Fucoidan and its application

Abstract

The present invention belongs to the technical field of food processing, and specifically relates to a fucoidan and its application. The present invention limits the molecular weight, monosaccharide type and sulfate of fucoidan, and provides a fucoidan that has a significant effect on reducing tumor markers, filling the technical gap in the research related to fucoidan and tumor markers, and providing a new idea for the reduction and exploration of tumor markers.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide application, and specifically relates to fucoidan and application thereof. Background Art

[0002] Fucoidan, also known as fucoidan sulfate, fucoidan, brown algae polysaccharide sulfate, etc., is mainly derived from brown algae. It is a type of polysaccharide containing fucose and sulfate groups. It has multiple biological functions and is widely used in the medical field and modern food industry.

[0003] Tumor markers, also known as tumor markers, refer to substances that characteristically exist in malignant tumor cells, or are abnormally produced by malignant tumor cells, or are produced by the host's response to tumor stimulation. They can reflect the occurrence and development of tumors and monitor the tumor's response to treatment. The level of tumor markers reflects the health status of the human body. Better control of tumor markers can be manifested as better control of the body's health status.

[0004] Some researchers have studied the anti-tumor activity of fucoidan on mice with Lewis lung adenocarcinoma, confirming that fucoidan has certain anti-tumor and tumor-suppressing properties. However, there is not a one-to-one correspondence between tumors and tumor markers. Some people with inflammatory diseases, proliferative diseases or smoking habits may also have abnormal markers. Therefore, when the results of tumor markers exceed the reference range, it does not necessarily mean that a tumor has formed. When the tumor marker test is elevated, it is necessary to consider it clinically in combination with other means such as symptoms and imaging examinations.

[0005] There are few literature reports on whether there is a corresponding relationship between fucoidan and tumor markers, and whether all fucoidans have such a relationship with tumor markers. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a fucoidan and its application. The present invention limits the proportion of heterogeneous molecular weight of fucoidan and the types and proportions of different monosaccharides, so that the obtained fucoidan can reduce tumor markers, filling the current technical gap on the relationship between fucoidan and tumor markers.

[0007] The technical solution of the present invention is as follows:

[0008] In the first aspect of the present invention, a fucoidan is provided. According to the mass percentage, among the fucoidan, the fucoidan with a molecular weight between 380 and 1000 kDa accounts for 4.3%, the fucoidan with a molecular weight between 150 and 380 kDa accounts for 26.1%, the fucoidan with a molecular weight between 30 and 150 kDa accounts for 20.3%, and the fucoidan with a molecular weight between 20 and 30 kDa accounts for 49.3%.

[0009] Preferably, the sulfate content in the fucoidan is 5-18% by mass; more preferably 10-16.5%.

[0010] Preferably, the fucoidan includes the following monosaccharides in terms of mass percentage:

[0011] Fucose 32.34%; xylose 13.79%; glucose 0.59%; mannose 5.25%; rhamnose 0.44%; galactose 1.72%; uronic acid 3.48%.

[0012] Preferably, the fucoidan is selected from at least one of bio-enzymatically hydrolyzed laminarin, sargassum polysaccharide, laminarin and Sargassum polysaccharide.

[0013] Preferably, the fucoidan is obtained by enzymatic hydrolysis with an acidic enzyme and an alkaline enzyme in sequence, followed by separation and purification, and is specifically prepared by the following method:

[0014] S1, pretreatment: washing, removing impurities and crushing the seaweed to obtain seaweed fragments;

[0015] S2, acid enzymatic hydrolysis: adding water to the seaweed pieces, adjusting the solid-liquid mass volume ratio of the seaweed pieces to water to 1:25-35, adjusting the pH to 4.8-5.0, adding acidic enzyme, and performing enzymatic hydrolysis at 45-55°C for 1-2h to obtain an enzymatic hydrolyzate;

[0016] S3, alkaline enzymolysis: adjusting the pH of the enzymolysis solution obtained in S2 to 8.0-9.0, then adding alkaline enzyme, and performing enzymolysis reaction at 55-60°C for 1-2h to obtain a secondary enzymolysis solution, centrifuging the secondary enzymolysis solution, and collecting the supernatant;

[0017] S4, calcium precipitation: add calcium chloride to the enzymatic supernatant obtained in S3, filter out the precipitate, and collect the supernatant;

[0018] S5, gradient alcohol precipitation: concentrate the supernatant obtained in step S4, remove the precipitate by centrifugation, add 70% ethanol to precipitate, centrifuge to remove the precipitate to obtain crude fucoidan; then add 30% ethanol to precipitate, centrifuge to remove the precipitate; finally add 100% ethanol to precipitate, centrifuge to obtain the precipitate, and dry to obtain fucoidan.

[0019] In the second aspect of the present invention, there is provided a use of the above-mentioned fucoidan or the fucoidan prepared by the above-mentioned preparation method in the preparation of a product for reducing tumor markers.

[0020] Preferably, the product is selected from at least one of tablets and capsules.

[0021] Preferably, the application method is:

[0022] Fucoidan is made into powder and mixed with food supplements in a ratio of 1:0.5 to 5 for use in products that reduce tumor markers.

