combinations thereof; the fucoidan trisaccharide is selected from one or more of ΔGG, ΔGM, ΔMM, and ΔMG; and the fucoidan tetrasaccharide is selected from one or more of ΔGGG, ΔGGM, ΔGMG, ΔGMM, ΔMMG, ΔMMM, ΔMGG, and ΔMGM. All oligosaccharides are monosaccharides linked by glycosidic bonds at positions 1 and 4; G represents α-L-guluronic acid; M represents β-D-mannuronic acid; β-elimination occurs at positions 4 and 5 of guluronic acid and / or mannuronic acid, generating unsaturated monosaccharides with non-reducing conjugated double bonds at positions 4 and 5, denoted by Δ. The structures of the corresponding monosaccharides are shown below:

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

[0006] Acute kidney injury (AKI), formerly known as acute renal failure, is a clinical syndrome caused by a rapid decline in kidney function over a short period of time due to various etiologies. It is characterized by a rapid increase in serum creatinine and a decrease in urine output. Despite increasing attention from the nephrology community, there is currently no specific treatment for AKI, and its morbidity and mortality rates remain high. AKI has become a global public health problem threatening human health. Acute kidney injury causes immense suffering for patients; therefore, the prevention and treatment of this disease are urgent issues to be addressed.

[0007] Chronic kidney disease (CKD) is a serious threat to human health with a high incidence rate. In developed countries, more than 10% of adults have CKD to varying degrees. Although the initial development of CKD is related to a variety of causes, such as genetics, autoimmune-related infections, environmental factors, diet, and medications, progressive kidney disease often leads to renal fibrosis, eventually resulting in kidney failure.

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

[0009] Inflammatory bowel disease (IBD) is more common in Western countries, with a relatively low incidence in developing countries. However, in the past 20 years, the incidence of IBD in China has shown a significant upward trend. IBD includes ulcerative colitis (UC) and Crohn's disease (CD). UC is a chronic ulcerative intestinal disease with a relapsing tendency, occurring only in the colon, and usually has a slow, insidious onset. CD is a chronic nonspecific inflammatory bowel disease with an unclear etiology; it is a chronic granulomatous inflammatory disease. It is most common in the terminal ileum and adjacent colon, but can also involve all segments of the digestive tract from the mouth to the anus, exhibiting segmental and skip-progression. Clinically, it is mainly characterized by abdominal distension and pain, diarrhea, weight loss, abdominal masses, and intestinal obstruction, and may be accompanied by fever and other extraintestinal injuries such as to the joints of the limbs, skin, mouth, and eyes. It is proliferative and transmural, capable of invading any part of the gastrointestinal tract in a discontinuous manner. Due to changes in living environment, diet, and work and rest schedules, the number of Crohn's disease patients in my country has increased dramatically in the past 20 years.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] Providing effective treatments for these diseases is also an urgent problem to be solved.

[0014] Invention Summary

[0015] To address the above problems, the present invention aims to provide a dehydroxylated alginate oligosaccharide derivative, its preparation method, and its uses. Based on previous research, the inventors subjected the obtained homogeneous alginate oligosaccharides to a series of derivatization reactions, including reduction and oxidation, to synthesize a class of dehydroxylated alginate oligosaccharide derivatives and their derivatives with their reducing ends oxidized to acids. The dehydroxylated alginate oligosaccharide derivatives and dehydroxylated alginate oligosaccharide acid derivatives designed and synthesized in this invention have shown good therapeutic effects on inflammatory diseases such as acute kidney injury, chronic kidney disease, gout, ulcerative colitis, Crohn's disease, oral ulcers, rhinitis, and liver damage.

[0016] In this invention, for ease of description, dehydroxylated alginate oligosaccharide derivatives and dehydroxylated alginate oligosaccharide acid derivatives are collectively referred to as dehydroxylated alginate oligosaccharide derivatives.

[0017] The objective of this invention is achieved by providing the following technical solutions.

[0018] In a first aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0019] Where n is an integer, and n = 0-8, preferably n = 0-3.

[0020] 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.

[0021] According to some embodiments of the present invention, in the compound represented by formula (I), 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.

[0022] 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 and The ratio of the total number of ends of the configuration is 1-5:1, preferably 2-4:1.

[0023] 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.

[0024] Preferably, in the compound represented by formula (I), The total number of ends of the configuration and and The ratio of the total number of ends of the configuration is 3-5:1.

[0025] 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.

[0026] 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:

[0027] In the presence of a catalyst, the compound of formula (III) or its pharmaceutically acceptable salt undergoes a C=C double bond hydrogenation reaction with H2 to obtain the compound of formula (I) or its pharmaceutically acceptable salt.

[0028] According to some embodiments of the present invention, the preparation method includes the following steps:

[0029] 1) Dissolve the compound of formula (III) or its pharmaceutically acceptable salt in a solvent, then add a catalyst, and react in the presence of hydrogen at 0-40°C, preferably 20-30°C, for 10-18 hours, preferably 12-15 hours, to obtain the reaction product;

[0030] 2) Filter the reaction product and then dry the filtrate to obtain the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0031] Preferably, in step 1), the solvent is selected from one or more of water, methanol, ethanol, n-propanol and isopropanol.

[0032] Preferably, in step 1), the catalyst is selected from one or more of palladium on carbon, platinum on carbon, rhodium on carbon, ruthenium on carbon, and Raney nickel.

[0033] Preferably, in step 1), the pressure of the hydrogen gas is 0.01-2 MPa, and more preferably atmospheric pressure.

[0034] Preferably, in step 1), the concentration of the compound represented by formula (III) or its pharmaceutically acceptable salt in the solvent is 0.01-0.2 g / mL, more preferably 0.1 g / mL.

[0035] Preferably, in step 1), the weight ratio of the compound represented by formula (III) or its pharmaceutically acceptable salt to the catalyst is 1:0.01-1, more preferably 1:0.05-0.1.

[0036] Thirdly, the present invention provides a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0037] Where n is an integer, and n = 0-8, preferably n = 0-3.

[0038] Preferably, in the compound represented by formula (II), 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; 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.

[0039] Preferably, in the compound represented by formula (II), 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.

[0040] Preferably, in the compound represented by formula (II), Structural units of configuration and The ratio of the number of structural units in the configuration is 1.0-2.5:1.

[0041] Preferably, in the compound represented by formula (II), The ends of the configuration and The ratio of the number of ends of the configuration is 3-5:1.

[0042] 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.

[0043] Fourthly, the present invention provides a method for preparing a compound of formula (II) according to the third aspect of the present invention, or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0044] In the presence of an oxidant and a solvent, a compound of formula (I) or a pharmaceutically acceptable salt thereof, according to a first aspect of the present invention, undergoes a ring-opening oxidation reaction to obtain a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0045] According to some embodiments of the present invention, the preparation method includes the following steps:

[0046] 1) Dissolve the compound of formula (I) 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;

[0047] 2) The reaction product is filtered, and then the filtrate is separated and purified to obtain the compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0048] 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.

