CN115671118A

CN115671118A - Application of brown algae oligosaccharide

Info

Publication number: CN115671118A
Application number: CN202110831844.0A
Authority: CN
Inventors: 高河勇; 刘振德
Original assignee: Haitang Jiangsu Biomedical Technology Co ltd
Current assignee: Haitang Jiangsu Biomedical Technology Co ltd
Priority date: 2021-07-22
Filing date: 2021-07-22
Publication date: 2023-02-03
Anticipated expiration: 2041-07-22
Also published as: CN115671118B

Abstract

The invention provides an application of brown algae oligosaccharide or pharmaceutically acceptable salt thereof in preparing a medicament for treating gout, wherein the brown algae oligosaccharide is brown algae disaccharide, brown algae trisaccharide or brown algae tetrasaccharide. Researches show that on an Acute Gouty Arthritis (AGA) mouse model, the brown algae disaccharide, the brown algae trisaccharide and the brown algae tetrasaccharide can obviously improve the symptoms of joint swelling and mechanical hyperalgesia of a mouse. Gait ethology research of mice also shows that the brown algae disaccharide, trisaccharide and tetrasaccharide can obviously improve the gait ethological abnormality of the mice. The brown algae oligosaccharide has a strong effect of treating acute gout. The brown algae oligosaccharide medicine provided by the invention is derived from marine algae, has no toxic or side effect, and can be used for a long time.

Description

Application of brown algae oligosaccharide

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to application of brown algae oligosaccharide.

Background

Carbohydrates (carbohydrates) are combined with nucleic acids, proteins and are called three major life substances. Algin is mainly present in the cell walls of kelp, gulfweed and kelp, and is a straight-chain, unbranched and negatively charged polysaccharide compound. Algin is a binary linear block compound consisting of β -D- (1,4) -Mannuronic acid (M) and α -L- (1,4) -Guluronic acid (Guluronic acid, G).

In recent years, due to the unique structure of alginate-derived oligosaccharides, the activity research becomes a hotspot in the research of carbohydrate drugs, and the research on the biological activity of alginate-derived oligosaccharides has made an important progress. It has been found that algin oligosaccharide and its derivatives have various biological activities, such as antioxidant, antitumor, anticoagulant, immunoregulatory, neuroprotective, antiinflammatory, antiviral, anti-senile dementia, anti-lithangiuria, anti-diabetic, etc.

Carbohydrates are a highly complex class of biological macromolecules with simultaneously varying multiple ends. It is difficult to separate oligosaccharides or polysaccharides having a uniform degree of polymerization regardless of chemical cleavage or enzymatic cleavage. To date, almost all of the studies have used a mixture of a series of sugars with close degrees of polymerization, which has made it very difficult to study the activity, metabolism, toxicology, and drug quality.

Aiming at the difficulty of the current saccharide research, the inventor develops a series of alginate lyase with strong specificity, and can decompose the alginate into the fucoidan, trisaccharide or tetrasaccharide with high purity, conjugated double bond at the non-reducing end and uniform polymerization degree; after enzyme inactivation and centrifugation, supernatant fluid is taken out and concentrated, and then is further purified into the fucoidan disaccharide, the trisaccharide or the tetrasaccharide with uniform polymerization degree by a gel column or ion exchange resin. The brown algae disaccharide is a combination of two structures of delta G and/or delta M and any proportion thereof; the brown algae trisaccharide is a combination of four structures of delta GG, delta GM, delta MM and delta MG in any proportion; brown algae tetrasaccharide is eight structures of delta GGG, delta GGM, delta GMG, delta GMM, delta MMG, delta MMM, delta MGG and delta MGM and the combination of any proportion thereof; all oligosaccharides are monosaccharide 1,4 glycosidic linkages; g represents alpha-L-guluronic acid; m represents beta-D-mannuronic acid; delta represents that beta-elimination occurs at 4,5 of alpha-L-guluronic acid and/or beta-D-mannuronic acid to generate unsaturated monosaccharide with 4,5 of non-reducing end as conjugated double bond; the structure of each monosaccharide is shown below:

taking Δ GM as an example, the structure of the corresponding fucoidan is as follows:

levels of Serum Uric Acid (SUA) in excess of 420 μmol/L are measured 2 times a day, both in men and women, and are referred to as hyperuricemia (Hyper-Uricimia, HUA). Blood uric acid, above its saturation in blood or tissue fluid, can locally form Monosodium Urate crystals (MSU) and deposit in the joints, inducing local inflammatory reactions and tissue destruction, i.e. gout (gout); acute nephropathy, chronic interstitial nephritis or kidney stones may be caused by deposition in the kidney, which is called uric acid nephropathy.