[0023] The beneficial effects of the present invention are:

[0024] (1) The present invention limits the molecular weight, monosaccharide types and sulfate groups of fucoidan, and provides fucoidan that can reduce tumor markers, providing a new idea for the study of the structure-activity relationship of fucoidan;

[0025] (2) The fucoidan provided by the present invention has a significant effect on reducing tumor markers, filling the technical gap in the research related to fucoidan and tumor markers, and providing a new idea for the reduction and exploration of tumor markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effects of different treatments on AFP in mice;

[0027] Figure 2 The effects of different treatments on CEA in mice;

[0028] Figure 3 The effects of different treatments on CA19-9 in mice;

[0029] Figure 4 The effects of different treatments on CA72-4 in mice;

[0030] Figure 5 The effects of different treatments on CA129 in mice;

[0031] Figure 6 Effects of different treatments on the body weight of mice;

[0032] Figure 7 The effects of different treatments on the tumor volume of mice. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0034] Example 1

[0035] A fucoidan, wherein, by mass percentage, the fucoidan has a molecular weight between 380 and 1000 kDa accounting for 4.3%, between 150 and 380 kDa accounting for 26.1%, between 30 and 150 kDa accounting for 20.3%, between 20 and 30 kDa accounting for 49.3%, and, by mass percentage, includes the following monosaccharides: 32.34% fucose; 13.79% xylose; 0.59% glucose; 5.25% mannose; 0.44% rhamnose; 1.72% galactose; and 3.48% uronic acid.

[0036] The fucoidan provided in this embodiment is a bio-enzymatically hydrolyzed Laminaria japonica polysaccharide, which is prepared by the following method:

[0037] S1, pretreatment: washing, removing impurities and crushing kelp (Laminaria japonica) to obtain kelp fragments;

[0038] S2, acid enzymatic hydrolysis: add water to the kelp pieces, adjust the solid-liquid mass volume ratio of the kelp pieces to water to 1:30, adjust the pH to 5.0, add 2.5% acid pectinase (based on the weight of the kelp, the same below), 0.3% acid cellulase, and perform enzymatic hydrolysis at 50°C for 2h to obtain an enzymatic hydrolyzate;

[0039] S3, alkaline enzymolysis: adjust the pH of the enzymolysis solution obtained in S2 to 8.0, then add 0.3% alkaline pectinase, and perform enzymolysis reaction at 60°C for 2h to obtain a secondary enzymolysis solution, centrifuge the secondary enzymolysis solution, and collect the supernatant;

[0040] S4, calcium precipitation: add calcium chloride (35% of the mass of kelp) to the enzymatic hydrolysis supernatant obtained in S3, filter out the precipitate, and collect the supernatant;

[0041] S5, gradient alcohol precipitation: concentrate the supernatant obtained in step S4, remove the precipitate, add 70% ethanol to precipitate, centrifuge to remove the precipitate to obtain crude fucoidan; then add 30% ethanol to precipitate, centrifuge to remove the precipitate; finally add 100% ethanol to precipitate, centrifuge to obtain the precipitate, and dry to obtain fucoidan.

[0042] Example 2

[0043] The fucoidan provided in this embodiment is a bio-enzymatically hydrolyzed Laminaria japonica polysaccharide, which is prepared by the following method:

[0044] S1, pretreatment: washing, removing impurities and crushing kelp (Laminaria japonica) to obtain kelp fragments;

[0045] S2, acid enzymatic hydrolysis: add water to the kelp pieces, adjust the solid-liquid mass volume ratio of the kelp pieces to water to 1:30, adjust the pH to 6.0, add 2.5% acid pectinase and 0.3% acid cellulase, and perform enzymatic hydrolysis at 55°C for 2h to obtain an enzymatic hydrolyzate;

[0046] S3, alkaline enzymolysis: adjust the pH of the enzymolysis solution obtained in S2 to 7.8, then add 0.3% alkaline pectinase, and perform enzymolysis reaction at 65°C for 2h to obtain a secondary enzymolysis solution, centrifuge the secondary enzymolysis solution, and collect the supernatant;

[0047] S4, calcium precipitation: add calcium chloride (35% of the mass of kelp) to the enzymatic hydrolysis supernatant obtained in S3, filter out the precipitate, and collect the supernatant;

[0048] S5, gradient alcohol precipitation: concentrate the supernatant obtained in step S4, remove the precipitate, add 70% ethanol to precipitate, centrifuge to remove the precipitate to obtain crude fucoidan; then add 30% ethanol to precipitate, centrifuge to remove the precipitate; finally add 70% ethanol to precipitate, centrifuge to obtain the precipitate, and dry to obtain fucoidan.