[0049] Preferably, in step 1), the concentration of the compound represented by formula (I) or its pharmaceutically acceptable salt in the solvent is 10-100 mg / mL, preferably 50 mg / mL.

[0050] Preferably, in step 1), the oxidant is selected from one or more of copper hydroxide, NaClO, bromine water, and H2O2.

[0051] Preferably, in step 1), the molar ratio of the compound represented by formula (I) or its pharmaceutically acceptable salt to the oxidant is 1:1-5, more preferably 1:2-3.

[0052] Preferably, in step 2), the separation and purification are performed using a gel column.

[0053] Fifthly, 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 and / or a compound of formula (II) according to the third aspect of the present invention or a pharmaceutically acceptable salt thereof.

[0054] Preferably, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and / or the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is the sole active ingredient in the pharmaceutical composition.

[0055] Preferably, the pharmaceutical composition further comprises an active ingredient other than the compound of formula (I) or a pharmaceutically acceptable salt thereof and the compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0056] Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0057] 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.

[0058] In a sixth aspect, the present invention provides the use of a compound of formula (I) according to the first aspect of the invention or a pharmaceutically acceptable salt thereof and / or a compound of formula (II) according to the third aspect of the invention or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of inflammatory diseases.

[0059] Preferably, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and / or the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is the sole active ingredient in the pharmaceutical composition.

[0060] Preferably, the inflammatory disease is selected from one or more of acute kidney injury, chronic kidney disease, gout, ulcerative colitis, Crohn's disease, oral ulcers, rhinitis, and liver injury.

[0061] More preferably, the symptoms of the chronic kidney disease are characterized by elevated levels of protein in the urine.

[0062] The present invention has at least the following beneficial effects:

[0063] This invention synthesizes novel dehydroxylated alginate oligosaccharide derivatives and dehydroxylated alginate oligosaccharide acid derivatives. Compared to unsaturated alginate oligosaccharides, the dehydroxylated alginate oligosaccharide derivatives and dehydroxylated alginate oligosaccharide acid derivatives synthesized in this invention exhibit better stability under different acidic and alkaline conditions, which is more beneficial for drug preservation and use, and helps to prolong the retention time of drugs in vivo and improve utilization.

[0064] The dehydroxylated alginate oligosaccharide derivatives and dehydroxylated alginate oligosaccharide acid derivatives designed and synthesized in this invention have good therapeutic effects on inflammatory diseases such as acute kidney injury, chronic kidney disease, gout, ulcerative colitis, Crohn's disease, oral ulcers, rhinitis, and liver damage.

[0065] Compared to unsaturated fucoidan oligosaccharides, the dehydroxylated fucoidan oligosaccharide derivatives and dehydroxylated fucoidan oligosaccharide acid derivatives designed and synthesized in this invention have better therapeutic effects on liver damage.

[0066] Brief description of the attached figures

[0067] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0068] Figure 1 shows the mass spectrum (LRMS(ESI)) of 1d alginate pentose;

[0069] Figure 2 shows the mass spectrum (LRMS(ESI)) of dehydroxylated fucoidan 2a;

[0070] Figure 3 shows the mass spectrum (LRMS(ESI)) of dehydroxylated alginate 2b;

[0071] Figure 4 shows the mass spectrum (LRMS(ESI)) of dehydroxylated alginate 2c;

[0072] Figure 5 shows the mass spectrum (LRMS(ESI)) of dehydroxylated alginate pentasaccharide 2d;

[0073] Figure 6 shows the 1H NMR spectrum of dehydroxylated fucoidan 2a. 1 HNMR, solvent D2O);

[0074] Figure 7 shows the 1H NMR spectrum of dehydroxylated alginate 2b. 1 HNMR, solvent D2O);

[0075] Figure 8 shows the 1H NMR spectrum of dehydroxylated alginate 2c. 1 HNMR, solvent D2O);

[0076] Figure 9 shows the mass spectrum (LRMS(ESI)) of dehydroxylated alginate 3a;

[0077] Figure 10 shows the mass spectrum (LRMS(ESI)) of dehydroxylated alginate 3b;

[0078] Figure 11 shows the mass spectrum (LRMS(ESI)) of dehydroxylated alginate 3c;

[0079] Figure 12 shows the mass spectrum (LRMS(ESI)) of dehydroxylated brown alginic acid 3d;

[0080] Figure 13 shows the 1H NMR spectrum of dehydroxylated alginate 3a. 1 HNMR (solvent: D2O);

[0081] Figure 14 shows the 1H NMR spectrum of dehydroxylated alginate 3b. 1 HNMR (solvent: D2O);

[0082] Figure 15 shows the 1H NMR spectrum of dehydroxylated alginate 3c. 1 HNMR (solvent: D2O);

[0083] Figure 16 shows the effect of dehydroxylated brown algae oligosaccharide derivatives on ischemia-reperfusion (I / R) inhibition on serum creatinine levels in rats;

[0084] Figure 17 shows the establishment and evaluation of a mouse model of acute gouty arthritis;

[0085] Figure 18 shows the effect of dehydroxylated brown algae oligosaccharide derivatives on inhibiting joint pain symptoms in AGA model mice;

[0086] Figure 19 shows the effect of dehydroxylated brown algae oligosaccharide derivatives on inhibiting DSS-induced ulcerative colitis;

[0087] Figure 20 shows the effects of dehydroxylated brown algae oligosaccharide derivatives on the colon of TNBS-induced mice;

[0088] Figure 21 shows the effect of pH on the stability of different types of brown algae oligosaccharides.

[0089] The best way to implement an invention

[0090] 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.

[0091] The homogeneous fucoidan 1 (n = 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.

[0092] The ratio of guluronic acid (G) to mannuronic acid (M) in fucoidan 1 (n=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 2-4:1. 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.

[0093] As can be seen, the homogeneous fucoidan 1, the dehydroxylated fucoidan derivative 2, and the dehydroxylated fucoidan acid derivative 3 were all 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.

