Application of fucoidin in preparation of medicine for treating radiation-induced leucopenia

Abstract

The invention discloses an application of fucoidin in preparation of a medicine for preventing or treating radiation-induced leucopenia. The effect of the fucoidin for preventing and treating radiation-induced leucopenia is investigated by measuring the influence of the fucoidin on the five-classification counting of peripheral blood leucocytes, thymus and spleen indexes, the number of bone marrow nucleated cells, the activity of bone marrow cells and the formation number of bone marrow cell colonies of a mouse with radiation-induced leucopenia. A test result shows that the fucoidin has a remarkable curative effect on preventing and treating radiation-induced leucopenia and has no toxic or side effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and specifically relates to the application of fucoidan in the preparation of a medicament for treating radiation-induced leukopenia. Background Art

[0002] Radiotherapy is to kill tumor cells by radioactive rays and is one of the main treatment means for malignant tumors at present. While killing tumor cells, radiotherapy may also cause bone marrow suppression, thereby leading to the occurrence of leukopenia. Leukopenia can reduce the immunity of patients, increase the risk of opportunistic infections, and thus affect the continuous progress of radiotherapy, increasing the risk of tumor cell metastasis in patients. Therefore, slowing down the occurrence of leukopenia is a key link for the continuous progress of tumor radiotherapy. Currently, the medicaments for treating leukopenia clinically are mainly divided into three categories, including traditional leukocyte-increasing medicaments such as leucojin and batyl alcohol, traditional Chinese medicines such as Diyushengbai Tablets and Yupingfeng Powder, and biological agents such as granulocyte colony-stimulating factor. However, they all have certain limitations. The curative effects of traditional leukocyte-increasing medicaments are unstable, traditional Chinese medicines lack clear material bases and elucidation of action mechanisms, and biological agents are expensive. Therefore, there is still a need to develop an economical leukocyte-increasing medicament with a clear action mechanism.

[0003] Fucoidan is a water-soluble sulfated heteropolysaccharide, and its main components are fucose and sulfate groups. This polysaccharide has a variety of biological activities and is widely used in the fields of medicine and food. Research shows that fucoidan can protect immune cells from the influence of radioactive radiation, relieve DNA damage of immune cells, and reduce the phenomenon of a large number of apoptotic immune cells caused by rays. However, there is currently no report on the application of fucoidan in the prevention and treatment of radiation-induced leukopenia.

[0004] Currently, there is no report on the prevention and treatment of radiation-induced leukopenia in mice by fucoidan. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of fucoidan in the preparation of a medicament for treating radiation-induced leukopenia.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect of the present invention, there is provided an application of fucoidan in the preparation of a medicament for preventing or treating radiation-induced leukopenia.

[0008] The dosage of the fucoidan is 50 - 200 mg / kg (preferably 50, 100, 200 mg / kg).

[0009] The medicament for preventing or treating radiation-induced leukopenia takes fucoidan as the only active ingredient.

[0010] The radiation is 137 Cs γ-ray.

[0011] The irradiation dose of the radiation is 1.0 Gy.

[0012] The radiation is through 137 Whole-body irradiation with Cs γ-ray, continuously irradiated for 3 days, with each irradiation dose of 1.0 Gy and each irradiation for 1 min.

[0013] The preparation method of the fucoidan includes the following steps:

[0014] Grind dry kelp into powder and sieve (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) and centrifuge to remove the precipitate; add ethanol to the supernatant and centrifuge to remove the precipitate; add ethanol again to the supernatant and filter by suction, wash the precipitate with ethanol, and freeze-dry to obtain the fucoidan.

[0015] When adding hot water, the material-liquid ratio of the powder to hot water is 1:2 - 20 (preferably 1:10).

[0016] The temperature of the hot water extraction is 90 - 100 °C (preferably 95 °C), and the time is 1 - 3 h (preferably 2 h).

[0017] The concentration of the aqueous calcium chloride solution is 1 - 10 mol / L (preferably 8 mol / L).

[0018] Adding ethanol to the supernatant and centrifuging to remove the precipitate means adding 95% ethanol to the supernatant to make the final volume fraction reach 20%.