The prevalence rate of hyperuricemia in different ethnic groups is 2.6-36%, the prevalence rate of gout is 0.03-15.3%, and middle-aged and elderly men and postmenopausal women are high-incidence people of the hyperuricemia and show obvious rising and young trend in recent years. Current epidemiological studies show that approximately 10% of hyperuricemia progresses to gout, and approximately 80-90% of gout patients are hyperuricemia. Meta analysis showed that the overall prevalence of hyperuricemia in China was 13.3% and gout was 1.1%. Global hyperuricemia has become yet another common metabolic disease following diabetes.

With the advance of understanding, more and more scholars realize that hyperuricemia and gout are a continuous pathological process. The 'hyperuricemia/gout patient practice guideline' in 2020 also provides that hyperuricemia and gouty arthritis are different states of the same disease, and the hyperuricemia and the gouty arthritis are divided into 8 states of asymptomatic hyperuricemia, asymptomatic urate deposition, asymptomatic hyperuricemia with urate deposition, gout, tophaceous gout, erosive gout, initial gout attack and recurrent gout attack.

The drug treatment of gout is divided into two categories, one is anti-inflammatory analgesic treatment, and when the gout is suddenly and acutely attacked, anti-inflammatory analgesic drugs can be adopted to improve the life quality of a patient, for example, non-steroidal anti-inflammatory drugs, colchicine and glucocorticoid can be used in a targeted manner within 24 hours to help the patient relieve the pain. The second is a medicament for reducing uric acid for assisting in regulating the content of uric acid.

Uric acid lowering drugs are mainly classified into 3 types: 1) Xanthine oxidase inhibitors, such as allopurinol, febuxostat and topiroxostat, are used to inhibit uric acid synthesis, maintaining uric acid at a normal level; 2) The medicine for promoting uric acid excretion, such as probenecid, benzbromarone, lei Xina de, dortinolein and the like, can play a role in accelerating uric acid excretion in vivo. The sodium bicarbonate is also beneficial to the excretion of uric acid, and can be used together with other uric acid lowering drugs or used for patients with mild uric acid rise. 3) Recombinant uricases, such as labyrinase and purekesin (also known as pegolose); uricase can degrade uric acid into water-soluble allantoin, and is easier to be discharged through the kidney. Researches show that the change of the intestinal flora of gout patients can play an important role in the occurrence and development of gout through the immunoregulation effect.

Patients with gout are easy to damage the liver due to long-term taking of the medicine, so that liver diseases such as hypertension, hyperlipidemia and the like are easy to occur. Besides gouty arthritis, joint deformity, uric acid nephropathy, type 2 diabetes, cardiovascular diseases and lipid metabolism disorder are important hazards of gout, and bring about no small economic burden to individuals, families and society.

The invention further researches the brown algae oligosaccharide and provides the application of the brown algae oligosaccharide.

Disclosure of Invention

Therefore, the invention aims to provide the application of the brown algae oligosaccharide.

The purpose of the invention is realized by the following technical scheme:

the invention provides an application of brown algae oligosaccharide or pharmaceutically acceptable salt thereof in preparing a medicament for treating gout, wherein the brown algae oligosaccharide is brown algae disaccharide, brown algae trisaccharide and/or brown algae tetrasaccharide.

In certain embodiments of the invention, the brown algae oligosaccharide is composed of monosaccharides G, M and/or Δ linked by a glycosidic linkage at

position

1,4; wherein G represents alpha-L-guluronic acid, M represents beta-D-mannuronic acid, and Delta represents the beta-elimination at

position

4,5 of alpha-L-guluronic acid or beta-D-mannuronic acid, to generate 4,5 unsaturated monosaccharide with conjugated double bond.

In certain embodiments of the invention, the fucoidan is selected from ag, am, or a combination thereof.

In certain embodiments of the invention, the fucoidan is selected from one or more of Δ GG, Δ GM, Δ MM, and Δ MG.