[0094] Example 1: Preparation and structural identification of dehydroxylated brown algae oligosaccharide derivatives

[0095] This invention utilizes a hydrogenation reduction reaction to hydrogenate and saturate the double bond at the Δ end of fucoidan 1, which is composed of Δ, G, and M, to synthesize a class of fucoidan derivatives 2. Compared with common fucoidan oligosaccharides, its non-reducing end monosaccharide is a 4-position dehydroxylated monosaccharide, which is simply referred to as dehydroxylated fucoidan oligosaccharide. Its synthetic route is shown in Scheme 1:

[0096] Taking the preparation of dehydroxylated fucoidan 2b as an example, fucoidan 1b (2.0 g, 3.37 mmol in sodium salt form) was dissolved in 20 mL of water, and then 10% palladium on carbon (100 mg, 5% (w / w)) or 5% platinum on carbon (100 mg, 5% (w / w)) or Raney nickel (200 mg, 10% (w / w)) was added. After purging the reaction vessel with hydrogen, the reaction was stirred at 25°C and atmospheric pressure for 15 hours. After the reaction was completed, the mixture was filtered, and the filtrate was freeze-dried to obtain the target compound. Here, 5% platinum on carbon refers to the weight percentage of platinum metal adsorbed on activated carbon being 5%, and 5% (w / w) refers to the 5% platinum on carbon added to the reaction system being 5% of the weight of the reactants. Using the same method, 2a was prepared from 1a, 2c from 1c, and 2d from 1d.

[0097] The resulting compound 1 The H-NMR and MS data are as follows:

[0098] 2a: MS (ESI): m / z=353.2[(MH) - The mass spectrum is shown in Figure 2. 1 The H-NMR spectrum is shown in Figure 6.

[0099] 2b: MS(ESI): m / z=529.2[(MH) - The mass spectrum is shown in Figure 3. 1 The H-NMR spectrum is shown in Figure 7.

[0100] 2c: MS(ESI): m / z=705.3[(MH) - The mass spectrum is shown in Figure 4. 1 The H-NMR spectrum is shown in Figure 8.

[0101] 2d: MS(ESI): m / z=881.2[(MH) - The mass spectrum is shown in Figure 5.

[0102] Example 2: Preparation and structural identification of dehydroxylated alginic acid derivatives

[0103] The present invention performs ring-opening oxidation on the reducing end of the above-mentioned dehydroxylated alginate oligosaccharide derivative 2 to synthesize a series of dehydroxylated alginate oligosaccharide acid derivatives 3, the synthetic route of which is shown in Scheme 2.

[0104] Taking the preparation of dehydroxylated alginate 3b as an example, dehydroxylated alginate 2b (250 mg, 0.42 mmol in sodium salt form) was dissolved in 5 mL of 0.5 M Na2HPO4-HCl buffer (concentrated hydrochloric acid was added dropwise to the pre-prepared 0.5 M Na2HPO4 solution to adjust the pH to about 8.5), and then an oxidant was added. The reaction was carried out at 25 °C for 3 hours. After the reaction was completed, the insoluble solids were removed by filtration, and the filtrate was purified by gel column chromatography to obtain the target compound.

[0105] The oxidant was selected from freshly prepared copper hydroxide (1.26 mmol, 3.0 eq), 2% NaClO solution (0.84 mmol, 2.0 eq), 3% bromine water (4.92 mL, 0.92 mmol, 2.2 eq) or 1% H2O2 (0.84 mmol, 2.0 eq).

[0106] Using the same method, 3a was prepared from 2a, 3c from 2c, and 3d from 2d. The resulting compounds... 1 The H-NMR and MS data are as follows:

[0107] 3a: MS (ESI): m / z=369.2[(MH) - The mass spectrum is shown in Figure 9; 1 The H-NMR spectrum is shown in Figure 13.

[0108] 3b: MS(ESI): m / z=545.2[(MH) - The mass spectrum is shown in Figure 10. 1 The H-NMR spectrum is shown in Figure 14.

[0109] 3c: MS(ESI): m / z=721.1[(MH) - The mass spectrum is shown in Figure 11. 1 The H-NMR spectrum is shown in Figure 15.

[0110] 3d: MS(ESI): m / z=897.2[(MH) - The mass spectrum is shown in Figure 12.

[0111] Example 3: Activity study of dehydroxylated brown algal oligosaccharide derivatives in the treatment of acute kidney injury

[0112] Ischemia-reperfusion, endotoxin-phospholipid polysaccharide, and cisplatin-induced kidney injury are commonly used animal models to simulate acute kidney injury caused by clinical ischemia, infection, and drugs, respectively. The pharmacological activities of dehydroxylated alginate oligosaccharides and dehydroxylated alginate oligosaccharide acid derivatives (collectively referred to as dehydroxylated alginate oligosaccharide derivatives) against acute kidney injury were tested using these three animal models.

[0113] I. Effects of dehydroxylated brown algae oligosaccharide derivatives on acute kidney injury (AKI) induced by ischemia / reperfusion (I / R) in rats

[0114] Male Sprague Dawley rats weighing 220-250 grams were selected, with six rats per group. Urine output was collected 24 hours prior to surgery and no abnormalities were observed. The rats were 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+3b, I / R+3c, and I / R+3d; due to limitations on the number of animals that could be operated on, only six compounds were tested). Ten minutes before surgery, the drugs were administered by gavage. The drug administration group received 0.1 g / kg / day, while 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 the left and right kidneys were exposed through an abdominal incision. In the sham-operated group, only the kidneys were examined. The wound was then sutured layer by layer to complete the surgery. In both the model group and the drug administration group, the renal pedicles of both kidneys were clamped with large arterial clamps, and then the kidneys were repositioned. The wounds were covered with gauze and a small amount of physiological saline was instilled for rehydration. After 45 minutes, the arterial clamps were released, and the wounds were sutured layer by layer to complete the surgery. After surgery, the rats were placed on a 37°C heating pad until recovery, after which they were returned to their metabolic cages. During this period, the rats' body weight, food intake, water intake, and urine output were monitored. Routine feeding was performed post-surgery, and the rats were sacrificed 24 hours later for tissue collection. Blood was collected from the inferior vena cava of the rats, and the supernatant serum was collected after centrifugation. Serum creatinine was then measured using a creatinine assay kit. The experimental results were statistically analyzed using the p-value method. The results are shown in Figure 16.

[0115] Figure 16 shows that renal filtration function is impaired after acute ischemia-reperfusion (I / R) surgery, and the injury causes a significant increase in serum creatinine levels in rats. Compared with the sham-operated group, the creatinine level in the I / R group was significantly higher. After administration of dehydroxylated alginate oligosaccharide derivatives, serum creatinine was reduced to varying degrees, suggesting a certain degree of recovery of glomerular function. In particular, after 3 days, serum creatinine levels essentially returned to normal, indicating that dehydroxylated alginate oligosaccharide derivatives have a significant therapeutic effect on acute kidney injury. Data were analyzed using the p-value test, and the results in Figure 16 are shown below. * This means that when comparing this group of data with the Sham group, p < 0.05. # This means that compared with the I / R group, p < 0.05.

[0116] The 24-hour urine output (UO) collected after the operation is shown in Table 1.

[0117] Table 1. Protective effect of dehydroxylated brown algae oligosaccharide derivatives on I / R-induced increase in urine volume in rats. The data were analyzed using the p-value test. (Table) * This indicates that compared to the Sham group, p < 0.05. # This indicates that the data in this group were compared with the I / R group, p < 0.05. Sham: sham surgery group, I / R: ischemia-reperfusion group.

