CN115671118B

CN115671118B - Application of brown alginate oligosaccharides

Info

Publication number: CN115671118B
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: 2024-02-27
Anticipated expiration: 2041-07-22
Also published as: CN115671118A

Abstract

The invention provides an application of brown alginate oligosaccharides or pharmaceutically acceptable salts thereof in preparing a medicament for treating gout, wherein the brown alginate oligosaccharides are brown alginate disaccharide, brown alginate trisaccharide or brown alginate tetrasaccharide. It was found that the brown algae disaccharides, trisaccharides and tetrasaccharides of the invention significantly improve the joint swelling and mechanical hyperalgesia symptoms of mice in an Acute Gouty Arthritis (AGA) mouse model. The gait behavioral studies of mice also show that the brown algae disaccharides, trisaccharides and tetrasaccharides of the invention can significantly improve the gait behavioral abnormalities of the mice. The brown alginate oligosaccharides of the invention have strong 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.

Description

Application of brown alginate oligosaccharides

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of brown alginate oligosaccharides.

Background

Saccharides (carbohydrates) are together with nucleic acids, proteins and are called tri-living materials. Algin is mainly present in the cell walls of kelp, gulfweed and kelp, and is a class of linear, unbranched, negatively charged polysaccharide compounds. Algins are binary linear block compounds consisting of β -D- (1, 4) -Mannuronic acid (M) and α -L- (1, 4) -Guluronic acid (G).

In recent years, the activity research of alginate oligosaccharides has become a hotspot in the research of saccharide medicaments due to the unique structure, and the biological activity research of alginate oligosaccharides has been greatly progressed. It has been found that alginate oligosaccharides and derivatives thereof have various biological activities such as oxidation resistance, antitumor, anticoagulation, immunomodulation, neuroprotection, anti-inflammatory activity, antiviral activity, anti-senile dementia, anti-lithangiuria, antidiabetic, etc.

Saccharides are a highly complex and widely varying class of biological macromolecules. It is difficult to separate oligosaccharides or polysaccharides having a uniform degree of polymerization, whether by chemical or enzymatic cleavage. To date, almost all of the oligosaccharides or polysaccharides employed in the studies are mixtures of a series of sugars with similar degrees of polymerization, which present great difficulties for their activity studies, metabolism, toxicology, and quality studies of drugs.

The previous research of the inventor aims at the difficulty of the current saccharide research, develops a series of alginate lyase with stronger specificity, and can respectively decompose the alginate into the brown algae disaccharide, trisaccharide or tetrasaccharide with higher purity, conjugated double bond at the non-reducing terminal and uniform polymerization degree; enzyme inactivation, centrifugation to obtain supernatant, concentration and further purification with gel column or ion exchange resin to obtain brown algae disaccharide, trisaccharide or tetrasaccharide with homogeneous polymerization degree. 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; the brown algae tetrasaccharide is composed of 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 connected by monosaccharide 1, 4-glycosidic bonds; g represents alpha-L-guluronic acid; m represents beta-D-mannuronic acid; delta represents that beta-elimination occurs at 4,5 positions of alpha-L-guluronic acid and/or beta-D-mannuronic acid, and unsaturated monosaccharide with conjugated double bonds at 4,5 positions of a non-reducing end is generated; the structure of each monosaccharide is as follows:

taking Δgm as an example, the structure of the corresponding brown algae trisaccharide is as follows:

whether male or female, the non-daily 2-time blood Uric Acid (SUA) level exceeds 420. Mu. Mol/L, referred to as Hyperuricemia (HUA). The saturation of uric acid in blood or interstitial fluid beyond that of uric acid can locally form Monosodium Urate crystals (MSU) in joints and deposit, inducing local inflammatory reactions and tissue destruction, namely gout (gout); acute kidney disease, chronic interstitial nephritis or kidney stones can be caused by kidney deposition, and are called uric acid kidney disease.