In certain embodiments of the invention, the brown algae tetrasaccharide is selected from one or more of Δ GGG, Δ GGM, Δ GMG, Δ GMM, Δ MMG, Δ MMM, Δ MGG and Δ MGM.

In certain embodiments of the invention, the pharmaceutically acceptable salt is a sodium, potassium, calcium, magnesium and/or ammonium salt.

The brown algae disaccharide, trisaccharide and tetrasaccharide with uniform polymerization degree provided by the invention have revolutionary progress on analytical researches such as quality control, pharmacology and toxicology of saccharide bulk drugs.

Researches show that on an Acute Gouty Arthritis (AGA) mouse model, the brown algae disaccharide, the brown algae trisaccharide and the brown algae tetrasaccharide can obviously improve the symptoms of joint swelling and mechanical hyperalgesia of a mouse. Gait ethology research of mice also shows that the brown algae disaccharide, trisaccharide and tetrasaccharide can obviously improve the gait ethological abnormality of the mice. Therefore, the brown algae disaccharide, trisaccharide and tetrasaccharide have stronger efficacy of treating acute gout. The brown algae oligosaccharide medicine provided by the invention is derived from marine algae, has no toxic or side effect, and can be used for a long time.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows a high performance liquid chromatogram of fucoidan at a wavelength of 230 nm;

FIG. 2 shows nuclear magnetic hydrogen spectrum of brown algae disaccharide (A) ¹ HNMR, solvent D ₂ O)；

FIG. 3 shows a high resolution mass spectrum (HRMS (ESI)) of fucoidan;

FIG. 4 shows a high performance liquid chromatogram of fucoidan at a wavelength of 230 nm;

FIG. 5 shows nuclear magnetic hydrogen spectrum of fucoidan (A) ¹ HNMR with solvent D ₂ O)；

FIG. 6 shows a high resolution mass spectrum (HRMS (ESI)) of fucoidan;

FIG. 7 shows a high performance liquid chromatogram of fucoidan at a wavelength of 230 nm;

FIG. 8 shows the nuclear magnetic hydrogen spectrum of fucoidan (M) ¹ HNMR with solvent D ₂ O)；

FIG. 9 shows a high resolution mass spectrum (HRMS (ESI)) of fucoidan;

FIG. 10 shows the establishment and evaluation of a mouse model of acute gouty arthritis;

FIG. 11 is a graph showing the effect of fucoidan on the inhibition of arthritic pain in mice of the AGA model;

FIG. 12 is a graph showing the effect of fucoidan on inhibiting the symptoms of joint pain in a mouse model of AGA;

FIG. 13 is a graph showing the effect of fucoidan on the symptoms of joint pain in mice of the AGA model;

FIG. 14 shows mass spectra of mixed alginate-derived oligosaccharides with degree of polymerization ranging from 2 to 8;

FIG. 15 shows a study of the inhibitory effect of fucoidan on the joint pain in mouse model AGA by fucoidan, mixed sugar and indomethacin;

figure 16 shows the effect of fucoidan on AGA model mouse gait behaviours.

Detailed Description

The present invention is further illustrated below with reference to examples, which are illustrative and explanatory only and are not meant to limit the scope of the present invention in any way.

Example 1Preparation of fucoidan with uniform polymerization degree and structural identification thereof

Dissolving 100g of purchased algin (purchased from Qingdao Mingyue algae group, ltd.) in water, adding fucoidan lyase (obtained from China oceanic university) at a certain temperature, performing pyrolysis for a certain period of time, centrifuging with a high-speed centrifuge, and collecting supernatant. Purifying the clear liquid with gel column to remove small amount of oligosaccharide, polysaccharide and non-saccharide impuritiesImpurities, 60g of brown algae disaccharide sodium salt is obtained. The purity of the brown algae disaccharide sodium salt is detected by high performance liquid chromatography (HPLC, 230 nm), and nuclear magnetic hydrogen spectrum is used ( ¹ HNMR) and high-resolution mass spectrometry (HRMS-ESI).

