Fucoidin composition and application thereof in preparation of medicine for treating functional constipation

Abstract

The invention discloses an application of fucoidin in preparation of a medicine for treating functional constipation. The effect of the fucoidin or the fucoidin composition on treating functional constipation is investigated by measuring the influence of the fucoidin or the fucoidin composition on defecation parameters and various biochemical indexes of mice with functional constipation. Test results prove that the fucoidin or the fucoidin composition has a remarkable curative effect on functional constipation and has no toxic or side effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and specifically relates to a fucoidan composition and its application in the preparation of drugs for treating functional constipation. Background Art

[0002] Functional constipation (FC) is a common digestive system disease, manifested as symptoms such as difficult defecation, reduced defecation frequency, or dry stools, and there is no obvious organic lesion. Its etiology is complex and may involve multiple factors such as intestinal motility disorders, intestinal sensory abnormalities, and intestinal flora imbalance, seriously affecting the quality of life of patients. At present, the drugs used to treat functional constipation on the market mainly include stool softeners, laxatives, secretagogues, and prokinetic drugs. However, these drugs mostly have defects such as poor efficacy and toxic side effects.

[0003] The symptom of constipation was first recorded in "Huangdi Neijing" and was called difficult defecation and posterior dyschezia. Traditional Chinese medicine believes that constipation is often related to qi stagnation. Fucoidan has the effect of promoting qi movement, which can help regulate qi movement and promote intestinal peristalsis, thus achieving the effect of defecation. Secondly, the water absorption of fucoidan can increase the water in the intestine, lubricate the intestine, and help soften the feces, which conforms to the treatment principle of moistening the intestine and promoting defecation in traditional Chinese medicine. Traditional Chinese medicine emphasizes the importance of the transportation and transformation function of the spleen and stomach in defecation. Fucoidan can indirectly improve the function of the spleen and stomach and help restore normal defecation habits by regulating the intestinal flora and promoting digestion and absorption.

[0004] At present, there is no report on the treatment of functional constipation with fucoidan. Summary of the Invention

[0005] The purpose of the present invention is to provide a fucoidan composition.

[0006] Another purpose of the present invention is to provide an application of the fucoidan composition in the preparation of drugs for treating functional constipation.

[0007] In order to achieve the above purposes, the technical scheme adopted by the present invention is as follows:

[0008] In the first aspect of the present invention, there is provided an application of fucoidan in the preparation of drugs for treating functional constipation.

[0009] The preparation method of the fucoidan includes the following steps:

[0010] Grind dry kelp into powder and sieve it (60 mesh), add hot water for extraction, filter to obtain a filtrate, add an aqueous calcium chloride solution to the filtrate, stir (for 2 h), centrifuge to remove the precipitate; add ethanol to the supernatant and centrifuge to remove the precipitate; add ethanol to the supernatant again and filter by suction, wash the precipitate with ethanol, and freeze-dry to obtain the fucoidan.

[0011] When adding hot water, the ratio of powder to hot water in the feed liquid is 1:2 to 20 (preferably 1:10).

[0012] The temperature for hot water extraction is 90 to 100 degrees (preferably 95 degrees), and the time is 1 to 3 hours (preferably 2 hours).

[0013] The concentration of the calcium chloride aqueous solution is 1 to 10 mol / L (preferably 8 mol / L).

[0014] Adding ethanol to the supernatant for centrifugation to remove precipitation means adding 95% ethanol to the supernatant to make its final volume fraction reach 20%.

[0015] Adding ethanol again to the supernatant for suction filtration means adding 95% ethanol to the supernatant to make its final volume fraction reach 75%.

[0016] The relative molecular weight of the fucoidan is 195 kDa.

[0017] The polysaccharide content in the fucoidan is 99.38%.

[0018] The glucuronic acid content in the fucoidan is 23.62%.

[0019] The mass percentage content of each monosaccharide in the fucoidan: fucose 28.28%, galactose 19.28%, rhamnose 2.16%, xylose 1.11%, mannose 1.59%, glucuronic acid 1.78%, glucose 0.36%, galacturonic acid 0.26%.

[0020] The drug for treating functional constipation uses fucoidan as the sole active ingredient, or fucoidan and at least one pharmaceutically acceptable excipient form a pharmaceutical composition.

[0021] In the second aspect of the present invention, a fucoidan composition is provided, which is made of the following components by mass percentage:

[0022] Fucoidan 10 to 40%, sorbitol 30 to 70%, microcrystalline cellulose 15 to 30%, magnesium stearate 0.5 to 2%.

[0023] The fucoidan composition is made of the following components by mass percentage:

[0024] Fucoidan 20%, sorbitol 59%, microcrystalline cellulose 20%, magnesium stearate 1%.

[0025] In the third aspect of the present invention, an application of the fucoidan composition in the preparation of a drug for treating functional constipation is provided.

[0026] In the fourth aspect of the present invention, a pharmaceutical preparation is provided, which is made of fucoidan.

[0027] The dosage form of the pharmaceutical preparation is selected from liquid pharmaceutical agents, tablets or capsules.

[0028] The administration method of the pharmaceutical preparation is oral administration, intravenous injection, intraperitoneal injection or enema.