Hyperuricemia has a prevalence of 2.6-36% in different families and gout of 0.03-15.3%, and men in middle-aged and elderly people and women after menopause are high-incidence people, and have obvious rising and younger trends in recent years. Current epidemiological studies have shown that about 10% of hyperuricemia develops into gout, and about 80-90% of gout patients are hyperuricemia. Meta analysis shows that the overall prevalence of Chinese hyperuricemia is 13.3% and gout is 1.1%. Global hyperuricemia has become another common metabolic disease following diabetes.

With the deep knowledge, more and more scholars are aware that hyperuricemia and gout are a continuous pathological process. In 2020, "hyperuricemia/gout patient practice guideline" also proposes that hyperuricemia and gouty arthritis are different states of the same disease, and the disease is divided into 8 states of asymptomatic hyperuricemia, asymptomatic urate deposition, asymptomatic hyperuricemia accompanied by urate deposition, gout, tophaceous gout, erosive gout, primary gout attacks and recurrent gout attacks.

The drug treatment of gout is divided into two main types, one is anti-inflammatory and analgesic treatment, and when the drug is used for treating the sudden acute episode of gout, the anti-inflammatory and analgesic drugs can be used for improving the life quality of patients, for example, nonsteroidal anti-inflammatory drugs, colchicine and glucocorticoid are used for pertinently within 24 hours to help patients to relieve the pains. The second is uric acid lowering drugs used to help regulate uric acid levels.

Uric acid lowering drugs are mainly classified into 3 classes: 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 uric acid excretion promoting drugs such as probenecid, benzbromarone, leifenesin, and domenode can play a role in accelerating the in vivo uric acid excretion. Sodium bicarbonate also facilitates uric acid excretion, and can be used together with other uric acid lowering drugs or singly for patients with mild uric acid elevation. 3) Recombinant uricases, such as, for example, labyrinase and primiki (also known as pegolozyme); uricase may degrade uric acid to water-soluble allantoin, which is more readily excreted via the kidneys. It has also been found that changes in the intestinal flora of gout patients can play an important role in the development and progression of gout through immunomodulation.

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. In addition to gouty arthritis, joint deformity, uric acid nephropathy, type 2 diabetes, cardiovascular diseases and lipid metabolism disorders are also important hazards of gout, and bring about a small economic burden to individuals, families and society.

The invention further researches on brown alginate oligosaccharides and provides an application of the brown alginate oligosaccharides.

Disclosure of Invention

Accordingly, an object of the present invention is to provide a use of brown alginate oligosaccharides.

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

the invention provides an application of brown alginate oligosaccharides or pharmaceutically acceptable salts thereof in preparing a medicament for treating gout, wherein the brown alginate oligosaccharides are brown alginate disaccharides, brown alginate trisaccharides and/or brown alginate tetrasaccharides.

In certain embodiments of the present invention, the brown alginate oligosaccharides are composed of monosaccharides G, M and/or Δ linked by glycosidic linkages at the 1,4 positions; wherein G represents alpha-L-guluronic acid, M represents beta-D-mannuronic acid, delta represents alpha-L-guluronic acid or beta-D-mannuronic acid, beta-elimination occurs at the 4,5 positions of the alpha-L-guluronic acid or the beta-D-mannuronic acid, and unsaturated monosaccharide with conjugated double bonds at the 4,5 positions is generated.

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

In certain embodiments of the invention, the fucoidan is selected from one or more of Δgg, Δgm, Δmm, and Δmg.

In certain embodiments of the invention, the brown algae tetrasaccharide is selected from one or more of Δggg, Δggm, Δgmg, Δgmm, Δmmg, Δmmm, Δmgg, and Δmgm.

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

The brown algae disaccharide, trisaccharide and tetrasaccharide with uniform polymerization degree have revolutionary progress for the quality control, pharmacology, toxicology and other analytical researches of the carbohydrate bulk drug.