HPLC: purity 99.06%, RT =13.6min (see fig. 1 for the relevant spectrum);

¹ the HNMR spectrogram is shown in figure 2;

HRMS(ESI)m/z:C ₁₂ H ₁₅ O ₁₂ {(M-H) ^- }, calcd 351.0569, found 351.0572 (M-H) ^- (the correlation spectrum is shown in FIG. 3);

the theoretical content of sodium ions in the sodium alginate disaccharide sodium salt is 11.58 percent if two carboxyl groups in a molecule are sodium salts; the content of sodium ions is 10.3 percent by actual ion chromatography detection. If the residue is detected by a residue on ignition method, sodium ions exist in the form of sodium sulfate, and the theoretical residue proportion is 35.77 percent; detecting actual residues on ignition, wherein the residue is 34.3%; the results obtained by the two detection methods are relatively close, which indicates that the carboxylic acid functional group of the compound is in a sodium salt form. However, the observed values are slightly smaller than the theoretical values, probably because the sodium salt is a weak acid strong base salt and a small part of the carboxylic acid is still in a free state.

Example 2Preparation of brown algae trisaccharide with uniform polymerization degree and structure identification thereof

Dissolving 100g of purchased algin in water, adding fucoidan lyase (obtained from China ocean university) at a certain temperature, performing pyrolysis for a certain time, centrifuging with a high-speed centrifuge, and collecting supernatant. And purifying the clear liquid by using a gel column to remove a small amount of impurity oligosaccharide, polysaccharide and non-saccharide impurities to obtain 70g of brown algae trisaccharide sodium salt. The purity of the brown algae trisaccharide sodium salt is detected by high performance liquid chromatography (HPLC, 230 nm), and nuclear magnetic hydrogen spectrum is used ( ¹ HNMR) and high resolution mass spectrometry (HRMS-ESI).

HPLC: purity 100%, RT =17.43min (see fig. 4 for the relevant spectrum);

¹ the HNMR spectrum is shown in FIG. 5;

HRMS(ESI)m/z:C ₁₈ H ₂₃ O ₁₈ {(M-H) ^- }, calcd 527.0890, found 527.0891 (M-H) ^- (see FIG. 6 for a correlation spectrum);

the brown algae trisaccharide sodium salt has the theoretical content of sodium ions of 11.59 percent if three carboxyl groups in the molecule are sodium salts; the content of sodium ions is 9.9 percent by actual ion chromatography detection. If the residue is detected by a glowing residue method, sodium ions exist in the form of sodium sulfate, and the theoretical residue proportion is 35.80%; the residue of incandescence was found to be 33.01%. The results obtained by the two detection modes are relatively close, which indicates that the carboxylic acid functional group of the compound is in a sodium salt form. However, the observed values are slightly smaller than the theoretical values, probably because the sodium salt is a weak acid strong base salt and a small part of the carboxylic acid is still in a free state.

Example 3Preparation of brown algae tetrasaccharide with uniform polymerization degree and structural identification of brown algae tetrasaccharide

Dissolving 100g of purchased algin in water, adding fucoidan lyase (obtained from China oceanic university) at a certain temperature, cracking for a certain time, centrifuging with a high-speed centrifuge, and taking supernatant. And purifying the clear liquid by using a gel column to remove a small amount of impurity oligosaccharide, polysaccharide and non-saccharide impurities to obtain 55g of brown algae tetrasaccharide sodium salt. The brown algae tetrasaccharide sodium salt is subjected to purity detection by high performance liquid chromatography (HPLC, 230 nm), and nuclear magnetic hydrogen spectrum (A), (B), (C) and (D) ¹ HNMR) and high-resolution mass spectrometry (HRMS-ESI).

HPLC: purity 99.71%, RT =18.71min (see fig. 7 for the correlation spectrum);

¹ the HNMR spectrogram is shown in FIG. 8;

HRMS(ESI)m/z：C ₂₄ H ₃₁ O ₂₄ {(M-H) ^- }, calcd 703.1211, found 703.1207 (M-H) ^- (the correlation spectrum is shown in FIG. 9);

the sodium alginate tetrasaccharide salt has the theoretical content of sodium ions of 11.59% if four carboxyl groups in a molecule are sodium salts; the content of sodium ions is 9.8 percent through actual ion chromatography detection. If the residue is detected by a glowing residue method, sodium ions exist in the form of sodium sulfate, and the theoretical residue proportion is 35.80%; residue on ignition was found to be 32.5%. The results obtained by the two detection modes are relatively close, which indicates that the carboxylic acid functional group of the compound is in a sodium salt form. However, the observed values are slightly smaller than the theoretical values, probably because the sodium salt is a weak acid strong base salt and a small part of the carboxylic acid is still in a free state.