[0029] Fucoidan is a kind of dietary polysaccharide containing fucose and sulfate groups, which is derived from kelp consumed in daily life. It has a variety of biological functions, such as improving gastrointestinal diseases, anticoagulation, anti-tumor, anti-thrombosis, antiviral, antioxidant and enhancing the body's immune function, etc., and has no toxic and side effects.

[0030] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0031] The present invention examines the effect of fucoidan or fucoidan composition on the defecation parameters and various biochemical indexes of mice with functional constipation, and investigates its effect on treating functional constipation. The test results prove that fucoidan or fucoidan composition has a significant effect on treating functional constipation and has no toxic and side effects.

[0032] The present invention intragastrically administers fucoidan to mice with a functional constipation model. The time of the first black stool of the mice is significantly reduced, and the number of feces, fecal weight, fecal water content, small intestine propulsion rate and gastric emptying rate are all significantly increased. The contents of motilin, gastrin and acetylcholinesterase in the serum are significantly increased, and the content of vasoactive intestinal peptide is significantly reduced, indicating that the constipation symptoms are significantly improved. Therefore, fucoidan can be used to treat functional constipation and can be used as a drug for functional constipation.

[0033] The present invention intragastrically administers a fucoidan composition to mice with a functional constipation model. The time of the first red stool of the mice is significantly reduced, and the number of feces, fecal weight, fecal water content, small intestine propulsion rate and gastric emptying rate are all significantly increased. The contents of motilin, gastrin and acetylcholinesterase in the serum are significantly increased, and the content of vasoactive intestinal peptide is significantly reduced, indicating that the constipation symptoms are significantly improved. Therefore, the fucoidan composition can be used to treat functional constipation and can be used as a drug for functional constipation. Description of the Drawings

[0034] Figure 1 It is a schematic diagram of the relative molecular weight spectrum of fucoidan.

[0035] Figure 2 It is a schematic diagram of the monosaccharide composition spectrum of fucoidan.

[0036] Figure 3 It is a schematic diagram of the infrared spectrum of fucoidan.

[0037] Figure 4 It is a schematic diagram of the ultraviolet-visible absorption spectrum of fucoidan aqueous solution.

[0038] Figure 5 It is a schematic diagram of the effects of fucoidan on the first black stool time, the number of feces within 6 h, the weight of feces within 6 h, and the water content of feces in functional constipation model mice.

[0039] Figure 6 It is a schematic diagram of the effects of fucoidan on the small intestine propulsion distance and gastric emptying in functional constipation model mice.

[0040] Figure 7 It is a schematic diagram of the effects of fucoidan on the contents of motilin, gastrin, acetylcholinesterase, and vasoactive intestinal peptide in the serum of functional constipation model mice.

[0041] Figure 8 It is a schematic diagram of the effects of fucoidan composition on the first red stool time, the number of feces within 6 h, the weight of feces within 6 h, and the water content of feces in functional constipation model mice.

[0042] Figure 9 It is a schematic diagram of the effects of fucoidan composition on the small intestine propulsion distance and gastric emptying in functional constipation model mice.

[0043] Figure 10 It is a schematic diagram of the effects of fucoidan composition on the contents of motilin, gastrin, acetylcholinesterase, and vasoactive intestinal peptide in the serum of functional constipation model mice. Detailed implementation manners

[0044] To illustrate the present invention more clearly, the present invention will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0045] Example 1

[0046] The preparation method of fucoidan includes the following steps:

[0047] First step, extraction: Take dry kelp, grind it into powder, pass through a 60-mesh sieve, weigh 100 grams of the powder, add hot water (the temperature of the hot water is 100 °C) for extraction (the solid-liquid ratio is 1:10, 95 °C, 2 h) twice, filter with gauze, and obtain 2 L of filtrate.

[0048] Step 2, Purification: Add 0.5 L of calcium chloride aqueous solution with a concentration of 8 mol / L to the above-mentioned 2 L filtrate, stir for 2 h, and centrifuge to remove the precipitate; add 95% ethanol to the supernatant to make the final volume fraction reach 20%, let it stand for 1 hour to promote the precipitation of fucoidan, and centrifuge to remove the precipitate; add 95% ethanol to the supernatant to make the final volume fraction reach 75%, filter by suction, wash the precipitate with ethanol, and freeze-dry to obtain 4.78 g of fucoidan.

[0049] Structural characterization of fucoidan:

[0050] Molecular weight: The relative molecular weight of fucoidan is 195 kDa. The spectrum is shown in Figure 1 as follows, Figure 1 which is a schematic diagram of the relative molecular weight spectrum of fucoidan.

[0051] Monosaccharide composition: Fucoidan is a heteropolysaccharide, and fucose is its characteristic monosaccharide. See the specific content in Figure 2 as follows, Figure 2 which is a schematic diagram of the monosaccharide composition spectrum of fucoidan. The mass percentage content of each monosaccharide in fucoidan: fucose 28.28%, galactose 19.28%, rhamnose 2.16%, xylose 1.11%, mannose 1.59%, glucuronic acid 1.78%, glucose 0.36%, galacturonic acid 0.26%. Excluding the above components, the remaining components are sulfate groups, proteins, water, etc.

[0052] The infrared absorption spectrum attribution is shown in Figure 3 and Table 1, Figure 3 which is a schematic diagram of the infrared spectrum of fucoidan.