It was found that the brown algae disaccharides, trisaccharides and tetrasaccharides of the invention significantly improve the joint swelling and mechanical hyperalgesia symptoms of mice in an Acute Gouty Arthritis (AGA) mouse model. The gait behavioral studies of mice also show that the brown algae disaccharides, trisaccharides and tetrasaccharides of the invention can significantly improve the gait behavioral abnormalities of the mice. Therefore, the brown algae disaccharides, trisaccharides and tetrasaccharides have strong 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 present invention are described in detail below with reference to the attached drawing figures, wherein:

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

FIG. 2 shows the nuclear magnetic resonance spectrum of brown algae disaccharide 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 brown algae trisaccharide at a wavelength of 230 nm;

FIG. 5 shows brown algaeNuclear magnetic hydrogen spectrum of trisaccharide ¹ HNMR, solvent D ₂ O)；

FIG. 6 shows a high resolution mass spectrum (HRMS (ESI)) of brown algae trisaccharide;

FIG. 7 shows a high performance liquid chromatogram of brown algae tetrasaccharide at a wavelength of 230 nm;

FIG. 8 shows nuclear magnetic resonance hydrogen spectrum of brown algae tetrasaccharide ¹ HNMR, solvent D ₂ O)；

Fig. 9 shows a high resolution mass spectrum (HRMS (ESI)) of brown algae tetrasaccharide;

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 inhibiting symptoms of joint pain in AGA model mice;

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

FIG. 13 is a graph showing the effect of brown algae tetrasaccharide on inhibiting symptoms of joint pain in AGA model mice;

FIG. 14 is a mass spectrum of mixed brown alginate oligosaccharides with a degree of polymerization of 2-8;

fig. 15 shows a study of the effect of fucoidan, mixed saccharide and indomethacin on inhibiting joint pain in the AGA model mice;

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

Detailed Description

The invention is further illustrated below in connection with examples which are merely illustrative and are not meant to limit the scope of the invention in any way.

Example 1Preparation of brown algae disaccharide with uniform polymerization degree and structural identification thereof

100g of purchased algin (purchased from Qingdao Mingya seaweed group Co., ltd.) was dissolved in water, and at a certain temperature, fucoidin lyase (obtained from China ocean university) was added, and after a certain time of lysis, the mixture was centrifuged by a high-speed centrifuge to obtain a supernatant. Purifying the clear solution by a gel column to remove a small amount of impurity oligosaccharide, polysaccharide and non-saccharide impurities, and obtaining 60g of brown algae disaccharide sodium salt. The obtained product is then processedThe purity of the sodium salt of brown algae disaccharide is detected by high performance liquid chromatography (HPLC, 230 nm), and nuclear magnetism hydrogen spectrum is used ¹ HNMR) and high resolution mass spectrometry (HRMS-ESI).

HPLC: purity 99.06%, rt=13.6 min (correlation spectrum see fig. 1);

¹ HNMR spectra are shown in fig. 2;

HRMS(ESI)m/z:C ₁₂ H ₁₅ O ₁₂ {(M-H) ^- calculated as 351.0569, found as 351.0572 (M-H) ^- (see FIG. 3 for a correlation profile);

the theoretical content of sodium ions of the sodium salt of the brown algae disaccharide is 11.58 percent if two carboxyl groups in the molecule are sodium salts; the content of sodium ions was 10.3% as measured by actual ion chromatography. If the method is used for detecting the glowing residues, sodium ions exist in the form of sodium sulfate, and the theoretical residue proportion is 35.77%; detecting actual burning residues, wherein the residues are 34.3%; the results obtained by both detection methods are relatively close, indicating that the carboxylic acid functional group of the compound is indeed in the sodium salt form. However, the observed values are slightly less than the theoretical values, probably because the sodium salt is a weak acid and strong alkali salt, and a small part of carboxylic acid is still in a free state.