[0019] Adding ethanol again to the supernatant and filtering by suction means adding 95% ethanol to the supernatant to make the final volume fraction reach 75%.

[0020] The relative molecular weight of the fucoidan is 195 kDa.

[0021] The polysaccharide content in the fucoidan is 99.38%.

[0022] The glucuronic acid content in the fucoidan is 23.62%.

[0023] 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%.

[0024] In the second aspect of the present invention, there is provided a fucoidan composition, which is a pharmaceutical composition composed of fucoidan and at least one pharmaceutically acceptable excipient.

[0025] In the third aspect of the present invention, there is provided an application of the fucoidan composition in the preparation of a medicament for preventing or treating radiation-induced leukopenia.

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

[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 immune function of the body, and has no toxic and side effects.

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

[0031] The present invention examines the preventive and therapeutic effects of fucoidan on radiation-induced leukopenia by measuring the effects of fucoidan on the five-classification count of peripheral blood leukocytes, thymus and spleen indices, the number of nucleated bone marrow cells, the viability of bone marrow cells, and the number of bone marrow cell colony formations in mice with radiation-induced leukopenia. The test results show that fucoidan has significant curative effects on preventing and treating radiation-induced leukopenia and has no toxic and side effects.

[0032] The present invention uses 1.0 Gy 137 Cs γ-rays to irradiate the whole body of mice continuously for 3 days to prepare a mouse model of radiation-induced leukopenia. The experimental results show that fucoidan can promote the faster recovery of leukocytes in mice caused by radiation to the normal level. At the same time, fucoidan can effectively improve the abnormal bone marrow image caused by low-dose especially low-dose radiation, increase the number of nucleated cells, enhance cell viability, stimulate colony formation, and increase thymus index and spleen index. Therefore, fucoidan can be used as a medicament for preventing and treating radiation-induced leukopenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic diagram of the effect of fucoidan on peripheral blood leukocytes in mice irradiated with 1.0 Gy γ-rays.

[0034] Figure 2 It is a schematic diagram of the effect of fucoidan on the five-classification of peripheral blood leukocytes in mice irradiated with 1.0 Gy γ-rays.

[0035] Figure 3Schematic diagram of the effect of fucoidan on the spleen index of mice irradiated with 1.0 Gy γ-rays.

[0036] Figure 4 Schematic diagram of the effect of fucoidan on the thymus index of mice irradiated with 1.0 Gy γ-rays.

[0037] Figure 5 Schematic diagram of the effect of fucoidan on the number of nucleated cells in the bone marrow of mice irradiated with 1.0 Gy γ-rays.

[0038] Figure 6 Schematic diagram of the effect of fucoidan on the viability of bone marrow cells in mice irradiated with 1.0 Gy γ-rays.

[0039] Figure 7 Schematic diagram of the effect of fucoidan on the colony formation of bone marrow cells in mice irradiated with 1.0 Gy γ-rays.

[0040] Figure 8 Schematic diagram of the relative molecular weight spectrum of fucoidan.

[0041] Figure 9 Schematic diagram of the monosaccharide composition spectrum of fucoidan.

[0042] Figure 10 Schematic diagram of the infrared spectrum of fucoidan.

[0043] Figure 11 Schematic diagram of the ultraviolet-visible light absorption spectrum of the aqueous solution of fucoidan. 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] The present invention studies the therapeutic effect on radiation-induced leukopenia in mice by observing the effects of fucoidan on the peripheral blood picture, thymus and spleen indices, the number of nucleated cells in the bone marrow, and the number of colony-forming units of mononuclear cells in the bone marrow of mice irradiated with 1.0 Gy 137 Cs γ-rays for 3 consecutive days.

[0046] Example 1

[0047] 1 Experimental materials and methods

[0048] 1.1 Instruments and reagents

[0049] Sysmex XN-1000V animal blood analyzer; CELLPACK DCL (Sysmex) whole blood diluent, CELLPACK DST (Sysmex) concentrated whole blood diluent, SULFOLYSER (Sysmex) hemolytic agent, Lysercell WNR (Sysmex) hemolytic agent, Lysercell WDF (Sysmex) hemolytic agent, CELLCLEAN AUTO (Sysmex) cleaning solution, 1.5 ml EDTA anticoagulant tube, quantitative capillary, disposable syringe, iodophor, scissors, sterile cotton swab, micropipette.