[0053] Table 1

[0054] Ultraviolet absorption spectrum: Ultraviolet spectrum analysis shows that the sample has ultraviolet absorption in neutral aqueous solution. The maximum absorption peak wavelength is 254.0 nm, the absorbance is 0.211, and the molar absorption coefficient , see Figure 4 as follows, Figure 4 which is a schematic diagram of the ultraviolet-visible absorption spectrum of fucoidan aqueous solution.

[0055] Polysaccharide purity: According to the method for detecting the content of acidic polysaccharides by methylene blue colorimetry, the polysaccharide content is 99.38%.

[0056] Uronic acid content: According to the metahydroxybiphenyl colorimetry method for detecting uronic acid content, the uronic acid content is 23.62%.

[0057] Example 2

[0058] Therapeutic effect of fucoidan on mice with functional constipation model

[0059] 1. Instruments and Materials

[0060] 1.1 Instruments

[0061] Ultraviolet-visible full-wavelength multifunctional microplate reader ( ), bench-top refrigerated centrifuge (Hitachi, Japan), electronic analytical balance (Sartorius Scientific Instruments Co., Ltd.), electrothermal constant temperature forced-air drying oven (Shanghai Huibai Instrument Co., Ltd.), surgical scissors, surgical forceps, ruler, 1 mL syringe.

[0062] 1.2 Reagents

[0063] Mosapride Citrate Tablets (National Medicine Approval No. H20203264, Fujian Haixi New Drug Innovation Co., Ltd., batch number 240503), Loperamide Hydrochloride Capsules (National Medicine Approval No. H10910085, Janssen Pharmaceutical Co., Ltd., Xi'an, batch number PHJ7511).

[0064] 1.3 Animals

[0065] SPF-grade C57BL / 6J mice (body weight 18 - 22 g), purchased from Shanghai Pajielo Technology Co., Ltd. The mice were kept in a comfortable and quiet environment, at room temperature of 20 - 24 °C, relative humidity of 40 - 60%, and the mice could drink and eat freely without any other adverse factors.

[0066] 2. Animal Grouping

[0067] After one week of adaptive feeding, the mice were randomly divided into 6 groups, with 16 mice in each group. Fucoidan was dissolved in normal saline and administered to the mice by gavage at doses of 25 mg / kg, 50 mg / kg, and 100 mg / kg respectively. At the same time, a blank group, a model group, and a positive control group were set up, and the positive drug was mosapride.

[0068] 3. Construction of Functional Constipation Mouse Model and Administration

[0069] Gavage was continued for 10 days. Starting from the first day, at 9:00 every morning, the model group, low-dose group, medium-dose group, high-dose group, and positive control group were respectively given loperamide (10 mg / Kg) by gavage once, the blank group was given normal saline, and the gavage dose was 10 mL / kg. Half an hour later, the positive control group was given mosapride (3.6 mg / kg). The low-dose group, medium-dose group, and high-dose group were respectively given 25 mg / kg, 50 mg / kg, and 100 mg / kg, and the blank group and model group were given normal saline, and the gavage dose was 10 mL / kg.

[0070] 4. Index Determination

[0071] 4.1 Determination of the time of the first black stool, the number of feces within 6 h, the weight of feces within 6 h, and the water content of feces

[0072] After the administration ended on the 10th day, the mice were fasted but allowed to drink water for 16 h. Then, 10 mice were randomly selected from each group for the following experiment: Each mouse in the group was intragastrically administered 0.2 mL of 10% activated carbon aqueous solution, and then the mice were transferred to clean empty cages, one mouse per cage, and allowed to eat and drink freely. Record the time of the first black feces excretion in the mice, the number of feces within 6 h, the weight of feces within 6 h, weigh the wet weight, dry it in an oven at a constant temperature of 56 °C for 24 h, weigh the dry weight, and calculate the water content of the feces.

[0073]

[0074] 4.2 Determination of small intestine propulsion rate and gastric emptying rate

[0075] After the administration ended on the 10th day, the mice were fasted but allowed to drink water for 16 h. Then, 6 mice were randomly selected from each group for the following experiment: Each mouse in the group was intragastrically administered 0.2 mL of 10% activated carbon aqueous solution. After 25 minutes, the animals were immediately decapitated. The abdominal cavity of the mice was opened with sterile forceps and surgical scissors. After ligating the cardiac and pyloric orifices of the stomach, the stomach was quickly removed and placed on filter paper and weighed, which was recorded as the total gastric weight. The weighed stomach was opened along the greater curvature, and the gastric contents were rinsed with phosphate buffer solution with a pH of 7.4. The filter paper was used to absorb the excess tissue fluid and phosphate buffer solution, and the net weight of the stomach was recorded. At the same time, the intestinal tract from the pylorus at the upper end to the ileocecal junction at the lower end was quickly excised and placed on a tray. The small intestine was gently straightened, and the length of the intestinal tract was measured as the total length of the small intestine, and the length from the pylorus to the activated carbon front was the activated carbon propulsion length. The calculation formulas for the small intestine propulsion rate and gastric emptying rate are as follows:

[0076] 4.3 Determination of motilin (MTL), gastrin (Gas), acetylcholinesterase (AChE), and vasoactive intestinal peptide (VIP) in serum

[0077] After the whole blood of the mice was allowed to stand at room temperature, it was centrifuged at 3000 revolutions per minute for 10 minutes to separate the serum. The contents of MTL, Gas, AChE, and VIP in the serum were measured according to the instructions of the corresponding ELISA kit. The methods and calculation formulas used for each index strictly followed the instructions of the kit.

