Application of fucoidan polysaccharide sulfate in reduction of TMAO content

Abstract

The invention discloses application of fucoidan polysaccharide sulfate in reduction of TMAO content. According to the scheme provided by the invention, the research finds that the fucoidan polysaccharide sulfate (a natural polysaccharide compound derived from fucoidan) has the capability of remarkably reducing the content of trimethylamine-N-oxide (TMAO) in blood by accident. The discovery opens up a new way and method for exploring and treating various diseases related to the increase of the TMAO level. As a metabolite generated by metabolism of intestinal microorganisms, TMAO is indicated to be closely related to health problems such as cardiovascular diseases, kidney diseases and metabolic disorders by many studies in recent years. Therefore, the invention not only enriches the understanding of the bioactivity of the fucoidan polysaccharide sulfate, but also provides a scientific basis and a practice direction for developing a novel treatment strategy for TMAO-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of brown algae polysaccharide sulfate in reducing TMAO content. Background Art

[0002] In recent years, with the continuous deepening of scientific research, the close connection between trimethylamine N-oxide (TMAO), a metabolite of intestinal flora, and a series of major chronic diseases such as cardiovascular disease, chronic kidney disease, metabolic syndrome and type 2 diabetes has gradually been revealed. Patients with these diseases have significantly elevated TMAO levels, making it a key factor in promoting the progression of the disease and increasing all-cause mortality, and also providing a new potential target for the treatment of these diseases. In order to find effective methods or intervention drugs to reduce TMAO levels, researchers are actively exploring various approaches.

[0003] In this scientific context, fucoidan sulfate, a complex sulfated polysaccharide derived from brown algae such as kelp, has been increasingly receiving widespread attention from the scientific community due to its unique bioactive properties. Differences in its bioactivity may stem from a variety of factors, including but not limited to its source, molecular weight, fine structural characteristics, types of monosaccharide composition, degree of sulfation, and specific distribution patterns of sulfate groups. It is worth noting that fucoidan sulfate can not only function in the gastrointestinal environment, but can also be effectively absorbed through the intestines into the blood circulation system. Its excellent adsorption capacity allows it to capture and remove a variety of harmful and toxic substances ranging from small molecules, medium molecules to large molecules.

[0004] Currently, there are products on the market that use fucoidan sulfate as the main active ingredient, such as Haikun Shenxi Capsules, which have shown significant therapeutic effects in the clinical application of chronic kidney disease. This practical achievement not only confirms the biological activity effect of fucoidan sulfate, but also opens up new research perspectives and broad prospects for its application in regulating TMAO (trimethylamine oxide) levels in the body and potentially treating other chronic diseases. However, it should be pointed out that despite the promising prospects, there is currently a lack of direct scientific research evidence on the specific effects and mechanisms of fucoidan sulfate directly used to reduce TMAO levels. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a new use of fucoidan sulfate, which is unexpectedly found to be used to reduce TMAO.

[0006] Use of the brown algae polysaccharide sulfate according to the first aspect of the present invention in the preparation of a product with reduced TMAO content.

[0007] According to the application of the embodiment of the present invention, there are at least the following beneficial effects: The present invention scheme unexpectedly found in the study that brown algae polysaccharide sulfate (a natural polysaccharide compound derived from brown algae) has the ability to significantly reduce the content of trimethylamine-N-oxide (TMAO) in the blood. This discovery has opened up new ways and methods for exploring and treating various diseases associated with elevated TMAO levels. TMAO, as a metabolite produced by intestinal microbial metabolism, has been pointed out by many studies in recent years to be closely related to health problems such as cardiovascular disease, kidney disease and metabolic disorders. Therefore, the present invention not only enriches the understanding of the biological activity of brown algae polysaccharide sulfate, but also provides a scientific basis and practical direction for the development of new treatment strategies for TMAO-related diseases.

[0008] According to some embodiments of the present invention, the method for preparing fucoidan sulfate comprises the following steps:

[0009] S1. Soak the brown algae in water and concentrate the soaking liquid;

[0010] S2, preparing the soaked kelp into a homogenate;

[0011] S3, adjust the pH value of the homogenate to 11.5±0.3, stir and let stand for more than 6 hours;

[0012] S4, adjust the pH value to 1.8-2.2, stir again and let stand (7-8h, preferably 7.5h), let stand for more than 10h, and filter by adsorption;

[0013] S5, the filtrate is combined with the concentrated solution in step S1 for desalination, and the pressure of the dilute and concentrated water reaches 0.06-0.08MPa; the desalted solution is neutralized to neutral;

[0014] S6, concentrate the liquid medicine and decolorize it;

[0015] S7, adding potassium chloride to the concentrate, adding alcohol solvent so that the volume ratio of alcohol exceeds that of water, leaving it to stand to obtain a precipitate, separating the solid and liquid, and collecting the solid phase;

[0016] S8. The solid phase is washed, dried and crushed to obtain the product.