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

100g of purchased algin is dissolved in water, at a certain temperature, fucoidin lyase (obtained from China university of ocean) is added, after a certain time of pyrolysis, the mixture is centrifuged by a high-speed centrifuge, and the supernatant is taken. Purifying the clear solution by a gel column to remove a small amount of impurity oligosaccharide, polysaccharide and non-saccharide impurities, thus obtaining 70g of brown algae trisaccharide sodium salt. Detecting purity of the brown algae trisaccharide sodium salt by high performance liquid chromatography (HPLC, 230 nm), and using nuclear magnetism hydrogen spectrum ¹ HNMR) and high resolution mass spectrometry (HRMS-ESI).

HPLC: purity 100%, rt=17.43 min (correlation spectrum see fig. 4);

¹ HNMR spectra are shown in fig. 5;

HRMS(ESI)m/z:C ₁₈ H ₂₃ O ₁₈ {(M-H) ^- calculated as 527.0890, foundFor 527.0891 (M-H) ^- (see FIG. 6 for a correlation profile);

the theoretical content of sodium ions is 11.59% if three carboxyl groups in the molecule are sodium salts; the content of sodium ions was 9.9% as measured by actual ion chromatography. If the method is used for detecting the glowing residues, sodium ions exist in the form of sodium sulfate, and the theoretical residue proportion is 35.80%; the glowing residue was found to be 33.01%. The results obtained by both detection modes are relatively close, which means that the carboxylic acid functional group of the compound is in the form of sodium salt. However, the observed values are slightly less than the theoretical values, probably because the sodium salt is a weak acid and strong alkali salt, and a small part of carboxylic acid is still in a free state.

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

100g of purchased algin is dissolved in water, at a certain temperature, fucoidin lyase (obtained from China university of ocean) is added, after a certain time of pyrolysis, the mixture is centrifuged by a high-speed centrifuge, and the supernatant is taken. Purifying the clear solution by a gel column to remove a small amount of impurity oligosaccharide, polysaccharide and non-saccharide impurities, thus obtaining 55g of brown algae tetrasaccharide sodium salt. Detecting purity of the brown algae tetrasaccharide sodium salt by high performance liquid chromatography (HPLC, 230 nm), and using nuclear magnetism hydrogen spectrum [ ] ¹ HNMR) and high resolution mass spectrometry (HRMS-ESI).

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

¹ HNMR spectra are shown in fig. 8;

HRMS(ESI)m/z：C ₂₄ H ₃₁ O ₂₄ {(M-H) ^- calculated as 703.1211, found as 703.1207 (M-H) ^- (see FIG. 9 for a correlation profile);

the sodium salt of brown algae tetrasaccharide, if four carboxyl groups in the molecule are all sodium salts, the theoretical content of sodium ions is 11.59%; the content of sodium ions was 9.8% as measured by actual ion chromatography. If the method is used for detecting the glowing residues, sodium ions exist in the form of sodium sulfate, and the theoretical residue proportion is 35.80%; the burning residue was found to be 32.5%. The results obtained by both detection modes are relatively close, which means that the carboxylic acid functional group of the compound is in the form of sodium salt. However, the observed values are slightly less than the theoretical values, probably because the sodium salt is a weak acid and strong alkali salt, and a small part of carboxylic acid is still in a free state.

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

Healthy, clean-grade male C57BL/6 mice were divided into control (Veh), model (MSU) groups of 6 animals each. After sterilization with medical alcohol, the model group was penetrated into the ankle cavity at 30-40 ° along the inner side of the achilles tendon at the rear side of the right ankle of the mouse, 1mg/20 μl sodium urate (MSU) solution was injected, and the control group was injected with an equal amount of Phosphate Buffer (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 determination: and (3) detecting the ankle joint diameter of the affected side of the mouse at four time points by using a vernier caliper for 2h, 6h, 24h and 48h after molding before molding, continuously measuring three times, and taking the average value as a final reading. Ankle swelling = current measured diameter-pre-mold measured diameter. Mechanical pain behavior determination: the mice were placed in a transparent plastic box over overhead wire mesh and covered with transparent organic glass to fit the environment for 45min. The pain threshold of the right hindpaw near the ankle of the mice was then measured using von Frey filaments of different gauges at four time points, 2h, 6h and 24h and 48h after the postmold before the mold, following the "Up and Down" method. And calculating to obtain the claw shrinkage threshold value by using a formula.