[0078] Statistical analysis: GraphPad 10.1.2 (324) statistical software was used for data analysis. The results of the data were expressed in the form of mean ± standard error. One-way analysis of variance was used for the comparison of means among multiple groups, and the T-test was used for the comparison of statistical differences between groups. P < 0.05 was considered to have certain statistical significance.

[0079] 5. Experimental results

[0080] 5.1 Time of the first black feces, number of feces within 6 h, weight of feces within 6 h, and water content of feces

[0081] Slow transmission of colonic contents caused by colonic dyskinesia is one of the main causes of functional constipation. Slow colonic motility, increased first black stool time, and decreased fecal quantity, weight, and water content are the main manifestations of colonic motility changes in patients with functional constipation. By observing the first black stool time, the number of feces within 6 h, the fecal weight within 6 h, the fecal water content, the small intestine propulsion rate, and the gastric emptying rate in mice with functional constipation, the effect of fucoidan on intestinal motility and the therapeutic effect in mice with functional constipation were judged.

[0082] Figure 5 It is a schematic diagram of the effect of fucoidan on the first black stool time, the number of feces within 6 h, the fecal weight within 6 h, and the fecal water content in mice with functional constipation model. Figure 5 In a, it is a schematic diagram of the effect of fucoidan on the first black stool time in mice with functional constipation model (n = 10). It can be seen from the figure that by measuring the first black stool time of mice, compared with the blank group, the first black stool time of mice in the model group was significantly increased (P < 0.001), indicating successful modeling; compared with the model group, the first black stool time of mice in the low-dose group was reduced, and the first black stool time of mice in the positive control group, the medium-dose group, and the high-dose group was significantly reduced (P < 0.001, P < 0.001, P < 0.001).

[0083] Figure 5 In b, it is a schematic diagram of the effect of fucoidan on the number of feces within 6 h in mice with functional constipation model (n = 10). It can be seen from the figure that by measuring the number of feces within 6 h of mice, compared with the blank group, the number of feces within 6 h of mice in the model group was significantly reduced (P < 0.001), indicating successful modeling; compared with the model group, the number of feces within 6 h of mice in the low-dose group, the medium-dose group, the high-dose group, and the positive control group was significantly increased (P < 0.001, P < 0.01, P < 0.001, P < 0.001).

[0084] Figure 5 In c, it is a schematic diagram of the effect of fucoidan on the fecal weight within 6 h in mice with functional constipation model (n = 10). It can be seen from the figure that by measuring the fecal weight within 6 h of mice, it was found that compared with the blank group, the fecal weight within 6 h of mice in the model group was significantly reduced (P < 0.001), indicating successful modeling; compared with the model group, the fecal weight within 6 h of mice in the low-dose group increased, and the fecal weight within 6 h of mice in the medium-dose group, the high-dose group, and the positive control group was significantly increased (P < 0.05, P < 0.05, P < 0.05).

[0085] Figure 5Figure d shows the schematic results of the effect of fucoidan on the fecal water content in functional constipation model mice (n = 10). It can be seen from the figure that by measuring the fecal water content of mice, compared with the blank group, the fecal water content of model group mice was significantly reduced (P < 0.05), indicating successful modeling; compared with the model group, the fecal water content of mice in the low-dose group, high-dose group and positive control group increased slightly, and the fecal water content of mice in the medium-dose group increased significantly (P < 0.05).

[0086] 5.2 Determination of small intestine propulsion rate and gastric emptying rate

[0087] Figure 6 Figure shows the schematic results of the effect of fucoidan on the small intestine propulsion distance and gastric emptying in functional constipation model mice. Among them, Figure 6 Figure a shows the schematic results of the effect of fucoidan on the small intestine propulsion distance in functional constipation model mice (n = 6). Figure 6 Figure b shows the schematic results of the effect of fucoidan on the gastric emptying in functional constipation model mice (n = 6).

[0088] By measuring the small intestine propulsion rate of mice, compared with the blank group, the small intestine propulsion rate of model group mice was significantly reduced (P < 0.001), indicating successful modeling; compared with the model group, the small intestine propulsion rates of mice in the low-dose group and high-dose group increased to varying degrees, and the small intestine propulsion rates of mice in the positive control group and medium-dose group increased significantly (P < 0.01, P < 0.001).

[0089] By measuring the gastric emptying rate of mice, compared with the blank group, the gastric emptying rate of model group mice was significantly reduced (P < 0.001), indicating successful modeling; compared with the model group, the gastric emptying rate of mice in the low-dose group increased slightly, and the gastric emptying rates of mice in the medium-dose group, high-dose group and positive control group increased significantly (P < 0.01, P < 0.001, P < 0.01).

[0090] 5.3 Motilin (MTL), gastrin (Gas), acetylcholinesterase (AChE) and vasoactive intestinal peptide (VIP)

[0091] A variety of gastrointestinal hormones secreted by digestive tract endocrine cells participate in the regulation of colonic motility, such as MTL, Gas, etc. Abnormal expression of enteric neurotransmitters in the colonic wall may be the main pathogenesis of functional constipation. When the enteric nervous system functions normally, neurotransmitters are in a balanced state, but when neurotransmitters lose coordination, it can lead to intestinal neuromuscular dysfunction, thus affecting defecation. In the present invention, by measuring the contents of MTL, Gas, AChE, VIP in the serum of mice, the effect of fucoidan on the intestine of functional constipation mice and the therapeutic effect are judged.