[0017] According to some embodiments of the present invention, the volume ratio of water to brown algae in step S1 is 1:5-15.

[0018] According to some embodiments of the present invention, the soaking time in step S1 is 6 to 12 hours; and / or the temperature is 10-30°C.

[0019] According to some embodiments of the present invention, the soaking liquid in step S1 is concentrated to 1 / 2 to 1 / 8 of the original volume.

[0020] According to some embodiments of the present invention, in step S3, the pH is adjusted to 11.5.

[0021] According to some embodiments of the present invention, the stirring time in step S3 is 0.1 to 1 h.

[0022] According to some embodiments of the present invention, the stirring time in step S3 is 0.1 to 0.3 h.

[0023] According to some embodiments of the present invention, the standing time in step S3 is 8 to 12 hours, such as 10 hours.

[0024] According to some embodiments of the present invention, diatomaceous earth drum adsorption filtration is used in step S4.

[0025] According to some embodiments of the present invention, the diatomaceous earth drum adsorption filtration includes the following parameters: the drum rotation speed is 0.1 rpm to 10 rpm; and / or the absolute pressure of the operating vacuum is 0.25×10 Pa to 0.8×10 Pa.

[0026] According to some embodiments of the present invention, the desalination in step S5 is performed by electrodialysis desalination.

[0027] According to some embodiments of the present invention, the desalination in step S5 includes the following parameters: the rated output is 1 to 10 m 3 / h.

[0028] According to some embodiments of the present invention, activated carbon is used for decolorization in step S6.

[0029] According to some embodiments of the present invention, the amount of potassium chloride added in step S7 is 5-10% of the mass of the concentrated solution.

[0030] According to some embodiments of the present invention, the alcohol solvent in step S7 is ethanol.

[0031] According to some embodiments of the present invention, the amount of alcohol solvent added in step S7 is such that the volume proportion of the alcohol solvent reaches 60% to 70%.

[0032] According to some embodiments of the present invention, the washing solvent in step S8 is aqueous ethanol.

[0033] According to some embodiments of the present invention, the volume content of ethanol in the aqueous ethanol is 95%.

[0034] According to some embodiments of the present invention, the drying temperature in step S8 is 50-60°C.

[0035] According to some embodiments of the present invention, the brown algae polysaccharide is extracted from at least one of the following brown algae: Laminaria japonica, Undaria pinnatifida, Sargassum fusiformis, Fucus vesiculosus, Ascophyllum nodosum, Kelp, Sargassum, Salvia thunbergii and Sea millet.

[0036] According to some embodiments of the present invention, the product includes at least one of a reagent, a kit, and a medicine.

[0037] According to some embodiments of the present invention, the product is a drug, and the dosage form of the drug is an oral preparation or an injection preparation. The drug can enter the body through the following routes: oral ingestion, direct injection by injection, application in the form of a spray, penetration, absorption process, or physical and chemical means to guide to areas such as muscle tissue, under the surface of the skin, subcutaneous fat layer, venous system and mucous membranes; in addition, the drug can also be mixed with other substances or introduced into the body as a package.

[0038] According to some embodiments of the present invention, the medicament comprises a therapeutically effective amount of the fucoidan sulfate and a pharmaceutically acceptable carrier.

[0039] For drugs containing brown algae polysaccharide sulfate as an active ingredient, one or more carrier substances that are pharmaceutically recognized as safe can be further added according to needs. These carriers widely cover diluents, molding agents, filling materials, adhesives, wetting aids, disintegration accelerators, absorption enhancers, surfactants, adsorptive carriers and lubricants commonly used in pharmaceutical practice. The drug has various routes of administration, including but not limited to oral routes, intravenous injections, intramuscular injections, etc., and its dosage form can be flexibly designed into various forms such as injections, oral tablets, powdered preparations, granules, capsules and oral liquids. All of the above dosage forms of drugs can be produced in accordance with the pharmaceutical standard preparation process.

[0040] According to some embodiments of the invention, the product comprises a health product.