Fig. 10 shows the establishment of a mouse model for acute gouty arthritis and evaluation of effect. Fig. 10A is a graph showing swelling of the right ankle joint of the group of age-model mice after injection of urinary acid salt (MSU) for crystallization. After 24h of urinary acid salt (MSU) crystallization, the ankle of the mice of the group of the AGA model showed significant swelling compared to the ankle of the mice of the control group injected with solvent Phosphate Buffer (PBS). Fig. 10B is an alignment of sections after H & E staining of the ankle joints of control and model mice. It can be observed that a large number of inflammatory cell infiltrates occurred in the ankle section of the mice of the AGA model group compared to the control group. Fig. 10C shows the change in ankle diameter of mice after right ankle injection of urinary acid salt crystallization as compared to mice with ankle injection of PBS. It can be seen that the ankle joint of the control mice had only slight swelling within a few hours after the PBS injection, but resolved rapidly and was essentially unchanged after recovery from normal. The ankle joint of the mice injected with the urate crystals is rapidly swelled, and the mice still have obvious swelling phenomenon after 48 hours. Figure 10D shows a comparison of the mechanical pain allergy phenomenon in the right hind paw of two groups of mice, showing that the mechanical pain threshold was significantly reduced in the mice after MSU injection. The results are consistent with the previous reports, so that the AGA mouse model is suggested to be successfully established.

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

In the present invention, fucoidan is simply referred to as "AOS2". AGA model mice were prepared as described in example 4. Mice were randomly divided into 5 groups: control (Veh+Veh), model (MSU+Veh), MSU+AOS2 dose groups (400 mg/kg,200mg/kg,100 mg/kg). The MSU+AOS2 dose groups were intraperitoneally injected with different concentrations of AOS2, and administered 1h before the mold, 5h after the mold, 23h, and 47 h; the control and model groups were intraperitoneally injected with the same volume of control solvent PBS (fig. 11A). Mechanical pain and swelling levels were measured before and after molding for 2, 6, 24 and 48 hours. The specific detection method was the same as in example 4.

Fig. 11 shows the effect of fucoidan AOS2 on inhibiting the symptoms of joint pain in the AGA model mice. Fig. 11A is a schematic illustration of dosing time points. FIG. 11B is a time chart of the effect of different doses of AOS2 (100 mg/kg,200mg/kg,400mg/kg, i.p.) on ankle mechanical pain in AGA model mice. 200 and 400mg/kg AOS2 released mechanical pain in AGA model mice to varying degrees after different doses of AOS2 were administered. FIG. 11C is an area chart statistical diagram under the curve of 11B. FIG. 11D shows a time chart of the effect of different doses of AOS2 (100 mg/kg,200mg/kg,400mg/kg, i.p.) on ankle swelling in AGA model mice. Ankle swelling was significantly reduced after different doses of AOS2, and the greater the dose, the less swelling. Figure E is an area statistic plot under the curve of figure D. The experimental results show that AOS2 can effectively inhibit ankle joint pain and swelling of mice with AGA model, and the effect shows a certain dose dependence.

Example 6Study of analgesic Effect of fucoidan (AOS 3) on AGA model mice

AGA model mice were prepared as described in example 4. In the present invention, fucoidan is simply referred to as "AOS3". Mice were randomly divided into 5 groups: control (Veh+Veh), model (MSU+Veh), MSU+AOS3 (400 mg/kg,200mg/kg,100 mg/kg). The MSU+AOS3 dose groups were intraperitoneally injected with different concentrations of AOS3, and administered 1h before the mold, 5, 23, and 47h after the mold, respectively; the control and model groups were intraperitoneally injected with the same volume of control solvent PBS (fig. 12A). Mechanical pain and swelling levels were measured before and after molding for 2, 6, 24 and 48 hours. The specific detection method was the same as in example 4.