[0092] Figure 7It is a schematic diagram of the results of the effects of fucoidan on the contents of motilin, gastrin, acetylcholinesterase, and vasoactive intestinal peptide in the serum of functional constipation model mice. Among them, Figure 7 a in it is a schematic diagram of the effect of fucoidan on the motilin content in the serum of functional constipation model mice (n = 6), Figure 7 b in it is a schematic diagram of the effect of fucoidan on the gastrin content in the serum of functional constipation model mice (n = 6), Figure 7 c in it is a schematic diagram of the effect of fucoidan on the acetylcholinesterase content in the serum of functional constipation model mice (n = 6), Figure 7 d in it is a schematic diagram of the effect of fucoidan on the vasoactive intestinal peptide content in the serum of functional constipation model mice (n = 6).

[0093] By measuring the motilin in the serum of mice, compared with the blank group, the motilin content in the serum of model group mice was significantly decreased (P < 0.01), indicating that the model was successfully established; compared with the model group, the motilin content in the serum of low-dose group mice increased slightly, and the motilin contents in the serum of medium-dose group, high-dose group, and positive control group mice were all significantly increased (P < 0.05, P < 0.001, P < 0.05).

[0094] By measuring the gastrin in the serum of mice, compared with the blank group, the gastrin content in the serum of model group mice was significantly decreased (P < 0.001), indicating that the model was successfully established; compared with the model group, the gastrin contents in the serum of low-dose group, medium-dose group, high-dose group, and positive control group mice were all significantly increased (P < 0.001, P < 0.05, P < 0.001, P < 0.001).

[0095] By measuring the acetylcholinesterase in the serum of mice, compared with the blank group, the acetylcholinesterase content in the serum of model group mice was significantly decreased (P < 0.01), indicating that the model was successfully established; compared with the model group, the acetylcholinesterase contents in the serum of low-dose group and positive control group mice increased slightly, and the acetylcholinesterase contents in the serum of medium-dose group and high-dose group mice were all significantly increased (P < 0.001, P < 0.05).

[0096] By measuring the vasoactive intestinal peptide in the serum of mice, compared with the blank group, the vasoactive intestinal peptide content in the serum of model group mice was significantly decreased (P < 0.001), indicating that the model was successfully established; compared with the model group, the vasoactive intestinal peptide content in the serum of low-dose group mice decreased slightly, and the vasoactive intestinal peptide contents in the serum of medium-dose group, high-dose group, and positive control group mice were all significantly decreased (P < 0.01, P < 0.001, P < 0.01).

[0097] In summary, in the present invention, fucoidan was intragastrically administered to mice with functional constipation model. The time of the first black stool in mice was significantly reduced, and the number of feces, fecal weight, fecal water content, small intestine propulsion rate and gastric emptying rate were all significantly increased. The contents of motilin, gastrin and acetylcholinesterase in serum were significantly increased, and the content of vasoactive intestinal peptide was significantly decreased, indicating that the constipation symptoms were significantly improved. Therefore, fucoidan can be used for the treatment of functional constipation and can be used as a drug for functional constipation.

[0098] Example 3

[0099] The method for preparing tablets of fucoidan composition comprises the following steps:

[0100] Put fucoidan with a mass percentage of 20%, sorbitol with a mass percentage of 59%, microcrystalline cellulose with a mass percentage of 20% and magnesium stearate with a mass percentage of 1% into a mixer, stir at a speed of 120 revolutions per minute for 30 minutes to mix evenly; press into candy tablets with a pressure of 8 KN to obtain fucoidan composition tablets, 500 mg per tablet.

[0101] Therapeutic effect of fucoidan composition tablets on mice with functional constipation model

[0102] 1. Instruments and materials

[0103] 1.1 Instruments

[0104] Ultraviolet-visible full-wavelength multifunctional microplate reader (BMG LABTECH), bench-top refrigerated centrifuge (Hitachi, Japan), electronic balance (Sartorius Scientific Instruments Co., Ltd.), electrothermal constant temperature forced air drying oven (Shanghai Huita Instrument Co., Ltd.), surgical scissors, surgical forceps, ruler, 1 mL syringe.

[0105] 1.2 Reagents

[0106] Mosapride citrate tablets (National Medicine Approval No. H20203264, Fujian Haixi New Drug Innovation Co., Ltd., batch number 240503), Loperamide hydrochloride capsules (National Medicine Approval No. H10910085, Janssen Pharmaceutical Co., Ltd., batch number PHJ7511).

[0107] 1.3 Animals

[0108] SPF-grade C57BL / 6J mice (body weight 18 - 22 g), purchased from Shanghai Pajielo Technology Co., Ltd. The mice were raised in a comfortable and quiet environment, with a room temperature of 20 - 24 °C and a relative humidity of 40 - 60%. The mice could drink water and eat freely without any other adverse factors.

[0109] 2. Animal grouping

[0110] After one week of adaptive feeding, the mice were randomly divided into 6 groups with 16 mice in each group. The fucoidan polysaccharide composition tablets were dissolved in distilled water and gavaged to the mice at doses of 125 mg / kg, 250 mg / kg, and 500 mg / kg of the fucoidan polysaccharide composition tablets respectively. At the same time, a blank group, a model group, and a positive control group were set up, and the positive drug was mosapride.