[0050] 1.2 Experimental animals

[0051] SPF-grade male BALB / C mice (weighing 18 - 22 g), purchased from Shanghai Pajielo Technology Co., Ltd. The mice were raised in a comfortable and quiet environment, with room temperature at 20 - 24 °C and relative humidity at 40 - 60%. The mice could drink water and eat freely without any other adverse factors.

[0052] 1.3 Experimental methods

[0053] Forty-eight male BALB / C mice were used in the experiment. After being raised for 3 - 5 days, their weights were measured first. Then, they were 137 irradiated with Csγ-rays throughout the body at a dose of 1.0 Gy for 3 consecutive days, with a dose rate of 100 cGy / min and each irradiation lasting for 1 min. After the first day of irradiation, intragastric administration could be carried out for 14 consecutive days. The blank group and the model group were given an equal volume of double-distilled water at the corresponding time. The peripheral blood white blood cell counts of each group were detected on the 1st, 4th, 7th, 11th, 14th, 17th, and 21st days after irradiation.

[0054] According to different administrations, they were divided into the following six groups:

[0055] Blank group: Not irradiated, given distilled water at the corresponding time, with an intragastric administration dose of 10 mL / kg, once a day for 14 consecutive days;

[0056] Model group: After being 137 irradiated with Csγ-rays throughout the body for 3 consecutive days, with each irradiation dose of 1.0 Gy and each irradiation lasting for 1 min. No drug was given after each irradiation. Distilled water was given at the corresponding time, with an intragastric administration dose of 10 mL / kg, once a day. Starting from the 4th day, no irradiation was carried out and no drug was given directly. Distilled water was given at the corresponding time, with an intragastric administration dose of 10 mL / kg, once a day. It lasted for 14 consecutive days in total.

[0057] Leucogen control group (positive control group): After being 137The mice were given whole-body irradiation with Cs γ-rays for 3 consecutive days, with a dose of 1.0 Gy each time, for 1 minute each time. After each irradiation, intragastric administration was performed. The drug was leucogen (tablet), with a dose of 20 mg / kg, once a day. Starting from the 4th day, irradiation was stopped, and only intragastric administration of leucogen (tablet) at a dose of 20 mg / kg once a day was carried out. This continued for 14 consecutive days.

[0058] Low-dose fucoidan group: 137 The mice were given whole-body irradiation with Cs γ-rays for 3 consecutive days, with a dose of 1.0 Gy each time, for 1 minute each time. After each irradiation, intragastric administration was performed. The drug was fucoidan, with a dose of 50 g / kg, once a day. Starting from the 4th day, irradiation was stopped, and only intragastric administration of fucoidan at a dose of 50 g / kg once a day was carried out. This continued for 14 consecutive days.

[0059] Medium-dose fucoidan group: 137 The mice were given whole-body irradiation with Cs γ-rays for 3 consecutive days, with a dose of 1.0 Gy each time, for 1 minute each time. After each irradiation, intragastric administration was performed. The drug was fucoidan, with a dose of 100 g / kg, once a day. Starting from the 4th day, irradiation was stopped, and only intragastric administration of fucoidan at a dose of 100 g / kg once a day was carried out. This continued for 14 consecutive days.

[0060] High-dose fucoidan group: 137 The mice were given whole-body irradiation with Cs γ-rays for 3 consecutive days, with a dose of 1.0 Gy each time, for 1 minute each time. After each irradiation, intragastric administration was performed. The drug was fucoidan, with a dose of 200 g / kg, once a day. Starting from the 4th day, irradiation was stopped, and only intragastric administration of fucoidan at a dose of 200 g / kg once a day was carried out. This continued for 14 consecutive days.

[0061] 2 Detection of peripheral blood picture

[0062] There were 8 mice in each group. On the 1st, 4th, 7th, 11th, 14th, 17th, and 21st days after irradiation, 20 μl of blood was taken from the tail vein of each mouse. The blood was added to an anticoagulant EP tube containing 480 μL of blood diluent and gently mixed. The blood picture was measured using an automatic blood cell analyzer, and the number of WBCs in the peripheral blood was used as the pharmacodynamic evaluation standard.