Example 4Establishment of Acute Gouty Arthritis (AGA) model mouse

Healthy, clean-grade male C57BL/6 mice were divided into control (Veh), model (MSU) groups of 6 mice each. After being sterilized with medical alcohol, the model group was inserted into the ankle cavity at 30-40 ° on the posterior side of the right ankle of the mouse along the medial side of the Achilles tendon, and 1mg/20 μ l of sodium urate (MSU) solution was injected, and the control group was injected with Phosphate Buffered Saline (PBS) in the same manner. After the injection is finished, the ankle joint swelling degree and the mechanical pain threshold change condition of the mice are observed. Ankle swelling assay: and (3) detecting the diameter of the ankle joint on the affected side of the mouse at four time points of 2h, 6h, 24h and 48h before and after the model is made by using a vernier caliper, continuously measuring for three times, and taking the average value as the final reading. Ankle swelling = current measured diameter-premold measured diameter. Mechanical pain behavior determination: the mice were placed in a transparent plastic box on an overhead wire mesh and covered with transparent plexiglass to adapt to the environment for 45min. The pain threshold of the mouse right hind paw near the ankle was then measured using von Frey filaments of different gauges at four time points, 2h, 6h and 24h and 48h post-mod, anterior to, and following the "Up and Down" procedure. And calculating to obtain a paw withdrawal threshold value by using a formula.

Fig. 10 shows the establishment of a mouse model of acute gouty arthritis and evaluation of the effect. FIG. 10A is a graph showing swelling of right ankle of mice in AGA model group after crystallization by injection of urate (MSU). After the right ankle of the mice in the AGA model group is injected with urate (MSU) for 24 hours to crystallize, the ankle of the mice in the AGA model group is obviously swollen compared with the ankle of the mice in the control group which is injected with Phosphate Buffer Solution (PBS). FIG. 10B is a comparison of sections after H & E staining of ankle joints of control and model mice. It was observed that a large amount of inflammatory cell infiltration occurred in the ankle section of the mice of the AGA model group compared to the control group. FIG. 10C is a graph of ankle diameter changes in right ankle mice injected with urate crystals compared to ankle mice injected with PBS. It can be seen that the ankle of the control mice had only slight swelling within a few hours after PBS injection, but rapidly resolved and had essentially no change after returning to normal. However, the ankle joint of the mice injected with the urate crystals rapidly swells, and the obvious swelling phenomenon still exists after 48 hours. FIG. 10D shows a comparison of the symptoms of mechanical pain sensitivity in the right hind paw in two groups of mice, showing that the mechanical pain threshold is significantly reduced in mice injected with MSU. The above results are consistent with previous reports, thus suggesting successful establishment of an AGA mouse model.

Example 5Study of Effect of fucoidan (AOS 2) on analgesic Effect of AGA model mice

In the present invention, fucoidan is abbreviated as "AOS2". AGA model mice were prepared as described in example 4. Mice were randomized into 5 groups: control group (Veh + Veh), model group (MSU + Veh), MSU + AOS2 dose groups (400 mg/kg,200mg/kg,100 mg/kg). Different concentrations of AOS2 are injected into the abdominal cavity of each dose group of MSU + AOS2, and the administration is carried out for 1h before the model, 5h after the model, 23h and 47h respectively; the control group and the model group were injected intraperitoneally with the same volume of PBS (fig. 11A). The detection of mechanical pain and swelling degree is carried out before the molding and 2, 6, 24 and 48 hours after the molding. The specific detection method was the same as in example 4.

FIG. 11 shows the effect of fucoidan AOS2 in inhibiting arthritic pain symptoms in mice of the AGA model. Fig. 11A is a schematic of the time points of administration. FIG. 11B is a time course plot of the effect of different doses of AOS2 (100 mg/kg,200mg/kg,400mg/kg, i.p.) on mechanical ankle pain in mouse model AGA. 200 and 400mg/kg AOS2 released mechanical pain in mice model AGA to different extents after administration of different doses of AOS 2. FIG. 11C is a histogram of the area under the curve of FIG. 11B. FIG. 11D is a graph showing the time course of the effect of different doses of AOS2 (100 mg/kg,200mg/kg,400mg/kg, i.p.) on ankle swelling in mice with the AGA model. After different doses of AOS2 were given, ankle swelling was significantly reduced, and the greater the dose, the less swelling. And the graph E is a statistical graph of the area under the curve of the graph D. The above experimental results show that AOS2 can effectively inhibit pain and swelling of ankle joints of mice of AGA model, and the effect shows a certain dose dependence.