[0111] 3. Construction of a functional constipation mouse model and administration of drugs

[0112] Continuous gavage was carried out for 14 days. Starting from the first day, the blank group and the model group were given distilled water at a gavage dose of 10 mL / kg. The positive control group was given mosapride (3.6 mg / kg), and the low-dose, medium-dose, and high-dose groups of the fucoidan polysaccharide composition tablets were given 125 mg / kg, 250 mg / kg, and 500 mg / kg respectively.

[0113] 4. Index determination

[0114] 4.1 Determination of the time of the first red feces, the number of feces within 6 h, the weight of feces within 6 h, and the water content of feces

[0115] After continuous gavage for 14 days, the mice in each group were fasted but allowed to drink water for 16 hours. The model group, the low-dose, medium-dose, and high-dose groups of the fucoidan polysaccharide composition tablets, and the positive control group were respectively gavaged with loperamide (10 mg / Kg) once, and the blank group was given distilled water at a gavage dose of 10 mL / kg. After 0.5 hour, the positive control group was given a carmine solution containing the positive test substance (take 1 g of carmine powder and add it to 49 ml of distilled water to prepare a carmine solution with a concentration of 2%, and the gavage dose is 10 ml / kg. Mix the carmine with the gastric lavage fluid to form a mixed solution). The low-dose, medium-dose, and high-dose groups of the fucoidan polysaccharide composition tablets were given a carmine solution containing the corresponding test nutrient, the model group and the blank group were given a 2% carmine solution, and the timing was started at the same time. When observing defecation, the animals were housed individually in cages and allowed to eat and drink freely. Record the time of the first red feces excretion of the mice, the number of feces within 6 h, the weight of feces within 6 h, weigh the wet weight, dry it in a constant temperature drying oven at 56 °C for 24 h, weigh the dry weight, and calculate the water content of feces.

[0116]

[0117] 4.2 Determination of small intestine propulsion rate and gastric emptying rate

[0118] After the administration on the 14th day ended, the animals were fasted but allowed to drink water for 16 h. Six animals were randomly selected from each group for the following experiment: the model group, the low-dose, medium-dose, and high-dose groups of the fucoidan composition tablets, and the positive control group were each intragastrically administered loperamide (10 mg / Kg) once, the blank group was given distilled water, and the intragastric administration dose was 10 mL / kg. After 0.5 h, the positive control group was given carmine solution containing the positive test substance, the low-dose, medium-dose, and high-dose groups of the fucoidan composition tablets were given carmine solution containing the corresponding test nutrient, the model group and the blank group were given carmine solution with a concentration of 2%, and the timing started simultaneously. After 25 minutes, the animals were immediately sacrificed by cervical dislocation. The abdominal cavity of the mice was opened with sterile forceps and surgical scissors. After ligating the cardiac and pyloric orifices of the stomach, the stomach was quickly removed and placed on filter paper, weighed, and recorded as the total stomach weight. The weighed stomach was opened along the greater curvature, and the gastric contents were rinsed with phosphate buffer solution with a pH of 7.4. The filter paper was used to absorb the excess tissue fluid and phosphate buffer solution, and the net weight of the stomach was recorded. At the same time, the intestinal tract from the pylorus at the upper end to the ileocecal junction at the lower end was quickly cut and placed on a tray. The small intestine was gently straightened, and the length of the intestinal tract was measured as the total length of the small intestine. The length from the pylorus to the carmine front was the carmine propulsion length.

[0119] The calculation formulas for the small intestine propulsion rate and gastric emptying rate are as follows:

[0120] 4.3 Determination of motilin (MTL), gastrin (Gas), acetylcholinesterase (AChE), and vasoactive intestinal peptide (VIP) in serum

[0121] After the whole blood of the mice was allowed to stand at room temperature, it was centrifuged at 3000 revolutions per minute for 10 minutes to separate the serum. The contents of MTL, Gas, AChE, and VIP in the serum were determined according to the instructions of the corresponding ELISA kit. The methods and calculation formulas used for each index were strictly in accordance with the kit instructions.

[0122] Statistical analysis: GraphPad 10.1.2 (324) statistical software was used for data analysis. The results of the data were expressed in the form of mean ± standard error. One-way ANOVA was used for the comparison of means among multiple groups, and the T-test was used for the comparison of statistical differences between groups. P<0.05 indicated that the difference had certain statistical significance.

[0123] 5. Experimental results

[0124] 5.1 Time of the first red feces, number of feces within 6 h, weight of feces within 6 h, and water content of feces

[0125] The slow transmission of colonic contents caused by colonic dyskinesia is one of the main causes of functional constipation. The main manifestations of colonic motility changes in patients with functional constipation are slow colonic motility, increased first red feces time, and decreased fecal quantity, weight, and water content. By observing the first red feces time, the number of feces within 6 h, the fecal weight within 6 h, the fecal water content, the small intestine propulsion rate, and the gastric emptying rate of mice with functional constipation, the effects of the fucoidan composition on intestinal motility and the therapeutic effect of functional constipation mice were judged.