Fig. 12 shows the effect of fucoidan AOS3 on inhibiting the symptoms of joint pain in the AGA model mice. Fig. 12A is a schematic illustration of dosing time points. FIG. 12B is a time chart of the effect of different doses of AOS3 (100 mg/kg,200mg/kg,400mg/kg, i.p.) on ankle mechanical pain in AGA model mice. Fig. 12C is an area statistic under the 12B curve. As shown in FIGS. 12B and 12C, 200mg/kg and 400mg/kg of AOS3 significantly released mechanical pain in AGA model mice after administration of different doses of AOS3, and 100mg/kg of AOS3 also had a partial release effect on mechanical pain, and a significant dose dependence was seen. FIG. 12D is a time chart 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 an area statistic under the curve of fig. 12D. Fig. 12D and 12E show the ankle swelling of mice over time. Ankle swelling was significantly reduced after different doses of AOS3, and the greater the dose, the less swelling. The experimental results show that AOS3 can effectively inhibit ankle joint pain and swelling of mice with AGA model, and the effect shows a certain dose dependence.

Example 7Study of analgesic Effect of fucoidan (AOS 4) on AGA model mice

AGA model mice were prepared as described in example 4. In the present invention, brown algae tetrasaccharide is simply referred to as "AOS4". Mice were randomly divided into 5 groups: control (Veh+Veh), model (MSU+Veh), MSU+AOS4 dose groups (400 mg/kg,200mg/kg,100 mg/kg). The MSU+AOS4 dose groups were intraperitoneally injected with different concentrations of AOS4, and administered 1h before the mold, 5, 23, and 47h after the mold, respectively; the control and model groups were intraperitoneally injected with the same volume of control solvent PBS (fig. 13A). Mechanical pain and swelling levels were measured before and after molding for 2, 6, 24 and 48 hours. The specific detection method was the same as in example 4.

Fig. 13 shows the effect of brown algae tetrasaccharide AOS4 on inhibiting the symptoms of joint pain in the AGA model mice. Fig. 13A is a schematic representation of the dosing time points. FIG. 13B is a time chart of the effect of different doses of AOS4 (100 mg/kg,200mg/kg,400mg/kg, i.p.) on ankle mechanical pain in AGA model mice. Fig. 13C is an area statistic diagram under the curve of fig. 13B. As shown in FIGS. 13B and 13C, the mechanical pain of AGA model mice was relieved to varying degrees by the respective dose levels after administration of different doses of AOS4, with the 200mg/kg and 400mg/kg effects being particularly pronounced. FIG. 13D is a time chart of the effect of different doses of AOS4 (100 mg/kg,200mg/kg,400mg/kg, i.p.) on ankle swelling in AGA model mice. Fig. 13E is an area statistic under the curve of fig. 13D. Fig. 13D and 13E show the ankle swelling of mice over time. Ankle swelling was significantly reduced after different doses of AOS4, and the larger the dose, the less swelling. The experimental results show that AOS4 can effectively inhibit ankle joint pain and swelling of mice with AGA model, and the effect shows a certain dose dependence.

Since AOS2, AOS3 and AOS4 all showed better analgesic activity against the AGA mouse model at a dose of 200 mg/kg. In subsequent experiments, the inventors therefore selected AOS3 at a concentration of 200mg/kg for further in vivo pharmacological activity studies.