Example 6Research on analgesic effect of fucoidan (AOS 3) on AGA model mouse

AGA model mice were prepared as described in example 4. In the present invention, fucoidan is abbreviated as "AOS3". Mice were randomized into 5 groups: control group (Veh + Veh), model group (MSU + Veh), MSU + AOS3 dose groups (400 mg/kg,200mg/kg,100 mg/kg). Different concentrations of AOS3 are injected into the abdominal cavity of each dose group of MSU + AOS3, and the doses are respectively administered in 1h before the model and 5, 23 and 47h after the model; the control group and the model group were injected intraperitoneally with the same volume of control solvent PBS (fig. 12A). The detection of mechanical pain and swelling degree is carried out before the molding and 2, 6, 24 and 48 hours after the molding. The specific detection method was the same as in example 4.

FIG. 12 shows the effect of fucoidan AOS3 in inhibiting arthritic pain in mice of the AGA model. Fig. 12A is a schematic of the time points of administration. FIG. 12B is a time course plot of the effect of different doses of AOS3 (100 mg/kg,200mg/kg,400mg/kg, i.p.) on mechanical ankle pain in mouse model AGA. FIG. 12C is a statistical plot of the area under the curve of 12B. As shown in FIGS. 12B and 12C, after different doses of AOS3, 200mg/kg and 400mg/kg of AOS3 significantly relieved mechanical pain of mice in AGA model, and AOS3 at a dose of 100mg/kg also has partial relieving effect on mechanical pain, and obvious dose dependence can be seen. FIG. 12D is a time course plot of the effect of different doses of AOS3 (100 mg/kg,200mg/kg,400mg/kg, i.p.) on ankle swelling in AGA model mice. FIG. 12E is a statistical plot of the area under the curve of FIG. 12D. FIGS. 12D and 12E show the swelling of the ankle joints of mice over time. After different doses of AOS3 were given, ankle swelling was significantly reduced, and the greater the dose, the less swelling. The experimental results show that AOS3 can effectively inhibit the pain and swelling of ankle joints of mice of an AGA model, and the effect shows a certain dose dependence.

Example 7Research on analgesic effect of fucoidan tetrasaccharide (AOS 4) on AGA model mouse

AGA model mice were prepared as described in example 4. In the present invention, fucoidan is abbreviated as "AOS4". Mice were randomized into 5 groups: control group (Veh + Veh), model group (MSU + Veh), MSU + AOS4 dose groups (400 mg/kg,200mg/kg,100 mg/kg). Different concentrations of AOS4 are injected into the abdominal cavity of each dose group of MSU + AOS4, and the doses are respectively administered in 1 hour before the model and 5, 23 and 47 hours after the model; the control group and the model group were injected intraperitoneally with the same volume of control solvent PBS (fig. 13A). The detection of mechanical pain and swelling degree is carried out before the molding and 2, 6, 24 and 48 hours after the molding. The specific detection method was the same as in example 4.

FIG. 13 shows the effect of fucoidan AOS4 on inhibiting arthritic pain in mice of the AGA model. Fig. 13A is a schematic of the time points of administration. FIG. 13B is a time course plot of the effect of different doses of AOS4 (100 mg/kg,200mg/kg,400mg/kg, i.p.) on mechanical ankle pain in mouse model AGA. FIG. 13C is a statistical plot of the area under the curve of FIG. 13B. As shown in FIGS. 13B and 13C, the AGA model mice were administered with different doses of AOS4, and the doses were at different levels to relieve mechanical pain in the AGA model mice, with the effects of 200mg/kg and 400mg/kg being particularly significant. FIG. 13D is a graph of the time course of the effect of different doses of AOS4 (100 mg/kg,200mg/kg,400mg/kg, i.p.) on ankle swelling in mice with the AGA model. FIG. 13E is a statistical plot of the area under the curve of FIG. 13D. FIGS. 13D and 13E show the swelling of the ankle joints of mice as a function of time. The ankle swelling was significantly reduced after different doses of AOS4 were given, with the greater the dose, the less swelling. The above experimental results show that AOS4 can effectively inhibit pain and swelling of ankle joints of mice of AGA model, and the effect shows a certain dose dependence.