[0063] 3 Experimental results

[0064] The changes in peripheral blood white blood cells of each group of mice were as Figure 1 shown, Figure 1Schematic diagram of the effect of fucoidan on peripheral blood leukocytes in mice irradiated with 1.0 Gy γ-rays. As can be seen from the figure, on the 1st day after radiation exposure, the number of peripheral blood leukocytes in the model group of mice was significantly lower than that in the blank group (P<0.001), indicating successful modeling; on the 1st, 11th, and 14th days after radiation modeling, the number of leukocytes in the medium-dose fucoidan group was significantly higher than that in the model group (P<0.01, P<0.05, P<0.05); on the 14th day after modeling, the number of leukocytes in the low-dose fucoidan group, the medium-dose fucoidan group, and the positive control group were all significantly higher than that in the model group (P<0.05, P<0.05, P<0.05). On the 14th day after radiation exposure, the effect of fucoidan on increasing white blood cells was the most stable, and the medium-dose effect of fucoidan was the best. Therefore, 100 mg / kg was selected as the optimal dosage, and according to the changes in peripheral blood leukocytes of each group of mice, the 14th day after the start of the experiment was determined as the sampling time.

[0065] Example 2

[0066] Effect of fucoidan on the immune system and bone marrow image of mice with radiation-induced leukopenia:

[0067] 1 Experimental materials and methods

[0068] 1.1 Instruments and reagents

[0069] Sysmex XN-1000V animal blood analyzer; CELLPACK DCL (Sysmex) whole blood diluent, CELLPACK DST (Sysmex) concentrated whole blood diluent, SULFOLYSER (Sysmex) lysing agent, Lysercell WNR (Sysmex) lysing agent, Lysercell WDF (Sysmex) lysing agent, CELLCLEAN AUTO (Sysmex) cleaning solution, 1.5 ml EDTA anticoagulant tube, quantitative capillary, disposable syringe, iodophor, scissors, sterile cotton swab, micropipette, DMEM medium, M3434 methylcellulose semi-solid medium, fetal bovine serum, PBS buffer, CO2 cell incubator, 96-well cell culture plate, cell culture laminar flow hood, inverted phase contrast microscope, micropipette, automatic microplate reader.

[0070] 1.2 Experimental animals

[0071] SPF-grade male BALB / C mice (weighing 18 - 22 g), purchased from Shanghai Pajielo Technology Co., Ltd. The mice were kept in a comfortable and quiet environment, at 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.

[0072] 1.3 Experimental methods

[0073] Thirty-six male BALB / C mice were used in the experiment. After being fed for 3 - 5 days, their body weights were measured first. Then, they were 137 whole-body irradiated with Cs γ-rays at a dose of 1.0 Gy, 1 min each time, for 3 consecutive days, and the dose rate was 100 cGy / min. Gastric gavage administration could be carried out after the first day of irradiation for 14 consecutive days. The blank group and the model group were given an equal volume of double-distilled water at the corresponding time. Samples were taken on the 14th day after the irradiation was completed.

[0074] According to different administrations, they were divided into the following six groups:

[0075] Blank group: Without irradiation, given distilled water at the corresponding time, the gastric gavage dose was 10 mL / kg, once a day, for 14 consecutive days.

[0076] Model group: Whole-body irradiated with 137 Cs γ-rays for 3 consecutive days, with a dose of 1.0 Gy each time, 1 min each time. No drug was given after each irradiation, and distilled water was given at the corresponding time, with a gastric gavage dose of 10 mL / kg, once a day. Starting from the 4th day, no irradiation was carried out and no drug was given, and distilled water was given at the corresponding time, with a gastric gavage dose of 10 mL / kg, once a day. It lasted for 14 consecutive days.

[0077] Leucogen control group (positive control group): Whole-body irradiated with 137 Cs γ-rays for 3 consecutive days, with a dose of 1.0 Gy each time, 1 min each time. Gastric gavage administration was carried out after each irradiation, and the drug was Leucogen (tablets), with a dose of 20 mg / kg, once a day. Starting from the 4th day, no irradiation was carried out and gastric gavage administration was directly carried out, and the drug was Leucogen (tablets), with a dose of 20 mg / kg, once a day. It lasted for 14 consecutive days.