Example 8Research on analgesic effect of AOS3 and AGA model mice mixed with sugar and contrast drug

AGA model mice were prepared as described in example 4. AOS3 was experimentally compared with mixed sugar (mixed brown alginate oligosaccharides with a degree of polymerization of 2-8, mass spectrum of which is shown in FIG. 14, obtained from China university of ocean, abbreviated as "AOS mix" in the present invention) and indomethacin (abbreviated as "Indo" in the present invention). Mice were randomly divided into 6 groups: control (veh+veh), model (MSU+veh), MSU+AOS3 (200 mg/kg), MSU+AOS3 (100 mg/kg), MSU+AOS mix (200 mg/kg), MSU+indomethacin (MSU+indo, 10 mg/kg). Administering a prescribed amount of the drug to the abdominal cavity of each dosage group, and administering the drug 1h before molding, 5, 23 and 47h after molding; the control and model groups were intraperitoneally injected with the same volume of control solvent PBS (fig. 15A). Mechanical pain and swelling levels were measured before and after molding for 2, 6, 24 and 48 hours. The specific detection method was the same as in example 4.

Fig. 15 shows the effect of AOS3, mixed sugar and the comparative drug indomethacin on inhibiting the symptoms of joint pain in the AGA model mice. Fig. 15A is a schematic illustration of dosing time points. FIG. 15B is a time chart of the effects of AOS3 (100 mg/kg,200mg/kg, i.p.), mixed sugars (200 mg/kg, i.p.) and indomethacin (10 mg/kg, i.p., effective dose in mice) on ankle mechanical pain in AGA model mice. Fig. 15C is an area statistic diagram under the curve of fig. 15B. As shown in FIGS. 15B and 15C, the mechanical pain of the AGA model mice can be relieved to different degrees after the AGA model mice are dosed, 200mg/kg of AOS3 has the most obvious effect, and 200mg/kg of mixed sugar has the worst effect compared with 100mg/kg of AOS3 and the comparative drug indomethacin (10 mg/kg). Fig. 15D is a time chart of the effect of different drugs on ankle swelling in the AGA model mice. Fig. 15E is an area statistic under the curve of fig. 15D. Fig. 15D and 15E show the ankle swelling of mice over time. After administration, ankle swelling is improved, and particularly, the swelling of an AOS3 (200 mg/kg) group is obviously reduced; after the AOS3 dose is reduced (100 mg/kg), the swelling improvement effect is poor and is close to that of indomethacin; the mixed sugar was the least effective. The experimental results show that AOS3 can effectively inhibit ankle joint pain and swelling of mice with AGA model, the effect shows a certain dose dependence, and the effect is better than that of mixing sugar and indomethacin.

Example 9Effect of AOS3 on improvement of AGA mouse gait behavioral abnormalities an AGA mouse model was prepared as described in example 4. Mice were randomly divided into 3 groups: control group (veh+veh), model group+solvent group (msu+veh), model group+aos 3 group (msu+aos 3). The MSU+AOS3 group was intraperitoneally injected with 200mg/kg dose of AOS3, and the same volume of control solvent PBS was intraperitoneally injected into the control group and the model group+solvent group, after 1h before the mold, 5h and 23h after the mold, respectively. As shown in fig. 16A, the experimental mice were first pre-adapted to the test environment for a period of time. Gait behavioural tests were performed at two time points of 8h and 24h after the mould, i.e. each group of mice was passed over the runway at constant speed for 10-15 seconds, the lower camera was acquiring gait images in real time, followed by DigiGait (Mouse Specifications, inc., U.S.SA) software analyzes gait behavioural parameters of footprint area and swing duration for the hind paw of the mice.