[0041] According to a second aspect of the present invention, a method for reducing TMAO content comprises the following steps: administering fucoidan sulfate to a subject in which TMAO content needs to be reduced.

[0042] According to the method of the embodiment of the present invention, there are at least the following beneficial effects: the scheme of the present invention utilizes brown algae polysaccharide sulfate (BAPS) as an effective bioactive substance to accurately regulate the intestinal flora, and the improvement of the intestinal microecology can indirectly affect the metabolic pathways in the host, especially reducing the conversion process of trimethylamine (TMA) to trimethylamine oxide (TMAO), thereby reducing the level of TMAO. The present invention utilizes the regulatory effect of brown algae polysaccharide sulfate on the intestinal flora to achieve the goal of effectively reducing the level of TMAO in the host.

[0043] According to some embodiments of the invention, the subject includes individuals having elevated levels of TMAO in blood, plasma, serum and / or urine.

[0044] According to some embodiments of the invention, the administering comprises oral administration or injection.

[0045] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0047] Figure 1 This is the effect of the sulfated fucoidan of the present invention on the serum TMAO level of mice. DETAILED DESCRIPTION

[0048] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. The test methods used in the embodiments are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can be reagents and materials obtained from commercial channels. Unless otherwise specified, the same parameter in each embodiment has the same value. The embodiments described below are exemplary and are only used to explain the present invention, and cannot be understood as limitations on the present invention.

[0049] In the description of the present invention, the description with reference to the term "some embodiments" or the like means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0050] In the description of the present invention, if there is a description of first, second, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0051] Although many studies have been devoted to exploring effective methods to reduce the content of trimethylamine oxide (TMAO) in the blood, in the current technical field, research on the development of products specifically used to reduce TMAO content is still relatively scarce. Most of the proposed methods face challenges such as low efficiency, significant side effects or uncertain long-term effects in practical applications. These problems not only limit the application of these methods in actual products, but also affect their effectiveness and acceptability as a means of reducing TMAO. In view of the increasing attention to the association between TMAO levels and increased risk of cardiovascular disease, it is particularly urgent to develop a product that can not only effectively reduce TMAO content but also has good safety and stability. Such a product will not only help improve individual health, but may also provide new therapeutic strategies for the prevention of cardiovascular disease. Therefore, the scientific research community and the industry urgently need to increase their efforts to jointly promote the research and development of safe, effective and easy-to-promote products that reduce TMAO content.

[0052] To this end, the present invention proposes an innovative application scheme, that is, using fucoidan sulfate (FPS) as an effective active ingredient to reduce the content of trimethylamine oxide (TMAO) in the blood. As a natural polysaccharide derived from the ocean, fucoidan sulfate has unique biological activity and pharmacological effects, and has received widespread attention in the biomedical field in recent years. Through in-depth research on the relationship between its structure and function, the present invention found that fucoidan sulfate can specifically interact with TMAO, thereby effectively promoting its metabolism and excretion, thereby reducing its concentration in the blood. This discovery provides a scientific basis for the development of new cardiovascular health products, and also provides a new idea for solving the problems of low efficiency and large side effects in the current methods for reducing TMAO content. Therefore, the present invention aims to utilize this unique property of fucoidan sulfate to develop a safe, effective and easy-to-promote product for reducing TMAO content to meet the urgent demand for cardiovascular health products on the market.

[0053] Example

[0054] This example provides the use of brown algae polysaccharide sulfate in the preparation of products with reduced TMAO content.

[0055] The preparation method of brown algae polysaccharide sulfate is as follows:

[0056] (1) Soak the dried kelp in 10 times the volume of water for 8 hours, and control the room temperature to about 20°C; take the soaking liquid and concentrate it to 1 / 4 of the original volume for later use;

[0057] (2) taking out the soaked kelp and chopping it into small pieces, and homogenizing it with a high-pressure homogenizer;

[0058] (3) The homogenate was adjusted to pH 11.5 with sodium hydroxide, stirred for 25 min, and allowed to stand for 10 h. The pH was adjusted to 1.8-2.2 with hydrochloric acid, stirred for 7.5 h, and allowed to stand for 11 h.