[0078] Low-dose fucoidan group: Whole-body irradiated with 137 Cs γ-rays for 3 consecutive days, with a dose of 1.0 Gy each time, 1 min each time. Gastric gavage administration was carried out after each irradiation, and the drug was fucoidan, with a dose of 50 g / kg, once a day. Starting from the 4th day, no irradiation was carried out and gastric gavage administration was directly carried out, and the drug was fucoidan, with a dose of 50 g / kg, once a day. It lasted for 14 consecutive days.

[0079] Medium-dose fucoidan group: Whole-body irradiated with 137 Cs γ-rays for 3 consecutive days, with a dose of 1.0 Gy each time, 1 min each time. Gastric gavage administration was carried out after each irradiation, and the drug was fucoidan, with a dose of 100 g / kg, once a day. Starting from the 4th day, no irradiation was carried out and gastric gavage administration was directly carried out, and the drug was fucoidan, with a dose of 100 g / kg, once a day. It lasted for 14 consecutive days.

[0080] High-dose fucoidan group: After 137 whole-body irradiation with Cs γ-rays for 3 consecutive days, with a dose of 1.0 Gy per irradiation, for 1 min each time. After each irradiation, intragastric administration was carried out. The drug was fucoidan, with a dose of 200 g / kg, once a day. Starting from the 4th day, irradiation was stopped, and only intragastric administration of fucoidan at a dose of 200 g / kg was carried out once a day for a total of 14 consecutive days.

[0081] 2 Index detection

[0082] 2.1 Five-class differential count of peripheral blood leukocytes

[0083] On the 14th day after radiation, 20 μl of blood was collected from the tail vein of each mouse. It was added to an anticoagulant EP tube containing 480 μL of blood diluent, gently mixed, and the blood picture was measured using an automatic blood cell analyzer.

[0084] 2.2 Determination of spleen and thymus indices

[0085] The thymus and spleen of BALB / C mice in each group were carefully removed with forceps, keeping the integrity of the organs. The surface liquid was blotted off, weighed on an analytical balance and photographed. The thymus index and spleen index were the ratios of their weights to the body weight, with the unit of mg / 10 g.

[0086] 2.3 Number of nucleated bone marrow cells

[0087] After blood collection, the mice were sacrificed by cervical dislocation, immediately soaked in 75% alcohol for 3 min, and the left femur was removed under sterile conditions. The attached muscle and connective tissue on the femur were carefully removed with sterile gauze as much as possible. The two ends of the femur were carefully cut open with ophthalmic scissors, and 1 mL of DMEM medium was aspirated with a 1 mL syringe. All bone marrow cells were flushed out of the bone marrow cavity into a 1.5 ml sterile EP tube, and the flushing was repeated 4 - 6 times until there was no obvious red color in the bone marrow cavity, and all bone marrow cells were flushed out as much as possible. Then, the bone marrow cells were pipetted with a 200 μL pipette gun to make a uniform single-cell suspension. 10 μL of the single-cell suspension was pipetted onto a cell automatic counting plate, and the number of nucleated bone marrow cells was measured using a cell automatic counter.

[0088] 2.4 Determination of bone marrow cell viability

[0089] After completing the counting of nucleated bone marrow cells, the bone marrow cell suspensions of each group were respectively combined into 15 ml sterile centrifuge tubes and pipetted and mixed with a 1000 μL pipette gun. 10 μL of the single-cell suspension was pipetted onto a cell automatic counting plate, and the number of nucleated bone marrow cells in each combined group was measured using a cell automatic counter. Then, an appropriate amount of the single-cell suspension was taken and the cell concentration was adjusted to 1×10 7mL. Use a multi-channel pipette to spread the single-cell suspension with adjusted concentration on a 96-well plate at 100 μL / well. Set 6 replicate wells for each group, then add 10 μL of CCK-8 solution to each well, incubate in an incubator at 37 °C for 4 hours, and finally measure the OD value at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0090] 2.5 Bone marrow cell colony formation assay