[0118] Table 1 shows that the renal urine concentration function was impaired after acute ischemia-reperfusion (I / R) surgery, and the urine volume increased significantly. After administration of dehydroxylated brown algae oligosaccharide derivatives, the urine volume of rats decreased significantly, suggesting that these compounds have a nephroprotective effect and that the renal tubular reabsorption function was restored to a certain extent.

[0119] II. Effects of dehydroxylated brown algal oligosaccharide derivatives on endotoxin phospholipid polysaccharide (LPS)-induced acute kidney injury (AKI) in mice.

[0120] Male C57 / Bl6 mice weighing 22-28 grams were selected, with 6 mice in each group. Urine output was collected 24 hours prior to surgery and no abnormalities were observed. The mice were randomly divided into four groups: control group (CTL), model group (LPS), modeling drug treatment groups (LPS+2a, LPS+2b, LPS+2c, LPS+2d, LPS+3a, LPS+3b, LPS+3c, and LPS+3d, with a dosage of 0.1 g / kg / day), and LPS+ positive control group (LPS+Dex, dexamethasone acetate 0.1 g / kg / day), with 6 mice in each group. One hour before modeling, the modeling drug treatment groups were administered the medication by gavage, while dexamethasone was administered intraperitoneally. The model and control groups were administered the same volume of physiological saline by gavage. LPS was used to induce septic acute kidney injury. At the time of modeling, the model group, the drug administration group, and the LPS+ positive control group were intraperitoneally injected with LPS 15 mg / kg, while the control group was intraperitoneally injected with an equal volume of physiological saline. Immediately after modeling, the mice were returned to their metabolic cages for observation, during which time their body weight, food intake, water intake, and urine output were measured. After 24 hours, the mice were sacrificed for sampling. Urine was collected, and blood was collected from the inferior vena cava. After centrifugation, the supernatant serum was collected, and the serum creatinine level was measured using a creatinine assay kit (P<0.05). Cr The experimental results were statistically analyzed using the p-value method, and the results are shown in Table 2.

[0121] Table 2. Effects of dehydroxylated brown algal oligosaccharide derivatives on LPS-induced serum creatinine levels in mice. * This indicates that compared to the CTL group, p < 0.05. # This indicates a comparison between this group and the LPS group, with p < 0.05. CTL: control group, LPS: model group.

[0122] Table 2 shows that LPS treatment significantly increased serum creatinine levels in mice, while dehydroxylated alginate oligosaccharide derivatives significantly reduced serum creatinine levels, exhibiting a similar effect to dexamethasone, suggesting that these compounds have a renal protective effect. This result indicates that compared to the control group, mice treated with intraperitoneal injection of LPS showed significantly increased serum creatinine, while the dehydroxylated alginate oligosaccharide derivative group showed a significant decrease in serum creatinine, essentially returning to normal levels. This demonstrates that dehydroxylated alginate oligosaccharide derivatives have a significant protective effect against LPS-induced renal function decline in mice.

[0123] III. Effects of dehydroxylated brown algal oligosaccharide derivatives on cisplatin-induced acute kidney injury in mice

[0124] Sixty male C57BL / J6 mice (22-28 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 (Cis), and three drug-induced modeling groups (Cis+2a, Cis+2b, Cis+2c, Cis+2d, Cis+3a, Cis+3b, Cis+3c, and Cis+3d, with a dosage of 0.1 g / kg / day). Cisplatin was used to induce drug-induced acute kidney injury. At the time of modeling, the model and drug-induced modeling groups were intraperitoneally injected with 20 mg / kg of cisplatin, while the control group was intraperitoneally injected with an equal volume of saline. Dehydroxylated alginate oligosaccharide derivatives were administered by gavage. Half an hour after modeling, the first administration was given, and both the model and control groups were administered the same volume of saline by gavage. 24 and 48 hours later, the same dose and specifications of dehydroxylated brown algae oligosaccharide derivative solution or physiological saline were administered by gavage again; the animals were sacrificed 72 hours later. Immediately after model establishment and drug administration, the mice were returned to their metabolic cages for observation, during which time their body weight, food intake, water intake, and urine output were measured. After 72 hours, the mice were sacrificed for sampling. Urine was collected, and blood was collected from the inferior vena cava. After centrifugation, the supernatant serum was collected, and the serum creatinine level was measured using a creatinine assay kit. The experimental results were statistically analyzed using the p-value method, and the results are shown in Table 3.

[0125] Table 3. Effects of dehydroxylated brown algal oligosaccharide derivatives on cisplatin-induced serum creatinine levels in mice. * indicates that this data set is compared with the CTL group, p < 0.05. # This indicates a comparison between this group and the Cis group, with p < 0.05. CTL: control group, Cis: model group.

[0126] Table 3 shows that cisplatin treatment significantly increased serum creatinine levels in mice, while dehydroxylated alginate oligosaccharide derivatives significantly reduced serum creatinine, suggesting a renal protective effect. This result indicates that, compared to the control group, mice treated with intraperitoneal injection of cisplatin showed significantly increased serum creatinine, while the dehydroxylated alginate oligosaccharide derivative group showed a significant decrease in serum creatinine, essentially returning to normal levels. This demonstrates that dehydroxylated alginate oligosaccharide derivatives have a significant protective effect against cisplatin-induced renal function decline in mice.

[0127] Example 4: Activity study of dehydroxylated brown algae oligosaccharide derivatives in the treatment of chronic kidney disease

[0128] The study used a mouse model of chronic kidney disease induced by aristolochic acid (AA).

[0129] Sixty 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+2a, AA+2b, AA+2c, AA+2d, AA+3a, AA+3b, AA+3c, and AA+3d, with a dosage of 0.1 g / kg / 48 hours). Aristolochic acid was used to induce chronic kidney disease. During modeling, mice in the model and treatment groups 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. The treatment group received aristolochic acid every two days via gavage for six weeks. 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. Blood was collected from the inferior vena cava under anesthesia, 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, and the results are shown in Tables 4 and 5.

[0130] Table 4. Effects of dehydroxylated brown algal oligosaccharide derivatives on aristolochic acid-induced serum creatinine levels in mice. * indicates that the p-value of this group is less than 0.05 compared to the CTL group (control group). # This indicates that compared with the AA group (model group), p < 0.05.

[0131] Table 4 shows that aristolochic acid treatment for three weeks significantly increased serum creatinine levels in mice, while dehydroxylated alginate oligosaccharide derivatives significantly reduced serum creatinine, 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, while the dehydroxylated alginate oligosaccharide derivative group showed a significant decrease in serum creatinine. Although not completely restored to normal levels, this suggests that dehydroxylated alginate oligosaccharide derivatives have a certain protective effect against decreased renal function in mice with chronic kidney disease.