As AOS2, AOS3 and AOS4 show better analgesic activity on an AGA mouse model at a dose of 200 mg/kg. In subsequent experiments, the inventors therefore chose AOS3 at a concentration of 200mg/kg for further in vivo pharmacological activity studies.

Example 8Study of the analgesic Effect of AOS3 and AGA model mice with Mixed sugar and control drugs

AGA model mice were prepared as described in example 4. AOS3 was experimentally compared with mixed saccharides (mixed alginate oligosaccharides with a degree of polymerization of 2-8, mass spectrum thereof is shown in FIG. 14, obtained from China's university of oceans, abbreviated as "AOS mix" in the present invention) and indomethacin (abbreviated as "Indo" in the present invention). Mice were randomized into 6 groups: control group (Veh + Veh), model group (MSU + Veh), MSU + AOS3 group (200 mg/kg), MSU + AOS3 group (100 mg/kg), MSU + AOS mixed group (200 mg/kg), MSU + indomethacin group (MSU + Indo,10 mg/kg). The medicine is administrated in each dosage group for intraperitoneal injection of a specified amount, and the medicine is administrated 1h before the model and 5, 23 and 47h after the model; the control group and the model group were injected intraperitoneally with the same volume of control solvent PBS (fig. 15A). The detection of mechanical pain and swelling degree is carried out before the molding and 2, 6, 24 and 48 hours after the molding. The specific detection method was the same as in example 4.

Figure 15 shows the effect of AOS3, mixed sugar and the comparative drug indomethacin on inhibiting arthritic pain symptoms in mice model AGA. Fig. 15A is a schematic of the time points of administration. FIG. 15B is a time course plot of the effect of AOS3 (100 mg/kg,200mg/kg, i.p.), mixed sugar (200 mg/kg, i.p.), and indomethacin (10 mg/kg, i.p., effective dose in mice) on mechanical ankle pain in AGA model mice. FIG. 15C is a statistical plot of the area under the curve of FIG. 15B. As shown in FIGS. 15B and 15C, after administration to mice in AGA model, mechanical pain was relieved to different extents in mice in AGA model, and 200mg/kg of AOS3 was most effective, and better than 100mg/kg of AOS3 and the comparative drug indomethacin (10 mg/kg), 200mg/kg of mixed sugar was the least effective. FIG. 15D is a graph of the time course of the effect of different drugs on ankle swelling in AGA model mice. FIG. 15E is a statistical plot of the area under the curve of FIG. 15D. FIGS. 15D and 15E show the swelling of the ankle joints of mice as a function of time. After administration, ankle swelling is improved, and particularly, the swelling of the AOS3 (200 mg/kg) group is obviously reduced; the swelling improvement effect becomes worse after the dose of AOS3 is reduced (100 mg/kg), which is close to the effect of indometacin; mixed sugars are the least effective. The experimental results show that AOS3 can effectively inhibit the ankle pain and swelling of mice of an AGA model, the effect is dose-dependent, and the effect is better than that of mixed sugar and indometacin.

Example 9Study of the improving Effect of AOS3 on gait behavioral abnormalities in AGA mice A mouse model for AGA was prepared as described in example 4. Mice were randomized into 3 groups: control group (Veh + Veh), model group + solvent group (MSU + Veh), model group + AOS3 group (MSU + AOS 3). The MSU + AOS3 group is intraperitoneally injected with AOS3 with a dose of 200mg/kg, and is respectively administered 1h before the model and 5h and 23h after the model, and the control group and the model group and the solvent group are intraperitoneally injected with a control solvent PBS with the same volume. As shown in fig. 16A, experimental mice were first pre-adapted for a period of testing environment. Gait ethology tests are carried out at two time points of 8h and 24h after the model, namely, each group of mice pass through the runway at constant speed without stopping within 10-15 seconds, and a camera below the mice collects gait images in real timeLike this, the Mouse hindpaw was then analyzed for gait behavioral parameters of footprinting area and swing duration using DigiGait (Mouse Specifics, inc., USA) software.