[0091] After completing the cell viability assay, filter the remaining single-cell suspensions of each group through a 70-μm cell strainer, and slowly add the single-cell suspensions of each group to the upper layer of an equal volume of Ficoll lymphocyte separation solution using a pipette. Then centrifuge at 2000 r / min and 4 °C for 20 min. The milky white cloud layer (middle layer) after centrifugation is the bone marrow mononuclear cell layer. Carefully aspirate the milky white middle layer cells into a 15-mL centrifuge tube using a pipette. Then add 5 volumes of DMEM medium to the centrifuge tube, thoroughly pipette and mix evenly, and centrifuge at 1000 r / min and 4 °C for 3 min. After pouring off the upper liquid, repeat the washing operation 2 times. Resuspend the bone marrow mononuclear cells with DMEM medium, then aspirate 10 μL of the single-cell suspension and count it on an automatic cell counter, and adjust the cell concentration to 3×10 5 ml. Add 500 μL of the cell suspension to 4.5 mL of M3434 methylcellulose semi-solid medium, vortex thoroughly to evenly disperse the cells in the semi-solid medium. At this time, the cell concentration is approximately 3×10 4 / mL. Use a pipette to evenly spread the M3434 medium containing bone marrow mononuclear cells on the middle 6 wells of a 24-well plate, 1 mL per well, and add 2 mL of PBS (containing 1% double antibody) to each of the surrounding 10 wells. Then culture in a cell incubator at 5% CO2 and 37 °C. After 10 days, carefully observe and count the number of cell clusters under a microscope. Cell clusters with more than 50 cells are counted as 1 cell colony.

[0092] 3 Experimental results

[0093] 3.1 Five-class differential count of peripheral blood white blood cells

[0094] The results of the five-class differential count of peripheral blood white blood cells are as Figure 2 shown, Figure 2 which is a schematic diagram of the effect of fucoidan on the five-class differential count of peripheral blood white blood cells in mice irradiated with 1.0 Gy γ-rays.

[0095] The results of the number of neutrophils in the peripheral blood of mice are as Figure 2As shown in Figure a, it can be seen from the figure that compared with the blank group, the number of neutrophils in the peripheral blood of mice in the model group was significantly reduced (P<0.001), indicating successful modeling; compared with the model group, the number of neutrophils in the peripheral blood of mice in the positive control group and the high-dose fucoidan group increased to varying degrees, and the number of neutrophils in the peripheral blood of mice in the low-dose fucoidan group and the medium-dose fucoidan group increased significantly (P<0.05, P<0.05).

[0096] The results of the number of lymphocytes in the peripheral blood of mice are as Figure 2 shown in Figure b. It can be seen from the figure that compared with the blank group, the number of lymphocytes in the peripheral blood of mice in the model group was significantly reduced (P<0.001), indicating successful modeling; compared with the model group, the number of lymphocytes in the peripheral blood of mice in the positive control group, the low-dose fucoidan group, the medium-dose fucoidan group, and the high-dose fucoidan group increased to varying degrees.

[0097] The results of the number of monocytes in the peripheral blood of mice are as Figure 2 shown in Figure c. It can be seen from the figure that compared with the blank group, the number of monocytes in the peripheral blood of mice in the model group was significantly reduced (P<0.05), indicating successful modeling; compared with the model group, the number of monocytes in the peripheral blood of mice in the positive control group, the low-dose fucoidan group, the medium-dose fucoidan group, and the high-dose fucoidan group increased to varying degrees.

[0098] The results of the number of eosinophils in the peripheral blood of mice are as Figure 2 shown in Figure d. It can be seen from the figure that compared with the blank group, the number of eosinophils in the peripheral blood of mice in the model group was significantly reduced (P<0.05), indicating successful modeling; compared with the model group, the number of eosinophils in the peripheral blood of mice in the positive control group, the low-dose fucoidan group, and the high-dose fucoidan group increased to varying degrees, and the number of eosinophils in the peripheral blood of mice in the medium-dose fucoidan group increased significantly (P<0.05).