[0132] Table 5. Effects of dehydroxylated brown algae oligosaccharide derivatives on aristolochic acid-induced UACR in mice. UACR: Urinary albumin to creatinine ratio; * indicates that the data in this group is significantly different from that in the CTL group (control group), p<0.05. # This indicates that compared with group AA (model group), p < 0.05.

[0133] Proteinuria is an important indicator of chronic kidney injury. Table 5 shows that three weeks of aristolochic acid treatment significantly increased the urinary albumin-to-creatinine ratio (UACR) in mice, while the dehydroxylated alginate oligosaccharide derivative significantly reduced the UACR, suggesting its renal protective effect. This result indicates that, compared with the control group, mice treated with intraperitoneal injection of aristolochic acid showed increased urinary protein excretion, and the UACR of mice treated with the dehydroxylated alginate oligosaccharide derivative significantly decreased, although it did not return to normal levels. However, this suggests that the dehydroxylated alginate oligosaccharide derivative has a good protective effect against the decline in renal function in mice with chronic kidney disease.

[0134] Example 5: Activity study of dehydroxylated brown algae oligosaccharide derivatives 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) via the same method. After injection, the degree of ankle swelling and changes in the mechanical pain threshold were observed.

[0137] Ankle swelling measurement: Using vernier calipers, the diameter of the affected ankle joint of mice was measured before modeling and at 2h, 6h, 24h, and 48h after modeling. Measurements were taken three times consecutively, and the average value was used as the final reading. Ankle swelling = current measured diameter - diameter measured before modeling.

[0138] Mechanical pain behavior assay: Mice were placed in a transparent plastic box on an elevated wire mesh and covered with transparent plexiglass for 45 minutes to acclimatize. Then, following the "Up and Down" method, the pain threshold near the ankle on the right hind paw was measured using von Frey wire of different specifications before modeling and at 2, 6, 24, and 48 hours after modeling. The paw retraction threshold was calculated using a formula.

[0139] Figure 17 illustrates the establishment and efficacy evaluation of the acute gouty arthritis mouse model. Figure 17A shows the swelling of the right ankle joint of mice in the AGA model group after injection of urate (MSU) crystals. 24 hours after injection of urate (MSU) crystals, the ankle joints of mice in the AGA model group showed significant swelling compared to those in the control group (injected with phosphate-buffered saline, PBS). Figure 17B compares H&E-stained sections of the ankle joints of mice in the control and model groups. It can be observed that, compared to the control group, the ankle joint sections of mice in the AGA model group showed extensive inflammatory cell infiltration. Figure 17C shows the change in ankle joint diameter between mice injected with urate crystals and those injected with PBS. It can be seen that the ankle joints of mice in the control group showed only slight swelling within hours after PBS injection, which quickly subsided and remained largely unchanged after returning to normal. However, the ankle joints of mice injected with urate crystals swelled rapidly, and significant swelling persisted even after 48 hours. Figure 17D 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.

[0140] II. Study on the analgesic effect of dehydroxylated brown algae oligosaccharide derivatives on AGA model mice

[0141] AGA model mice were prepared as described in "I. Establishment of an Acute Gouty Arthritis (AGA) Mouse Model" of this embodiment. Mice were randomly divided into 10 groups: control group (Veh+Veh), model group (MSU+Veh), and modeling drug administration groups (MSU+2a, MSU+2b, MSU+2c, MSU+2d, MSU+3a, MSU+3b, MSU+3c, and MSU+3d, with a dosage of 200 mg / kg). The modeling drug administration groups received intraperitoneal injections of the corresponding dehydroxylated alginate oligosaccharide derivatives at 1 hour before modeling and at 5, 23, and 47 hours after modeling. The control group and model group received an equal volume of the control solvent PBS intraperitoneally. Mechanical pain and swelling were assessed before modeling and at 2, 6, 24, and 48 hours 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.

[0142] Figure 18 shows the effect of dehydroxylated alginate oligosaccharide derivatives on inhibiting joint pain symptoms in AGA model mice. Figure 18A is a time-course graph showing the effect of the control group, model group, and model-induced drug administration group on ankle mechanical pain in AGA model mice. The time-course graphs show that compared with the control group, the withdrawal threshold was significantly reduced in the model group, indicating significantly severe mechanical pain; after administration of the dehydroxylated alginate oligosaccharide derivative, the withdrawal threshold was significantly increased, indicating significant relief of mechanical pain. Figure 18B is a time-course graph showing the effect of each group 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 the dehydroxylated alginate oligosaccharide derivative, the degree of ankle swelling in the mice was significantly reduced. The above experiments demonstrate that dehydroxylated alginate oligosaccharide derivatives can effectively inhibit ankle pain and swelling in AGA model mice.

[0143] Example 6: Activity study of dehydroxylated brown algae oligosaccharide derivatives in the treatment of ulcerative colitis

[0144] Dextran sulfate sodium (DSS) is commonly used to induce colitis models. Low concentrations of DSS can induce chronic ulcerative colitis in mice, while high concentrations can induce acute ulcerative colitis symptoms. DSS-induced ulcerative colitis in mice has a high success rate in model establishment and its symptoms are highly similar to those in humans, making it a commonly used animal model. 5-Aminosalicylic acid (5-ASA) is a commonly used drug for treating ulcerative colitis and is often used as a positive control. Using the classic DSS model and with 5-ASA as the positive control, the activity of dehydroxylated alginate oligosaccharide derivatives in treating ulcerative colitis was studied. The experimental procedure is as follows:

[0145] After one week of normal feeding, C57BL / 6 mice were randomly divided into 11 groups, with 6 mice in each group. These groups were: control group (NC), model group (Veh, DSS), positive control group (DSS+5-ASA, dose of 150 mg / kg), and modeling drug administration groups (DSS+2a, DSS+2b, DSS+2c, DSS+2d, DSS+3a, DSS+3b, DSS+3c, and DSS+3d, dose of 150 mg / kg). The experiment lasted for 7 days. All mice except the control group received 3% DSS solution in their drinking water. The control and model groups were administered 0.1 ml / 10g body weight of physiological saline via gavage. The positive control group received 300 mg of 5-ASA suspended in 20 ml of 0.5% CMC-Na solution via gavage at a dose of 0.1 ml / 10g body weight. The modeling groups received various dehydroxylated alginate oligosaccharide derivatives diluted to 15 mg / ml with 0.9% physiological saline via gavage at a dose of 10 ml / kg body weight. The mice's weight, mood, fur, and fecal condition were recorded daily. Mice were sacrificed after 7 days, and serum and tissue samples were collected for further research.