[0126] Figure 8 It is a schematic diagram of the effects of the fucoidan composition on the first red feces time, the number of feces within 6 h, the fecal weight within 6 h, and the fecal water content of mice with functional constipation model. Among them, Figure 8 a in it is a schematic diagram of the effect of the fucoidan composition on the first red feces time of mice with functional constipation model (n = 10), Figure 8 b in it is a schematic diagram of the effect of the fucoidan composition on the number of feces within 6 h of mice with functional constipation model (n = 10), Figure 8 c in it is a schematic diagram of the effect of the fucoidan composition on the fecal weight within 6 h of mice with functional constipation model (n = 10), Figure 8 d in it is a schematic diagram of the effect of the fucoidan composition on the fecal water content of mice with functional constipation model (n = 10).

[0127] By measuring the first red feces time of mice, compared with the blank group, the first red feces time of mice in the model group was significantly increased (P < 0.001), indicating that the model was successfully established; compared with the model group, the first red feces time of mice in the low-dose fucoidan composition tablet group was reduced, and the first red feces time of mice in the positive control group, the medium-dose fucoidan composition tablet group, and the high-dose fucoidan composition tablet group was significantly reduced (P < 0.05, P < 0.05, P < 0.05).

[0128] By measuring the number of feces within 6 h of mice, compared with the blank group, the number of feces within 6 h of mice in the model group was significantly reduced (P < 0.001), indicating that the model was successfully established; compared with the model group, the number of feces within 6 h of mice in the low-dose fucoidan composition tablet group increased, and the number of feces within 6 h of mice in the positive control group, the medium-dose fucoidan composition tablet group, and the high-dose fucoidan composition tablet group was significantly increased (P < 0.05, P < 0.001, P < 0.01).

[0129] By measuring the fecal weight of mice within 6 hours, compared with the blank group, the fecal weight of mice in the model group within 6 hours was significantly reduced (P<0.001), indicating successful modeling; compared with the model group, the fecal weights of mice in the positive control group, the low-dose fucan polysaccharide composition tablet group, the medium-dose fucan polysaccharide composition tablet group, and the high-dose fucan polysaccharide composition tablet group within 6 hours were all significantly increased (P<0.05, P<0.05, P<0.001, P<0.05).

[0130] By measuring the water content of mice feces, compared with the blank group, the water content of mice feces in the model group was significantly reduced (P<0.001), indicating successful modeling; compared with the model group, the water content of feces in the positive control group, the low-dose fucan polysaccharide composition tablet group, and the high-dose fucan polysaccharide composition tablet group increased slightly, and the water content of feces in the medium-dose fucan polysaccharide composition tablet group increased significantly (P<0.05).

[0131] 5.2 Determination of small intestine propulsion rate and gastric emptying rate

[0132] Figure 9 It is a schematic diagram of the results of the effect of fucan polysaccharide composition on the small intestine propulsion distance and gastric emptying of functional constipation model mice. Among them, Figure 9 a in it is a schematic diagram of the effect of fucan polysaccharide composition on the small intestine propulsion distance of functional constipation model mice (n = 6). Figure 9 b in it is a schematic diagram of the effect of fucan polysaccharide composition on the gastric emptying of functional constipation model mice (n = 6).

[0133] By measuring the small intestine propulsion rate of mice, compared with the blank group, the small intestine propulsion rate of mice in the model group was significantly reduced (P<0.001), indicating successful modeling; compared with the model group, the small intestine propulsion rates of mice in the low-dose fucan polysaccharide composition tablet group and the high-dose fucan polysaccharide composition tablet group increased to varying degrees, and the small intestine propulsion rates of mice in the positive control group and the medium-dose fucan polysaccharide composition tablet group increased significantly (P<0.05, P<0.01).

[0134] By measuring the gastric emptying rate of mice, compared with the blank group, the gastric emptying rate of mice in the model group was significantly reduced (P<0.001), indicating successful modeling; compared with the model group, the gastric emptying rates of mice in the positive control group, the low-dose fucan polysaccharide composition tablet group, and the high-dose fucan polysaccharide composition tablet group increased to varying degrees, and the gastric emptying rate of mice in the medium-dose fucan polysaccharide composition tablet group increased significantly (P<0.01).

[0135] 5.3 Motilin (MTL), gastrin (Gas), acetylcholinesterase (AChE) and vasoactive intestinal peptide (VIP)

[0136] Multiple gastrointestinal hormones secreted by gastrointestinal endocrine cells are involved in the regulation of colonic motility, such as MTL, Gas, etc. Abnormal expression of enteric neurotransmitters in the colonic wall may be the main pathogenesis of functional constipation. When the enteric nervous system functions normally, neurotransmitters are in a balanced state, but when neurotransmitters lose coordination, it can lead to intestinal neuromuscular dysfunction, thereby affecting defecation. In this invention, by measuring the contents of MTL, Gas, AChE, and VIP in the serum of mice, the effects and therapeutic effects of fucoidan compositions on the intestines of mice with functional constipation are judged.