[0059] (4) Diatomaceous earth vacuum drum adsorption filtration; the drum speed is 5 rpm; the absolute pressure of the operating vacuum is 0.6×10 Pa;

[0060] (5) The filtrate is combined with the concentrated solution in step (1) and desalinated by electrodialysis of special type 500, and the pressure of the dilute and concentrated water reaches 0.06-0.08MPa; the desalted solution is neutralized to neutrality by adding concentrated ammonia water; special type 500 means: the rated output is 5m 3 / h;

[0061] (6) Concentrating the liquid medicine and adding activated carbon for decolorization;

[0062] (7) adding 8 wt % potassium chloride to the concentrate and adding ethanol to make the alcohol content reach 65 vol %, leaving the concentrate to stand to obtain a precipitate, centrifuging it, and washing the filter cake with 95 vol % ethanol;

[0063] (8) The filter cake was dried under vacuum at 55°C for 5 hours;

[0064] (9) The dried product is pulverized to obtain a milky white or light yellow powder of fucoidan sulfate.

[0065] Experimental animals: healthy SPF-grade 8-week-old C57BL / 6J male mice, weighing about 20g, kept at room temperature (25°C), relative humidity of about 55%, and 12h of light and dark time per day. All mice were adapted to feeding for 1 week before the formal start of the experiment. During the feeding period, stable feeding conditions and sufficient feed and drinking water were ensured.

[0066] The experimental design is shown in Table 1. The specific steps are as follows: All 8-week-old C57BL / 6J male mice were randomly divided into 5 groups after being fed for 1 week: control group, model group, low-dose fucoidan sulfate group (HZ-L), high-dose fucoidan sulfate group (HZ-H), and positive control drug (3,3-dimethyl-1-butanol) group (DMB).

[0067] As shown in Table 1, there were 10-15 mice in each group. Except for the control group, all groups were fed with feed containing 1.0% choline. The fucoidan sulfate was dissolved in saline and administered by gavage for 8 weeks. The control group and the model group were gavaged with an equal amount of saline. The positive drug control group was given DMB in the drinking water of mice. After the experiment, the mice were killed and blood, intestinal contents and liver tissue samples were collected.

[0068] Table 1

[0069]

[0070] The specific operations are as follows:

[0071] (1) Sample collection

[0072] ① Plasma sample: Place the mouse on its side on a flat surface, and use the index finger and thumb to press the skin around the eye to make the eyeball protrude and become congested. Use ophthalmic forceps to quickly remove the eyeball, turn the mouse upside down with its head down, and massage the mouse body from the back of the body to the eye to allow the blood in the eye socket to flow into the centrifuge tube prepared in advance. After centrifugation at 4℃, separate the upper layer of serum and store it in a -80℃ refrigerator.

[0073] ② Cecal contents: Dissect the mouse to expose the cecum, use sterile forceps and scissors to carefully separate the cecum, and collect its contents into sterile cryotubes. Immediately place the cryotubes into a liquid nitrogen tank, and after collection, remove the cryotubes from the liquid nitrogen and transfer them to a -80℃ freezer.

[0074] ③ Liver tissue: After dissecting the mouse, remove the liver and place it in a sterile culture dish. Rinse it with saline and divide it into small portions and put it into cryopreservation tubes. Immediately put the cryopreservation tubes into a liquid nitrogen tank. After collection, remove the cryopreservation tubes from the liquid nitrogen and transfer them to a -80℃ refrigerator.

[0075] (2) Detection of TMAO concentration by HPLC-MS

[0076] ①Chromatographic conditions

[0077] Liquid chromatograph: Waters UPLCI-Class; chromatographic column: Waters ACQUITY UPLC BEHHILIK 1.7μm (2.1×50mm); online filter: Waters CRITICAL CLEAN TM; autosampler temperature: 10°C; column temperature: 40°C; mobile phase: phase A: 10mM ammonium acetate aqueous solution, phase B: acetonitrile; flow rate: 0.3mL / min; injection volume: 5μL. The gradient elution program for the analysis sample is as follows: 0-0.5min, 70% B; 0.5-2.0min, 10% B; 2.4-3.0min, 70% B.

[0078] ②Mass spectrometry conditions

[0079] The ion source selected was an electrospray ionization source, and the detection was performed in positive ion mode. The signal acquisition mode was MRM, the ion source temperature was 150°C, the capillary voltage was 0.5 kV, the cone voltage was 25 V, the desolvation gas flow rate was 1000 L / hr, and the desolvation gas temperature was 500°C.

[0080] ③Solution preparation and sample processing

[0081] Preparation of reference substance stock solution: Accurately weigh 1 mg of TMAO reference substance, transfer it to a volumetric flask, dissolve it in 50% methanol to make up the volume and shake well to obtain a 1 mg / mL reference substance stock solution, and store it in a -20°C refrigerator.