[0146] Figure 19 illustrates the effect of dehydroxylated alginate oligosaccharide derivatives on inhibiting DSS-induced ulcerative colitis in mice. Figure 19A shows the effect of dehydroxylated alginate oligosaccharide derivatives on mouse body weight. Compared with the control group, the body weight of mice in the model group gradually decreased as the experiment progressed; the decrease in body weight of mice in the positive control group was significantly smaller. After administration of each dehydroxylated alginate oligosaccharide derivative, the decrease in body weight of mice was less than that in the model group, and some of the dehydroxylated alginate oligosaccharide derivatives resulted in a lower degree of body weight reduction than the positive control group.

[0147] Mice were monitored daily for body weight, fecal characteristics, and rectal bleeding. A Disease Activity Index (DAI) score was calculated based on these three factors. Each parameter was scored from 0 to 4: weight loss (0, <5%; 2, 6–10%; 4, >10%); fecal viscosity (0, normal feces; 2, loose feces; 4, diarrhea); and rectal bleeding (0, normal feces; 2, blood streaks or dark brown feces; 4, reddish-brown or dark red bloody feces). DAI = fecal viscosity score + rectal bleeding score + weight loss score. Figure 19B shows the DAI scores after administration of dehydroxylated alginate oligosaccharide derivatives. It can be seen that the model group score increased significantly with DSS administration, while the DAI score decreased significantly compared to the model group after administration of the positive control drug and dehydroxylated alginate oligosaccharide derivatives.

[0148] Figures 19C and 19D show the effects of dehydroxylated alginate oligosaccharide derivatives on colon length and weight in mice. Compared with the control group, the colon length and weight of the model group animals were significantly reduced. After administration of the positive control and dehydroxylated alginate oligosaccharide derivatives, colon length increased slightly, but colon weight increased significantly. Some dehydroxylated alginate oligosaccharide derivatives restored colon length to near the control group. These data indicate that dehydroxylated alginate oligosaccharide derivatives have significant inhibitory activity against DSS-induced ulcerative colitis in mice, similar to the positive control drug 5-aminosalicylic acid, and even superior to some compounds in some cases.

[0149] Example 7: Activity study of dehydroxylated brown algal oligosaccharide derivatives in the treatment of Crohn's disease

[0150] The causes of Crohn's disease are complex, and its etiology and specific pathogenesis remain unclear. 2,4,6-Trinitrobenzenesulfonic acid (TNBS) is a hapten that can bind to high-molecular-weight tissue proteins to become an antigen, inducing a T-cell-mediated cellular immune response, thereby causing colonic inflammation. TNBS-induced colitis can be used to study acute colitis, intermediate-stage colitis, and chronic colitis, with chronic colitis exhibiting similar pathological changes to fibrous stricture-type Crohn's disease. This model has advantages such as simple model establishment, long model duration, low cost, and similar pathological changes to human Crohn's disease.

[0151] I. Preparation of a mouse model of Crohn's disease

[0152] After a week of acclimatization, BALB / c mice were shaved 1.5cm x 1.5cm on their backs on the morning of the first day of the experiment, and a 1% 2,4,6-trinitrobenzenesulfonic acid (TNBS) pre-sensitization solution was applied externally. On the morning of the eighth day, enemas were administered after a 12-hour fast, followed by anesthesia (isoflurane inhalation). 0.1mL of 40% TNBS anhydrous ethanol solution was administered via enema, inserted through the anus to a depth of 4-6cm, head down, and inverted for 1 minute. This was repeated weekly, with gradually increasing doses (0.75mg, 1.5mg, 2.5mg, 2.5mg, 2.5mg, 2.5mg, 2.5mg, 2.5mg) for a total of 7 weeks. After the enema, to prevent immediate leakage, the tail was lifted and the anus raised for 3 minutes to ensure even distribution of the solution throughout the colon, thus establishing the model. To aid recovery, a heat lamp was placed 20cm away from the cage to maintain warmth until the mice recovered well.

[0153] II. Therapeutic effects of dehydroxylated brown algae oligosaccharide derivatives on TNBS Crohn's disease model mice

[0154] BALB / c mice were randomly divided into 10 groups of 6 mice each: a control group (CTL), a model group (TNBS), and three model-inducing drug administration groups (TNBS+2a, TNBS+2b, TNBS+2c, TNBS+2d, TNBS+3a, TNBS+3b, TNBS+3c, and TNBS+3d). Administration was by gavage at a dose of 200 mg / kg. Each dehydroxylated alginate oligosaccharide derivative was diluted to 20 mg / ml with 0.9% physiological saline and administered by gavage at a dose of 0.1 ml / 10 g body weight. The control group was fed normally with free access to food and water. After modeling, the model groups were administered an equal volume of physiological saline by gavage daily. From the first gavage initiation, all groups received the drug once daily for 49 days.

[0155] The characteristics of mouse stool, coat color, activity, and mental state were observed daily, and the mice were weighed. After 49 days of drug administration, the mice were euthanized using carbon dioxide, and the colon was dissected. All colonic tissue was removed and quickly washed with PBS. The length, weight, and body weight of the colon were measured and recorded, and the ratios of colon weight to length and weight to body weight were calculated. The colon was excised approximately 1 cm from the anus, fixed in 10% formalin at room temperature, routinely embedded in paraffin, and sectioned to a thickness of 4–5 cm. Routine hematoxylin-eosin staining (HE staining) was performed for histopathological examination. The mouse colon was observed under a microscope, and the remaining tissue was immediately placed in an 80°C freezer for further analysis.

[0156] The control group mice were in good spirits, ate and drank normally, and excreted yellow, formed, soft stools. The model group mice were in poor spirits, had decreased appetite, and experienced diarrhea, bloody stools, intestinal obstruction, and weight loss. The mice in the treatment groups with various dehydroxylated algae oligosaccharide derivatives showed significantly improved spirits and appetite compared to the model group, with little or no diarrhea, bloody stools, or weight loss.

[0157] Figure 20 shows the effects of dehydroxylated alginate oligosaccharide derivatives on the colon of mice after TNBS modeling. Figure 20A shows the ratio of colon weight to length in TNBS-modeled mice in each experimental group; Figure 20B shows the ratio of colon weight to body weight in TNBS-modeled mice in each experimental group. Compared with the control group, the ratios of colon weight to length and colon weight to body weight in the model group mice were significantly increased. However, after intervention with dehydroxylated alginate oligosaccharide derivatives, the ratios of colon weight to length and colon weight to body weight in each treatment group were significantly decreased compared with the model group. This suggests that dehydroxylated alginate oligosaccharide derivatives have a certain therapeutic effect on Crohn's disease.

[0158] HE staining was used to observe the colon pathology of mice in each group. The colon tissue structure of mice in the control group was clear and there was no inflammatory cell infiltration. The colon tissue structure of mice in the model group was damaged, with inflammatory cell infiltration and colonic edema. After intervention with dehydroxylated brown algae oligosaccharide derivatives, the colonic epithelial cells were obviously normal in morphology and the inflammatory cells were slightly infiltrated.