[0137] Figure 10 It is a schematic diagram of the results of the effects of fucoidan compositions on the contents of motilin, gastrin, acetylcholinesterase, and vasoactive intestinal peptide in the serum of mice with a functional constipation model. Among them, Figure 10 a in it is a schematic diagram of the results of the effects of fucoidan compositions on the content of motilin in the serum of mice with a functional constipation model (n = 6), Figure 10 b in it is a schematic diagram of the results of the effects of fucoidan compositions on the content of gastrin in the serum of mice with a functional constipation model (n = 6), Figure 10 c in it is a schematic diagram of the results of the effects of fucoidan compositions on the content of acetylcholinesterase in the serum of mice with a functional constipation model (n = 6), Figure 10 d in it is a schematic diagram of the results of the effects of fucoidan compositions on the content of vasoactive intestinal peptide in the serum of mice with a functional constipation model (n = 6).

[0138] By measuring motilin in the serum of mice, compared with the blank group, the content of motilin in the serum of mice in the model group was significantly reduced (P < 0.001), indicating that the modeling was successful; compared with the model group, the content of motilin in the serum of mice in the low-dose group increased slightly, and the contents of motilin in the serum of mice in the positive control group, medium-dose group, and high-dose group were all significantly increased (P < 0.05, P < 0.001, P < 0.001).

[0139] By measuring gastrin in the serum of mice, compared with the blank group, the content of gastrin in the serum of mice in the model group was significantly reduced (P < 0.001), indicating that the modeling was successful; compared with the model group, the content of gastrin in the serum of mice in the low-dose group increased slightly, and the contents of gastrin in the serum of mice in the positive control group, medium-dose group, and high-dose group were all significantly increased (P < 0.01, P < 0.001, P < 0.001).

[0140] By measuring acetylcholinesterase in the serum of mice, compared with the blank group, the content of acetylcholinesterase in the serum of mice in the model group was significantly reduced (P < 0.001), indicating that the modeling was successful; compared with the model group, the content of acetylcholinesterase in the serum of mice in the positive control group and low-dose group increased slightly, and the contents of acetylcholinesterase in the serum of mice in the medium-dose group and high-dose group were all significantly increased (P < 0.001, P < 0.01).

[0141] By measuring vasoactive intestinal peptide in the serum of mice, compared with the blank group, the content of vasoactive intestinal peptide in the serum of mice in the model group was significantly reduced (P<0.001), indicating successful modeling; compared with the model group, the content of vasoactive intestinal peptide in the serum of mice in the positive control group and the low-dose group was slightly reduced, and the content of vasoactive intestinal peptide in the serum of mice in the medium-dose group and the high-dose group was significantly reduced (P<0.05, P<0.05).

[0142] In summary, in the present invention, the fucoidan composition was intragastrically administered to mice with a functional constipation model. The time of the first red stool in the mice was significantly reduced, and the number of feces, fecal weight, fecal water content, small intestine propulsion rate, and gastric emptying rate were all significantly increased. The contents of motilin, gastrin, and acetylcholinesterase in the serum were significantly increased, and the content of vasoactive intestinal peptide was significantly reduced, indicating that the constipation symptoms were significantly improved. Therefore, the fucoidan composition can be used to treat functional constipation and can be used as a drug for functional constipation.

[0143] The above are only the preferred embodiments of the present invention and do not limit the present invention in any form. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art of this patent can make some changes or modifications to equivalent embodiments by using the technical content prompted above within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change, and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. Use of fucoidan in the preparation of a medicament for treating functional constipation.

2. Use of fucoidan according to claim 1 in the preparation of a medicament for treating functional constipation, characterized in that, The preparation method of the fucoidan comprises the following steps: Grind dry kelp into powder and sieve it, add hot water for extraction, filter to obtain a filtrate, add an aqueous calcium chloride solution to the filtrate, stir and centrifuge to remove the precipitate; add ethanol to the supernatant and centrifuge to remove the precipitate; add ethanol to the supernatant again and filter by suction, wash the precipitate with ethanol, and freeze-dry to obtain the fucoidan.

3. Use of fucoidan according to claim 2 in the preparation of a medicament for treating functional constipation, characterized in that, The relative molecular weight of the fucoidan is 195 kDa; The polysaccharide content in the fucoidan is 99.38%; The uronic acid content in the fucoidan is 23.62%.

4. Use of fucoidan according to claim 2 in the preparation of a medicament for treating functional constipation, characterized in that, The mass percentage content of each monosaccharide in the fucoidan: fucose 28.28%, galactose 19.28%, rhamnose 2.16%, xylose 1.11%, mannose 1.59%, glucuronic acid 1.78%, glucose 0.36%, galacturonic acid 0.26%.

5. Use of fucoidan according to claim 1 in the preparation of a medicament for treating functional constipation, characterized in that, The medicament for treating functional constipation is made of fucoidan as the sole active ingredient, or a pharmaceutical composition is formed by fucoidan and at least one pharmaceutically acceptable excipient.

6. A fucoidan composition, characterized in that, It is made of components with the following mass percentage contents: Fucoidan 10 - 40%, sorbitol 30 - 70%, microcrystalline cellulose 15 - 30%, magnesium stearate 0.5 - 2%.

7. The fucoidan composition according to claim 6, wherein The fucoidan composition is made of components with the following mass percentage contents: Fucoidan 20%, sorbitol 59%, microcrystalline cellulose 20%, magnesium stearate 1%.

8. Use of the fucoidan composition according to claim 6 or 7 in the preparation of a medicament for treating functional constipation.

9. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation is made of fucoidan.

10. The pharmaceutical preparation according to claim 9, characterized in that, The dosage form of the pharmaceutical preparation is selected from liquid medicaments, tablets or capsules.

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