Figure 16 shows the effect of fucoidan AOS3 on AGA model mouse gait behaviours. Fig. 16A is a schematic representation of Catwalk mouse gait machine recording and analysis. Figure 16B is a representative graph of the area of the hindpaw footprints that passed the runway within 5 seconds for each group of mice. The foot paw blot shows that the area of the right hind paw of the model group and solvent group mice 24h after the model is remarkably reduced compared with that of the healthy lateral paw, and obvious lameness gait occurs. And the landing area of the right hind paw of the affected side of the mice of the model group and the AOS3 group is improved. Fig. 16C is a statistical graph of the relative percentage of the affected paw area to the healthy paw area at 8 and 24h post-molding for each group of mice. The foot and paw area of the affected side of the mouse of the model group and the solvent group is obviously reduced compared with that of the healthy side, and the foot and paw area of the affected side of the mouse of the model group and the AOS3 group is obviously improved. Fig. 16D is a statistical graph of the relative percentage of swing time of the affected side paw and the healthy side paw at 8h and 24h after molding for each group of mice. The data show that 24h after the model, the air swing time of the affected side paw of the model group and solvent group mice is remarkably prolonged, and the air swing time of the affected limb of the model group and AOS3 group mice is remarkably reduced compared with the model group and solvent group. The above results suggest that the AGA model mouse has lameness-like behaviors such as reduction in the landing area of the lateral paw and prolongation of the time of the paw swing in the air in gait ethology. This is consistent with lameness-like behavior in gout patients. And AOS3 can effectively improve the abnormal change of gait and ethology of the mouse of the AGA model.

Example 10Rat single AOS3 gavage acute oral toxicity test

SD rats are divided into 4 groups by taking 40 males and females respectively with weight of about 160-180g, wherein the groups are respectively a negative control group (normal saline), a brown algae trisaccharide AOS3 low dose group (0.25 g/kg body weight), a medium dose group (1.0 g/kg body weight) and a high dose group (2.0 g/kg body weight), the doses are respectively equivalent to 25 times, 100 times and 200 times of pharmacodynamic effective doses of the rats, 10 males and females in each group are fasted at night before experiments, and water is not forbidden. The condition of the animals is observed for 14 days continuously, the animals in each group have no obvious difference, the mental state is good, the respiration is normal, the behavior is normal, the activity is normal, and no abnormality is observed in walking gait. Toxic symptoms and death do not occur. There was no significant difference between the initial body weight and the final body weight of the rats in each group. Pathological sections of liver, pancreas, kidney, stomach, ovary, brain showed no lesions in each organ. The blood test index is normal. The above results indicate that AOS3 is a practically non-toxic grade.

And (4) experimental conclusion: AOS2, AOS3 and AOS4 can be used for dose-dependently relieving joint swelling and mechanical hyperalgesia symptoms of an Acute Gouty Arthritis (AGA) model mouse; the AOS3 can obviously improve gait behavioral abnormality of an AGA model mouse, and the AOS3 analgesic effect under the dosage of 200mg/kg is better than that of indometacin (the dosage of 10 mg/kg) and is also better than that of mixed sugar under the same dosage.

Finally, it should be pointed out here that: the above is only a part of the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention, and the insubstantial modifications and adaptations of the present invention by those skilled in the art based on the above description are intended to be covered by the present invention.

Claims

1. Use of a brown algae oligosaccharide or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating gout, wherein the brown algae oligosaccharide is brown algae disaccharide, brown algae trisaccharide and/or brown algae tetrasaccharide.

2. The use of claim 1, wherein the alginate oligosaccharide is composed of monosaccharides G, M and/or Δ linked by a glycosidic bond at position 1,4; wherein G represents alpha-L-guluronic acid, M represents beta-D-mannuronic acid, and Delta represents the beta-elimination at position 4,5 of alpha-L-guluronic acid or beta-D-mannuronic acid, to generate 4,5 unsaturated monosaccharide with conjugated double bond.

3. The use of claim 2, wherein the fucoidan is selected from Δ G, Δ M, or a combination thereof.

4. The use of claim 2, wherein the brown alginate triose is selected from one or more of Δ GG, Δ GM, Δ MM, and Δ MG.

5. The use of claim 2, wherein the fucoidan is selected from one or more of Δ GGG, Δ GGM, Δ GMG, Δ GMM, Δ MMG, Δ MMM, Δ MGG and Δ MGM.

6. The use of claim 1, wherein the pharmaceutically acceptable salt is a sodium, potassium, calcium, magnesium and/or ammonium salt.