[0159] The above results indicate that, compared with the model group, administration of dehydroxylated alginate oligosaccharide derivatives can significantly improve symptoms such as poor mental state, diarrhea, bloody stools, and weight loss in Crohn's disease mice. Dissection revealed that the weight and length of the mouse colon were reduced. HE staining of the colon showed that dehydroxylated alginate oligosaccharide derivatives had a significant therapeutic effect on TNBS-induced colonic edema, structural damage, and inflammatory cell infiltration.

[0160] Example 8: Activity study of dehydroxylated brown algae oligosaccharide derivatives in the treatment of oral ulcers

[0161] This study investigated the ulcer-causing effects of dehydroxylated brown algae oligosaccharide derivatives on adult oral ulcers (induced by chemotherapy). Preliminary experiments revealed that direct administration of the compound powder caused irritation, leading to intensified 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 experimental testing. Taking derivative 2a as an example, the formulation of a 6mm diameter, 60mg patch is shown in Table 6, where HPMC is hydroxypropyl methylcellulose; CP934 is carbomer 934; and MCC is microcrystalline cellulose.

[0162] Table 6 Oral Patch Prescriptions

[0163] According to the formulation ratio in Table 6, 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 dehydroxylated alginate oligosaccharide derivatives were also formulated into 6mm diameter oral patches according to the formulation in Table 6.

[0164] Oral ulcer patch irritation and healing promotion effect test

[0165] Adult patients with oral ulcers (induced by chemotherapy) were randomly divided into a control group (no medication) and a medication group (groups 2a, 2b, 2c, 2d, 3a, 3b, 3c, and 3d), with 10 patients in each group. Except for the control group, which received no medication, the other groups received oral patches made from dehydroxylated alginate oligosaccharide derivatives, three patches daily, applied to the ulcer until healing. Results showed that no patients experienced irritation or pain after applying the oral patches; pain from the ulcer itself significantly decreased after 5-10 minutes; the foreign body sensation in the mouth was minimal, and it did not affect drinking or swallowing. The healing time of oral ulcers in the medication groups was significantly shorter than that in the control group (see Table 7).

[0166] Table 7. Effect of oral patches on oral ulcer healing time. *In the control group, only 4 patients healed within 7 days, 3 of the remaining patients healed within 10 days, and 3 of the remaining patients did not heal after 10 days and were treated with other methods.

[0167] As shown in Table 7, in the control group without medication, only 4 patients healed spontaneously within 7 days, and only 1 patient healed spontaneously by the 5th day. Furthermore, 3 patients still had not healed after 10 days. In contrast, in the oral patch groups treated with various dehydroxylated alginate oligosaccharide derivatives, some patients began to heal on the 2nd day, most healed by the 3rd day, and all patients healed within 5 days. Compared to the control group, the healing rate in each treatment group was significantly shorter, indicating that dehydroxylated alginate oligosaccharide derivatives have a significant therapeutic effect on oral ulcers.

[0168] Example 9: Activity study of dehydroxylated brown algae oligosaccharide derivatives in the treatment of rhinitis

[0169] 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 relatively common type of rhinitis, caused by allergens.

[0170] A guinea pig model was established using nasal drops of 2,4-toluene diisocyanate (TDI) and olive oil solution to investigate the inhibitory effect of dehydroxylated brown algae oligosaccharide derivatives on allergic rhinitis.

[0171] I. Establishment of a guinea pig allergic rhinitis model induced by 10% TDI olive oil solution

[0172] 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).

[0173] II. The effect of dehydroxylated brown algae oligosaccharide derivatives in inhibiting allergic rhinitis

[0174] Guinea pigs were randomly divided into 10 groups of 10 animals each: a control group (CTL), a model group (TDI), and three drug administration groups (TDI+2a, TDI+2b, TDI+2c, TDI+2d, TDI+3a, TDI+3b, TDI+3c, and TDI+3d). Drug administration began on day 7 after model establishment. Each dehydroxylated brown algae 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.

[0175] 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 8). 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 9.

[0176] Table 8. Nasal Appearance Symptoms and Scoring Criteria in TDI Model Guinea Pigs

[0177] Table 9 Grading Criteria for Local Mucosal Irritation Response

[0178] The statistical results of allergic rhinitis symptoms in each group of guinea pigs are shown in Table 10.

[0179] Table 10 Effects of each drug administration group on TDI-induced nasal appearance symptoms in guinea pigs (x±s, n=10)

[0180] As shown in Table 10, 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 dehydroxylated alginate oligosaccharide derivatives began to significantly improve from the second day after administration. On the fifth day, except for the TDI+2a and TDI+3a groups which had scores above 4, the scores in the other groups were between 2 and 3. This indicates that the dehydroxylated alginate oligosaccharide derivatives all have a certain therapeutic effect on allergic rhinitis.

[0181] Table 11 Effects of each drug administration group on the nasal mucosal irritation response score of TDI-treated guinea pigs (x±s, n=10)

[0182] Table 11 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 dehydroxylated alginate oligosaccharide derivatives were significantly lower. Except for TDI+2a and TDI+3a, whose scores were slightly higher (around 0.5), the scores of the other treatment groups were around 0.2 to 0.3, slightly higher than the control group, indicating that these compounds have a good repair effect on nasal mucosal irritation in the guinea pig model of allergic rhinitis.

[0183] Example 10: Activity study of dehydroxylated brown algal oligosaccharide derivatives in the treatment of liver injury

[0184] Chemical-induced liver injury is a very important type of liver injury. CCl4 is a commonly used modeling agent for chemical-induced liver injury. This invention uses a CCl4-induced mouse model of acute liver injury to study the protective effect of partially dehydroxylated alginate oligosaccharide derivatives against acute liver injury, and compares it with unsaturated alginate oligosaccharide 1.

[0185] Sixty C57BL / 6 mice, weighing 18–22 g (half male and half female), 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+2b, CCL4+2c, CCL4+2d, CCL4+3b, CCL4+3c, and CCL4+3d). Administration was by gavage at a dose of 200 mg / kg. Unsaturated fucoidan 1 and dehydroxylated fucoidan derivatives 2 and 3 were diluted with 0.9% saline to 20 mg / ml and administered by gavage at a dose of 0.1 ml / 10 g body weight (200 mg / kg). The blank control and model control groups received 0.1 ml / 10 g body weight saline daily, while the model-induced drug administration groups received the drug by 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 in the mice was harvested, 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 homogenates were stored at -20℃ for later analysis. All indicators in the serum and liver were detected using an automated biochemical analyzer, following the instructions for each kit. The results of detecting alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum are shown in Table 12.