[0082] Preparation of internal standard reference substance stock solution: Accurately weigh 1 mg of D9-TMAO reference substance, transfer it to a volumetric flask, dissolve it in 50% methanol to make up to volume and shake well to obtain a 1 mg / mL internal standard reference substance stock solution, and store it in a -20°C refrigerator.

[0083] Preparation of standard curve working solution: Take the TMAO reference substance stock solution (concentration is 1 mg / mL), dilute it with 50% methanol water, mix the TMAO reference substance to a final concentration of 100.000 μg / mL, and use it as the standard curve working solution after stepwise dilution.

[0084] Preparation of internal standard working solution: Take D9-TMAO internal standard reference substance stock solution (concentration is 1 mg / mL), mix it with acetonitrile to obtain an internal standard working solution containing 50.000 ng / mL of D9-TMAO.

[0085] Take 50 μL of the sample to be tested, add 150 μL of internal standard working solution, oscillate for 30 s, centrifuge (18400×g, 6 min), take 50 μL of the supernatant, add 250 μL of ultrapure water, vortex to mix, and inject 5 μL.

[0086] ④TMAO detection results

[0087] The results are shown in Table 2 and Figure 1 shown.

[0088] Table 2 Results of serum TMAO content in each group

[0089]

[0090]

[0091] Table 2 and Figure 1 The symbols in the figure are as follows: Compared with the control group, #### P<0.0001; compared with the model group, * P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001.

[0092] From Table 2 and Figure 1 It can be seen that compared with the control group, the serum TMAO level of the model group mice fed with choline feed was significantly increased; compared with the model group, the TMAO levels of the low and high dose groups of fucoidan sulfate were significantly reduced (P < 0.0001), and there was no significant difference compared with the positive drug control DMB group, indicating that fucoidan sulfate can reduce the TMAO level of choline-fed mice.

[0093] This shows that brown algae polysaccharide has good application prospects in reducing TMAO content.

[0094] The embodiments of the present invention are described in detail above, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. Application of brown algae polysaccharide sulfate in the preparation of products with reduced TMAO content.

2. The use according to claim 1, characterized in that: The preparation method of the fucoidan sulfate comprises the following steps: S1. Soak the brown algae in water and concentrate the soaking liquid; S2, preparing the soaked kelp into a homogenate; S3, adjust the pH value of the homogenate to 11.5±0.3, stir and let stand for more than 6 hours; S4, adjust the pH value to 1.8-2.2, stir again and let stand for more than 10 hours, and then filter by adsorption; S5, the filtrate is combined with the concentrated solution in step S1 for desalination, and the pressure of the dilute and concentrated water reaches 0.06-0.08MPa; the desalted solution is neutralized to neutral; S6, concentrate the liquid medicine and decolorize it; S7, adding potassium chloride to the concentrate, adding alcohol solvent so that the volume ratio of alcohol exceeds that of water, leaving it to stand to obtain a precipitate, separating the solid and liquid, and collecting the solid phase; S8. The solid phase is washed, dried and crushed to obtain the product.

3. The use according to claim 1, characterized in that: The soaking in step S1 includes at least one of the following conditions: 1) the volume ratio of water to brown algae is 1:5-15; 2) the soaking time is 6-12 hours; and / or the temperature is 10-30°C.

4. The use according to claim 1, characterized in that: The brown algae polysaccharide sulfate is extracted from at least one of the following brown algae: kelp, undaria pinnatifida, hijiki, fucus, ascophyllum nodosum, kelp, sargassum, sargassum and sea millet.

5. The use according to any one of claims 1 to 4, characterized in that: The product includes at least one of a reagent, a kit, and a medicine.

6. The use according to claim 5, characterized in that: The product is a medicine, and the dosage form of the medicine is an oral preparation or an injection preparation.

7. The use according to claim 5, characterized in that: The product is a medicine, which contains an effective therapeutic amount of the fucoidan sulfate and a pharmaceutically acceptable carrier.

8. The use according to any one of claims 1 to 4, characterized in that: The products include health products.

9. A method for reducing TMAO content, characterized in that: The method comprises the following steps: administering brown algae polysaccharide sulfate to a subject whose TMAO content needs to be reduced.

10. The method according to claim 8, characterized in that: Such subjects include individuals having elevated levels of TMAO in blood, plasma, serum and / or urine.

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