[0099] The results of the number of basophils in the peripheral blood of mice are as Figure 2 shown in Figure e. It can be seen from the figure that compared with the blank group, the number of basophils in the peripheral blood of mice in the model group was significantly reduced (P<0.001), indicating successful modeling; compared with the model group, the number of basophils in the peripheral blood of mice in the positive control group, the low-dose fucoidan group, the medium-dose fucoidan group, and the high-dose fucoidan group increased to varying degrees.

[0100] 3.2 Spleen and thymus indices

[0101] The results of the spleen index of mice are as Figure 3 shown, Figure 3Schematic diagram of the effect of fucoidan on the spleen index of mice irradiated with 1.0 Gy γ-rays. It can be seen from the figure that compared with the blank group, the spleen index of mice in the model group was significantly reduced (P<0.01), indicating successful modeling; compared with the model group, the spleen indices of mice in the positive control group and the low-dose fucoidan group increased to varying degrees, and the spleen indices of mice in the medium-dose fucoidan group and the high-dose fucoidan group increased significantly (P<0.05, P<0.01).

[0102] The results of the thymus index of mice are as Figure 4 shown Figure 4 Schematic diagram of the effect of fucoidan on the thymus index of mice irradiated with 1.0 Gy γ-rays. It can be seen from the figure that compared with the blank group, the thymus index of mice in the model group was significantly reduced (P<0.05), indicating successful modeling; compared with the model group, the thymus indices of mice in the low-dose fucoidan group, the medium-dose fucoidan group, and the high-dose fucoidan group increased to varying degrees.

[0103] 3.3 Nucleated bone marrow cell count

[0104] The results of the nucleated bone marrow cell count of mice are shown in Figure 5 the figure Figure 5 Schematic diagram of the effect of fucoidan on the nucleated bone marrow cell count of mice irradiated with 1.0 Gy γ-rays. It can be seen from the figure that compared with the blank group, the nucleated bone marrow cell count of mice in the model group was significantly reduced (P<0.001), indicating successful modeling; compared with the model group, the nucleated bone marrow cell counts of mice in the low-dose fucoidan group and the high-dose fucoidan group increased to varying degrees, and the nucleated bone marrow cell counts of mice in the positive control group and the medium-dose fucoidan group increased significantly (P<0.01, P<0.001).

[0105] 3.4 Determination of bone marrow cell viability

[0106] The results of the viability of mouse bone marrow cells are shown in Figure 6 the figure Figure 6 Schematic diagram of the effect of fucoidan on the viability of mouse bone marrow cells irradiated with 1.0 Gy γ-rays. It can be seen from the figure that compared with the blank group, the viability of mouse bone marrow cells in the model group was significantly reduced (P<0.001), indicating successful modeling; compared with the model group, the viability of mouse bone marrow cells in the high-dose fucoidan group increased somewhat, and the viabilities of mouse bone marrow cells in the positive control group, the low-dose fucoidan group, and the medium-dose fucoidan group increased significantly (P<0.001, P<0.001, P<0.01).

[0107] 3.5 Bone marrow cell colony formation assay

[0108] The results of bone marrow cell colony formation in mice are shown in Figure 7 the figure Figure 7Schematic diagram of the effect of fucoidan on the colony formation of bone marrow cells in mice irradiated with 1.0 Gy γ-rays. It can be seen from the figure that compared with the blank group, the number of bone marrow cell colonies formed in the model group decreased (P<0.001), indicating that the model was successfully established; compared with the model group, the number of bone marrow cell colonies formed in the positive control group, the low-dose fucoidan group, and the high-dose fucoidan group increased to varying degrees, and the number of bone marrow cell colonies formed in the medium-dose fucoidan group increased significantly (P<0.05).

[0109] In summary, fucoidan can promote the faster recovery of irradiated mouse white blood cells to normal levels. At the same time, fucoidan can effectively improve the abnormal bone marrow image caused by low-dose especially low-dose radiation, increase the number of nucleated cells, enhance cell viability, stimulate colony formation, and increase the thymus index and spleen index. Therefore, fucoidan can be used as a drug for the prevention and treatment of radiation-induced leukopenia.

[0110] In this invention, mice were irradiated for 3 days, and drug treatment was started on the first day of mouse irradiation. Therefore, the experimental results can illustrate the preventive and therapeutic effects of fucoidan on radiation-induced leukopenia.