[0049] Comparative Example 1

[0050] The fucoidan provided in this embodiment is a water-extracted Laminaria japonica polysaccharide, which is prepared by the following method:

[0051] S1, pretreatment: washing, removing impurities and crushing kelp (Laminaria japonica) to obtain kelp fragments;

[0052] S2, water extraction: adding water to the kelp pieces, adjusting the solid-liquid mass volume ratio of the kelp pieces to water to 1:30, and hydrolyzing the pieces at 90°C for 4 hours to obtain a water extract;

[0053] S3, calcium precipitation: add calcium chloride (35% of the mass of kelp) to the water extraction supernatant obtained in S3, filter out the precipitate, and collect the supernatant;

[0054] S4, gradient alcohol precipitation: concentrate the supernatant obtained in step S3, remove the precipitate, add 70% ethanol to precipitate, centrifuge to remove the precipitate to obtain crude fucoidan; then add 30% ethanol to precipitate, centrifuge to remove the precipitate; finally add 100% ethanol to precipitate, centrifuge to obtain the precipitate, and dry to obtain fucoidan.

[0055] Comparative Example 2

[0056] The difference from Example 1 is that the molecular weight of the fucoidan used is: 6.6% between 470 and 2000 kDa, 51.4% between 150 and 470 kDa, and 42% between 30 and 150 kDa.

[0057] Experimental Example 1: Investigating the Effects of Fucoidan on Five Tumor Markers

[0058] 5 × 10 6 / mL of H22 cell suspension was administered for seven days to establish an H22 tumor-bearing mouse model. The fucoidan was prepared into a solution and gavaged into H22 tumor-bearing mice continuously for 14 days. Blood was collected from the cheek every three days, and after standing for half an hour, it was centrifuged at 3000rpm and 4℃ for 10 minutes to obtain serum for the measurement of tumor markers carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), carbohydrate antigen 125 (CA125), carbohydrate antigen 724 (CA724), and carbohydrate antigen 199 (CA199).

[0059] ICR male mice were 6 weeks old and randomly divided into groups according to body weight. The cultured H22 cells were diluted with PBS to a concentration of 5.0×10 6 The suspension of cells was inoculated subcutaneously in the axilla of the right forelimb of mice (0.2 mL per mouse) to establish a mouse tumor model. A blank group, a model group, and a sample group (fucoidan 1 group) were set up and divided into the following groups:

[0060] Blank group (NC): gavage with 200 μL normal saline daily;

[0061] Model group (M): cells were injected on day 0 and 200 μL of normal saline was gavaged daily;

[0062] Fucoidan group 1 (FUC): cells were injected on day 0, and 200 μL of fucoidan solution was gavaged daily.

[0063] Blood was collected from the cheek every three days, allowed to stand for half an hour, and then centrifuged at 3000 rpm for 10 minutes at 4°C to obtain serum for measuring the levels of tumor markers carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), carbohydrate antigen 125 (CA125), carbohydrate antigen 724 (CA724), and carbohydrate antigen 199 (CA199).

[0064] The results are as follows Figure 1 to Figure 5 As shown by Figure 1 to Figure 5 It can be seen that fucoidan treatment can effectively reduce the levels of five tumor markers.

[0065] After 14 days of oral administration of fucoidan, blood was collected to measure the levels of tumor markers carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). The comparative results of the experimental examples are shown in Tables 1 and 2.

[0066] Table 1 Serum carcinoembryonic antigen (CEA) levels

[0067]

[0068] Table 2 Serum alpha-fetoprotein (AFP) levels

[0069]

[0070] Test Example 2

[0071] Monitor the weight of the mice and measure their weight every three days using an electronic scale. Peel and adjust to zero before weighing. After verifying the number, place the mouse in a peeled beaker and record the weight. Then put the mouse back into the original feeding box and continue weighing the next mouse. Figure 6 As shown by Figure 6 It can be seen that after fucoidan treatment, there was no significant change in the weight of mice, indicating that fucoidan had no significant effect on the physiological characteristics of mice.

[0072] Test Example 3

[0073] Monitor the changes in tumor volume. Every three days, measure the tumor volume of the experimental mice with a vernier caliper. Fix the mouse with the left hand, and then use the vernier caliper to measure the longest diameter of the tumor and the shortest horizontal diameter perpendicular to the diameter. The tumor volume can be calculated by the formula V=abb / 2, where V represents the tumor volume, a represents the long diameter of the tumor, and b represents the short diameter of the tumor.

[0074] The results are as follows Figure 7 As shown by Figure 7 It can be seen that fucoidan treatment can significantly inhibit tumor growth in tumor-bearing mice.

Claims

1. A fucoidan, characterized in that: According to the mass percentage, the molecular weight of the fucoidan is between 380 and 1000 kDa, accounting for 4.3%, between 150 and 380 kDa, accounting for 26.1%, between 30 and 150 kDa, accounting for 20.3%, and between 20 and 30 kDa, accounting for 49.3%; The sulfate content in the fucoidan is 5-18%; The fucoidan includes the following monosaccharides: Fucose 32.34%; xylose 13.79%; glucose 0.59%; mannose 5.25%; rhamnose 0.44%; galactose 1.72%; uronic acid 3.48%.

2. The fucoidan according to claim 1, characterized in that The fucoidan is selected from at least one of kelp polysaccharide, sargassum polysaccharide, laminarin and Sargassum polysaccharide produced by bio-enzymatic hydrolysis.

3. The use of the fucoidan according to any one of claims 1 to 2 in the preparation of a product for reducing tumor markers, characterized in that: The products are tablets and capsules.

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