[0186] Table 12 Effects of the drug on serum ALT and AST in mice (x±s, n=6)

[0187] 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 12, compared to the control group, the model group showed significantly elevated serum ALT and AST levels, indicating hepatocyte damage. In the dehydroxylated alginate oligosaccharide derivative groups 2 and 3 (CCl4+2b, CCl4+2c, CCl4+2d, CCl4+3b, CCl4+3c, and CCl4+3d), 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 alginate oligosaccharide group 1 (CCl4+1b and CCl4+1c), serum ALT and AST levels were slightly lower than in the model group, but the change was minimal, indicating that these compounds had little protective effect against liver injury in mice.

[0188] The results of the detection of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) in mouse liver tissue are shown in Table 13.

[0189] Table 13 Effects of the drug on GSH-Px, SOD and MDA in mouse liver tissue (x±s, n=6)

[0190] 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 13 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 dehydroxylated alginate oligosaccharide derivative groups 2 and 3 (CCl4+2b, CCl4+2c, CCl4+2d, CCl4+3b, CCl4+3c, and CCl4+3d) 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 slightly increased, but the changes were not significant, indicating that these compounds have a weak protective effect on the liver of mice.

[0191] Malondialdehyde (MDA) is a product of the reaction between oxygen free radicals and unsaturated fatty acids on the cell membrane surface, and it is a cause of cell swelling and necrosis. Measuring the MDA content in liver tissue can reflect the degree of oxidative damage to the tissue when cells are attacked by free radicals. Table 13 shows that compared with the control group, the model group had a significantly higher MDA level in mouse liver tissue, indicating liver damage. After drug administration, the dehydroxylated alginate oligosaccharide derivatives 2 and 3 (CCl4+2b, CCl4+2c, CCl4+2d, CCl4+3b, CCl4+3c, and CCl4+3d) showed varying degrees of decrease in MDA in mouse liver tissue compared with the model group, indicating that these two compounds can significantly improve CCl4-induced liver damage. However, the MDA level in the liver tissue of mice in the unsaturated alginate oligosaccharide 1 group (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.

[0192] Example 11: Stability Study of Different Types of Fucoidan

[0193] 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.

[0194] For unsaturated and dehydroxylated alginate oligosaccharides, one type from each class (1b, 2b, and 3b) was dissolved in purified water to prepare a 100 mg / ml solution. The pH of the solution was adjusted to 3, 5, 7, 9, 11, and 13 using 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 21. Figure 21A shows that unsaturated alginate oligosaccharide 1b has good stability at moderate acidity / alkalinity (pH 5–9). At pH values ​​of 3 and 11, the acidity / alkalinity is relatively strong, and about 10% of the substance is destroyed after 9 days. The degradation rate accelerates significantly with increasing pH, especially above pH 11. Figures 21B and 21C show that saturated dehydroxylated alginate oligosaccharide derivatives 2b and 3b have significantly enhanced stability under the same acidity / alkalinity conditions. Within the pH range of 3–13, the degradation is less than 5% after 9 days at room temperature. 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.

[0195] 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 = 0-8, preferably n = 0-3.

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 selected from One or more of the following; 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 total number of ends of the configuration and The ratio of the total number of ends in the configuration is 3-5:1; 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.

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 a catalyst, the compound of formula (III) or its pharmaceutically acceptable salt undergoes a C=C double bond hydrogenation reaction with H2 to obtain the compound of 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 (III) or its pharmaceutically acceptable salt in a solvent, then add a catalyst, and react in the presence of hydrogen at 0-40°C, preferably 20-30°C, for 10-18 hours, preferably 12-15 hours, to obtain the reaction product; 2) Filter the reaction product and then dry the filtrate to obtain the compound of formula (I) or a pharmaceutically acceptable salt thereof; Preferably, in step 1), the solvent is selected from one or more of water, methanol, ethanol, n-propanol, and isopropanol; Preferably, in step 1), the catalyst is selected from one or more of palladium on carbon, platinum on carbon, rhodium on carbon, ruthenium on carbon, and Raney nickel; Preferably, in step 1), the pressure of the hydrogen gas is 0.01-2 MPa, preferably atmospheric pressure; Preferably, in step 1), the concentration of the compound represented by formula (III) or a pharmaceutically acceptable salt thereof in the solvent is 0.01-0.2 g / mL, preferably 0.1 g / mL; Preferably, in step 1), the weight ratio of the compound represented by formula (III) or its pharmaceutically acceptable salt to the catalyst is 1:0.01-1, more preferably 1:0.05-0.

1.

5. The compound represented by formula (II) or a pharmaceutically acceptable salt thereof, in, n is an integer, and n = 0-8, preferably n = 0-3.

6. The compound of formula (II) according to claim 5, or a pharmaceutically acceptable salt thereof, wherein, In the compound shown in formula (II), the structural unit The configuration is and / or end The configuration is and / or Preferably, in the compound represented by formula (II), 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 (II), 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 (II), 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 one or more of sodium salts, potassium salts, calcium salts, magnesium salts, iron salts, zinc salts, and ammonium salts.

7. A method for preparing the compound of formula (II) as claimed in claim 5 or 6, or a pharmaceutically acceptable salt thereof, comprising the following steps: In the presence of an oxidizing agent and a solvent, the compound of formula (I) according to claim 1 or 2 or a pharmaceutically acceptable salt thereof undergoes a ring-opening oxidation reaction to obtain the compound of formula (II) or a pharmaceutically acceptable salt thereof.

8. The preparation method according to claim 7, wherein, The preparation method includes the following steps: 1) Dissolve the compound of formula (I) 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 (II) 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 (I) or its pharmaceutically acceptable salt in the solvent is 10-100 mg / mL, preferably 50 mg / 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 (I) or its pharmaceutically acceptable salt to the oxidant is 1:1-5, preferably 1:2-3; Preferably, in step 2), the separation and purification are performed using a gel column.

9. 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 and / or the compound of formula (II) according to claim 5 or 6 or a pharmaceutically acceptable salt thereof; Preferably, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and / or the compound represented by formula (II) 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 of formula (I) or a pharmaceutically acceptable salt thereof and the compound of formula (II) 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, or gel. Patch, spray, external solution, external foam, suppository or enema.

10. Use of the compound of formula (I) as claimed in claim 1 or 2 or a pharmaceutically acceptable salt thereof and / or the compound of formula (II) as claimed in claim 5 or 6 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of inflammatory diseases; Preferably, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and / or the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is the sole active ingredient in the pharmaceutical composition; Preferably, the inflammatory disease is selected from one or more of acute kidney injury, chronic kidney disease, gout, ulcerative colitis, Crohn's disease, oral ulcers, rhinitis, and liver injury; More preferably, the symptoms of the chronic kidney disease are characterized by elevated levels of protein in the urine.

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