[0111] Example 3

[0112] The preparation method of fucoidan includes the following steps:

[0113] 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 material-liquid ratio is 1:10, 95 °C, 2 h) twice, filter with gauze to obtain 2 L of filtrate.

[0114] Second step, purification: Add 0.5 L of calcium chloride aqueous solution with a concentration of 8 mol / L to the above 2 L of filtrate, stir for 2 h, and centrifuge to remove the precipitate; add 95% ethanol to the supernatant to make its 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 its final volume fraction reach 75%, filter by suction, wash the precipitate with ethanol, and freeze-dry to obtain 4.78 g of fucoidan.

[0115] Structural characterization of fucoidan:

[0116] Molecular weight: The relative molecular weight of fucoidan is 195 kDa. The spectrum is shown in Figure 8 as shown, Figure 8 which is a schematic diagram of the relative molecular weight spectrum of fucoidan.

[0117] Monosaccharide composition: Fucoidan is a heteropolysaccharide, and fucose is its characteristic monosaccharide. See specifically in Figure 9 as shown, Figure 9It is a schematic diagram of the monosaccharide composition spectrum of fucoidan. The mass percentage 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.

[0118] The infrared absorption spectrum attribution is shown in Figure 10 and Table 1, Figure 10 It is a schematic diagram of the infrared spectrum of fucoidan.

[0119] Table 1

[0120] Ultraviolet absorption spectrum: Ultraviolet spectrum analysis shows that the sample has ultraviolet absorption in neutral aqueous solution, the wavelength of the maximum absorption peak is 254.0 nm, the absorbance is 0.211, and the molar absorption coefficient , as shown in Figure 11 shown, Figure 11 It is a schematic diagram of the ultraviolet-visible absorption spectrum of the fucoidan aqueous solution.

[0121] Polysaccharide purity: According to the method for detecting the content of acidic polysaccharides by methylene blue colorimetry, the polysaccharide content is 99.38%.

[0122] Uronic acid content: According to the metahydroxybiphenyl colorimetry method for detecting the uronic acid content, it is 23.62%.

[0123] The above are only the preferred embodiments of the present invention, and there is no limitation in any form to the present invention. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art of this patent, without departing from the scope of the technical solution of the present invention, may make some changes or modifications using the technical content prompted above as equivalent embodiments of equivalent changes. 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 present invention.

Claims

1. Use of fucoidan in the preparation of a medicament for preventing or treating radiation-induced leukopenia.

2. Use of fucoidan according to claim 1 in the preparation of a drug for preventing or treating radiation-induced leukopenia, characterized in that, The dosage of the fucoidan is 50-200 mg / kg.

3. Use of fucoidan according to claim 1 in the preparation of a medicament for preventing or treating radiation-induced leukopenia, characterized in that, The medicament for preventing or treating radiation-induced leukopenia takes fucoidan as the sole active ingredient.

4. Use of fucoidan according to claim 1 in the preparation of a medicament for preventing or treating radiation-induced leukopenia, characterized in that, The radiation is 137 Cs γ-ray.

5. Use of fucoidan according to claim 1 in the preparation of a medicament for preventing or treating radiation-induced leukopenia, characterized in that, The preparation method of the fucoidan comprises the following steps: Grind dry kelp into powder, 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 again to the supernatant for suction filtration, wash the precipitate with ethanol, and freeze-dry to obtain the fucoidan.

6. A fucoidan composition, characterized in that, It is a pharmaceutical composition composed of fucoidan and at least one pharmaceutically acceptable excipient.

7. Use of the fucoidan composition according to claim 6 in the preparation of a medicament for preventing or treating radiation-induced leukopenia.

8. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation is made of fucoidan and a medically acceptable excipient.

9. The pharmaceutical preparation according to claim 8, characterized in that, The dosage form of the pharmaceutical preparation is selected from liquid pharmaceutical agents, tablets or capsules.

10. The pharmaceutical preparation according to claim 8, characterized in that, The administration method of the pharmaceutical preparation is oral administration, intravenous injection, intraperitoneal injection or enema.

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