Figure 16 shows the effect of fucoidan AOS3 on AGA model mice gait behaviours. Fig. 16A is a schematic of Catwalk mouse gait meter recording and analysis. Fig. 16B is a representative graph of hindpaw print area through the runway within 5 seconds for each group of mice. Paw blots showed that the hindpaw area of model group + solvent group mice was significantly reduced compared to healthy side paw at 24h post-model, with a clear lameness-like gait. Whereas the model group + AOS3 mice showed an improvement in the area of right hindpaw footprint on the affected side. Fig. 16C is a graph showing the relative percentage of the paw area of the affected side versus the paw area of the healthy side for each group of mice at 8 and 24h after molding. The area of the paw on the affected side of the mice in the model group + the solvent group was significantly reduced compared to the healthy side, while the area of the paw on the affected side of the mice in the model group + the AOS3 group was significantly improved. Fig. 16D is a graph showing the relative percentage of time that the affected side paw was swung in air versus the healthy side paw swing time for each group of mice at 8 and 24h after molding. The data show that 24h after the model group + solvent group mice had significantly longer paw air swing time, while model group + AOS3 group mice had significantly less limb air swing time than model group + solvent group. The above results suggest that the AGA model mice exhibit lameness-like behavior such as reduced foot strike area on the affected side and prolonged paw swing time in gait behaviours. This is consistent with lameness in gout patients. And AOS3 can effectively improve abnormal change of gait behaviours of the AGA model mouse.

Example 10Rat single AOS3 gastric lavage acute oral toxicity test

The method comprises the steps of taking 40 SD rats, wherein the weights of the rats and the males are about 160-180g, randomly dividing the rats and the females into 4 groups, namely a negative control group (normal saline), a low-dose group (0.25 g/kg body weight) of fucoidan AOS3, a medium-dose group (1.0 g/kg body weight) and a high-dose group (2.0 g/kg body weight), wherein the doses are respectively equivalent to 25 times, 100 times and 200 times of pharmacodynamic effective doses of the rats, and each group of 10 females and males is fasted overnight before experiments and is not forbidden. The animal conditions were observed for 14 consecutive days, and the animals in each group were not significantly different, had good mental status, were normal in breathing, were behaving normally, were active, and were not observed any abnormality in walking gait. No toxic symptoms and death occurred. There was no significant difference between the initial body weight and the final body weight of each group of rats. Pathological sections of the liver, pancreas, kidney, stomach, ovary, brain show no lesions in each organ. The blood test index is normal. The above results demonstrate that AOS3 is a practically non-toxic grade.

Conclusion of experiment: AOS2, AOS3 and AOS4 can dose-dependently alleviate symptoms of Acute Gouty Arthritis (AGA) model mice joint swelling and mechanical hyperalgesia; AOS3 can remarkably improve gait behavioural abnormality of AGA model mice, and the AOS3 analgesic effect at the dosage of 200mg/kg is better than that of indomethacin (10 mg/kg dosage) and is also better than that of mixed sugar at the same dosage.

Finally, it is 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 some insubstantial modifications and adaptations of the present invention based on the foregoing are within the scope of the present invention.

Claims

1. Use of brown alginate-derived oligosaccharide or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating acute gout, wherein the brown alginate-derived oligosaccharide is brown alginate-derived oligosaccharide, brown alginate-derived oligosaccharide or brown alginate-derived oligosaccharide.

2. The use of claim 1, wherein the brown alginate oligosaccharides are comprised of monosaccharides G, M and/or Δ linked by glycosidic linkages at the 1,4 positions; wherein G represents alpha-L-guluronic acid, M represents beta-D-mannuronic acid, delta represents alpha-L-guluronic acid or beta-D-mannuronic acid, beta-elimination occurs at the 4,5 positions of the alpha-L-guluronic acid or the beta-D-mannuronic acid, and unsaturated monosaccharide with conjugated double bonds at the 4,5 positions is generated.

3. The use of claim 2, wherein the fucoidan is selected from Δg, Δm, or a combination thereof.

4. The use of claim 2, wherein the fucoidan is selected from one or more of Δgg, Δgm, Δmm, and Δmg.

5. The use of claim 2, wherein the brown algae tetrasaccharide is selected from one or more of Δggg, Δggm, Δgmg, Δgmm, Δmmg, Δmmm, Δmgg, and Δmgm.

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